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Membrane-bound regulation of hematopoietic stem cells
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Membrane-bound regulation of hematopoietic stem cells
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Content
MEMBRANE-BOUND REGULATION OF
HEMATOPOIETIC STEM CELLS
by
Jia Hao
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOMEDICAL ENGINEERING)
August 2022
Copyright 2022 Jia Hao
ii
Dedication
This dissertation is dedicated to my daughter, Presley Cai. You are my love and delight
every day. May you never stop learning.
iii
Acknowledgments
This dissertation would not have been possible without the financial support of the National
Institutes of Health, the USC Viterbi School of Engineering, an Eli and Edythe Broad Innovation
Award, a Rose Hills Fellowship, a STOP CANCER Marni Levine Memorial Research Career
Development Award, the Phi Beta Psi Charity Trust, and the USC Diversity, Inclusion and Access
(DIA) Ph.D. Fellowship. I would like to express my warmest gratitude to my advisor, Dr. Keyue
Shen. Thank you for offering me the excellent opportunity to pursue my passion for science. Your
personal and professional guidance carried me through all the stages of my Ph.D. Your persistence
and diligence inspire me and will serve as an encouragement for a lifetime. My dissertation
committee members, Dr. Rong Lu, Dr. Megan McCain, Dr. Stacey Finley, and Dr. Yong-Mi
Kim, thank you for your contributions to my development and growth from a student to a scientist.
I am grateful to my best labmates and friends, who have provided me with warmhearted
help, professional insights into my research, and excellent collaboration. Hao Zhou, Kristen
Nemes, Daniel Yen, Hydari Begum, Winfield Zhao, Bowen Wang, Bramlett Charles, Jeong
min Oh, and Irene Li. You have made the past five years of my life a journey filled with laughter.
Nobody has been more important to my achievement than my family members. I am
forever indebted to my parents, Chunyan Yu ( 于 春艳) and Liming Hao ( 郝立明). No words will
be enough to express my gratitude for being your child. Your years of continued love and support
are the foundation for my completion of this degree. Most importantly, I wish to thank Henry Cai
( 蔡雷鸣), my loving and caring husband, thank you for always being there for me during my ups
and downs. Presley Cai ( 蔡星苒), my baby girl, thank you for your arrival; the best thing ever
happened to me.
iv
Table of Contents
Dedication ................................................................................................................................. ii
Acknowledgments ................................................................................................................... iii
List of Tables .......................................................................................................................... vii
List of Figures........................................................................................................................ viii
Abstract .....................................................................................................................................x
Chapter 1: Introduction ............................................................................................................1
1.1 Hematopoietic stem cells (HSCs) and their niches ......................................................................................... 1
1.1.1 Cellular components of HSC niches ....................................................................................................... 4
1.1.2 Soluble factors....................................................................................................................................... 6
1.1.3 Membrane-bound factors ..................................................................................................................... 10
1.1.4 Adhesive signaling and ECM matrix .................................................................................................... 16
Immunoglobulin superfamily (IgSF) cell adhesion molecules (CAMs) ........................................................... 18
1.2 Niche regulation on cell functions ............................................................................................................... 20
1.2.1 HSC morphology and cell migration .................................................................................................... 20
1.2.2 HSC fate decisions .............................................................................................................................. 22
1.3 Existing methods for studying HSC-niche cell interaction ............................................................................ 25
1.3.1 Animal models .................................................................................................................................... 25
1.3.2 Cellular models ................................................................................................................................... 28
1.3.3 Surface immobilization ........................................................................................................................ 29
1.3.4 Supported lipid bilayer (SLB) model .................................................................................................... 30
1.4 Overview .................................................................................................................................................... 32
Chapter 2: Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of
HSCs ........................................................................................................................................ 33
2.1 Rationale .................................................................................................................................................... 33
2.2 Materials and Methods ............................................................................................................................... 36
2.2.1 Mice .................................................................................................................................................... 36
2.2.2 Cell isolation and flow cytometry ........................................................................................................ 36
2.2.3 Quantitative PCR................................................................................................................................. 40
2.2.4 Fabrication of lipid bilayer and cell loading chambers .......................................................................... 40
2.2.5 Preparation of small unilamellar vesicles and supported lipid bilayers .................................................. 40
2.2.6 Protein capturing on lipid bilayer ......................................................................................................... 41
2.2.7 Cell seeding and incubation ................................................................................................................. 41
2.2.8 Fluorescence microscopy ..................................................................................................................... 42
2.2.9 Total Internal Reflection Fluorescence (TIRF) microscopy ................................................................... 42
2.2.10 Scanning electron microscopy (SEM) ................................................................................................ 42
2.2.11 Shear flow and adhesion analysis ....................................................................................................... 43
2.2.12 Treatments with inhibitors ................................................................................................................. 44
2.2.13 Immunofluorescence staining............................................................................................................. 45
2.2.14 Image analysis ................................................................................................................................... 45
v
2.2.15 Statistics ............................................................................................................................................ 45
2.3 Results........................................................................................................................................................ 46
2.3.1 Membrane-bound SCF is the only factor recruited/clustered by HSCs in a screening ............................ 46
2.3.2 HSCs are highly efficient in recruiting mSCF with a distinct cell morphology ...................................... 51
2.3.3 VCAM-1 promotes polarized morphology and mSCF recruitment pattern in HSCs ............................... 55
2.3.4 mSCF and VCAM-1 synergistically promote HSC adhesion ................................................................ 60
2.3.5 mSCF redistribution and morphological transition require cytoskeletal remodeling ............................... 62
2.3.6 mSCF-VCAM-1 synergy involves PI3K signaling ............................................................................... 66
2.3.7 mSCF-VCAM-1 synergy promotes nuclear FOXO3a retention............................................................. 71
2.3.8 Soluble SCF competitively disrupts mSCF-VCAM-1 synergy .............................................................. 73
2.4 Discussion .................................................................................................................................................. 76
Chapter 3: Adhesion-based cell selection on artificial stromal membrane enriches
hematopoietic stem cells .......................................................................................................... 83
3.1 Rationale .................................................................................................................................................... 83
3.2 Materials and methods................................................................................................................................ 85
3.2.1 Mice. ................................................................................................................................................... 85
3.2.2 Cell isolation and flow cytometry ........................................................................................................ 85
3.2.3 Preparation of small unilamellar vesicles and supported lipid bilayers. ................................................. 85
3.2.4 Protein capturing on lipid bilayer ......................................................................................................... 86
3.2.5 Cell seeding and incubation ................................................................................................................. 86
3.2.6 Fluorescence microscopy ..................................................................................................................... 86
3.2.7 Design, fabrication, and assembly of the microfluidic device ................................................................ 87
3.2.8 COMSOL simulation of flow rate distribution ...................................................................................... 87
3.2.9 Shear flow and adhesion analysis ......................................................................................................... 87
3.2.10 Statistics ............................................................................................................................................ 88
3.2.11 CFU assay ......................................................................................................................................... 88
3.3 Results........................................................................................................................................................ 89
3.3.1 Adhesion profiling of HSCs and MPPs ................................................................................................ 89
3.3.2 Experimental setup of scaled-up cell enrichment device ....................................................................... 91
3.3.3 HSC markers can be enriched using snake device ................................................................................. 93
3.3.4 Adhesion-enriched cells contain cells similar to colony forming HSCs ................................................. 96
3.3 Discussion .................................................................................................................................................. 98
Chapter 4: Engineering a pillar-free diffusion device for studying cell chemotaxis on lipid
bilayers .................................................................................................................................. 100
4.1 Rationale .................................................................................................................................................. 100
4.2 Materials and Methods ............................................................................................................................. 102
4.2.1 Design, fabrication, and assembly of the microfluidic device .............................................................. 102
4.2.2 Formation of gel diffusion barrier and workflow of the device ............................................................ 102
4.2.3 COMSOL simulation and characterization of transport phenomena with FITC-Dextran ...................... 103
4.2.4 Preparation of supported lipid bilayers and protein tethered surfaces .................................................. 104
4.2.5 ICAM-1 capturing on lipid bilayer and immobilization....................................................................... 104
4.2.6 Cell seeding and incubation ............................................................................................................... 105
4.2.7 Imaging, cell tracking, and data analysis ............................................................................................ 105
4.2.8 Statistics ............................................................................................................................................ 106
4.3 Results...................................................................................................................................................... 106
vi
4.3.1 A multichannel device design allows for separate lipid bilayer and chemoattractant gradient formation
.................................................................................................................................................................. 106
4.3.2 Channel height and surface treatment are key to liquid pinning-based hydrogel barrier formation ....... 108
4.3.3 Coating hydrogel channel is necessary to prevent leakage of soluble factors ....................................... 110
4.3.4 Gradient profiles within the device can be optimized through COMSOL simulation ........................... 112
3.3.5 Fluorescence recovery after photobleaching confirms lipid bilayer formation and mobility in the device
.................................................................................................................................................................. 116
4.3.6 Jurkat cells have different chemotactic profiles on membrane bound vs. immobilized ICAM-1 ........... 119
4.3.7 mSCF-VCAM-1 regulate HSC motility but not migration .................................................................. 122
4.4 Discussion ................................................................................................................................................ 123
Chapter 5: Concluding Remarks and Future Directions .................................................... 125
References.............................................................................................................................. 127
vii
List of Tables
Table 1-1. Factors in bone marrow niches contributing to HSC activities. ................................................ 7
Table 1-2. Mouse models established to study membrane bound stem cell–niche interactions. ............... 26
Table 2-1. Cell surface markers for sorting hematopoietic populations. .................................................. 36
Table 2-2. Antibodies used for immunofluorescence staining. ................................................................ 38
Table 2-3. Diffusion coefficients of membrane-bound factors on supported lipid bilayer. ....................... 47
viii
List of Figures
Figure 1-1. Development of the hematopoietic system. ............................................................................ 3
Figure 1-2 Cellular components in the bone marrow HSC niche............................................................... 6
Figure 1-3. Alternative splicing of SCF.................................................................................................. 12
Figure 1-4. Membrane-bound regulation of stem cell functions. ............................................................. 16
Figure 1-5. Asymmetrical segregation of AP2A2 during mitosis. ........................................................... 24
Figure 1-6. Extrinsic and intrinsic regulation of stem cell polarity and asymmetric division. .................. 25
Figure 1-7. Supported lipid bilayer as a model of the cell membrane. ..................................................... 30
Figure 1-8. Modeling immunological synapse (IS) with supported lipid bilayer (SLB) model................. 31
Figure 2-1. Measuring diffusion coefficients of the membrane-bound factors on supported lipid bilayers
with fluorescence recovery after photobleaching (FRAP). ...................................................................... 47
Figure 2-2. Gating for hematopoietic and stromal populations from bone marrow. ................................. 49
Figure 2-3. Membrane-bound SCF is the only factor recruited/clustered by HSCs in a screening. ........... 50
Figure 2-4. HSCs are highly efficient in recruiting mSCF with distinct cell morphology. ....................... 54
Figure 2-5. Morphological maintenance of HSCs and the role of VCAM-1 in HSC adhesion. ................ 55
Figure 2-6. VCAM-1 promotes polarized morphology and mSCF recruitment pattern in HSCs. ............. 58
Figure 2-7. Dynamic regulation of HSC-mSCF interaction by VCAM-1 and importance of lateral mobility
in mSCF-VCAM-1 synergy. .................................................................................................................. 59
Figure 2-8. mSCF and VCAM-1 synergistically promote HSC adhesion. ............................................... 61
Figure 2-9. mSCF redistribution and morphological transition require cytoskeletal remodeling. ............. 64
Figure 2-10. Regulation of HSC adhesion and mSCF distribution by cytoskeletal and Akt inhibitors. .... 65
Figure 2-11. mSCF-VCAM-1 synergy involves PI3K-Akt signaling. ..................................................... 69
Figure 2-12. Regulation of HSC adhesion and mSCF distribution by cytoskeletal and Akt inhibitors. .... 70
Figure 2-13. mSCF-VCAM-1 synergy promotes nuclear FOXO3a retention. ......................................... 72
Figure 2-14. Soluble SCF competitively disrupts mSCF-VCAM-1 synergy. ........................................... 76
Figure 3-1. A lipid bilayer model tethered with SCF and VCAM-1 used for adhesion profiling of HSCs and
MPPs. ................................................................................................................................................... 91
ix
Figure 3-2. Platform designed for adhesion-based cell selection from bone marrow. .............................. 92
Figure 3-3. Adhesion towards SCF-VCAM-1 enriches Hoxb5-expressing cells. ..................................... 93
Figure 3-4. Adhesion towards mSCF-VCAM-1 enriches phenotypic HSC-like cells. ............................. 96
Figure 3-5. Enriched cells are slow colony forming units similar to HSCs. ............................................. 97
Figure 4-1 Design of a microchannel diffusion device for chemotactic studies. .................................... 108
Figure 4-2. Dependence of liquid pinning on device parameters and surface treatment. ........................ 110
Figure 4-3. Effect of hydrogel type and poly-D-lysine precoating. ....................................................... 112
Figure 4-4. Characterization of diffusion with COMSOL Multiphysics® and FITC-Dextran diffusion. 116
Figure 4-5 Lipid bilayer formation and confirmation of lateral mobility of the membrane-bound ICAM-1.
............................................................................................................................................................ 118
Figure 4-6. Jurkat cell chemotaxis towards CXCL12 on membrane bound vs immobilized ICAM-1 in the
diffusion microdevice. ......................................................................................................................... 121
Figure 4-7. mSCF-VCAM-1 promote HSC local motility but not migration. ........................................ 122
x
Abstract
In vivo, adult stem cells reside in specific tissue locations known as their niches, which
contain certain combinations of factors that maintain and regulate the stem cell functions.
Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized
cues and regulate the long-term functions of adult stem cells. Yet little is known about the
underlying mechanisms. Elucidating those factors and their underlying mechanisms is thus
instrumental to stem cell biology and regenerative medicine. In this dissertation, we used a
tethered supported lipid bilayer (SLB) model to recapitulate the membrane-bound interactions
between hematopoietic stem cells (HSCs) and niche stromal cells, and investigated the effects of
membrane-bound factors on HSC morphology and adhesion, using mouse cells as a model. HSCs
cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a
polarized morphology with aggregated mSCF under a large protrusion through a synergy with
vascular cell adhesion molecule 1 (VCAM-1) on the bilayer, which drastically enhances HSC
adhesion, instead of migration, as assessed in a microfluidic model. These features are unique to
mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF-
VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal
reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC
quiescence, and minimizes its loss induced by soluble SCF. As a proof-of-concept, we further
demonstrated the feasibility of applying the observed synergy to HSC enrichment from a pool of
progenitors. Our work thus established a novel mechanism of mSCF and VCAM-1 in
synergistically regulating HSC behaviors (protrusion, adhesion, proliferation) in the bone marrow
niche, which can potentially be translated to human HSCs and benefit basic/clinical research and
applications.
1
Chapter 1: Introduction
Adult stem cells exist in many tissues, particularly those characterized by rapid cell
turnover rates, such as the blood, epidermis, and intestine [1]. They hold great therapeutic potential
due to their ability to self-renew and differentiate into mature somatic cells for tissue/organ repair
and regeneration. Adult stem cells often reside in specific tissue microenvironments known as the
niches, which regulate the survival, proliferation, and differentiation of stem cells through
localized signals produced by niche stromal cells [2-4]. Among those, the membrane-bound factors,
defined here as the membrane-tethered isoforms of growth factors or cytokines/chemokines and
the cell-cell adhesion molecules expressed by stromal cells, constitute a unique class of niche
signals. Unlike the soluble factors that can act from a distance, the membrane-bound factors require
physical contact between stem and stromal cells to function. This mode of interaction may dictate
the locality of stem cell niches and explain the distinct behaviors of stem cells within and outside
the niches. Elucidating those factors and their underlying mechanisms is thus instrumental to stem
cell biology and regenerative medicine.
1.1 Hematopoietic stem cells (HSCs) and their niches
Hematopoietic stem cells (HSCs) give rise to and regenerate the whole blood and immune
systems. At each step of the HSC development from embryonic to adult stages, local niche stromal
cells and associated signals play a crucial role in recruiting HSCs and supporting their functions.
The development of HSCs is initiated in the aorta-gonad-mesonephros (AGM) region of the
embryo body [5]. Embryonic AGM-derived stromal cells support HSCs in contact- and
noncontact-mediated manners [6-8], and activate various signals such as stem cell factor (SCF),
Wnt, bone morphogenetic protein (BMP), and Notch to promote HSC maturation and early
2
expansion [9-12]. Later in the fetal development, hematopoiesis transiently localizes to the fetal
liver, which provides an optimal niche that dramatically expands HSCs before they eventually
migrate to and reside in the bone marrow [13]. Quantitative analysis shows that, compared to the
bone marrow, the fetal liver stroma contain more N-cadherin and osteopontin (OPN)-expressing
cells, have greater proliferative capacity, and involve high Wnt and lower Notch signaling [14].
Such niche stromal characteristics and differences likely contribute to the distinct HSC expansion
in the fetal liver vs. quiescent state in the bone marrow [14, 15].
Bone marrow-derived self-renewing and multipotent hematopoietic stem cells (HSCs) are
responsible for the homeostasis of the whole blood and immune system throughout life [16-18].
HSCs are defined functionally by their ability to reconstitute and maintain the entire adult blood
system after transplantation into recipient bone marrow at the clonal level [19]. Clinically, the
unique properties of HSCs are utilized in the form of bone marrow transplantation (HSC
transplantation or HSCT) that represents the only curative therapy for a range of blood and immune
diseases, including bone marrow failure, immunodeficiencies, and hematopoietic malignancies [20,
21]. Currently, more than 40,000 cases receive HSCT in the world each year [22]. However, it
does carry a burden of possible morbidity and mortality [23]. For example, primary graft failure
remains a significant contributor to morbidity and mortality following allogeneic HSCT; whose
reported incidence is as high as 11-25% depending on the type of conditioning, type of donor,
human leukocyte antigen (HLA)-disparity between donor and recipient, and type of graft [24, 25].
Treatment approaches have been developed accordingly to combat engraftment failure following
HSCT, including growth factors, manipulation of immunosuppressant doses, or infusion of
additional autologous or allogeneic stem cells with or without preparative regimens [26]. Another
strategy is to culture and expand HSCs ex vivo before transplantation, hoping increasing cell
3
number could increase the engraftment success rate. Over the past decades, studies have been
developed to phenotype HSCs and study the genetic, epigenetic, and metabolic regulation of HSC
function and hematopoiesis [27-30]. However, despite substantial interest from scientific and
clinical perspectives, existing ex vivo culture conditions have been clinically unsuccessful because
of the generation of insufficient cell numbers or improper differentiation of the HSPC starting cell
population [31, 32]. These pieces of evidence suggest that our understanding of basic
hematopoiesis biology, including how HSCs’ proliferation, differentiation, and engraftment are
regulated by their microenvironment, is not yet complete.
Figure 1-1. Development of the hematopoietic system. In the adult, all blood cell types are produced from
hematopoietic stem cells (HSCs) that reside in the bone marrow (BM). The pool of HSCs is maintained in
specific niches (endosteal and perivascular), where they are in close contact with the surrounding
microenvironment. Adult HSCs are generated during embryonic development. First detected in the aorta-gonad-
mesonephros (AGM) region and in the vitelline/umbilical vessels, HSCs are then found in other highly
vascularized tissues, including the yolk sac, placenta, and fetal liver. At mid-gestation, HSCs massively expand
4
in the placenta and fetal liver, the latter of which becomes the central hematopoietic tissue until BM colonization
at birth [33].
In adults, HSCs are mostly present in the bone marrow, but the development of HSCs in
the embryonic state is mediated by the yolk sac and the aorta-gonad-mesonephros (AGM) region
[5]. More specifically, primitive hematopoiesis starts in the yolk sac, and definitive hematopoiesis
occurs at the AGM [5, 13, 34]. Embryonic stromal cells naturally express N-cadherin and
Osteopontin (OPN) at more than twice the amount of bone marrow stromal cells and activate the
Wnt signal to provide a unique niche for HSCs [15, 35]. Although HSCs develop and expand in
the placenta at similar times when HSCs develop at AGM [36-38], fetal liver provides an optimal
embryonic niche environment for a dramatic expansion of HSCs [13]. HSC reduction in the fetal
liver occurs at E15.5 days, resulting in HSC migration to the spleen. Finally, released stromal cell-
derived factor 1 (SDF-1) from bone marrow stromal cells attracts HSC CXCR4 expression into
the bone marrow (Fig. 1-1) [39, 40]. HSCs that reach the bone marrow are preserved and kept in
the quiescent state rather than in proliferation. HSCs are appropriately maintained and regulated
by various cells and factors in the bone marrow microenvironment, termed “bone marrow niches”
(Fig. 1-2). [41-44]. In addition, when tissue damage occurs, niches are also feedback systems for
communicating information about a tissue's state back to the related stem cells [45, 46].
1.1.1 Cellular components of HSC niches
Until less than a decade ago, the bone marrow niche was divided into two major types
depending on the location of HSCs: osteoblastic (endosteal) niche or vascular (sinusoidal) niche.
Historically, most studies have focused on the effect of osteoblasts on HSCs. For example,
osteoblasts have been known to regulate the pool size of HSCs [47, 48] and play essential roles in
the production and maintenance of HSCs and the osteoblastic niche by producing growth factors
5
such as C-X-C motif chemokine 12 (CXCL12), angiopoietin-1 (Ang-1), bone morphogenetic
protein (BMP), and Notch ligands [49, 50]. However, an expanded concept of the niche was
introduced in 2013 in which a new niche, the vascular niche, was included [51]. The osteoblastic
niche supports a microenvironment required for quiescence and maintenance of HSCs, while when
the differentiation signal is transmitted to the BM niche, HSCs encounter a new microenvironment
in the vascular niche (near bone marrow sinusoidal vessels) involved in the mobilization,
differentiation, and maturation of HSCs [49, 52]. At this stage, 60% of HSCs express the signaling
lymphocyte activation molecule (SLAM) (CD150
+
CD244
−
CD48
−
), which is a positive marker of
HSCs residing in BM sinusoidal vessels [52, 53]. Many studies have focused on mesenchymal
stromal cells (MSCs) surrounding blood vessels, and CD271 [54, 55] and CD146 [56] were
identified as a mesenchymal stromal cell marker in human. In recent years, as technology advances,
there has been a transition in our knowledge in bone marrow niches. Notably, deep imaging of
bone marrow has revealed that nearly all HSCs are in contact with leptin receptor positive (LepR+)
MSCs near the sinusoidal blood vessel [57], suggesting vascular niches are dominating over
osteoblastic niches. There are many types of cells around the sinusoidal blood vessels (perivascular
stromal cells) and play essential roles in regulating HSC activities. Bone marrow imaging and
genetic manipulation of crucial regulatory factors has enabled the identification of several
candidate cell types regulating the niche, including both non-hematopoietic (for example,
perivascular mesenchymal stem and endothelial cells [58-61] and HSC-derived cells (for example,
megakaryocytes, macrophages, and regulatory T cells) [41, 43, 62]. The niche cells provide
environmental cues for HSC regulation, i.e., growth factors/cytokines known for HSC retention
and maintenance, such as stem cell factor (SCF), chemokine (C-X-C motif) ligand 12 (CXCL12),
and angiopoietin-1 (Ang-1) of which the functions on HSC maintenance and proliferation have
6
been studied extensively both qualitatively and quantitatively. These stromal cell derived cues can
be further classified in secreted/soluble form and membrane-bound form, which will be discussed
respectively.
Figure 1-2 Cellular components in the bone marrow HSC niche. Created with BioRender.com.
1.1.2 Soluble factors
HSCs are regulated by a number of growth factors/cytokines derived from stromal cells in
the bone marrow niches [63]. Many hematopoietic cytokines were identified and purified on the
basis of their abilities to support in vitro formation of hematopoietic colonies from progenitors
[31]; the functions of many of these cytokines and their receptors were confirmed and extended
through studies of genetically modified mice. Subsequently, many cytokines were shown to bind
directly to receptors on hematopoietic stem cells (HSCs) and regulate many HSC functions,
including quiescence, self-renewal, differentiation, apoptosis, and mobility [64].
7
Bone marrow-derived stem cell factor (SCF) has been proposed to be a candidate key
player actively involved in HSC-niche interaction. SCF, as the name suggests, is a major HSC
growth factor, and potently induces HSC proliferation [65]. SCF binds to and activates the KIT
receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including
melanogenesis, gametogenesis, and hematopoiesis [66]. Phosphorylation of specific tyrosine (p-
Tyr) residues in the cytoplasmic tail of c-kit are involved in the recruitment of signaling proteins
that activate processes leading to proliferation, actin remodeling, and migration [67].
Angiopoietin-1 (Ang-1) is another growth factor in the bone marrow that has been long recognized.
Through binding to its receptor Tie2, Ang-1 enhances HSC quiescence and adhesion to niches,
resulting in protection of the HSC compartment from myelosuppressive stress [68]. Tie2/Ang-1
signaling activates its key downstream targets, β1-integrin and N-cadherin in lineage-negative,
Sca-1, c-kit double-positive (LSK), and Tie2-positive cells, and promotes HSC interactions with
extracellular matrix and cellular components of the niche. This interaction is sufficient to maintain
the quiescence and enhanced survival of HSCs by preventing cell division [68, 69].
Thrombopoietin (TPO) initiates signaling by binding to its receptor myeloproliferative leukemia
virus proto-oncogene (MPL) expressed on HSCs [70, 71] modulates HSC cell-cycle progression
at the endosteal surface [72, 73]. The Flt3 ligand, otherwise referred to as fetal liver kinase-2 (Flk2),
is another essential factor for HSC expansion [74, 75]. A list of other factors contributing to HSC
activities in the BM are summarized below (Table 1).
Table 1-1. Factors in bone marrow niches contributing to HSC activities.
Factor Source Function Reference
8
SCF LepR+
CAR cell
Nes-GFP
dim
HSC maintenance [60]
[76]
[77]
[78]
VCAM-1 Endothelial cells
LepR+
Macrophages
HSC homing
HSC engraftment
[79]
CXCL12
(SDF-1)
LepR+
CAR cell
Sinusoidal Nes-GFP
dim
Arterioles, Nes-GFP
bright
NG2+
HSC mobilization
HSC maintenance
[59]
[80]
[81]
[82]
[80]
[81]
[82]
TPO Liver
Kidney
Osteoblastic niche
expansion
HSC maintenance
[72]
[83, 84]
[85]
Ang-1 HSCs
cKit+ hematopoietic
progenitors
Megakaryocytes
HSC quiescence
HSC maintenance
Hematopoiesis
[86]
[87]
9
LepR+
FGF family Megakaryocytes
BM stromal cells
HSC niche recovery
HSC and
osteoblastic niche
expansion
[88]
[89]
[70]
[90]
[91]
[92]
TGF-β Non-myelinating Schwann
cells
Megakaryocytes
Bone (osteoblast)
formation
HSC quiescence
[93]
[91]
OPN Osteoblastic niche Negative role in
HSCs
[94]
G-CSF Monocyte
Macrophage bone marrow
stromal cells
HSC mobilization [84]
[95]
[96]
IGF-1 Liver
Osteoblasts
Osteoblastic niche
expansion (promote
osteoblast
proliferation and
differentiation)
HSC maintenance
[72]
[97]
[98]
[83]
[85]
Flt3L Bone marrow fibroblasts
and other adherent cells
HSC proliferation
and differentiation
[99]
[100]
10
IL-3 Bone marrow cells
Activated T cells
HSC proliferation
and differentiation
[101]
IL-6 T cells HSC survival and
differentiation
Hematopoiesis
recovery
[102]
1.1.3 Membrane-bound factors
Some growth factors exist in both membrane bound and soluble forms through alternative
splicing (Fig. 1-3). However, the signaling differences between the two remain largely unknown.
Here, we summarize the roles of membrane bound form of growth factors in regulating stem cell
functions that have become known to the field.
Stem cell factor (SCF)
SCF is the ligand for the receptor tyrosine kinase c-Kit. In mouse, they are encoded by the
Steel (Sl) and dominant-white spotting (W) loci, respectively [103, 104]. Analyses of mice with
SCF and c-Kit mutations suggest important roles of SCF/c-Kit signaling in hematopoiesis,
including maintaining the number of progenitor cells, long-term reconstitution, and mature cell
pools, which have been reviewed previously [67]. SCF is expressed in both soluble and membrane-
bound isoforms by stromal cells. Soluble SCF primarily serves as an essential growth factor that
promotes HSC survival and sustains HSC self-renewal [105, 106], and is commonly used in
cultures for HSC maintenance and proliferation [107]. When used in combination with other
cytokines such as interleukin-11, it can also facilitate the long-term repopulating or colony forming
11
capability of bone marrow cells [108, 109]. On the other hand, soluble SCF also acts as a mobilizer
of primitive hematopoietic cells from the bone marrow into the peripheral blood in synergy with
granulocyte colony-stimulating factor (G-CSF) [110]. Similarly, soluble c-Kit can also mobilize
HSCs to circulation supposedly by blocking the binding sites for SCF [111]. In addition, SCF is
involved in stem cell morphology. HSCs incubated with SCF, SDF-1, or their combination induce
the formation of membrane extensions or podia that point toward stromal cells in culture [112].
12
Figure 1-3. Alternative splicing of SCF. Alternative splicing of the sixth exon of SCF mRNA produces two
membrane-bound forms, SCF248 and SCF220. SCF248 is cleaved by proteases in the domain encoded by exon
6 (→) to produce a soluble 165 amino acid protein (SCF165 or sSCF). SCF220 or mSCF remains membrane-
bound as it lacks the proteolytic cleavage site encoded by exon 6, but it may also be shed in the region encoded
by exon 7 to produce a soluble form [113].
In contrast, the membrane-bound SCF demonstrates functions distinct from those of the
soluble SCF. Sl
d
mutant mice express only the secreted form of SCF due to the deletions of the
transmembrane and intracellular domains [114]. As a result, HSC population is depleted from the
bone marrow of the Sl
d
mice [115]. HSC transplantation in mutant mice also shows that membrane-
bound SCF is required for the initial lodgment of HSCs in the endosteal marrow region [3]. MMP-
9 cleaves membrane-bound SCF into soluble SCF, which is correlated with the release of HSCs
from bone marrow and their recruitment into the circulation [116], suggesting that membrane-
bound SCF anchors HSCs to their niches. In the absence of membrane-bound SCF, soluble SCF
inhibits long-term clonal growth of primary or established human CD34
+
cord blood hemopoietic
cells; in contrast, membrane-bound SCF induces long-term proliferation of these cells [117].
MSCs overexpressing membrane-bound SCF have been found to expand cord blood CD34
+
HSCs
to the greatest extent [118]. Takagi et al. created a strain of NSG mice expressing membrane-
bound human SCF and found substantially increased levels of human HSC engraftment and their
myeloid differentiation [119].
Notch ligands
Notch signaling is involved in stem cell renewal and differentiation in many tissue types
[120]. It is mediated through membrane-bound interactions between Notch receptors (Notch1~4)
on the signal receiving cells and Notch ligands (Delta-like ligands, DLL1, 3, 4 and Jagged1, 2) on
the signal-sending cells [121]. While Notch signaling is required for HSC development [122], its
13
role in the bone marrow niches remains controversial. Notch signaling is active in HSCs but
downregulated during differentiation, which has been confirmed with transgenic Notch reporter
mice [123]. In bone marrow, Notch ligands expressed by sinusoidal cells are essential for HSC
repopulation during recovery from myeloablation [124]. In vitro, Delta1, a canonical Notch ligand,
was reported to support the expansion and inhibit myeloid differentiation of hematopoietic
progenitors in an immobilized but not soluble form in vitro [125]. Activating Notch signaling by
exposing primitive hematopoietic cells to soluble Jagged1 also promotes stem cell expansion [126].
However, in vivo, HSC self-renewal or differentiation is not impaired by inactivation of the
Jagged1 gene in bone marrow progenitors and/or stromal cells [127]. Moreover, mice with both
Jagged1 and Notch1 inactivation in the bone marrow survived normally after 5-fluorouracil (5-
FU)-based challenge, and Notch1-deficient HSCs can still reconstitute mice under competitive
conditions with Jagged1 inactivated in the bone marrow stroma [127]. More recently, it was shown
that, while the endothelial cell-derived Jagged 2 is dispensable for steady-state hematopoiesis, it
promotes hematopoietic recovery under myelosuppressive conditions [128].
Angiopoietin-1
Angiopoietin 1 (Ang-1) is one of the many extracellular proteins expressed in the bone
marrow, which binds to Tie2, a receptor tyrosine kinase expressed on HSCs. Ang-1 has been
previously thought to be expressed by bone marrow osteoblasts and play a role in HSC adhesion
and quiescence, as well as maintenance of an immature phenotype of HSCs in coculture with
stromal cells in vitro [68]. However, a later study by Sacchetti et al. shows that, in human bone
marrow, Ang-1 expression is restricted to the adventitial reticular cells which form the
subendothelial layer in sinusoids, whereas the bone surfaces (where osteoblasts reside) are absent
of Ang-1 immunostaining [129]. Recently, Morrison group has further demonstrated a dispensible
14
role of Ang-1 in the HSC niche [87]. It was found that Ang-1 is not expressed by osteoblasts.
Instead, it is most highly expressed by HSCs, followed by c-Kit
+
hematopoietic progenitors,
megakaryocytes, and Leptin Receptor
+
(LepR
+
) stromal cells. Importantly, the deletion of Ang-1,
whether it is global and cell-type specific in osteoblasts, LepR
+
cells, Nes-cre-expressing cells,
megakaryocytes, endothelial cells or hematopoietic cells, does not affect any normal hematopoietic
behaviors including hematopoiesis, HSC maintenance, and HSC quiescence [87]. Although there
is no specific report of Ang-1 as a membrane-bound factor, its presence in the bone marrow has
been illustrated as a membrane-bound one [130], likely due to the restricted nature of Ang-1
expression in the stromal subpopulations [129].
CXCL12
Chemokines are a family of small, secreted proteins that stimulate cell migration. The C-
X-C motif chemokine ligand 12 (CXCL12), also known as the stromal-derived factor 1 (SDF-1),
is one of the most well-studied chemokines. CXCL12 binds to C-X-C chemokine receptor 4 and
7 (CXCR4 and CXCR7) [131]. In bone marrow, CXCL12 is highly expressed by osteoblasts,
endothelial cells, and a subpopulation of MSCs termed CXCL12-abundant reticular (CAR) cells,
which most HSCs are in close contact with. CXCL12-CXCR4 signaling plays an essential role in
maintaining HSCs in their bone marrow niches. Antagonizing CXCR4 leads to mobilization of
HSPCs to the peripheral blood [132], while induced deletion of CXCR4 results in severe reduction
of HSCs in the bone marrow [133], indicating CXCL12’s role in retaining HSCs in their bone
marrow niches. The unique function of CXCL12 in HSC migration has been clearly demonstrated
in vitro, where HSCs migrated exclusively to CXCL12 (SDF-1α) in a panel of 15 evaluated
chemokines [134].
15
In humans, CXCL12 is expressed as secreted proteins in six splicing variants, which differ
only at the C-terminus and are regulated of binding affinity to other molecules/receptors through
proteolytic degradations [135, 136]. Although there is no evidence of a membrane-tethered
isoform of CXCL12 with a transmembrane domain like that in the membrane-bound SCF, the
positive charge at the C-terminus of some CXCL12 isoforms enables their strong binding to the
cell membrane through the negatively charged glycoseaminoglycans (GAGs) [137]. This may also
be referred to as “membrane-bound” in some literature. Kollet et al. reported that such membrane-
bound CXCL12 plays a role in HSC anchorage in the niche, and its cleavage by cathepsin K (CTK)
leads to a loss of CXCL12 and mobilization of HSPCs from the endosteal niche [138].
Fms-like tyrosine kinase 3 ligand (Flt3L)
The fms-like tyrosine kinase 3 ligand (Flt3L) is a growth factor that regulate early
hematopoiesis and is crucial for the proliferation of primitive hematopoietic progenitor cells. Flt3L
is the ligand for Flt3/Flk2, a cell surface receptor tyrosine kinase that is selectively expressed in
HSCs [139]. In vitro studies show that Flt3L, when used alone, has a limited effect on the
proliferation of hematopoietic progenitors; however, it becomes highly potent in promoting the
growth of immature progenitors when combined with other cytokines, such as SCF, IL-3, IL-6,
IL-11, Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), G-CSF, and
thrombopoietin (TPO) [140-144]. In vivo, mice lacking Flt3L were found to have abnormal
hematopoiesis, with reduced leukocyte cellularity in bone marrow, peripheral blood, lymphnodes,
and spleen, as well as significantly reduced numbers of myeloid and B-lymphoid progenitors in
the bone marrow [145].
Flt3L shares structural homology with SCF and cology stimulating factor-1 (CSF-1), and
is first produced as a membrane-bound protein before being cleaved by metalloproteases to
16
become soluble [146]. In mouse, an unusual isoform of Flt3L was also found to only exist in the
membrane-bound form without giving rise to the soluble form [147]. However, whether these
isoforms play different roles in HSC niches and hematopoiesis remain to be investigated.
Figure 1-4. Membrane-bound regulation of stem cell functions. (A) Different types of membrane-bound factors.
(B) Functions regulated by membrane-bound interactions. Created with BioRender.com
1.1.4 Adhesive signaling and ECM matrix
Niche signals often function within a short-range, allowing cells in the niche to self-renew
while their daughters outside the niche differentiate [148]. Thus, adhesion molecules, including
those for cell-cell and cell-matrix interactions, are often required for stem cell anchorage in the
niche. They can also elicit intracellular signaling that regulates stem cell functions [148-152].
HSCs express a diverse array of adhesive receptors, such as N-cadherin, P-selectin, integrins, etc.
[153].
N-cadherin
17
N-cadherin is a type of calcium-dependent transmembrane proteins that homotypically
interact with their counterparts on the neighboring cells. The presence of N-cadherin on HSCs and
its role in the regulation of HSC functions have been controversial. On one hand, a series of studies
have found N-cadherin playing a role in HSC adhesion and functional maintenance. For instance,
long-term HSCs were found attached to the spindle-shaped N-cadherin+ osteoblastic (SNO) cells
in the bone marrow, where N-cadherin was asymmetrically localized toward the HSC-SNO contact
interface [154]. In vitro, HSCs cultured with bone-derived osteoblasts expressing high levels of
N-cadherin maintained the long-term reconstitution activity of HSCs [155], while upregulated N-
cadherin expression in HSCs and stromal cells correlated with the improved HSC maintenance
[68]. Forced overexpression of N-cadherin in HSCs enhanced cell adhesion and inhibited HSC
division in vitro, and also increased HSC lodgment and preserved the long-term reconstitution
capacity of HSCs during serial transplantations [156]. Conversely, knocking-down N-cadherin
expression in HSCs accelerated HSC division in culture and diminished the lodgment of donor
HSCs to the endosteal niche and reduced their long-term engraftment in vivo [69, 157]. In contrast,
several other groups found N-cadherin to be dispensible in HSC niches. First, it was demonstrated
that N-cadherin is not expressed on purified HSCs and that osteoblasts are dispensable for HSC
maintenance [158]. Second, the conditional deletion of N-cadherin in HSCs did not affect
hematopoiesis, nor did its specific deletion in osteoblasts [159-161]. Therefore, the role of N-
cadherin in HSC maintenance has been revisited both in terms of methodology and the expression
in HSC subsets [69, 162]. It also led to a more recent discovery of two classes of HSCs: reserved
vs. primed HSCs, where N-cadherin mediated adhesion plays an important role in protecting the
reserved HSCs from chemotherapy [15].
18
Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and
cell-pathogen interactions [163]. Ligand binding to integrins mediates cell adhesion and transmits
“outside-in” signals that lead to cell spreading, migration, and proliferation [164]. Integrin
engagement of cells with the microenvironment induces activation of multiple intracellular signal
transduction pathways, involving protein phosphorylation, that regulates various cellular functions
[165, 166].
Immunoglobulin superfamily (IgSF) cell adhesion molecules (CAMs)
The IgSF CAMs are cell adhesion molecules characterized by their extracellular domains
containing Ig-like sequences with two cysteines separated by 55 to 75 amino acids [167]. Several
IgSF CAMs are expressed on stromal cells in the bone marrow and involved in the stem cell
retention and engraftment, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular
adhesion molecule-1 (ICAM-1), as well as activated leukocyte cell adhesion molecule (ALCAM
or CD166), endothelial cell-selective adhesion molecule (ESAM), and junctional adhesion
molecules (JAMs).
Vascular cell adhesion molecule 1 (VCAM-1), or CD106, is a membrane bound cell–cell
adhesion molecule that regulates HSC homing and retention in the bone marrow [168, 169].
VCAM-1 is expressed by vascular endothelial cells and BMSCs in the bone marrow niches.
Human hematopoietic progenitor cells adhere to stromal cells mainly through VLA-4/VCAM-1
[170]. In mouse, this interaction is required for the homing of HSPCs to the bone marrow [151].
Down-regulation or functional blockade of VCAM-1 or its receptor, integrin α4β1 (or Very Late
Antigen 4, VLA-4), mobilizes long-term hematopoietic progenitor cells [171]. Ablating β1 in adult
HSCs prevents their engraftment in the irradiated recipient mice upon transplantation [172], while
conditional deletion of α4 integrin results in mobilization of hematopoietic progenitors into the
19
blood circulation [171, 173]. Notably, human cord blood CD34+ progenitor cells were shown to
have altered preference in adhesion to fibronectin vs. VCAM-1 during the S phase transit of cell
cycle, where their adhesion to fibronectin was increased and that to VCAM-1 was decreased
transiently [174]. Researchers also revealed that VCAM-1 expression is impaired in the bone
marrow stromal cells from patients with myelodysplastic syndromes and acute leukemias [175].
Such results illustrate a less understood role of VCAM-1 in affecting stem cell fates and niche
dynamics during HSC maintenance and bone marrow disease progression.
Intercellular adhesion molecule 1 (ICAM-1), also known as CD54, is a cell-cell adhesion
molecule that engages lymphocyte function-associated antigen 1 (LFA-1), an integrin widely
expressed in hematopoietic cells [176]. ICAM-1 deficiency in the niche leads to expansion of
phenotypic long-term HSCs with impaired quiescence and repopulation capacity [177]. In addition,
ICAM-1 deletion caused failure to retain HSCs in the bone marrow and changed the expression
profile of CXCL12, possibly representing the mechanism for defective HSCs in ICAM-1−/− mice.
Treatment of human CD34+ cells with anti-LFA-1 antibody was shown to significantly reduce
HSCs engraftment [178].
Junctional adhesion molecules (JAMs) have been identified as a family of novel mediators
of HSC-niche interactions. JAMs have the ability to form homophilic (with the identical molecule
as itself) and heterophilic (with other JAM family molecules) interactions [187]. Enzymatic JAM-
A cleavage on HSCs impaired homing potential of cells [179]. JAM-B deficient mice showed
decreased pool of quiescent HSCs [180], while JAM-C deficient mice showed low viability with
surviving animals demonstrating increased bone marrow cellularity and myelopoiesis [181].
Furthermore, JAM-B/JAM-C blockade with antibody partially reduced HSC homing efficiency
and caused induced HSC mobilization [182]. A few other JAMs reported to be expressed by
20
HSPCs and potentially play roles in membrane bound regulation of HSCs include JAM-A, JAM4,
and endothelial-cell selective adhesion molecule 1 (ESAM-1) [183-190]. However, the functional
role of the above molecules remains not defined.
Despite the extensive amount of interest and research in the field, it remains largely unclear
how HSCs interact with niche stromal cells through membrane-bound factors and intercellular
adhesion molecules, and how such interactions regulate HSC cellular signaling at the single-cell
level. Furthermore, how HSCs respond to soluble vs membrane-bound cues (e.g., soluble SCF vs
membrane-bound SCF) differently, especially in downstream intracellular signaling and cell fate
decisions, remains mysterious, given that both forms of SCF signal through binding to cKit
receptor. Understanding these will not only improve our knowledge of hematopoiesis in health
and diseases [63] but also provide new insights into the long-term maintenance and expansion of
HSCs ex vivo for transplantation [20, 21] and the treatment of hematopoietic malignancies [21].
1.2 Niche regulation on cell functions
Throughout the life-long hematopoiesis, stem cells and progenitor cells migrate, proliferate,
and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. Cell
migration and morphogenesis occur in response to paracrine signaling mediated via growth factors
and chemokines [191, 192]. The specific presentation of these factors could critically influence
cell functions, including migration and cell fate decisions (quiescence, proliferation vs. self-
renewal or survival) [193].
1.2.1 HSC morphology and cell migration
HSCs constancy traffic between their niches in the bone marrow and peripheral circulation
[194], a process involves the front-to-back polarity of the cells. Over the past few years, immense
progress has been made in understanding cell migration, including the establishment of polar
21
structures, the regulation of the dynamic processes of actin and microtubule polymerization, and
the regulation of spatial and temporal signal transduction. Polarization is often accompanied by
sensitization of receptors at the leading edge, thus favoring continued movement in the same
direction [195]. The morphology of HSCs is traditionally considered to vary between that of a
transitional lymphocyte or a blast cell [112]. Also, HSC was demonstrated to have two different
pseudopodia types on a stem cell-enriched population and a hematopoietic progenitor cell line
[196]. Ever since then, HSC morphology has begun to gain interests from the field. In vitro
cultured HSCs exhibit various surface microspikes, occasionally with branching [112]. Cytokine-
induced pseudopodial membrane extensions (termed “proteopodia”) formed by HSCs were also
observed. HSCs that had been incubated with SCF, SDF-1, or a combination of both, formed more
and larger proteopodia than cells not incubated in cytokines (Frimberger et al., 2001). Further
analysis on the association of the observed HSC morphology to homing showed that HSCs might
home to stromal cells either by burrowing beneath or adhering to the top surface. Once homed,
stem cells do not express proteopodia, indicating a role of cytokine-induced membrane protrusions
in “searching” for the niche. Fruehalf et al. reported that the podia formed by CD34+ hematopoietic
stem cells (HSC) on the BM stroma component fibronectin were characteristic of lamellipodia at
the leading edge and uropodia at the trailing edge, cytoskeletal structures that have previously been
shown to be responsible for cell locomotion of lymphocytes [197]. Short cytoplasmic protrusions
were observed in 15–20% of the CD34+ HSC. SEM revealed the formation of uropodia at the one
pole and lamellipodia at the opposite, i.e., on the contact surface. The CD34+ cells (5–7%) showed
formation of long protrusions (magnupodia) at one or both poles. Locomotion was also associated
with cells demonstrating short podia [197].
22
The balance of HSPCs present in their bone marrow niches or the circulating blood can be
drastically shifted towards the periphery by treating patients with chemotherapeutic drugs like
cyclophosphamide and/or a variety of growth factors or chemokines such as granulocyte-colony
stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-8 or
erythropoietin [198]. Given their different kinetics and efficacy of HSC mobilization towards the
circulating blood, it is still an unresolved issue whether the different mobilizing agents act
mechanistically in a similar way. Soluble c-kit receptor has been reported to mobilize HSCs to
circulation [111]; based on our findings, it is probably due to its binding to niche mSCF and
disrupts HSC anchorage. Treatment of mice with soluble SCF was also reported to mobilize
primitive hematopoietic cells from the BM into the blood and the spleen [110]. Soluble SCF also
inhibited the adhesion of M07E cells to VCAM-1-transfected CH0 cells in a dose-dependent
manner [199]. CXCL12 has been found to facilitate the BM migration of immature human CD34+
cells across subendothelial basal membranes by regulating the production of matrix-degrading
MMPs, such as MMP-2 and MMP-9 [200]; MMP-9 induces cleavage of mSCF into sSCF, which
correlated with the release of HSCs from bone marrow and their recruitment into the circulation
[116], suggesting that mSCF anchors HSCs to their niche. However, despite the established
association between HSC polarization and cell motility, our understanding of the exact regulation
of HSC polarity and migration is not complete, and a universal mechanism and reconcile these
different HSPC mobilization mechanisms is lacking.
1.2.2 HSC fate decisions
HSC self-renewal occurs in two ways; either through symmetrical cell division (SCD)
where two new stem cells are formed or through asymmetrical cell division (ACD), giving rise to
one stem cell and one cell programmed to proliferate or differentiate [201, 202]. This self-renewal
23
process is controlled by intrinsic genetic pathways subject to regulation by extrinsic signals from
the microenvironment (called niche) in which stem cells reside [203]. Cells have been shown to
be polarized during ACD and to unequally localize cell fate-determining molecules, which are
asymmetrically inherited in the daughter cells [204], such as AP2A2 (Fig. 1-5). In recent years,
the mechanistic details of HSC renewal have been related to the asymmetric architecture of HSC-
niche interaction (Fig. 1-6) [152, 204]. The interaction between the niche cells and quiescent HSCs
creates a physical “in-and-out” polarity of HSCs, with one side of the stem cell receiving signals
through direct contact with niche cells and the other side of the cell receiving only soluble factors
[204]. For example, HSC asymmetric division has been visually confirmed in live imaging
experiments [205, 206] using a GFP-reporter for the Notch signaling pathway, which is active
within HSCs but downregulated during differentiation. They revealed the asymmetric segregation
of Numb in daughter cells results in differential levels of Notch signaling. The cell polarity of the
same notion has been seen in the immunological synapse (IS) formation at the interface between
T-lymphocytes and antigen-presenting cells (APCs), where the observed polarized actomyosin
cytoskeleton and surface receptor clustering patterns regulate T-cell activation [207, 208]. Both
the orientation of stem cell division and the polarized distribution of cell fate determinants follow
an axis of polarity that is already determined in interphase [204]. However, how HSC polarity is
set up in the bone marrow niche, and the precise correlation between the cell-niche interaction and
the asymmetric outcome of HSC divisions remains unclear.
24
Figure 1-5. Asymmetrical segregation of AP2A2 during mitosis. CD150
+
48
-
LSK cells were transduced with an
Ap2a2Cherry fusion gene. Still frame from live-cell video microscopy of dividing cells: DNA (FITC-green);
AP2A2 (Cherry-orange). Hematopoietic cell (right) with AP2A2-polarized clustering and asymmetric
segregation into only one daughter cell (asterisk). The other daughter cell (no asterisk) reexpressed small vesicles
of AP2A2 from time 3:58:04 onward [210].
25
Figure 1-6. Extrinsic and intrinsic regulation of stem cell polarity and asymmetric division. Stem cells can set
up an axis of polarity during interphase and use it to localize cell fate determinants asymmetrically in mitosis.
Orientation of the mitotic spindle along the same polarity axis ensures the asymmetric segregation of
determinants into only one of the two daughter cells. Extrinsic: Stem cells may depend on a signal coming from
the surrounding niche for self-renewal. By orienting their mitotic spindle perpendicularly to the niche surface,
they ensure that only one of the two daughter cells continues to receive this signal and maintains the ability to
self-renew [211].
1.3 Existing methods for studying HSC-niche cell interaction
1.3.1 Animal models
Gene knock-out mouse models have been traditionally generated to disrupt or inactivate
one or more genes of interest through gene targeting (Table 1-2). The loss of gene activity often
causes changes in the expression of a particular factor, allowing in vivo studies of the biological
roles of the niche factor. Reverse to the loss-of-function approach, i.e., gain-of-function approach
(GOF), has also been explored to generate transgenic mice that overexpress a factor of interest
under the control of the gene promoters. Over the past years, analysis of genetically modified or
mutant mouse models has confirmed and defined the physiological roles for specific cytokines in
hematopoietic stem cell production and function. Phenotypes of W and Sl mutations (e.g., Sl
d
, W
41
and W/W
v
) suggest roles for Kit/SCF signaling in hematopoiesis, including maintaining the
number of progenitor cells, long-term reconstitution, and mature cell pools, which have been
reviewed previously [67]. SCF promotes HSC survival [105, 212] and sustained HSC self-renewal
[106]. Furthermore, SCF in combination with interleukin-11 is shown to favor the long-term
repopulating activity of BM cells [108], and SCF also synergizes with other cytokines to support
colony growth of both granulocyte-macrophage and erythroid units [109]. In addition, using
various cre mice, such as Cxcl12-GFP [133], Nes-Cre [59, 213], Prx1-Cre [214], LepR-GFP [60],
26
Osx-Cre [214-216], Sp7-Cre [214], and Mx1-cre [217], have been constructed to reveal the
relationship between bone marrow MSC characteristics and HSC.
Table 1-2. Mouse models established to study membrane bound stem cell–niche interactions.
Model
name
Gene modification Notable phenotypes Refs
Sl
d
Removing the
transmembrane and
intracellular domain of scf
Impairment of development of
hematopoietic cells, melanocytes and
germ cells
[114]
Sl/Sl Lethal deletion of scf gene Mice die in utero with a severe anemia [218]
W/W
v
Single residue change in the
ATP binding domain of ckit
Mouse embryos do not die but generate
viable but severely anemic animals
[219]
W/W Lethal deletion of ckit
expression
Mice die in utero with a severe anemia [219]
W
41
;W
42
Single residue change in the
ckit intracellular
phosphotransferase domain
Severe defects in reconstituting
peripheral blood lineages and bone
marrow of irradiated recipients
[220]
MxCre-
CXCR4
f/null
Cxcr4 deletion in bone
marrow
Reduced HSC number;
Reduced LTC-IC number;
Enhanced exit from G0 phase
[133]
Cxcl12-
GFP
GFP knockin into Cxcl12
locus
CAR cells were scattered in
intertrabecular space;
HSCs are associated with CAR cells;
[133]
27
CXCR4 is dispensable for the contact of
HSCs with CAR cells
Cxcl12
fl
crossed
with Nes-
Cre
Conditionally delete Cxcl12
in nestin+ cells
~30% reduction in HSCs and
hematopoietic progenitors
[59,
213]
Prx1-Cre Conditionally delete Cxcl12
in nestin- MSCs
loss of HSCs, longterm repopulating
activity, HSC quiescence and common
lymphoid progenitors
[214]
LepR-GFP,
Scf-GFP;
Tie2-cre;
Lepr-cre
GFP knockin into Scf or
LepR;
Conditionally delete Scf
from endothelial cells or
lepr-expressing cells
Scf was primarily expressed by
perivascular cells in BM;
HSCs were depleted when Scf was
deleted from endothelial cells or leptin
receptor (Lepr)-expressing perivascular
stromal cells
[60]
Osx
-/-
Deletion of osteoblasts and
osteolineage cells
Fetal bone marrow cells formed
multilineage hematopoietic colonies in
vitro;
KSL cells exhibited hyperproliferation
and dysregulation of cell-cycle genes
and failed to engraft transplant
recipients.
[215,
216]
28
Osx-Cre Conditionally delete Cxcl12
in osteoblasts
Deletion of Cxcl12 from mineralizing
osteoblasts has no effect on HSCs or
lymphoid progenitors.
[214]
Because it is much more laborious to generate transgenic mouse lines and extremely
challenging to perform live imaging in mice, combined approaches using zebrafish and mice have
been most successful in demonstrating niche cell function. The use of zebrafish to study the
hematopoietic niche has enabled the discovery of novel cell-cell interactions and chemical and
genetic regulators of hematopoietic stem cells [221]. For example, stromal and endothelial cell
populations in the mouse bone marrow and stromal cells in the zebrafish CHT and kidney are not
identical but play remarkably similar roles producing cytokines, tethering HSCs, and responding
to local metalloproteinases [116, 222-228]. There are some critical differences between the murine
and zebrafish niches. HSC birth from aortic endothelial cells occurs in the extravascular space in
zebrafish vs. the aortic lumen in mice [229-231], and there are cell types in the bone marrow niche
that have not yet been identified in the zebrafish kidney niche including Nestin1 cells and cells of
the sympathetic nervous system [59, 232].
1.3.2 Cellular models
Quantification of putative HSCs in the bone marrow or cord blood (CB) is traditionally
assessed by its potential to mediate long-term, multilineage engraftment when transplanted into
immunodeficient murine recipients [233]. In vitro surrogate assays, e.g., long-term culture
initiating cell (LTC-IC) assay, are attractive due to their relative ease of implementation, lower
cost, and improved throughput of results. In recent years, the direct cell-cell contact mediated
interactions are regaining attention. Studies have demonstrated the vital role of a stroma feeder
29
layer to maintain hematopoietic progenitor cell (HPC) function in vitro [234-236]. Direct cell-cell
contact with AFT024 stroma maintained the self-renewing ability of HPCs in vitro [237]. For
example, OP9 and M2-10B4 cell lines have been shown to maintain HSCs in long-term culture,
acting like a hematopoietic niche [238]. HSCs sought direct contact with stromal cells as shown
in the study, CD34+/CD38− cells actively migrated toward and sought contact with stroma cells,
and 30% of them adhered firmly to AFT024 stroma through the uropod [237].
1.3.3 Surface immobilization
Protein immobilization onto inorganic surfaces (e.g., glass coverslips or hydrogels) has
been long used in various applications [239]. Current protein immobilization strategies include
physical, covalent, and bioaffinity immobilization for the fabrication of surface-coated substrates.
In the past two decades, much effort has focused on the immobilization of proteins in defined
patterns resulting in micro- or nano-scale arrays [240], termed “micro- or nano-patterning.” These
protein patterning strategies have enabled biophysical study and modulation of cellular processes,
including cellular adhesion [241], surface antigen presentation-related signaling pathways in T-
cells [242].
Immobilization of biomolecules has improved our understanding of the biological roles of
receptor-ligand interaction in HSC niches. FN, laminin, and collagen types I and IV are frequently
used as surface coatings for studying the effects on HSC activities. FN-coated surface promotes
HSPC differentiation into erythroids ex vivo, while laminin supports the expansion of
megakaryocyte progenitors [243]. HSC-ECM protein interaction is mediated by the nanometer-
scaled lateral distance between conjugated ligands. Ligand presentation with a specific distance
regulates integrin-involved lipid raft clustering for signal complex formation, a process dependent
on ligand type [244]. However, immobilized proteins fail to mimic the lateral mobility of natural
30
cell-membrane bound proteins, which have limited the usage of this method in studying the
dynamics and receptor-ligand interaction (e.g., clustering and recruitment).
1.3.4 Supported lipid bilayer (SLB) model
Figure 1-7. Supported lipid bilayer as a model of the cell membrane. (A) Supported lipid bilayer is composed of
amphiphilic lipid molecules containing both a hydrophilic tail and a hydrophobic head [245]. (B-C) Preparation
methods of lipid bilayers. (B) The Langmuir-Blodgett (LB) technique and (C) The fusion of lipid vesicles [246].
Mastery of niche components may improve therapeutic efforts to direct differentiation of
hematopoietic stem cells from pluripotent cells, sustain stem cells in culture, or improve stem cell
transplant [221]. However, there is still a lack of data on the dynamic molecular and signaling
activities resulting from membrane-bound interactions at the cellular level due to the complexity
of in vivo niches [130]. Supported lipid bilayer (SLB) technology has been widely used to study
the properties of cellular membranes (Fig. 1-7) [247]. Phospholipid bilayers closely resemble cell
membranes in some key respects. For example, they retain two-dimensional fluidity and can be an
excellent environment for presenting membrane proteins. Model bilayer systems allow for the
investigation of biological processes that occur at the cellular level, providing information about
ligand-receptor interactions [248-251], viral attack [252, 253], and cellular signaling events [254-
31
256]. Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful
tool for studying cell-cell interfaces, particularly T cell antigen-presenting cell (APC) interactions,
using fluorescence microscopy (Fig. 1-8) [247, 257-259]. The lipid bilayer system allows these
processes to be visualized in detail, although the trade-off is that it is artificial. The methodology
was adopted and further developed by Dustin and colleagues, who used it to study the
immunological synapse, the two-dimensional interface formed between the T cell and an APC,
presenting a cognate antigen [260-263]. These studies highlighted the usefulness of this
reductionist approach because: (a) one could have quantitative control over the density of
molecules incorporated into the bilayer, (b) the flat interface was ideal for optical imaging, and (c)
the resulting lipid bilayers elicit immune function of the T cell. SLB research offers the possibility
to observe time-dependent processes such as the interaction of lipid membranes with proteins [264],
peptides [265] and drugs [266], and the growth of single lipid domains [267]. Therefore, the SLB
model is a versatile and promising tool for studying the molecular events in HSC-niche interactions.
Figure 1-8. Modeling immunological synapse (IS) with supported lipid bilayer (SLB) model. (A) The
intercellular junction between a T cell and an antigen-presenting cell (APC) is known as the immunological
synapse. Micrometer-scale protein patterns emerge at the interface between the two cells. (B) Formation of
32
micrometer-scale patterns from the time point of contact with an activating APC. TCRs recognize pMHCs and
form small clusters (dark green) that are driven by the actin cytoskeleton to the center of the immunological
synapse (top). After 5 minutes, most of the TCRs are in the central zone of the immunological synapse. The T
cell integrin LFA1 recognizes ICAM1, and the conjugates form an enriched ring, peripheral to the TCR central
zone (bottom) [259]. (C-D) T cell forming immunological synapse pattern on a lipid bilayer model [268].
1.4 Overview
HSCs receive membrane-bound signals from the bone marrow microenvironment through
direct cell-cell contact with stromal cells, and these signals are indispensable for a wide range of
HSC functions, including quiescence, self-renewal, and migration. However, the mechanistic
signaling details of individual or combination of membrane-bound factors and the effect on HSC
activities remain largely unclear, which hinders efficient utilization of HSCs in clinical
applications. In this dissertation, we used a tethered supported lipid bilayer model and investigated
the effects of membrane-bound regulation in HSC morphology and adhesion (chapter 2 and 3).
We also innovated a multifactor microfluidic device for studying cell migration on tethered lipid
bilayers. The knowledge gained from this dissertation provided insights and tools for the research
and utilization of HSCs.
33
Chapter 2: Membrane-bound SCF and VCAM-1 synergistically regulate the
morphology of HSCs
2.1 Rationale
Adult stem cells are responsible for maintaining and repairing adult tissuesand organs, and
thus a crucial cell source for regenerative medicine. They often reside in specific tissue locations
(also known as the niches) comprised of distinct sets of stromal cells and biomolecules, which
support vital stem cell functions including migration/homing, quiescence, and self-renewal [269].
While adult stem cells can move in and out of their niches, e.g., during transplantation or in
response to tissue injury, their physical presence in the niche and localized interactions with
surrounding stromal cells are generally required for their long-term functions [269]. It is thus
critical to elucidate the key factors and mechanisms underlying these localized stem-stromal
interactions.
The membrane-bound factors expressed on stromal cell surfaces constitute a unique class
of localized niche cues, which act through direct stem-stromal contact [270]. Membrane-bound
Notch ligands, for example, are involved in stem cell renewal and differentiation in many tissue
types [120]. In the bone marrow niches of hematopoietic stem cells (HSCs), the most studied and
clinically-used adult stem cell [271], several soluble factors exist in the membrane-tethered forms,
which work distinctly from their soluble counterparts [130]. Instead of promoting growth, the
membrane-bound stem cell factor (mSCF) is crucial for HSC lodgment, maintenance and
hematopoiesis [272, 273]; the membrane-bound version of C-X-C motif chemokine ligand 12
(CXCL12) plays a role in HSC anchorage in the niche rather than chemotaxis [274]. Vascular cell
adhesion molecule 1 (VCAM-1), a cell-cell adhesion molecule expressed on stromal cells [275],
34
regulates HSC homing and retention in the bone marrow [168, 169]. However, it remains unknown
on how these membrane-bound factors regulate stem cell behaviors in a localized manner at the
cellular level.
Importantly, some membrane-bound factors have shown great promise in enhancing the
long-term functions and/or expansion of HSCs ex vivo, the “holy grail” for bone marrow
transplantation [276]. It has been shown that HSCs cultured on extracellular matrix with
immobilized SCF, or stromal cells with overexpressed mSCF have improved maintenance or
expansion in vitro [277, 278]. Surface-coated fibronectin, which engages the very late antigen-4
(VLA-4), the receptor for VCAM-1, also promotes the ex vivo expansion of human and mouse
hematopoietic stem/progenitor cells (HSPCs) [20, 279]. Yet, to date, such applications have been
sporadic and only with limited success. Further advances in this area demand a mechanistic
understanding of the molecular pathways underlying the membrane-bound factors, their crosstalk
with each other, and their downstream targets involved in the long-term functions of HSCs.
The membrane-bound factors in HSC niches have been largely studied with genetic
knockdown/knockout or fluorescently-tagged models, or immuno-staining of bone marrow tissues
[60, 272, 274, 280]. These in vivo studies provide critical insights on the identity, function, and
localization of these factors in the HSC niches. However, no prior knowledge exists on the
dynamic molecular and signaling activities in HSCs induced by membrane-bound interactions at
the cellular level, due to the limits of intravital imaging and the rarity and molecular complexity
of the HSC niches [130, 281]. Lately, several in vitro HSC-stromal co-culture studies have
demonstrated interesting morphological, migratory, and division patterns of HSPCs physically in
contact with stromal cells, which suggests a role of stromal contact for HSC functions [282, 283].
Yet, whether these cellular behaviors are mediated by specific membrane-bound factors remains
35
unanswered. Other in vitro studies showed adhesive behavior of hematopoietic progenitor cells on
substrates with immobilized growth factors [284]. However, immobilization eliminates the lateral
mobility of membrane-bound factors, a crucial property for membrane-bound interactions that
allows for micro/nano-clustering and spatial reorganization of receptors and signaling complexes,
which can drastically amplify the membrane-bound signals and achieve distinct cellular functions
[285].
In this study, we utilized a supported lipid bilayer (SLB) platform to mimic the stromal cell
surface and membrane-bound interactions in the context of HSC perivascular niche in the bone
marrow (Fig. 2-3A). SLB preserves the lateral mobility of membrane-bound proteins and has
served as a powerful tool for studying cell-cell interfaces, such as the immunological synapse
between T cells and antigen presenting cells (APCs) [285, 286]. Here we demonstrated a unique
recruitment pattern and cell morphology taken by HSCs in the interaction with mSCF in the
presence of VCAM-1, compared to other membrane-bound factors and hematopoietic progenitor
cells. We further revealed a synergy between mSCF and VCAM-1 in regulating HSC morphology
and mSCF recruitment pattern, which dramatically enhances the strength of HSC adhesion on SLB,
the degree of which is not seen with multipotent progenitors. This synergy involves actin
cytoskeleton and PI3K signaling, and promotes nuclear FOXO3a retention in contrast to soluble
SCF (sSCF). Our work thus reveals a unique role and a new signaling mechanism of membrane-
bound factors in mediating stem-stromal interactions, cell adhesion, and stem cell maintenance in
the adult stem cell niche.
36
2.2 Materials and Methods
2.2.1 Mice
C57BL/6J mice were purchased from Jackson Laboratories. All mice were bred at the
Research Animal Facility of the University of Southern California. Animal care and euthanasia
protocols were approved by the Institutional Animal Care and Use Committee of the University
of Southern California. Mice were provided continuously with sterile food, water, and bedding.
2.2.2 Cell isolation and flow cytometry
Bone marrow cells were obtained from the crushed bones of 4~6-month-old mice and
immunostained and sorted for Lin+CD45+ cells (BMLs), or enriched by cKit and then
immunostained for HSC, MPPs and OPPs (Fig. 2-2 and Table 2-1,2). Bone marrow mesenchymal
stromal cells were harvested from mouse bone marrow [60], immunostained, negatively enriched
(CD45-, TER119-) using EasySep™ mouse mesenchymal stem/progenitor cell enrichment kit
following vendor’s instructions (Stemcell Technologies, Catalog #19771), and sorted based on
PDGFRα expression. FACS sorting was carried out with a BD FACS™Aria II cell sorter (BD
Biosciences, San Jose, CA) at 4°C.
Table 2-1. Cell surface markers for sorting hematopoietic populations.
Cell type Surface antigens
HSC *Lin- cKit+ Sca1+ Flk2- CD34- Slamf1+
MPP-Flk2- Lin- cKit+ Sca1+ Flk2- CD34+
MPP-Flk2+ Lin- cKit+ Sca1+ Flk2+
CLP Lin- IL7rα+ Flk2+
37
CMP Lin- cKit+ Sca1- FcγR- CD34+
MEP Lin- cKit+ Sca1- FcγR- CD34-
GMP Lin- cKit+ Sca1- FcγR+
Lin-CD45+ Lin- CD45.2+
CD45+ CD45.2+
*Lin (Lineage) markers include B220, CD3, CD4, CD8, Gr1, Mac1 and Ter119.
38
Table 2-2. Antibodies used for immunofluorescence staining.
Antigen Conjugation Vendor Catalog# Clone
B220 PerCP-Cy5.5 eBioscience 45-0452-82 6B2
CD3 PerCP-Cy5.5 eBioscience 45-0031-82 KT31.1
CD34 e660 eBioscience 50-0341-82 RAM34
CD4 PerCP-Cy5.5 eBioscience 45-0042-82 GK1.5
CD8 PerCP-Cy5.5 eBioscience 45-0081-82 53-6.7
cKit APC-EF780 eBioscience 47-1171-82 2B8
FcγR Biotin eBioscience 13-0161-85 93
Flk2 PE-Cy5 eBioscience 15-1351-81 A2F-10
Gr1 PerCP-Cy5.5 Affymetrix 45-5931-80 8C5
IL7rα PE-Cy7 Biolegend 135013 A7R34
CD45.2 A700 Biolegend 109822 104
CD45.1 PE-eFluor 610 Affymetrix 61-0453-82 A20
Mac1 PerCP-Cy5.5 eBioscience 45-0112-82 M1/70
Sca1 BV711 Biolegend 108131 D7
Slamf1 PE Biolegend 115904 TC15-12F12.2
Biotin PE-Cy5.5 Life technologies SA1018
39
Ter119 PerCP-Cy5.5 eBioscience 45-5921-82 TER-119
Ter119 APC eBioscience 17-5921-81 TER-119
CD45 APC eBioscience 17-0451-82 30-F11
CD31 APC eBioscience 17-0311-80 390
CD140a PE eBioscience 12-1401-81 APA5
SCF - Santa Cruz sc-13126 G-3
Phosphotyrosine - MilliporeSigma 051050MI 4G10
FoxO3a - Cell Signaling 2497 75D8
40
2.2.3 Quantitative PCR
The phenotypic identity of the stromal cells was confirmed by their high expression of SCF
(Kitl), CXCL12, VCAM-1 and PDGFRα using TaqMan® real-time PCR assays following
vendor’s instructions. Assay ID: SCF (Mm00442972_m1), VCAM-1 (Mm01320970_m1),
CXCL12 (Mm00445553_m1), Lepr (Mm00440181_m1), PGFRa (Mm00440701_m1), CD31
(Mm01242576_m1), β-actin (Mm02619580_g1).
2.2.4 Fabrication of lipid bilayer and cell loading chambers
The loading chambers for lipid bilayer and cells were manufactured by pouring
polydimethylsiloxane (PDMS) mixed at 10:1 base to curing agent ratio (Sylgard 184 elastomer kit;
Dow Corning) into a custom-milled polycarbonate mold with a rectangular plateau of 1.5 mm
(width) x 6 mm (length) x 1 mm (height) raised from a flat bottom surface. PDMS was cured at
80°C for 3h, peeled off, and cut into individual devices. Circular inlet and outlet with 2 mm
diameter were punched at both ends of the PDMS chamber. Glass coverslips (24 mm x 40 mm,
Fisher scientific, Hampton, NH) were cleaned with piranha solution (36 M H2SO4:30% H2O2 =
3:1, by volume), extensively rinsed with deionized water, baked overnight at 400°C, and treated
with plasma for 5 min before permanently bound with PDMS chamber (Harrick Plasma, Model
PDC-001-HP). The PDMS chambers were treated with plasma for 50s and permanently bound to
the plasma-treated glass coverslips for the subsequent lipid bilayer formation.
2.2.5 Preparation of small unilamellar vesicles and supported lipid bilayers
Lipid components (18:1 (Δ9-Cis) PC (DOPC), 18:1 PS (DOPS), 18:1 DGS-NTA(Ni) and
18:1 Biotinyl Cap PE) dissolved in chloroform were purchased from Avanti Polar Lipids and
mixed at mol % indicated in the main text. The lipids were dried in round-bottom flasks under a
stream of N2 for 5 min and desiccated for 2 h with house vacuum pump in a chemical fume hood.
41
The lipid mixture was resuspended by bath sonication in 1X PBS at a final concentration of 2.5
mg/ml and extruded 10 times through a membrane with 50-nm pore size (Avanti Polar Lipids) into
small unilamellar vesicles (SUVs). The SUV solutions were then diluted 1:1 in 1X PBS (pH 7.4)
before being loaded onto the glass coverslip through the loading chamber, and incubated for 2 min
to spontaneously form the lipid bilayers. The chambers were then washed with a 10X excess
volume of 1X PBS. For SEM, indium-tin-oxide (ITO) coated coverslips (SPI supplies) were used
to form lipid bilayers, where DOPC was replaced with 18:1 PS (DOPS) [287].
2.2.6 Protein capturing on lipid bilayer
DOPC lipids supplemented with 0.1 mol % Biotinyl-Cap-PE and 5 mol % of DGS-NTA(Ni)
were used to form SLBs. The bilayer was blocked with 0.1 mg/ml BSA for 1 hr and incubated
with 10 µg/ml streptavidin for 20 min. The bilayer was then washed extensively with PBS (pH 7.4)
followed by 30 min incubation with the Alexa Fluor
®
488-labeled mono-biotinylated protein (5
µg/ml CXCL12; 7 µg/ml SCF; 7 µg/ml Flt3-L; 15 µg/ml TPO; 20 µg/ml Ang-1; 20 µg/ml sDLL1)
(Peprotech), and DAPI/Cy5 labeled VCAM-1 with a 6-histidine tail (2 µg/ml) (R&D Systems).
The entire chamber was then washed with 10X excess volumes of 1X PBS. Fluorescence recovery
after photo-bleaching (FRAP) techniques were used to examine the lateral mobility, and the
diffusion coefficients of the captured proteins were calculated through a custom fitting program in
MATLAB (MathWorks) using Fast Fourier Transform (FFT) analysis of the time evolution of the
lateral fluorescence profile [288].
2.2.7 Cell seeding and incubation
FACS sorted cells were kept in PBS with 2% FBS on ice. Before being seeded onto bilayers,
cells were pelleted at 300x g and resuspended in either extracellular buffer (ECB; 130 mM NaCl,
5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 1 mg/ml BSA, and 5 mM glucose, pH
42
7.4) or StemSpan SFEM (STEMCELL Technologies Inc., Vancouver, Canada) supplemented with
50 ng/ml TPO. The chamber containing lipid bilayers was equilibrated with the same buffer, and
the resuspended cells were then injected into the chamber and incubated for 1 hr in a humidified
incubator maintained at 37 °C and 5% CO2.
2.2.8 Fluorescence microscopy
A Nikon Eclipse Ti-E inverted fluorescence microscope (Nikon, Tokyo, Japan) was used
for live-cell imaging, which is equipped with an OKOLab incubation box (OKOLAB, Italy)
controlling for temperature (37°C) and CO2 concentration (5%). The chamber containing lipid
bilayers was equilibrated with ECB, and the resuspended cells were then injected and live imaged
on the pre-warmed fluorescence microscope. Interference reflection microscopy (IRM) was used
to visualize the adhesion-substratum interface of cells as described previously [289]. Images were
taken using a 60x 1.40 NA Oil objective. In live-cell tracking and motility analysis, cells were
using a 10x 0.45 NA Air objective, at 5 min interval, for 1 hr.
2.2.9 Total Internal Reflection Fluorescence (TIRF) microscopy
The TIRF microscopy was performed on a Delta Vision OMX system equipped with Ring
TIRF illumination optics (GE Healthcare, Issaquah, WA, USA) using a 100x 1.49 NA Oil TIRF
objective. Laser wavelengths of 405 nm (for F-actin), 488 nm (for mSCF) and 642 nm (for cKit)
were used for excitation, and the images were collected at the emission wavelengths of 442/30 nm,
532/56 nm, and 683/40 nm, respectively.
2.2.10 Scanning electron microscopy (SEM)
Cell samples were fixed with 4% paraformaldehyde in 1X PBS, serially dehydrated with
ethanol (10%, 25%, 50%, 70%, 80%, 95, 100%, 100%; 5 min at each concentration), critical-point
dried with a Tousimis 815 Critical Point Dryer (Tousimis, Rockville, MD, USA), and sputter
43
coated with Pt:Pd for 90 seconds using sputter coater 108 (Cressington Scientific Instruments,
United Kingdom), to reach a thickness of 3 nm. The SEM images was acquired on a Nova
NanoSEM 450 (FEI, Hillsboro, OR). Settings used for imaging were as follows: voltage at 3 kV;
spot size at 3.5; working distance at 10.0 mm. Beam deceleration (BD) mode with a 200 V bias
voltage were used to reduce the charging effect. Micrographs were taken with 6 μs scanning time
at 12000x magnification. For angled images, samples were tilted at 45 degree and imaged with the
same settings.
2.2.11 Shear flow and adhesion analysis
We created the microfluidic device in-house using a micromilling platform, design and
fabrication protocols, and soft-lithography techniques [290] for shear flow and adhesion analysis.
Within each device, SLBs were formed in two geometrically identical (mirrored), parallel
microfluidic channels separated by a 250 µm barrier (Fig. 2-9A inset). The design and toolpaths
for the double channel microdevice (channel height 1 mm, channel width 2 mm, length 16 mm)
were created in Autodesk Fusion 360 (San Rafael, CA) and custom-milled (Shapeoko, Carbide 3D,
Torrance, CA) out of polycarbonate. The final device was manufactured by pouring
polydimethylsiloxane (PDMS) mixed at 10:1 base to curing agent ratio (Sylgard 184 elastomer kit;
Dow Corning). PDMS was cured at 80°C for 3 h, peeled off, and cut into individual devices.
Channel inlets and outlets with 0.75 mm diameter were punched at both ends of microfluidic
channels. The PDMS devices were permanently bound to the detergent-cleaned glass coverslips
after plasma treatment for 50 seconds (Harrick Plasma, Model PDC-001-HP) for the subsequent
lipid bilayer formation and substrate modification.
A dual-channel syringe pump (New Era Pump Systems, NY) was used to apply controlled
shear flow to the two channels through 5 mL glass syringes (inner diameter 10.3 mm) and tubing
44
connections. Cells were incubated with membrane bound factors on SLBs for 1hr, before a shear
flow of StemSpan SFEM media at controlled flow rates (ramping up from 0 to 15 mL/min, with 5
s holding of each flow rates in a stepwise fashion) under a 37
o
C environment. The design enables
real-time imaging and direct comparison of cell adhesion on two different SLBs under the same
flow rates. BF images were taken once every second using a 2x objective (CFI60 Plan Apochromat
Lambda Lens, NA 0.1, WD 8.5mm). The remaining cells under each flow rate were normalized as
a percentage by the starting cell numbers in the same regions of interest (ROIs). Each ROI is a
300x300 µm square containing 50-100 cells randomly selected along the center of the channel.
Shear stress at the SLB surface (bottom of channel) was calculated at
https://www.elveflow.com/microfluidic-calculator/, where the fluidic properties were assumed the
same as water at 37
o
C considering the serum-free nature of the StemSpan media. For
morphological analysis under flow, cells were imaged with 60x 1.40 NA Oil objective.
2.2.12 Treatments with inhibitors
Cells were treated with BIO 5192 (20 nM, 45 min) (R&D Systems) to inhibit the VLA4
integrin; Latrunculin A (1 µM, 1 hr) (Tocris Bioscience) to inhibit actin polymerization;
Blebbistatin (50 µM, 10 min) (MedChemExpress) to inhibit non-muscle myosin II; Y-27632
dihydrochloride (10 µM, 1 hr) (Tocris Bioscience) to inhibit ROCK signaling; LY294002 (5 µM,
1 hr) (MedChemExpress), BAY 80-6946 (Copanlisib) (64 nM, 1 hr), CAL-101 (Idelalisib) (50 nM,
1 hr), MLN1117 (Serabelisib) (150 nM, 1 hr), TGX-221 (85 nM, 1 hr) to inhibit Phosphoinositide
3-kinase (PI3K); MK2206 (5 µM, 1 hr) (MedChemExpress), GSK2141795 (1 µM, 1 hr) to inhibit
Akt activities. Vehicle solvents were used accordingly as controls.
45
2.2.13 Immunofluorescence staining
For the detection of phosphorylated tyrosine, phosphorylated Akt, or FOXO3a, cells were
fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton-X-100 in PBS for 15
min, and blocked with 1% BSA in PBS for 1 hr. 1 mM sodium orthovanadate were added
throughout the fixation and staining process for the phosphorylation staining. Cells were then
incubated overnight at 4°C with primary antibodies in PBS with 1% BSA. On the following day,
cells were rinsed extensively and incubated for 2 h at room temperature with secondary antibodies
in PBS with 1% BSA. Primary antibodies are listed in Table 2-4. F-actin was visualized with
Alexa Fluor
®
Plus 405 Phalloidin (Invitrogen). Myosin IIA or IIB was visualized with Myosin IIA
antibody (3C7) (NovusBio, Catalog#H00004627-M01) and Myosin IIB antibody (D8H8) (Cell
Signaling, Catalog# 8824). Cell nucleus was visualized with DAPI staining. The Alexa Fluor
®
405,
488, 568 conjugated goat anti-mouse, donkey anti-rabbit, donkey anti-mouse secondary antibodies
were purchased from Invitrogen.
2.2.14 Image analysis
Images were analyzed using ImageJ (U.S. National Institutes of Health, Bethesda, MD,
USA; http://rsb.info.nih.gov/ij) supplemented with customized macro codes including clustering
parameters, adhesion, cell size, and fluorescence intensity (mean cluster intensity after background
subtraction) from images taken with identical optical configurations and exposure settings.
Colocalization was quantified as Pearson’s correlation coefficient of pixel intensities between two
images, measured with an ImageJ plugin, Coloc2.
2.2.15 Statistics
The HSC-LepR+ MSC coculture experiment has been repeated twice. All other
experiments have been repeated at least three times. All data are presented in mean ± SD. n
46
represents cell number analyzed in each experiment, as detailed in figure legends. One-way
ANOVA or two-tailed Student’s t-tests were used for evaluating the significance of difference
unless otherwise indicated. Statistical analyses and plots generation were performed using
GraphPad Prism 8 software (GraphPad Software, Inc). N.D.: not detected; n.s.: not significant (p >
0.05), *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
2.3 Results
2.3.1 Membrane-bound SCF is the only factor recruited/clustered by HSCs in a screening
Membrane-bound receptor-ligand engagements often involve recruitment of ligands into
micro-clusters which precedes downstream cell signaling [291]. To determine which membrane-
bound factors are recruited/clustered during the HSC-stromal cell interactions, we performed a
factor screening using a supported lipid bilayer (SLB) system (Fig. 2-3A), which recapitulates the
lateral mobility and dynamic recruitment of the tethered molecules on natural cell membranes
[292]. We chose the perivascular niche as the context/background of the factor screening, as most
HSCs are found in this niche in the bone marrow [60]. The chosen ligands include SCF, CXCL12,
Ang-1, delta-like 1 (DLL1) and FMS-like tyrosine kinase 3 ligand (Flt3-L), which can be found in
membrane-bound form in the bone marrow [60, 68, 125, 274, 293]; and thrombopoietin (TPO), a
systemic, soluble HSC maintenance factor [294]. These ligands were conjugated with a
fluorophore, mono-biotinylated, and tethered to the Biotinyl-Cap-PE lipid in the SLBs through a
streptavidin “bridge” (Fig. 2-3B). VCAM-1 appears abundantly on endothelial cells and leptin
receptor positive (LepR+) mesenchymal stem/stromal cells (MSCs) [60]. We thus included
VCAM-1 in all the SLBs in the initial factor screening. VCAM-1 was tethered to the SLBs through
the chelation of 6-histidine tail to the DGS-NTA(Ni+) lipid for HSC adhesion (Fig. 2-3B). We
confirmed the lateral mobility and measured the diffusion coefficients of the tethered biomolecules
47
(Fig. 2-1A, Table 2-3). We also confirmed that the density of fluorescent molecules on the SLBs
was proportional to the fluorescence intensity (Fig. 2-1B), thus allowing for quantitative
interpretation of the fluorescence intensity as the number of recruited biomolecules.
Figure 2-1. Measuring diffusion coefficients of the membrane-bound factors on supported lipid bilayers with
fluorescence recovery after photobleaching (FRAP). (A) Measuring diffusion coefficients of the membrane-
bound factors on supported lipid bilayers with fluorescence recovery after photobleaching (FRAP). Green and
purple: fluorescently labeled SCF and VCAM-1. Left: right after photobleaching. Right: the same area after 600
seconds of recovery using the same acquisition settings. (B) Linear relationship between fluorescence intensity
of lipid bilayer and the percentage of fluorescent lipid molecules. Lipid bilayers were composed of DOPC and
0%, 0.125%, 0.25%, 0.5%, 1% and 2% fluorescent DHPE. Error bars: standard deviation.
Table 2-3. Diffusion coefficients of membrane-bound factors on supported lipid bilayer.
Membrane-bound factor Diffusion coefficient, Mean ± SD (µm
2
/s)
VCAM-1 0.315 ± 0.040
SCF 0.382 ± 0.022
CXCL12 0.378 ± 0.149
Ang-1 0.287 ± 0.077
48
sDLL-1 0.280 ± 0.070
TPO 0.255 ± 0.084
Flt3L 0.254 ± 0.051
Fluorescence activated cell sorting (FACS) was used to sort HSCs (Lin-cKit+Sca1+Flk2-
CD34-Slamf1+) from the bone marrow of adult mice (4–6 months old) (Fig. 2-2). The freshly
isolated HSCs were incubated on SLBs tethered with both VCAM-1 and one of the six screened
ligands at 37
o
C for 1 hr, and live-imaged on an inverted microscope with a heated incubation
chamber (37
o
C). Interference reflection microscopy (IRM) was used to assess the site of cell
adhesion on SLBs, with the area and intensity (darkness) indicating the extent and strength of
adhesion to the surface [285]. While HSCs adhered to all the bilayers with a screened ligand +
VCAM-1, we found that only the membrane-bound SCF (mSCF) was recruited and clustered by
HSCs at the HSC-SLB interface (Fig. 2-3B,C).
49
Figure 2-2. Gating for hematopoietic and stromal populations from bone marrow. (A) Gating scheme for
hematopoietic populations from bone marrow. Sorting starts from DAPI- singlets. Following the arrows: (a)
Lin+ and Lin- populations; (b) oligopotent progenitors (OPPs) and cKit+Lin-Sca1+ cells (KLS); (c) Flk2+
multipotent progenitors (MPP+) and Flk2- cells (d) HSCs and Flk2- MPPs (MPP-). Numbers indicate the
percentage of the gated populations to the parent population. (B) Gating scheme for niche stromal cells (LepR+
MSCs) [295]. (C) The phenotypic identity of the stromal cells was confirmed by their high expression of SCF
(Kitl), CXCL12, VCAM-1 and PDGFRα compared to PDGFRα-negative cells using quantitative PCR (qPCR).
To verify if such recruitment/clustering recapitulates the real HSC-stromal cell interactions
in the bone marrow niche, we FACS-sorted LepR+ MSCs from the bone marrow [296] (Fig. 2-
2B). The identity of these stromal cells was confirmed by their high mRNA levels of SCF,
CXCL12, VCAM-1 and platelet-derived growth factor receptor α (PDGFRα) (Fig. 2-2C). LepR+
MSCs and HSCs were randomly mixed, incubated for 1 hr, fixed, immuno-stained, and imaged
for the heterotypic HSC-LepR+ cell pairs. In ~80% of HSC-MSC pairs, the mSCF on stromal cells
and its receptor cKit on HSCs were clustered at the HSC-stromal cell interface, as compared to
their diffusive patterns on the individual MSCs and HSCs, respectively (Fig. 2-3D,E). Thus, we
proceeded with the SLBs to further investigate the HSC-stromal cell interactions mediated through
the mSCF and VCAM-1, and their downstream signaling activities.
50
Figure 2-3. Membrane-bound SCF is the only factor recruited/clustered by HSCs in a screening. (A) Schematic
illustration of membrane-bound HSC-niche cell interaction and its recapitulation on supported lipid bilayer (SLB)
with tethered membrane-bound factors. (B) Representative images of HSCs after incubation with VCAM-1 with
or without membrane-bound factors (mb-factor, with a fluorescent label, background subtracted) on SLB for 1
h. DIC: differential interference contrast; IRM: interference reflection microscopy (dark regions: cell-substrate
contact/adhesion). (C) Total mb-factors recruited by single HSCs assessed by the total fluorescence from all
pixels under each cell. n = 37 cells per condition. Data are presented as mean ± SD. ****p<0.0001 by ANOVA
with Tukey’s test. (D) Representative images of HSCs, LepR+ MSCs (niche stromal cell) and HSC + LepR+
MSC pairs forming physical contact; all cells or cell pairs were incubated for 1 h. Arrows: dispersed cKit or SCF
localization on the surface of single HSC or LepR+ MSC, respectively, or clustered SCF/cKit at the HSC-MSC
interface. (E) Frequency of cKit/mSCF clustering on one side of the cell in single cells or in HSC-MSC pairs. n
= 20 single cells or cell pairs for each condition.
51
2.3.2 HSCs are highly efficient in recruiting mSCF with a distinct cell morphology
The cKit receptor is expressed on HSCs as well as on multipotent progenitors (MPPs) and
oligopotent progenitors (OPPs) [297], which are believed to be all supported by LepR+ stromal
cells [41]. To investigate whether HSCs and these cKit-expressing progenitors share similar
patterns of mSCF recruitment, we FACS sorted HSCs, MPPs (MPP-:
Lin-cKit+Sca1+CD34+Flk2-
and MPP+: Lin-cKit+Sca1+CD34+Flk2+), a subset of OPPs (Lin-cKit+Sca1-, which includes the
common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and
megakaryocyte-erythrocyte progenitor (MEP)), and the lineage-committed bone-marrow
leukocytes (BML: Lin+CD45+ cells, which have no cKit expression and serve as the negative
control) (Fig. 2-4A). The cKit expressions by the five populations were determined by FACS.
Among those, HSCs had an intermediate cKit level between MPPs and OPPs (Fig. 2-4A, right
panel). Upon incubation on SLBs with mSCF+VCAM-1 at 37
o
C for 1 hr, all the cKit+ cells (HSCs,
MPPs, and OPPs) recruited mSCF in clusters at the cell-SLB interface, while the BMLs did not
have any detectable mSCF recruitment (Fig. 2-4B). Interestingly, HSCs recruited the most mSCF
per cell (Fig. 2-4C), suggesting that HSCs were more efficient than the MPPs in mSCF recruitment.
Most strikingly, unlike the other cKit+ cells, HSCs often had mSCF clusters polarized to
one side of the cell body, which colocalized with distinct large protrusions in the bright field (Fig.
2-4B, white arrow). We first quantified the polarization of mSCF recruitment, defined as the
distance between the geometric center of cells in bright field and the mass center of mSCF clusters
(Fig. 2-4D, left). HSCs had the most polarized mSCF clusters of all cells, often extending beyond
the radii of the cells (~4 µm) (Fig. 2-4D). Under scanning electron microscopy (SEM), we
confirmed that most HSCs indeed had a polarized morphology, with long protrusions extended
from the cell body toward one side of the cell beyond the immediate adhesion/contact area on SLB
(Fig. 2-4E). The frequency of cells showing distinguishable membrane protrusions (extending
52
from the cell periphery for ≥2 µm) was significantly higher in HSCs (88.0 ± 6.0%) than the more
differentiated cells, which ranged from 0% (BML) to 13.3% (MPP-) with no statistical differences
between them (Fig. 2-4F). To assess the stability of the polarized morphology, we cultured HSCs
on SLBs with mSCF+VCAM-1 and fixed them at 1, 2, 6 and 12 hours. We found that the
proportion of HSCs with the polarized morphology remained stable over time (Fig. 2-5A,B). Our
data indicate that HSCs have a unique interaction pattern with SLBs with mSCF and VCAM-1,
which features membrane protrusions and clustering of mSCF at the sites of protrusions.
53
54
Figure 2-4. HSCs are highly efficient in recruiting mSCF with distinct cell morphology. (A) Quantification of
cKit expression by five hematopoietic populations measured by flow cytometry. Data quantified from 50,000
events in total. MPP-: Flk2- multipotent progenitors; MPP+: Flk2+ multipotent progenitors; OPP: oligopotent
progenitors; BML: bone marrow lineage+ cells. (B) Representative images of mSCF and cKit clustering patterns
under the five hematopoietic cell types on SLB tethered with mSCF and VCAM-1. Cells were incubated for 1
h. Arrows: HSC has a polarized morphology with mSCF cluster recruited to the cell protrusion on one side. (C)
Quantification of total mSCF recruited by each cell type. n = 100-108 single cells per condition. N.D.: not
detected. (D) The polarity of mSCF distribution is defined as the distance between the mass center of mSCF
clusters and the cell center. HSCs have the highest polarity of mSCF distribution among all the cKit+ HSPCs. n
= 70 single cells per condition. (E) Representative scanning electron microscopy (SEM) micrographs of the five
hematopoietic cells incubated on SLB with mSCF+VCAM-1. HSCs show unique polarized membrane
protrusions. (top row: top view; bottom row: 45° side view). (F) Frequency of cells with membrane protrusions
of 2 µm or longer in SEM. n = 3-5 field of views per condition, with 3-10 single cells per field of view. All scale
bars = 10 µm. Data are presented as mean ± SD. ****p < 0.0001 by ANOVA with Tukey’s test.
55
Figure 2-5. Morphological maintenance of HSCs and the role of VCAM-1 in HSC adhesion. (A) HSCs maintain
polarized morphology on an SLB with mSCF+VCAM-1 at 1, 2, 6, and 12 hour in the culture. (B) The percentages
of HSCs with protrusions remain stable over 12 hours. n.s.: p>0.05 by ANOVA and Tukey’s test. (C) VCAM-1
promotes HSC adhesion to lipid bilayers. Representative DIC, BF and IRM images of HSCs incubated with a
blank lipid bilayer or a lipid bilayer tethered with VCAM-1. (D) Quantification of cell adhesion areas measured
from IRM images. n = 39 (Blank) and 52 (VCAM-1) single cells per condition. **p < 0.01 by unpaired Student’s
t test (two-tailed). Scale bar: 10 µm.
2.3.3 VCAM-1 promotes polarized morphology and mSCF recruitment pattern in HSCs
We next investigated the contributions of mSCF and VCAM-1 in forming the polarized
HSC morphology and mSCF recruitment pattern. Freshly isolated HSCs were incubated for 1 hr
on SLBs with VCAM-1 or mSCF alone, or with both factors (mSCF+VCAM-1), and examined
for their adhesion and morphology with light microscopy and SEM (Fig. 2-6A). With VCAM-1
alone, HSCs had a non-characteristic, rounded morphology in bright field and formed continuous
contact with the SLB in IRM (Fig. 2-6A), the area of which was slightly larger than those on a
blank SLB (Fig. 2-5C,D). In contrast, HSCs on mSCF-alone SLB were featured with small
multifocal adhesion footprints in IRM. When both mSCF and VCAM-1 were present, the polarized
morphology emerged, with distinct protrusion(s) toward one side of the cell body (Fig. 2-6A).
Strikingly, under SEM, cells on VCAM-1 alone had little or no protrusions from their surface;
with mSCF alone, slim protrusions could be observed pointing in all directions, as opposed to the
large, polarized protrusion seen with mSCF+VCAM-1 (Fig. 2-6A, bottom). We found that the
normalized cell adhesion, defined as the adhesive area in IRM divided by the cell area in bright
field, was the lowest with mSCF alone while being similar between the VCAM-1-containing
conditions, demonstrating a role of VCAM-1 in promoting HSC adhesion. Quantitative analysis
56
of the SEM images shows that HSCs had minimal protrusions on SLB with VCAM-1 alone,
measured by both the percentage of cells with protrusions ≥ 2 µm in length and the number of such
protrusions per cell (Fig. 2-6C,D). Interestingly, mSCF was sufficient to induce cell protrusions
in HSCs with or without VCAM-1 (Fig. 2-6C), while the number of protrusions per cell was
reduced (Fig. 2-6D) and their width increased (Fig. 2-6E) by the presence of VCAM-1. The data
here suggest that the large protrusion and polarized morphology are synergistically induced by
VCAM-1 and mSCF.
Notably, the number of protrusions per cell and the protrusion width were negatively
associated in the mSCF and mSCF+VCAM-1 conditions (Fig. 2-7D,E). We hypothesized that the
polarized morphology of HSCs on mSCF+VCAM-1 was preceded in time by a multifocal
morphology similar to that with mSCF alone. To test the hypothesis, we examined HSCs on SLBs
with mSCF+VCAM-1 fixed after 10 min or 1 hr incubation (Fig. 2-7F). The HSC morphology on
mSCF+VCAM-1 SLBs at 10 min indeed resemble that on mSCF-alone SLBs at 1 hr. Importantly,
the number of protrusions (≥ 2 µm) per cell reduced significantly after 1 hr of incubation (Fig. 2-
7F,G), while their average width was more than doubled from <0.5 µm to over 1.2 µm (Fig. 2-
7H). The polarity of mSCF distribution also increased significantly over time (Fig. 2-7F,I). In
addition, we noted a transition in the distribution of mSCF clusters from a diffused, multifocal
pattern to a more concentrated one (Fig. 2-7F). This was confirmed by measuring the distribution
of mSCF clusters (Fig. 2-7J), which features a reduction of total area of mSCF distribution (Fig.
2-7K) and an increase of areal fraction of mSCF clusters within the distribution area (Fig. 2-7L).
With time lapse microscopy, we further observed that early on, HSCs gained more adhesion and
mSCF recruitment in a smaller footprint on mSCF+VCAM-1 SLBs than those on mSCF alone
surfaces over time (Fig. 2-8A-E). Overall, our data here indicate that VCAM-1 dynamically and
57
synergistically regulates the morphology of HSCs and the interaction pattern of HSCs with mSCF,
and promotes a more polarized, clustered distribution of mSCF recruitment.
58
Figure 2-6. VCAM-1 promotes polarized morphology and mSCF recruitment pattern in HSCs. (A) DIC, IRM
and SEM images of HSCs after 1 hr incubation on SLBs with VCAM-1 alone, mSCF alone or mSCF+VCAM-
1. (B) Comparison of normalized cell adhesion areas in microscopy (adhesion measured in IRM divided by cell
area measured in DIC). n = 21-83 cells per condition. (C-E) Analyses of HSC morphology from SEM images.
(C) Frequency of cells showing membrane protrusions ≥ 2 µm in SEM. n = 3 SEM area per condition with 20-
40 single cells per area. (D) Number of protrusions ≥ 2 µm per cell in SEM. n = 20-30 single cells per condition.
(E) Width of protrusions measured at the middle point. n = 30-82 single cells per condition. (F) SEM and
microscopic images of HSCs incubated on mSCF+VCAM-1 SLBs for 10 min and 1 hr. Corresponding
quantifications of (G) number of membrane protrusions per cell and (H) average width of membrane protrusions
under SEM. n = 18 (10 min) and n = 30 (1h) single cells per condition. (I) Polarity of recruited mSCF by HSCs.
n = 74 (10 min) and n = 120 (1h) single cells per condition. (J) Definition of the area of mSCF distribution.
Quantification of (K) area of mSCF distribution and (L) percentage of area occupied by mSCF clusters in the
total area of mSCF distribution. (K-L) n = 57 (10 min) and n = 94 (1h) single cells per condition. Scale bars =
10 µm. Error bars: SD. *: p˂0.05, **: p˂0.01, ***: p˂0.001, ****: p<0.0001 by ANOVA with Tukey’s test in
(B-D) and 2-tailed unpaired Student t test in (E, G-I, K-L).
59
Figure 2-7. Dynamic regulation of HSC-mSCF interaction by VCAM-1 and importance of lateral mobility in
mSCF-VCAM-1 synergy. (A) VCAM-1 is a dynamic regulator of HSC-mSCF interaction. Time lapse images
of HSCs seeded on supported lipid bilayers tethered with mSCF (a) with VCAM-1 or (b) without VCAM-1. (B)
Normalized cell adhesion assessed by the dark area in IRM channel divided by cell area. (C) Total recruitment
of cKit by HSCs assessed by the sum of fluorescence intensities of all pixels under the cells after background
subtraction. (D) Total recruitment of mSCF by HSCs. (E) Mass distribution of recruited mSCF. n = 10 single
cells per condition. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p < 0.0001 by Student’s t test. Scale bar: 10 µm.
(F) HSCs show similar morphology on coverslips with immobilized SCF, VCAM-1, or both factors. (G) HSCs
have similar adhesion strength on the three immobilized surfaces as indicated by the percentage of remaining
60
HSCs after a series of shear flow up to 15 mL/min. n = 5 ROIs per condition. Each ROI has 50-100 HSCs. n.s.:
p>0.05 by ANOVA and Tukey’s test.
2.3.4 mSCF and VCAM-1 synergistically promote HSC adhesion
Cell adhesion is crucial for HSC homing and maintenance in the niche [298]. The increased
normalized adhesion area of HSCs on mSCF+VCAM-1 SLB (Fig. 2-6B and Fig. 2-7B) suggest a
stronger cell-SLB interaction under the mSCF-VCAM-1 synergy. To measure the strength of such
interaction/adhesion, we designed a microfluidic device that imposes controlled shear stresses on
the adhered HSCs (Fig. 2-8A). HSCs and MPP- cells were loaded and incubated in the device for
1 hr to establish the adhesion on pairs of SLB surfaces, respectively (HSCs in Fig. 2-8B). As a
measure of the adhesion strength, we interpolated the flow rates at which 50% of the cells were
peeled off, and calculated the corresponding flow shear stresses at the SLB surface (Fig. 2-8C,D).
We found that mSCF alone provided little strength in cell adhesion, while VCAM-1 alone provided
similar, intermediate adhesive strength to both HSCs and MPPs. In contrast, mSCF+VCAM-1
dramatically increased the adhesion of HSCs by 50-fold from those on VCAM-1 alone (Fig. 2-
8C), compared to a 5-fold increase on mSCF+VCAM-1 vs. VCAM-1 for MPP- cells (Fig. 2-8D),
the progenitor closest to HSCs in the lineage hierarchy (Fig. 2-4A). Notably, we did not see a
synergistic effect of SCF and VCAM-1 on HSC adhesion when the two were directly immobilized
on the glass (Fig. 2-8F,G), where HSCs had similar adhesion on all the three surfaces under a high
flow rate (15 mL/min) that would peel most HSCs off SLBs (Fig 2-8C).
61
Figure 2-8. mSCF and VCAM-1 synergistically promote HSC adhesion. (A) Microfluidic setup for assessing the
strength of HSC adhesion to SLBs tethered with membrane-bound factors. (B) 2x bright field images of HSCs
seeded on SLBs with mSCF+VCAM-1 and VCAM-1 alone, respectively. Left: before shear flow; Right, after
the shear flow of 6 mL/min. Percentages of (C) HSCs and (D) MPP- cells remaining on the SLBs after a series
of increasing flow rates and the 50% wash-off flow rates & corresponding shear stresses on SLBs. n = 5 ROIs
per data point. (E-G) Effect of shear flow on the orientation of the HSC protrusions on the SCF+VCAM-1 SLBs.
(E) Definition of the protrusion angle (θ), and orientation changes in HSCs with different starting θ before and
after the shear flow (11 mL/min). (F) Protrusion angles of single HSCs before and after the shear flow. (G)
Changes of protrusion angles (θ after – θ before) in HSCs with starting θ greater or less than 90
o
. n = 22 single HSCs.
62
Error bars: SD. **: p˂0.01 by paired Student’s t test and unpaired 2-tailed Student’s t test in (F) and (G),
respectively.
We further investigated the role of the polarized morphology in HSC adhesion on the
mSCF+VCAM-1 SLBs. Under 60x magnification, we observed that HSCs remained largely
unchanged under most flow rates (low to medium). Under a high flow rate (11 mL/min), the
adhesion and orientation of the remaining cells started to change, albeit mildly. We defined the
protrusion angle (θ, 0
o
~180
o
) as the angle between the flow direction and the cell-to-protrusion
vector in HSCs (Fig. 2-8E). Under the 11 mL/min flow rate, HSCs had significant changes in θ
compared to the no-flow condition (Fig. 2-8F). Further examination revealed that, those with cell
bodies upstream of the protrusions (i.e., θ<90
o
, Fig. 2-8E, top row) would reorient the cell bodies
more to the downstream (i.e., larger Δθ), while those with cell bodies already at the downstream
(θ>90
o
, Fig. 2-8E, bottom row) often had a slight extension of the protrusion without much change
in θ (Fig. 2-8G). In both cases, the protrusion remained anchored while the cell body shifted. These
findings suggest that the protrusions formed a stronger adhesion on SLBs than the cell body.
Overall, our results here demonstrate a distinct functional role of the mSCF and VCAM-1 synergy
and the polarized morphology in promoting HSC adhesion.
2.3.5 mSCF redistribution and morphological transition require cytoskeletal remodeling
We next investigated the involvement of cytoskeletal components in shaping the HSC
morphology under the mSCF-VCAM-1 synergy. It was previously shown that microcluster
recruitment by T cells during the formation of immunological synapse require actin polymerization
and myosin contraction [299]. In human HSPCs, Myosin IIA and IIB are further differentially
regulated in cell polarization [300]. We first examined the subcellular distribution of F-actin,
63
myosin IIa, and myosin IIb in mouse HSCs with the polarized morphology on mSCF+VCAM-1
SLBs (Fig. 2-9A). Notably, F-actin and myosin IIa were more specifically enriched at the
protrusion. Myosin IIa also appeared more cortical than myosin IIb, the latter of which was
relatively uniformly distributed across the cell (Fig. 2-9B), suggesting more involvement of F-
actin and myosin IIa than myosin IIb in the peripheral reorganization of protrusions. To understand
their roles in forming the polarized morphology, we next treated HSCs upon seeding on
mSCF+VCAM-1 SLBs with inhibitors of myosin contraction (with Blebbistatin, or Blebb), actin
polymerization (with latrunculin-A, or Lat-A), and Rho-associated protein kinase (ROCK) (with
Y27632) for 1 hr before fixation and downstream analyses (Fig. 2-9C). All the three drugs
prevented the polarized morphology of HSCs while resulting in different morphological
characteristics (Fig. 2-9D). Lat-A abrogated almost all the protrusions from the cell surface,
resulting in a smooth, “dough”-like morphology. Blebb did not fully disrupt membrane protrusions,
but led to long, slim ones pointing in all directions (Fig. 2-9D). Those treated with ROCK inhibitor
Y27632 had spiky protrusions shorter than those under the DMSO and Blebb conditions (Fig. 2-
9D,F). Importantly, while HSCs still formed tiny mSCF clusters on SLBs (and cKit clusters on
cell membrane), their ability to make large mSCF and cKit clusters in polarized, tight distribution
was impaired by all the three inhibitors (Fig. 2-9E,G,H). Interestingly, all the inhibitions led to
minor changes in the normalized cell adhesion area except for ROCK inhibition (Fig. 2-10A),
while the total mSCF recruitment was not impacted (Fig. 2-10B). These results indicate that the
actomyosin cytoskeleton and ROCK activity play a primary role in forming large protrusions and
polarized/tightly distributed mSCF in the mSCF-VCAM-1 synergy, but are not required for
enhancing the total mSCF recruitment.
64
Figure 2-9. mSCF redistribution and morphological transition require cytoskeletal remodeling. (A)
Immunostaining of myosin IIa and myosin IIb and counterstained F-actin in an HSC forming polarized
morphology on an SLB with mSCF+VCAM-1. (B) Line-scan of fluorescent intensities of the three cytoskeletal
components as illustrated in (A). The yellow box highlights the region of the protrusion. (C) ROCK signaling
and cytoskeletal reorganization downstream of mSCF and VCAM-1 engagement and the corresponding
inhibitors. (D) SEM images of HSCs pre-treated with inhibitors for 1 hr, and then incubated with SLBs with
65
mSCF+VCAM-1 for 1 hr. White arrows indicate protrusions on HSCs. (E) Microscopic images of HSCs from
the corresponding treated or non-treated conditions in (D). Arrows: colocalization of membrane protrusion with
adhesion sites and clusters of cKit, mSCF and F-actin. (F) Frequency of cells showing membrane protrusions ≥
2 µm in SEM. n = 23-26 cells per condition. (G) Polarity of mSCF distribution under single HSCs. n = 50 cells
per condition. (H) Area of the mSCF distribution under single HSCs. n = 59-84 single cells per condition. Scale
bars: 10 µm. Error bars: SD. *: p˂0.05, ****: p<0.0001 by ANOVA with Tukey’s test (F-H).
Figure 2-10. Regulation of HSC adhesion and mSCF distribution by cytoskeletal and Akt inhibitors. (A) Impact
of cytoskeletal inhibition on the normalized adhesion area in HSCs, which is assessed by the dark adhesion area
in IRM divided by the cell area in DIC in each cell. n = 59-84 single cells per condition. (B) None of the
cytoskeletal inhibition affected the total recruitment of mSCF by HSCs assessed by the sum of fluorescence
intensities of all pixels under the single cells after background subtraction. n = 64-145 single cells per condition.
66
n.s. p>0.05, ****p < 0.0001 by ANOVA with Tukey’s test. (C) Images of HSCs on mSCF+VCAM-1 SLB
without treatment (NTX) or with Akt inhibitor GSK2141795 (GSK). Scale bar: 10 µm. Effect of GSK treatment
on (B) normalized cell adhesion, (C) polarity of mSCF distribution, and (D) area of mSCF distribution. n.s.:
p>0.05, *p<0.05, **p<0.01 by unpaired 2-tailed Student’s t test.
2.3.6 mSCF-VCAM-1 synergy involves PI3K signaling
Both mSCF/cKit and VCAM-1/VLA-4 engagements have been reported to activate
phosphatidylinositol 3-kinase (PI3K) signaling [301, 302], which is upstream of cytoskeletal
remodeling and ROCK activity (Fig. 2-9C, Fig. 2-11A). We thus investigated the involvement of
PI3K in the mSCF-VCAM-1 synergy in HSCs. HSCs were fixed after incubation on SLBs with
mSCF alone or mSCF+VCAM-1 for 15 min or 1 hr, and immunostained for intracellular PI3K.
Using total internal reflection fluorescence (TIRF) microscopy, we found that PI3K was
predominantly colocalized with cKit/mSCF clusters (Fig. 2-11B). Importantly, despite of an
overall decrease of the colocalization over time in both conditions, the PI3K/cKit colocalization
was independent of the presence of VCAM-1 (Fig. 2-11C), suggesting cKit as the major site of
intracellular PI3K recruitment.
PI3K activation during VCAM-1 engagement has been shown to phosphorylate the protein
kinase Akt [302] (Fig. 2-11A). We next investigated the role of the three sequential signaling steps,
i.e., VCAM-1 engagement, PI3K activation, and Akt phosphorylation, in the formation of the
distinct mSCF recruitment pattern and HSC morphology. We first used small molecule inhibitors
BIO5192 (BIO) to disrupt the interaction of VCAM-1 with VLA-4, LY294002 (LY) and MK2206
(MK) to inhibit PI3K activation and Akt phosphorylation, respectively. Non-treated HSCs were
incubated on SLBs with mSCF alone or mSCF+VCAM-1 as controls. Consistent with the earlier
67
findings (Fig. 2-6A), incubation of HSCs with mSCF alone resulted in multiple membrane
protrusions (white arrows) and multifocal adhesion with lipid bilayer (red arrows) at the site of
cKit/mSCF clusters (Fig. 2-11D). The presence of VCAM-1 promoted extended adhesion with a
polarized morphology of the cell (Fig. 2-11D), which was completely disrupted by BIO and LY
and partially by MK (Fig. 2-11D,E). All the three inhibitions significantly reduced the polarity of
mSCF distribution (Fig. 2-11F), yet under MK there were more residual polarized HSCs similar
to the untreated ones (red box, Fig. 2-11D,F). Moreover, only the BIO treatment led to a significant
increase of the area of mSCF distribution (Fig. 2-11G). Inhibiting Akt with an alternative,
GSK2141795 (or Uprosertib) had similar effects to MK (Fig. 2-12C-F). These results suggest that,
while all the three signaling steps (VLA4-PI3K-Akt) are involved in the cell adhesion and the
polarity of mSCF distribution, the redistribution of mSCF on SLBs is primarily regulated by
VCAM-1-mediated adhesion. The differential results of Akt and PI3K inhibitions also implies an
involvement of downstream targets other than Akt in VCAM-1 and PI3K activation.
LY294002 was reported to also target bromo and extra-terminal domain (BET) family
proteins [303]. Meanwhile, among the eight isoforms of PI3K, class IA PI3Ks are involved in the
signaling directly downstream of membrane-bound receptors [304]. Hematopoietic cells express
three isoforms of the p110 subunit of the class IA PI3Ks, the p110α, β, and δ [305]. To rule out
the off-target effect of LY294002, and to determine which of the three PI3K isoforms are primarily
involved in the mSCF-VCAM-1 synergy, we treated HSCs with another pan-PI3K inhibitor BAY
80-6946 (BAY), and three isoform-specific inhibitors to p110α (MLN1117), p110β (TGX-221),
and p110δ (CAL-101), respectively. All the four inhibitors resulted in a decrease of HSC adhesion
on SLBs with mSCF+VCAM-1, with p110δ and pan-PI3K (BAY) inhibitions causing greater
disruptions and resulting in a multifocal footprint (Fig. 2-11H,I). We also noticed that inhibiting
68
p110δ, but not p110α or β, led to a significant reduction in the polarity of mSCF distribution (Fig.
2-11J) to a level like the BAY treatment. Our findings here thus confirm the role of PI3K and
indicate the p110δ isoform as the major player in regulating the adhesion and formation of the
polarized mSCF distribution in the mSCF-VCAM-1 synergy.
69
Figure 2-11. mSCF-VCAM-1 synergy involves PI3K-Akt signaling. (A) PI3K-Akt signaling downstream of
HSC engagement with mSCF and VCAM-1 and the corresponding inhibitors. (B) TIRF images of PI3K
immunostaining in HSCs after 15 min or 1 hr incubation on SLBs with mSCF or mSCF+VCAM-1. Scale bar: 5
µm. (C) Colocalization of PI3K and cKit. n = 20 single cells per condition. (D) Microscopic images of HSCs on
SLB with mSCF alone (non-treated) or the non-treated and drug treated conditions on SLBs with
mSCF+VCAM-1. White arrows: protrusions visible in the bright field; yellow arrows: adhesion under cell body;
red arrows: adhesion through elongated protrusions. Corresponding quantifications of (E) normalized cell
adhesion, (F) polarity of mSCF distribution, and (G) area of mSCF distribution. (E,F) n = 100-104 single cells
per condition. (H) HSCs on SLBs under pan- and isoform-specific PI3K inhibitions, and the quantifications of
b normalized cell adhesion and (J) polarity of mSCF distribution. (I,J) n = 41-91 single cells per condition. (D,H)
Scale bars: 10 µm. Error bars: SD. n.s.: not significant, *: p˂0.05, **: p˂0.01, ****: p<0.0001 by ANOVA with
Tukey’s test (C,E-G,I-J).
70
Figure 2-12. Regulation of HSC adhesion and mSCF distribution by cytoskeletal and Akt inhibitors. (A) Impact
of cytoskeletal inhibition on the normalized adhesion area in HSCs, which is assessed by the dark adhesion area
in IRM divided by the cell area in DIC in each cell. n = 59-84 single cells per condition. (B) None of the
cytoskeletal inhibition affected the total recruitment of mSCF by HSCs assessed by the sum of fluorescence
intensities of all pixels under the single cells after background subtraction. n = 64-145 single cells per condition.
n.s. p>0.05, ****p < 0.0001 by ANOVA with Tukey’s test. (C) Images of HSCs on mSCF+VCAM-1 SLB
without treatment (NTX) or with Akt inhibitor GSK2141795 (GSK). Scale bar: 10 µm. Effect of GSK treatment
on (B) normalized cell adhesion, (C) polarity of mSCF distribution, and (D) area of mSCF distribution. n.s.:
p>0.05, *p<0.05, **p<0.01 by unpaired 2-tailed Student’s t test.
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2.3.7 mSCF-VCAM-1 synergy promotes nuclear FOXO3a retention
Next, we examined the effect of the mSCF-VCAM-1 synergy on downstream signaling
and nuclear FOXO3a (Fig. 2-13A). SCF can activate cKit phosphorylation to initiate PI3K/Akt
signaling [306]. We examined the colocalization of mSCF/cKit clusters with all phosphorylated
tyrosine residues (pY) by immunostaining and TIRF microscopy (Fig. 2-13B). The pY staining
was found largely colocalized with SCF/cKit clusters without or with VCAM-1 (Fig. 2-13B, top
two rows). Importantly, the presence of VCAM-1 promoted tight clustering of pY staining and its
colocalization with cKit, which can be reverted by VCAM-1 inhibition through BIO pre-treatment
(Fig. 2-13B,C). Our result suggests that VCAM-1 promotes the tyrosine phosphorylation that
directly associates with c-Kit in HSCs.
FOXO3a is a transcription factor that plays an important role in maintaining the HSC pool
through activities in nucleus [307]. We next studied the effects of the mSCF-VCAM-1 synergy on
nuclear retention of FOXO3a in HSCs. HSCs were incubated on SLBs with mSCF with or without
VCAM-1 for 12 h, and immunostained for FOXO3a. We found that HSCs on mSCF+VCAM-1
SLBs had significantly higher nuclear FOXO3a compared to those on mSCF alone (Fig. 2-13D,E).
In addition, pre-treatments of HSCs with the VCAM-1 and PI3K inhibitors (BIO and LY) reduced
nuclear FOXO3a levels (Fig. 2-13D,E). In contrast, pre-treatment of HSCs with the Akt inhibitor
MK further promoted the nuclear FOXO3a level compared to the non-treated group (Fig. 2-13D,E).
We further examined the effects of the p110α, β, and δ isoform-specific PI3K inhibitors (the same
ones used in Fig. 2-13H-J). The nuclear FOXO3a levels were significantly reduced by pan-PI3K
inhibition (by BAY and LY) but not the individual isoform inhibitors (Fig. 2-13G), indicating a
functional redundancy among the three isoforms for nuclear FOXO3a regulation. While the effect
of Akt inhibition on nuclear FOXO3a is expected, the result of PI3K inhibition was contrary to the
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known negative regulation of nuclear FOXO3a by the PI3K/Akt pathway, suggesting a previously
unknown, positive role of PI3K in promoting the maintenance of nuclear FOXO3a through the
mSCF-VCAM-1 synergy.
Figure 2-13. mSCF-VCAM-1 synergy promotes nuclear FOXO3a retention. (A) PI3K-FOXO3a signaling in
HSCs in an Akt-dependent or independent manner upon engagement with mSCF and VCAM-1. (B) TIRF
microscopy images of pan-phospho-tyrosine (pY) immunostaining (with 4G10) in HSCs after 1 hr incubation
on SLBs with mSCF alone, or with mSCF+VCAM-1 without or with pre-inhibition of VLA-4 for 1 hr. Scale: 5
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μm. (C) Colocalization of pY and cKit clusters under the three conditions. n = 9-41 single cells per condition.
(D) FOXO3a immunostaining (nuclei counter-stained) of HSCs incubated for 12 hr on SLBs with mSCF alone,
or mSCF+VCAM-1 without or with VLA-4, PI3K and Akt inhibitors. (E) Intensities of nuclear FOXO3a
immunostaining in single HSCs, normalized to the population average of the mSCF-alone condition. n = 100
single cell per condition. (F) FOXO3a immunostaining of HSCs incubated for 12 hr on SLBs with mSCF alone,
or mSCF+VCAM-1 without or with pan- and isoform-specific PI3K inhibitors, and corresponding quantification
of (G) intensities of nuclear FOXO3a immunostaining, normalized to the population average of the mSCF-alone
condition. n = 16-66 single cells per condition. (D,F) Dash lines: contour of nuclei used for nuclear FOXO3a
quantification. (D,F) Scale bars: 10 μm. Error bars: SD. *: p˂ 0.05, **: p˂0.01, ***: p˂0.001, by ANOVA with
Tukey’s test in (C, E and G).
2.3.8 Soluble SCF competitively disrupts mSCF-VCAM-1 synergy
The sSCF is an essential growth factor commonly used in HSC cultures for their
maintenance and proliferation [107]. Since both the soluble and membrane-bound forms of SCF
can engage cKit, we next investigated the role of sSCF in the mSCF-VCAM-1 synergy in HSCs.
HSCs were incubated with sSCF before (pre-TX) or 30-min after (post-TX) being seeded onto
mSCF+VCAM-1 SLBs. We found that the pre-TX HSCs recruited significantly less mSCF
compared to those without sSCF (non-treated, NTX); in contrast, the post-TX HSCs retained the
mSCF recruitment (Fig. 2-14A,B). This suggests that both sSCF and mSCF can occupy and
prevent the other from binding to cKit. Cell adhesion was found slightly impaired by sSCF under
both treatment conditions, with more disruption in the pre-TX group (Fig. 2-14C). Importantly,
we observed a striking difference in cell morphology with the pre-TX HSCs, which barely formed
any membrane protrusions (Fig. 2-14A,D). Such morphological disruption in the pre-TX HSCs
coincided with a loss of polarization and widened area of mSCF distribution compared to the NTX
condition (Fig. 2-14E,F). In contrast, the post-TX cells largely retained the polarized morphology
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with a large, distinct protrusion, with minimal changes in the overall distribution of mSCF clusters
on SLBs (Fig. 2-14E,F).
Next, we examined the downstream effect of sSCF on the maintenance of nuclear FOXO3a
by the mSCF-VCAM-1 synergy (Fig. 2-13D,E). We extended the incubation of HSCs on
mSCF+VCAM-1 SLBs without or with pre- or post-treatment of sSCF to 12h before fixation and
immunostaining for FOXO3a, and compared the results to HSCs incubated with mSCF or sSCF
alone (Fig. 2-14G). We found that the presence of sSCF impaired the maintenance of nuclear
FOXO3a in HSCs by the mSCF-VCAM-1 synergy, while the difference between pre- and post-
TX was not significant (Fig. 2-14H). Strikingly, even with sSCF, the nuclear FOXO3a levels
remained significantly higher in HSCs on the mSCF+VCAM-1 SLBs than those treated with sSCF
alone (Fig. 2-14H). Overall, the data here suggest a competitive/disruptive nature of sSCF against
mSCF for cKit engagement and morphological regulation, and the ability of mSCF in promoting
nuclear FOXO3a maintenance despite of the presence of sSCF (Fig. 2-14I).
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76
Figure 2-14. Soluble SCF competitively disrupts mSCF-VCAM-1 synergy. (A) HSCs incubated on SLBs with
mSCF+VCAM-1 for 1 hr. Pre-TX: pre-treated with 50 ng/mL sSCF on ice for 1h (no washing) before loading
onto SLB; post-TX: HSCs allowed to interact with SLB for 30 min before being treated with 50 ng/mL sSCF
for 30 min (post-TX). The total interaction time with SLBs was 1 hr in all conditions. Corresponding
quantifications of: (B) Total recruited mSCF by each cell. n = 103-109 single cells per condition. (C) Normalized
cell adhesion. n = 100-104 single cells per condition. (D) Frequency of cells showing large membrane protrusions
(in DIC). n = 19-21 images per condition with 6-23 cells per image. (E) Polarity of mSCF distribution. n = 105-
107 single cells per condition. (F) Area of mSCF distribution. n = 100 single cells per condition. (G) HSCs
immunostained for FOXO3a (nuclei counterstained) after 12 hr incubation on SLBs. (H) Intensities of nuclear
FOXO3a immunostaining in single HSCs, normalized to the population average of the mSCF-alone condition.
n = 100 single cells per condition. (I) Summarized mechanism of mSCF-VCAM-1 synergy and regulation of
HSC morphology, adhesion, and nuclear FOXO3a. Scale bars: 10 μm. Error bars: SD. n.s.: p > 0.05, *: p˂0.05,
**: p˂0.01, ****: p<0.0001 by ANOVA with Tukey’s test (B-F and H).
2.4 Discussion
Membrane-bound factors have long been recognized as a crucial component in HSC niches
in the bone marrow [308]. However, little is known about how HSCs interact with membrane-
bound factors and how these localized interactions in the niches contribute to HSC
phenotype/cellular activities at the single-cell level. Here we reported a clustered recruitment
pattern and a polarized morphology assumed by HSCs in the synergistic interaction with mSCF
and VCAM-1, which are unique to mSCF in the screened factors. For instance, we did not observe
any clustered patterns or morphological changes in HSCs with Ang-1, which binds to Tie2, another
receptor tyrosine kinase [68]. Similarly, HSCs did not form clusters with Delta-1 (sDLL1, Fig. 2-
3B), a notch ligand reported to support the expansion of hematopoietic progenitors in an
immobilized but not soluble form in vitro [125]. Our findings also coincide with reports on the
dispensable roles of Ang-1 and Notch signaling in HSC maintenance in vivo [87, 309]. Clustering
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may thus play an important functional role in the membrane-bound signaling in the stem cell niche.
Microcluster formation is central to the activation of T and B lymphocytes [285, 310]. But whereas
T and B cells cluster TCR or BCR into a bullseye shape in the presence of ICAM-1, HSCs form a
polarized morphology and pattern of cKit/mSCF clusters with VCAM-1, suggesting a distinct role
of mSCF/cKit microclusters in HSCs from those in lymphocyte activations. Notably, all the cKit-
expressing HSPCs examined in the study can form mSCF microclusters. In vivo, HSC niches are
likely accessible to all the cKit+ HSPC populations [311, 312]. The mSCF recruitment by cKit
may thus be a common route of HSPC-stromal interactions. Interestingly, the cKit+ populations
(HSCs, MPPs, OPPs) have varying efficiency in mSCF clustering (Fig. 2-4). As a receptor tyrosine
kinase, this efficiency may indicate the strength of the cKit signaling. That being said, cKit is
upstream of several signaling pathways that involve multiple functions including cell proliferation,
survival and differentiation [313]. As such, the recruitment efficiency can have different functional
implications, which warrants further investigation.
Most strikingly, the polarized morphology in response to mSCF+VCAM-1 is specific to
HSCs among all the HSPCs, including the closely related MPPs (MPP- and MPP+) (Fig. 2-4B).
Moreover, in our SLB model, the mSCF-VCAM-1 synergy drastically increases the strength of
HSC adhesion to a level that is about 10-fold that of MPPs, whereas the protrusion provides
additional adhesive strength (Fig. 2-8E-G). The synergy and specificity to HSCs provide a
potential cellular mechanism for the selectivity of bone marrow niche to recruit and anchor HSCs
over other cKit+ progenitors during HSC homing or transplantation. Conversely, disrupting the
mSCF-VCAM-1 synergy may disproportionally affect the retention of HSCs in their niches more
than their progenitor counterparts. In fact, such differential effect between HSCs and cKit+ MPPs
has been indirectly noted before, where a partial loss of cKit function led to more depletion of
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HSCs than MPPs in the bone marrow [314]. Our results here thus suggest a functional role of the
mSCF-VCAM-1 synergy and the resultant HSC morphology in HSC homing/anchorage in the
bone marrow niche. The findings and microfluidic tools may also be applied to enrich HSCs and
study their heterogeneities.
The membrane protrusions and the morphological transformation are among the most
prominent features of HSCs under the mSCF-VCAM-1 synergy. It has been reported that isolated
mouse HSCs exhibit surface microspikes, and soluble cytokines such as CXCL12 and SCF can
further induce the formation of membrane extensions or podia that point toward stromal cells in
culture [112]. Interestingly, these podia only exist on the motile HSCs in vitro or those lodged in
peripheral organs (except bone marrow) upon transplantation in vivo, but not on those adhered to
stromal cells (in vitro) or homed to bone marrow [112]. The membrane protrusions we observed
resemble these microspikes and/or podia, although under the distinct context of membrane-bound
HSC-stromal interactions which has not been described in mouse HSCs before. The existence of
long podia has also been seen in human CD34+ hematopoietic progenitor cells (HPCs) in culture
[237, 315]. Notably, the more primitive fraction of human CD34+/CD38- HPCs has a higher
frequency of polarized morphology [237], and these cells seek contact with a mouse stromal cell
line (AFT024) through protrusions [316]. However, unlike the mouse HSCs, the polarized human
HPC morphology seems to precede and is only slightly promoted by the direct contact with stromal
cells [316]. It was unclear whether the difference is due to the progenitors in the much less purified
HPC pool than the mouse HSCs. We foresee that the SLB model can be adapted to elucidate the
regulation of human HSC morphology by the molecules found in human HPC-stromal contact
[317], as well as mSCF and VCAM-1. Interestingly, HSCs formed the polarized morphology in a
non-polarized ligand field on SLBs. Similarly, T cells can spontaneously break and reestablish the
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symmetry of immunological synapse on a uniform SLB, through the opposing effects of PKCθ
and WASp [318]. Neutrophils can also self-organize into a polarized shape in uniform
chemoattractant concentrations [319]. Our cytoskeleton and PI3K inhibition data also suggest that
the HSC polarization on SLBs is due to the intrinsic signaling of HSCs in response to mSCF and
VCAM-1. Meanwhile, some intracellular molecules are distributed in a polarized manner in HSCs.
Cdc42 and tubulin have been found asymmetrically distributed in HSCs from young mouse [320].
We have previously observed a polarized distribution of metabolic coenzymes NAD(P)H in the
freshly isolated HSCs [321]. These molecules may also play a role in regulating HSC morphology.
Another key question is regarding the nature of the protrusion. In leukocytes, membrane
protrusions are often found associated with cell polarity and migration, which can be a protruding
structure at the leading edge (a pseudopod), or a contractile structure at the rear (a uropod). The
two structures differ in cytoskeletal composition: pseudopods are enriched with newly synthesized
actin, and the uropods are composed of contractile actin-myosin complexes [322, 323]. While
studies on HSC morphologies are relatively few, researchers have described the microspikes or
protrusions of mouse HSCs or human HPCs with various terms, e.g., magnupodia, tenupodia,
proteopodia, and uropods, which resemble morphological and functional features of pseudopods
and/or uropods [112, 315, 316]. However, little is known about the cytoskeletal nature of these
structures and the signaling components that regulate them in HSCs. We showed that the
protrusions formed by HSCs on mSCF+VCAM-1 SLBs are enriched for myosin II and F-actin,
and their formation is highly dependent on new actin polymerization, myosin contraction and
ROCK signaling (Fig. 2-10). Therefore, they have some characteristics of pseudopods (the
enriched F-actin and dependence on its polymerization) [324] but share more similarity with
uropods in shape and myosin II localization [323]. However, even though cells can migrate on
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SLBs [318], we did not observe obvious migratory behaviors in HSCs in the study. Instead, the
protrusions provide strong adhesion that resists high shear stresses (Fig. 2-8E). Therefore, they
may endow HSCs niche-anchoring abilities instead of migratory functions seen with a typical
uropod in motile leukocytes. Yet, it is still possible that the protrusions may play a role in migration
under other contexts (e.g., chemotaxis, extravasation, etc.) [325].
The importance of VLA-4/VCAM-1 interaction in HSPC and leukocyte adhesion has been
reported previously. VCAM-1 can synergize with B-cell receptor for tight adhesion and enhanced
signaling [326]. Human HPCs adhere to stromal cells mainly through VLA4/VCAM-1 [170]. In
mouse, this interaction is required for the homing of HSPCs to the bone marrow but not to spleen
[151]. Conversely, down-regulation or functional blockade of VLA4 or VCAM-1, or conditional
deletion of α4 integrin all result in mobilization of HPCs into the blood circulation [171, 327]. In
our SLB model, VCAM-1 slightly increases cell adhesion area underneath the cell body (Fig. 2-
7C,D) and provide a baseline adhesive strength to HSCs and MPPs (Fig. 2-98E,F). However,
compared to cell adhesion, we observed a more prominent role of VCAM-1 in supporting
mSCF/cKit recruitment, polarization of HSC morphology, and synergistic upregulation of HSC
adhesion strength with mSCF-1. Indeed, anti-VLA4/VCAM-1 induced HSC mobilization is found
dependent on the mSCF-mediated cKit signaling [79]. Our results thus support a central role of
the mSCF-VCAM-1 synergy in the homing and retention of HSCs in the bone marrow niche,
which may provide alternative strategies in HSC transplantation by strengthening or weakening
the synergy.
Our study demonstrated a crucial role of PI3K in the mSCF-VCAM-1 synergy. Consistent
with our in vitro results, PI3K activity has been implicated in HSC migration and bone marrow
homing in vivo [328]. However, PI3K is known to have many downstream targets, some of which
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may have opposing roles under different contexts. For example, activation of the PI3K/Akt
pathway in response to growth factor stimulation (e.g. sSCF) inhibits nuclear FOXO3a in HSCs
[329]. In contrast, our study shows a central role of PI3K in mediating the mSCF-VCAM-1
synergy, which promotes nuclear FOXO3a in HSCs through an Akt-independent manner (Fig. 2-
11A). It was recently reported in epithelial cells that PI3K can promote glycolysis through actin
cytoskeletal remodeling in an Akt-insensitive manner [330]. Since glycolysis is a key feature of
quiescent HSCs [331], and we have shown an involvement of actin cytoskeletal remodeling in
mSCF-VCAM-1 synergy, we postulate that the PI3K/actin pathway may be responsible for the
FOXO3a nuclear retention in our model, which will be investigated in future studies.
Among the three isoforms (p110α, β, and δ) of the class IA PI3Ks in hematopoietic cells,
p110α and β have been reported to have redundant or dispensable roles in HSC self-renewal [305,
332]. Our results indicate that p110α and β are involved in HSC adhesion on mSCF+VCAM-1
SLBs, but their roles in redistributing mSCF clusters into a polarized form are dispensable. In
contrast, inhibiting p110δ can recapitulate most of the effects on these features from pan-PI3K
inhibitions (Fig. 2-11H-J), highlighting the importance of p110δ in HSC-niche interactions.
Interestingly, PI3Kδ inhibitors combined with antibody therapies have shown promising effects
on chronic lymphocytic leukemia, by releasing leukemic cells from their protective niches in the
bone marrow [333]. It is thus crucial to understand the similarity and differences in the roles of
membrane-bound factors and PI3K isoforms between normal HSCs and leukemic cells, to
inform/improve niche-targeted leukemia treatments. Surprisingly, the maintenance of nuclear
FOXO3a is only significantly disrupted by the two pan-PI3K inhibitors but not the isoform-
specific ones (Fig. 2-11G), suggesting more functional redundancy of the three isoforms in the
downstream nuclear signaling than the cell adhesion/morphology. It also indicates the divergent
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roles of PI3K in regulating HSC maintenance vs. adhesion/morphology downstream of the mSCF-
VCAM-1 synergy (Fig. 2-14I).
The sSCF and mSCF have previously been shown to have diametric roles in HSCs, i.e.
sSCF induces HSC proliferation [65] while mSCF is required for the long-term HSC maintenance
[272]. It has been unclear, however, about how HSCs receive and interpret the two signals to
balance proliferation vs. quiescence in the bone marrow niche. We showed that sSCF competes
against mSCF by disrupting cKit clustering and HSC morphology, whereas the temporal order of
the two SCF forms makes a significant difference (Fig. 2-14B-F). On the other hand, the nuclear
FOXO3a in HSCs treated with both sSCF and mSCF+VCAM-1 remained higher than those with
sSCF alone, suggesting a more dominant role of mSCF in maintaining HSC quiescence. Overall,
our results suggest a stabilizing role of mSCF in HSC retention/maintenance in the bone marrow
niche. The mSCF and sSCF competition may also toggle the activation between the
PI3K/Akt/mTOR and the PI3K/actin pathways, leading to mitochondrial respiration or glycolysis,
respectively [330, 334], which also differentially regulate HSC activation or quiescence [331].
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Chapter 3: Adhesion-based cell selection on artificial stromal membrane
enriches hematopoietic stem cells
3.1 Rationale
Blood cells are short-lived. As a result, a continuous differentiation and proliferation is
required for multipotent hematopoietic stem and progenitor cells to renew and maintain the blood
system under regeneration stress [335]. A heterogeneous multipotent progenitor pool is
responsible for this job, which is composed of colonogenic long-term and short term self-renewing
HSCs and non-self-renewing multipotent progenitors (MPP) [336]. Developing methods for
accurate identification and precise isolation of HSC from the heterogeneous population has been
a continuous goal for many researchers.
Traditionally, a series of surface markers has been discovered, including cKit [337], CD34
[338], stem cell antigen-1 [339] , Slamf1[340], Hoxb5 [341], etc, which has been utilized for HSC
enrichment extensively in the means of immunophenotyping. So far, a marker uniformly expressed
by and unique to HSCs has not been discovered. Furthermore, HSC isolation based on phenotypic
expressions suffers from disadvantages such as cost, and unavailability of fluorescence assisted
cell sorting (FACS) equipment to most labs, which limits HSC research to only selected research
labs. Side population (SP) discrimination assay is another method using flow cytometry to isolate
stem cells based on the enhanced dye efflux properties of ABC (ATP-binding cassette)
transporters [342], first identified by Goodell et al [343]. In spite of the enrichment of SP in stem
cells, reports cautioned that not all stem cells show this characteristic, and this phenomenon is not
restricted to stem cells, either [342]. Exploring new ways of stem cell identification based on
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knowledge about HSC characteristics have been a continuous effort which spurs innovative work
across labs with different background and techniques.
HSC homing and engraftment is a multistep process that involves complex interactions
with bone marrow cells, including vascular endothelial cells, macrophages, and mesenchymal
stromal cells. It was first described several years ago as an active process that allows for the
migration of HSCs through the blood and vascular endothelium to different organs and BM niches.
It is now clear that the process of extravasation involves a range of adhesion molecules on both
HSCs and stromal cells, as well as extensive intracellular signaling that drives adhesion and
chemotaxis on the one hand and controls a transient modulation of stromal cell (e.g., endothelial
cells) integrity on the other [344]. The coordinated action of adhesive molecules and activation
processes triggered specifically by chemokines such as SDF-1 and vascular ligands, e.g., VCAM-
1, has been implicated in HSC migration. Cxcr4 gene deletion reduced the reconstitution of
immature murine hematopoietic cells in myeloablated recipient mice [345-347]. Moreover, HSCs
migrated exclusively to SDF-1 among a series of evaluated chemokines [134]. Although the
influence of SDF-1 on HSC chemotactic responses has been well established, its role in the
different molecular pathways underlying the early stages of homing remains a highly discussed
and contentious issue.
We have previously discovered that membrane bound form of two niche factors, SCF and
VCAM-1, synergistically induce HSC protrusions that provide strong adhesion against high shear
stresses in a supported lipid bilayer model [348]. In this work, we took a step forward and explored
the utilization of this discovery in stem cell enrichment from a heterogeneous stem and progenitor
cell population. HSPCs that are selected based on their greater adhesion strength towards
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membrane bound SCF and VCAM-1 show are phenotypically and functionally closer to FACS
sorted HSCs, compared to their weakly adhered counterparts.
3.2 Materials and methods
3.2.1 Mice.
C57BL/6J mice were purchased from Jackson Laboratories. Hoxb5-mCherry mice were a
generous gift from Rong Lu lab at USC stem cell. All mice were bred at the Research Animal
Facility of the University of Southern California. Animal care and euthanasia protocols were
approved by the Institutional Animal Care and Use Committee of the University of Southern
California. Mice were provided continuously with sterile food, water, and bedding.
3.2.2 Cell isolation and flow cytometry
Bone marrow cells were obtained from the crushed bones of 3~6-month-old mice, enriched
by cKit, immunostained and sorted for Lin+CD45+ cells (BMLs), HSCs and MPPs. FACS sorting
was carried out with a BD FACS™Aria II cell sorter (BD Biosciences, San Jose, CA) at 4°C.
3.2.3 Preparation of small unilamellar vesicles and supported lipid bilayers.
Lipid components (18:1 (Δ9-Cis) PC (DOPC), 18:1 PS (DOPS), 18:1 DGS-NTA(Ni) and
18:1 Biotinyl Cap PE) dissolved in chloroform were purchased from Avanti Polar Lipids and
mixed at mol % indicated in the main text. The lipids were dried in round-bottom flasks under a
stream of N2 for 5 min and desiccated for 2 h with house vacuum pump in a chemical fume hood.
The lipid mixture was resuspended by bath sonication in 1X PBS at a final concentration of 2.5
mg/ml and extruded 10 times through a membrane with 50-nm pore size (Avanti Polar Lipids) into
small unilamellar vesicles (SUVs). The SUV solutions were then diluted 1:1 in 1X PBS (pH 7.4)
before being loaded onto the glass coverslip through the loading chamber, and incubated for 2 min
to spontaneously form the lipid bilayers. The chambers were then washed with a 10X excess
86
volume of 1X PBS. For SEM, indium-tin-oxide (ITO) coated coverslips (SPI supplies) were used
to form lipid bilayers, where DOPC was replaced with 18:1 PS (DOPS) (Kumar et al, 2009).
3.2.4 Protein capturing on lipid bilayer
DOPC lipids supplemented with 0.1 mol % Biotinyl-Cap-PE and 5 mol % of DGS-NTA(Ni)
were used to form SLBs. The bilayer was blocked with 0.1 mg/ml BSA for 1 hr and incubated
with 10 μg/ml streptavidin for 20 min. The bilayer was then washed extensively with PBS (pH 7.4)
followed by 30 min incubation with the Alexa Fluor® 488-labeled mono-biotinylated SCF (7
μg/ml) (Peprotech), and VCAM-1 with a 6-histidine tail (2 μg/ml) (R&D Systems). The entire
chamber was then washed with 10X excess volumes of 1X PBS.
3.2.5 Cell seeding and incubation
FACS sorted cells were kept in PBS with 2% FBS on ice. Before being seeded onto bilayers,
cells were pelleted at 300x g and resuspended in StemSpan SFEM II (STEMCELL Technologies
Inc., Vancouver, Canada). The chamber containing lipid bilayers was equilibrated with the same
buffer, and the resuspended cells were then injected into the chamber and incubated for 1.5 hours
in a humidified incubator maintained at 37°C and 5% CO2.
3.2.6 Fluorescence microscopy
A Nikon Eclipse Ti-E inverted fluorescence microscope (Nikon, Tokyo, Japan) was used
for live-cell imaging, which is equipped with an OKOLab incubation box (OKOLAB, Italy)
controlling for temperature (37°C) and CO2 concentration (5%). The chamber containing lipid
bilayers was equilibrated with ECB, and the resuspended cells were then injected and live imaged
on the pre-warmed fluorescence microscope.
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3.2.7 Design, fabrication, and assembly of the microfluidic device
The design and toolpaths for the microdevice were created in Autodesk Fusion 360 (San
Rafael, CA) and custom-milled (Shapeoko, Carbide 3D, Torrance, CA) out of polycarbonate. The
final device was manufactured by pouring polydimethylsiloxane (PDMS) mixed at 10:1 base to
curing agent ratio (Sylgard 184 elastomer kit; Dow Corning). PDMS was cured at 80°C for 3 hours,
peeled off, and cut into individual devices. Channel inlets and outlets with 1.5 mm diameter were
punched at both ends of microfluidic channels. The PDMS devices were permanently bound to the
detergent-cleaned glass coverslips after plasma treatment for 50 seconds (Harrick Plasma, Model
PDC-001-HP) for the subsequent lipid bilayer formation and substrate modification.
3.2.8 COMSOL simulation of flow rate distribution
COMSOL Multiphysics® (Stockholm, Switzerland) was used to simulate the flow velocity
within the microdevice. Initial flow rate at inlets were set as 1 mL/min, with all other boundaries
set to be “no flux”. All geometries in the model were defined with an extremely fine mesh in
COMSOL Multiphysics.
3.2.9 Shear flow and adhesion analysis
We created the microfluidic device in-house using a micromilling platform, design and
fabrication protocols, and soft-lithography techniques for shear flow and adhesion analysis. Within
each device, SLBs were formed on the substrate. The design and toolpaths were created in
Autodesk Fusion 360 (San Rafael, CA) and custom-milled (Shapeoko, Carbide 3D, Torrance, CA)
out of polycarbonate. The final device was manufactured by pouring polydimethylsiloxane (PDMS)
mixed at 10:1 base to curing agent ratio (Sylgard 184 elastomer kit; Dow Corning). PDMS was
cured at 80°C for 3 h, peeled off, and cut into individual devices. Two inlets and one outlet with
0.75 mm diameter were punched in the microfluidic channel. The PDMS devices were
88
permanently bound to the detergent-cleaned glass coverslips after plasma treatment for 50 seconds
(Harrick Plasma, Model PDC-001-HP) for the subsequent lipid bilayer formation and substrate
modification. A dual-channel syringe pump (New Era Pump Systems, NY) was used to apply
controlled shear flow to the two channels through 10 mL glass syringes and tubing connections.
Cells were incubated with membrane bound factors on SLBs for 1.5 hr, before infusing Iscove's
Modified Dulbecco's Medium (IMDM) media at controlled flow rates (ramping up from 0 to 10
mL/min, with 5 s holding of each flow rates in a stepwise fashion) under a 37°C environment.
Flow through cells were collected from the outlet. The strongly adhered cells remained on the
substrate after applying shear flow were collected by three times of direct micropipetting out liquid
and filling the channel up with fresh IMDM media. and BF images were taken once every second
using a 2x objective (CFI60 Plan Apochromat Lambda Lens, NA 0.1, WD 8.5mm).
3.2.10 Statistics
All data are presented in mean ± SD. Statistical analyses and plots generation were
performed using GraphPad Prism 8 software (GraphPad Software, Inc). Statistical significance
was assessed using the Welch’s t-test (parametric) and ordinary 1-way ANOVA for comparison
between multiple (≥3) conditions. Data distribution was assumed to be normal, but this was not
formally tested. N.D.: not detected; n.s.: not significant (p > 0.05), *: p < 0.05, **: p < 0.01, ***:
p < 0.001, ****: p < 0.0001.
3.2.11 CFU assay
Enriched LSK cells were plated in MethoCult GF M3434, supporting myeloid and
erythroid differentiation (STEMCELL Technologies), supplemented with 0–20 ng/ml rmFlt-3L
and 0–100 ng/ml rmSCF (PeproTech), and cultured at 37°C and 5% CO 2. Colonies were counted
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and sized between days 7 and 10 after plating, and images were captured using an inverted
microscope at 2x (CFI60 Plan Apochromat Lambda Lens, NA 0.1, WD 8.5mm).
3.3 Results
3.3.1 Adhesion profiling of HSCs and MPPs
We utilized a supported lipid bilayer tethered with membrane bound SCF (mSCF) and
VCAM-1 (Fig. 3-1A) as a stromal membrane mimicking model as described previously [348]. To
measure the strength of cell adhesion, we used a microfluidic device that we have designed [348]
that imposes controlled shear stresses on the adhered cells (Fig. 3-1B). First, we FACS sorted
HSCs and MPPa-d [349] and profiled the adhesive characteristics of the cells (Fig. 3-1C).
Interestingly LT-HSCs showed highest adhesion strength as assessed by the percentage of cells
remained after a series of shear flow (Fig. 3-1D). MPPa, or ST-HSCs, showed intermediate
adhesion strength towards mSCF-VCAM-1 tethered lipid bilayer between LT-HSC and MPP b-c
(Fig. 3-1D). To show the heterogeneity of adhesion strength within each cell type, we defined
weakly adhered cells (lifted by shear flow if less than 2 mL/min), moderately adhered cells (lifted
by shear flow between 2 mL/min and 10 mL/min) and strongly adhered cells (remained on the
substrate after running a series of shear flow of up to 10 mL/min) based on the obtained adhesion
profiles. We found that the proportion of strongly adhered cells varied the most among HSCs and
MPPs, which is about 70 % for HSCs, less than 40% for MPPa, and less than 10 % for MPP b-d
(Fig. 3-1E).
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91
Figure 3-1. A lipid bilayer model tethered with SCF and VCAM-1 used for adhesion profiling of HSCs and
MPPs. (A) Illustration of HSC-niche interaction and niche membrane mimicking supported lipid bilayer model
tethered with SCF and VCAM-1. (B) Microfluidic setup for assessing the strength of HSC and MPP adhesion
to SLBs tethered with membrane-bound factors. (C) Gating strategies for HSCs and MPPs from the bone marrow.
Lineage, sca1 and cKit surface expression defines LSK cells, a mixed population of HSCs and MPPa-d with
different CD34, CD150 and Flk2 expression levels. (D) Adhesion profiles of HSCs and MPPa-d towards SCF-
VCAM-1 tethered membrane under shear flow of a series of flow rates. (E) HSCs and MPPa-d cells are
composed of cells with different adhesion strength towards the substrate. Weakly adhered: lifted by 2 mL/min
shear flow; moderately adhered: lifted by 2 mL/min to 10 mL/min shear flow; strongly adhered: remained
adhered after a 10 mL/min shear flow.
3.3.2 Experimental setup of scaled-up cell enrichment device
LSK (Lin-, sca1+, cKit+) markers are commonly used for the isolation of hematopoietic
stem and progenitor cells. Having shown the difference of adhesion strength towards mSCF-
VCAM-1 tethered lipid bilayer between HSCs and MPPs, we hypothesized strongly adhered LSK
cells enriched from the lipid bilayer system will include more HSCs. LSK cells were freshly
isolated from mouse bone marrow and FACS sorted (Fig. 3-2A). The device incorporates two
inlets, which are connected to a dual channel syringe pump, and one outlet, which connects to the
tube collecting the flow through cells. A snake shaped PDMS chip was designed and fabricated
using microfabrication techniques (Fig. 3-2B-D). The dual-inlet setup was purposed to shorten the
path from inlet to outlet and increase the efficiency of cell collection by two folds (Fig. 3-2C). We
simulated the flow rate distribution within the snake device and showed a uniform distribution
within the majority of the device area using 1 mL/min as the input flow rate (Fig. 3-2E).
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Figure 3-2. Platform designed for adhesion-based cell selection from bone marrow. (A) Workflow and
microfluidic setup for adhesion-based cell selection from bone marrow LSK cells. A dual channel syringe pump
93
was connected to the two inlets of a snake-shaped channel device which has a lipid bilayer tethered with SCF
and VCAM-1. The flow-through cells were collected from the outlet into a tube for phenotypic characterization
and colony formation assay. (B) CAD design of the PC mold; A micro-milled PC mold; A PDMS device replica-
molded from the PC mold and drilled with two inlets and one outlet. (C) An assembled PDMS on glass device
connected with the flow set-up through tubing and needle. A slab of PDMS was used to seal the outlet during
lipid bilayer formation and cell seeding before cell collection. (D-E) COMSOL Multiphysics simulation of flow
rates distribution within the device.
3.3.3 HSC markers can be enriched using snake device
Hoxb5 is a marker for long-term hematopoietic stem cells [341]. As a proof of concept, we
utilized the Hoxb5-mCherry mouse, a mouse strain that has expression of red fluorescent protein
(mCherry) in Hoxb5 expressing cells, to visualize cell enrichment from the LSK population. We
found Hoxb5-expressing cells tend to remain adhered to mSCF-VCAM-1 tethered bilayer than
non-Hoxb5-expressing cells under shear flow, as assessed by increasing purity of HSCs
(percentage of cells expressing Hoxb5) with increasing flow rates, while loosely adhered cells
were flushed off (Fig. 3-3A-B), indicating the potential for differential adhesion-based HSC
enrichment using mSCF-VCAM-1 tethered membrane.
Figure 3-3. Adhesion towards SCF-VCAM-1 enriches Hoxb5-expressing cells. (A) Example images of a
selected area that showed enrichment of Hoxb5 expressing cells on the SCF and VCAM-1 tethered lipid bilayer
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after a series shear flow of flow rates up to 20 m:/min. (B) Profiles of the resulted cell recovery (proportion of
cell remained on the substrate) and purity (proportion of cells expressing Hoxb5-mCherry).
We further examined the phenotypic identity of the weakly and strongly adhered
populations of LSK cells. We collected the two populations using differential adhesion and ran
flow cytometry, using non-enriched, FACS sorted HSC and LSK cells as controls. HSCs showed
clear upregulation for two established stem cell markers, Sca1 [339] and Slamf1 [340], compared
to the sorted LSKs (Fig. 3-4A). Furthermore, downregulation of Flk2 was observed in purified
HSCs compared to LSKs, shown as a negative skew in the histogram (Fig. 3-4A). Interestingly,
after adhesion-based cell separation, we found strongly adhered LSK was a population that has
lower expression level of cKit compared to weakly adhered LSK, negating the possibility that cells
adhere better to mSCF-VCAM-1 because they have higher cKit expression. A higher cKit
expression in HSCs has been correlated with impaired self-renewal and bias towards
megakaryocytic lineages [297]. In addition, strongly adhered LSKs showed clear upregulation of
Sca1 and Slamf1, and downregulation of negative HSC marker Flk2, compared to their weakly
adhered counterparts, indicating a more stem-like phenotype of these cells (Fig. 3-4B).
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96
Figure 3-4. Adhesion towards mSCF-VCAM-1 enriches phenotypic HSC-like cells. (A-B) Histograms of
expression levels of known stem cell markers of (A) FACS sorted HSCs and LSK cells and (B) strongly adhered
vs weakly adhered subpopulation of FACS sorted LSK cells assessed by flow cytometry. (C) Violin plots of
expression levels of stem cell markers for the four populations.
3.3.4 Adhesion-enriched cells contain cells similar to colony forming HSCs
Under steady state, HSCs remain quiescent in the bone marrow, and can be awakened by
external differentiation stimuli. When cultured in vitro, HSCs receive proliferating signals
(cytokines) from the culture media, enter cell cycle, and start proliferation [336, 350, 351]. HSC
quiescence and self-renewal mechanism requires proper balancing and is a highly regulated
process. Failure to be awakened can lead to lack of differentiation and hematopoiesis failure. On
the other hand, hypersensitive awakening response will lead to HSC exhaustion and poor
maintenance of the blood and immune system [352]. Colony forming assay is an in vitro clonal
assay to assess the ability of cells to proliferate and differentiate along the different hematopoietic
lineages [353]. We found FACS sorted HSCs in general late in clonal expansion and
consequentially form significantly smaller colonies compared to LSK cells (which are mostly
hematopoietic progenitor cells) in 7-day colony formation assay (Fig. 3-5A,C). Notably, we
observed similarly slow cycling cells (slow colony forming units), as assessed by formation of
small colonies within the strongly adhered compartment of LSK population (Fig. 3-5B,D). In
comparison, HSCs and LSKs (with or without adhesion enrichment) showed similar number of
CFUs per 100 cells. These results imply that stem cells differ from MPPs in proliferation dynamics.
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Figure 3-5. Enriched cells are slow colony forming units similar to HSCs. (A-B) Example images of colonies
formed by (A) FACS sorted HSCs and LSKs and (B) strongly adhered vs weakly adhered subpopulation of
FACS sorted LSK cells after 7 days of culture in the CFU media. (C-D) Quantification of the area of the
individual colonies formed by the four populations at day 7. (E) Number of colonies formed per 100 cells seeded
in the dish.
We hypothesized that the strongly adhered subpopulation of LSKs contain more HSCs with
long-term engraftment potential. Therefore, we isolated weakly vs strongly adhered LSKs and
performed in vivo transplantation using 5,000 cells for each recipient mouse. We expect to
continuously obtain data from the mice within the next 4 months.
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3.3 Discussion
This study mimics the initial process of HSC lodgment to bone marrow niche by presenting
freshly sorted HSCs with niche cell membrane mimicking supported lipid bilayer tethered with
VCAM-1 and mSCF. Up to date, people have been relying on the combination of surface marker
expressions to distinguish long term stem cells from progenitor cells. However, it has been
reported that MPPs contain ST-HSCs that can engraft the recipient and reconstitute the blood and
immune system, however with inferior capabilities [354], implying some phenotypically defined
MPPs are HSCs in nature. In this work, we have shown that HSCs contain the most cells that are
strongly adhered to mSCF-VCAM-1 tethered niche mimicking lipid bilayer. Interestingly, MPPa
cells, which is only one surface marker (CD34) different from HSCs, also contain a notable number
of strongly adhered cells. The percentage of strongly adhered cells are lower in MPPb-d, which
are differentiated progenies of MPPa cells. Taken into consideration the fact that the number of
MPPa are about 8 fold of HSCs (Fig. 3-1C), the strongly adhered population of LSK would
supposedly be composed of majorly MPPa cells, with HSCs and small amount of MPP b-d.
We isolated the strongly adhered cells from LSK population, which is a pool of all HSCs
and MPPs, and characterized the weakly vs strongly adhered cells phenotypically and functionally
(in vitro and in vivo). Adhesion enriched cells showed expression levels of positive and negative
stem cell markers consistent to previous reports. Notably, slamf1 expression level was
significantly higher in the LSK subpopulation that has higher adhesion strength towards membrane
bound SCF and VCAM-1. Surprisingly, cKit level was lower in strongly adhered LSK population
compared to the weakly adhered counterpart. It was reported that cKit level negatively correlates
with stemness of HSCs [297].
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The observation that HSCs form smaller colonies has not been reported to the best of our
knowledge. Future explorations and characterizations on the dynamics of HSC self-renewal and
differentiation in vitro would unravel the mechanism underlying the observed phenomenon.
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Chapter 4: Engineering a pillar-free diffusion device for studying cell
chemotaxis on lipid bilayers
4.1 Rationale
Cell migration plays a crucial role in physiological and pathophysiological processes, such
as tissue regeneration [355, 356], immunosurveillance [357, 358], and cancer metastasis [359-361].
During cell migration, cells typically respond to a gradient of chemoattractant, and migrate through
the extracellular matrix (ECM). Notably, cells can also migrate along the surfaces of surrounding
stromal cells in tissue microenvironments through membrane-bound receptor-ligand interactions.
For instance, homing and engraftment of hematopoietic stem cells (HSCs) involves complex
interactions of HSCs with vascular endothelial cells, macrophages, and mesenchymal stromal cells
through a range of cell-cell adhesion molecules on both stem and stromal cells [344] and
chemokines [134]. During immunosurveillance, circulating T cells exit and return to the blood
circulation through T cell interactions with vascular/lymphatic endothelial cell [362]. They also
migrate over the surface of antigen presenting cells and other somatic cells in search for pathogenic
antigens [363]. In cancer metastasis, cancer cells often lodge in the microvasculature in distant
organs and transmigrate through the endothelium via direct cell-cell adhesion to form
micrometastases [364, 365]. It is thus important to understand chemotaxis in the context of
membrane-bound interactions.
However, there remains a lack of models recapitulating chemotactic migration in such
context. The existing in vitro platforms designed for chemotactic studies, including Boyden
chamber and microfluidic assays have largely been designed for cell migration on ECM [366, 367].
Some studies investigating the role of membrane-bound factors, such as ICAM-1, have also been
101
modeled as immobilized factors on solid substrates [368, 369], which lacks the unique properties
associated with their membrane-bound natures such as lateral mobility and molecular orientation
[370]. Recently, researchers have developed cellular cultures in microfluidic channels to directly
evaluate cell migration through stromal cells (often endothelial cells) [371-374]. However, such
studies often lack a clearly defined chemoattractant and/or involve multiple membrane-bound
interactions that are difficult to delineate in the coculture systems.
Supported lipid bilayers (SLBs) tethered with biomolecules have been widely adopted as
a model of cell membranes, with extraordinary success in studying immune cell activation and
stem cell – niche interactions [348, 375, 376]. However, cell migration in response to a
chemoattractant gradient on SLBs tethered with membrane-bound factors, to the best of our
knowledge, has not been reported. There remains a lack of models that incorporate both
chemoattractant gradient and SLBs in the same system, partly due to the delicate nature of SLBs,
where flow in conventional microfluidic designs may interfere with established gradients, SLB, or
cell migration. As such, conventional microfluidic models are not compatible with studying
membrane-bound factors using SLBs.
Here, we engineered a multichannel device that orthogonally separates chemoattractant
channels from SLB and cell loading channel by pillar-free, hydrogel barriers, to enable precise
control of the timing and profile of chemokine gradients applied on cells interacting with SLBs.
Using this model, we analyzed the synergistic effects of membrane-bound ICAM-1 and chemokine
CXCL12 on Jurkat T cell migration. We further demonstrated an independent relationship between
HSC morphology formed under membrane-bound interactions and the orientation of cell
protrusions and the migration direction of HSCs under the CXCL12 gradient.
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4.2 Materials and Methods
4.2.1 Design, fabrication, and assembly of the microfluidic device
The design and toolpaths for the microdevice were created in Autodesk Fusion 360 (San
Rafael, CA) and custom-milled (Shapeoko, Carbide 3D, Torrance, CA) out of polycarbonate [377].
The final device was manufactured by pouring polydimethylsiloxane (PDMS) mixed at 10:1 base
to curing agent ratio (Sylgard 184 elastomer kit; Dow Corning). PDMS was cured at 80°C for 3
hours, peeled off, and cut into individual devices. Channel inlets and outlets with 1.5 mm diameter
were punched at both ends of microfluidic channels. The PDMS devices were permanently bound
to the detergent-cleaned glass coverslips after plasma treatment for 50 seconds (Harrick Plasma,
Model PDC-001-HP) for the subsequent lipid bilayer formation and substrate modification.
4.2.2 Formation of gel diffusion barrier and workflow of the device
A solution of Poly-D-lysine (PDL) (VWR, Radnor, PA) in Milli-Q® water in the
concentration of 0.5mg/mL was injected into the two gel channels of a freshly assembled device,
incubated at room temperature for 30 min, and aspirated with vacuum. 1X PBS was injected to the
gel channels and aspirated out to remove excess PDL. A solution of agarose (Catalog number:
16500100; Invitrogen, Carlsbad, CA) in the concentration of 0.8% wt/vol was freshly dissolved in
water by microwaving for 45 seconds. Hot agarose solution was injected to the pre-coated hydrogel
channels in the same fashion and allowed to solidify at RT for 30 min in a wet chamber to minimize
hydrogel dehydration. After the formation of two gel barriers, lipid bilayer formation and/or
protein capturing, cell seeding was performed in the center channel for chemotaxis studies (details
described in corresponding method sections below).
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4.2.3 COMSOL simulation and characterization of transport phenomena with FITC-Dextran
COMSOL Multiphysics® (Stockholm, Switzerland) was used to simulate the gradient
formation in the microdevice to guide the design of the gradient chamber geometry using the
implemented Transport of Diluted Species in Porous Media Module. The porous media was
approximated to be liquid water. Effective diffusivity model used was Millington and Quirk model.
Diffusion coefficient of a 10 kDa molecule within the hydrogel and media was approximated to
be 8.7 × 10
−11
m
2
s
−1
and 9.25 × 10
−11
m
2
s
−1
, respectively
[378]. Initial concentrations were set for
the inlet and outlet reservoirs to represent the sink and the source, respectively (C = 0 and C = 5 ×
10
-6
mol/m
3
), with all other boundaries set to be “no flux”. All geometries in the model were
defined with an extremely fine mesh in COMSOL Multiphysics. The model was then solved as a
time-dependent study up to 120 minutes (time step = 5 minute). For geometric parameter sweep,
each of the five chambers of the device was generated as a rectangular solid with variable
geometries (width, length and height). Simulated concentration gradients were obtained along a
line that traversed the center cell chamber on the bottom surface, representing the concentration
gradient experienced by cells seeded onto the membrane bound or immobilized ICAM-1.
To experimentally demonstrate diffusion across the device, we loaded a solution of 10 kDa
FITC-Dextran in PBS (2 μg/mL) into one reservoir chamber, PBS into the other chamber, and the
center channel was filled with PBS to visualize the transport of fluorescent Dextran across the
device by time-lapse imaging. Solutions of food colors were injected into the device, and the whole
device was photographed to demonstrate the wall-less liquid confinement and diffusion across the
platform.
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4.2.4 Preparation of supported lipid bilayers and protein tethered surfaces
Lipid components, 18:1 (Δ9-Cis) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and
5% 18:1 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]
(nickel salt) (DGS-NTA(Ni)), dissolved in chloroform were purchased from Avanti Polar Lipids
and mixed. The lipids were dried in round-bottom flasks under a stream of N2 for 5 min and
desiccated for 2 hours with house vacuum pump in a chemical fume hood. The lipid mixture was
resuspended by bath sonication in 1X PBS at a final concentration of 2.5 mg/ml and extruded 10
times through a membrane with 50 nm pore size (Avanti Polar Lipids) into small unilamellar
vesicles (SUVs). The SUV solutions were then diluted 1:1 in 1X PBS (pH 7.4) before being loaded
onto the detergent cleaned and dried glass coverslip through the loading chamber, and incubated
for 2 min to spontaneously form the lipid bilayers. The chambers were then washed with a 10X
excess volume of 1X PBS.
4.2.5 ICAM-1 capturing on lipid bilayer and immobilization
For protein capturing on lipid bilayer, a solution of 10 µg/mL Alexa Fluor 568 labeled
recombinant human ICAM-1 with poly-histidine and human Fc tag (Cat. 10346-H03H,
SinoBiological, Beijing, China), was injected to supported lipid bilayer, incubated at RT for 40
min and tethered to 18:1 DGS-NTA(Ni) through chelation. Tethered SLB was washed excessively
with 1X PBS before use. For the immobilization of ICAM-1, 100 µg/mL recombinant protein A
(Cat.101100, Thermo Fisher) in 1X PBS was injected to detergent cleaned and dried glass
coverslip, incubated for 30 min, washed with 1X PBS, before a same solution of human ICAM-1
was injected and incubated at RT for 40 min. The resulted substrate was then washed with 1X PBS
before use.
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4.2.6 Cell seeding and incubation
Jurkat cells were cultured in ATCC-formulated RPMI-1640 Medium (Cat. ATCC 30-2001)
supplemented with 10% Fetal Bovine Serum (FBS) (Cat. F2442, Sigma-Aldrich, St. Louis, MO,
USA) and 1% 100 U/ml penicillin-streptomycin (Sigma-Aldrich, MO, USA). Cells were sub-
cultured every 2-3 days and kept in sterile incubation conditions (37°C, 5% CO2 and 90%
humidity) according to ATCC protocols. Cells were labeled with Calcein AM (Cat. C1430,
Thermo Fisher) according to manufacturer’s protocol before loaded to the device. The chamber
containing lipid bilayers was equilibrated with the same media, and the resuspended cells were
then injected into the chamber and incubated for 1 h in a humidified incubator maintained at 37°C
and 5% CO2. Cell culture media with or without 50 ng/mL of CXCL12 (Cat. 250-20A, Peprotech)
was injected to the two reservoirs of the device, respectively. Then cells were imaged at 2X at an
interval of 5 min, for a total period of 1 h for chemotaxis analysis.
4.2.7 Imaging, cell tracking, and data analysis
A Nikon Eclipse Ti-E inverted fluorescence microscope (Nikon, Tokyo, Japan) was used
for live-cell imaging, which is equipped with an OKOLab incubation box (OKOLAB, Italy)
controlling for temperature (37°C) and CO2 concentration (5%). Images were taken using a 2x
objective (CFI60 Plan Apochromat Lambda, NA 0.1). Images were analyzed using ImageJ (U.S.
National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij). Two open-source
plugins, “Manual Tracking” and “Chemotaxis and migration tool 2.0” along with customized
MATLAB (MathWorks, Natick, MA, USA) codes were used to analyze time-lapse images and
cell tracking data.
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4.2.8 Statistics
All experiments have been repeated at least three times. All data are presented in mean ±
SD. n represents cell number analyzed in each experiment, as detailed in figure legends. One-way
ANOVA or two-tailed Student’s t-tests were used for evaluating the significance of difference
unless otherwise indicated. Statistical analyses were performed using GraphPad Prism 7 software.
Not specified: p > 0.05; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
4.3 Results
4.3.1 A multichannel device design allows for separate lipid bilayer and chemoattractant gradient
formation
To study the role of membrane-bound interactions in cell chemotaxis, we designed a
multichannel microdevice that contains both a chemoattractant gradient and a lipid bilayer for
chemotactic migration under the context of membrane-bound factors and interactions (Fig. 4-1A).
The device is geometrically symmetrical and composed of five channels: a center channel for cell
culture, two large reservoir channels serving as the source and sink of chemoattractants, and two
thin hydrogel barrier channels that separate the lipid bilayers and cell culture from the reservoir
channels (Fig. 1A,B). The five channels differ in heights and are laterally connected. The
chemoattractant gradient was established across the width of the central channel by the diffusion
of soluble factors from source to sink channel (Fig. 4-1B). Two hydrogel barriers were permeable
to chemoattractants but not cells, allowing for independent handling of lipid bilayer formation and
cell loading from the gradient generation (Fig. 4-1C). Typical microfluidic gel barriers often
involve micropillar structures to hold gel in the channels [379-381], which however can lead to
non-uniform chemoattractant distribution in the central channel due to the blockade of diffusion.
To avoid this, we employed a pillarless, liquid pinning strategy [382] which utilizes the capillary
107
force and surface tension to draw and hold gel solution in the barrier channels, thus allowing for a
simplified design of gel barrier channel without interfering with lateral diffusion profiles (Fig. 4-
1B). We then carried out a proof-of-concept fabrication workflow for the multichannel device. A
master mold of the device was designed in Autodesk Fusion 360 (Fig. 4-1D) and milled in
polycarbonate (PC) on a Carbide 3D Nomad desktop milling machine (Fig. 4-1E). The device was
then replica-molded in polydimethylsiloxane (PDMS), drilled with inlets and outlets with biopsy
punches to allow for downstream studies (Fig. 4-1F).
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Figure 4-1 Design of a microchannel diffusion device for chemotactic studies. (A) An isometric view of the
device design. (B) Schematics of the cross-section of microchannels. (C) A zoomed-in illustration of cell culture
chamber. (D) CAD design of the PC mold. (E) A micro-milled PC mold. (F) A PDMS device replica-molded
from the PC mold and drilled with inlets and outlets.
4.3.2 Channel height and surface treatment are key to liquid pinning-based hydrogel barrier
formation
The hydrogel barriers are a crucial component for separating the lipid bilayer/cell culture
channel from the chemoattractant channels in our device. We first investigated the design
parameters of the hydrogel channels that are key to their ability to pin liquids in order to form the
hydrogel barriers. To illustrate this concept, a simplified version of the hydrogel barrier channel
was designed, which contains a central liquid channel flanked by two taller air channels on both
sides for liquid pinning (Fig. 4-2A). The lateral dimensions of the center and side channels were
designed as 2 mm x 20 mm and 4 mm x 12 mm, respectively. We tested liquid pinning on the
center channel on different device designs, using water with blue food coloring for visualization.
The success of liquid pinning was defined as the retention of the aqueous solution in the center
channel without breakage or spillage into either of the side channels.
The first geometric parameter we examined was the difference in the heights of center and
side channels, which helps restrain the vertical advancement of liquid-air interface into the side
channels (Fig. 4-2A). With the height of center channel fixed at 300 µm, we varied the height of
side channels so that the height difference (ΔH) varied from 300~700 µm at a 100 µm step (Fig.
4-2B). We found that a minimum height difference of 500 µm was required to pin liquid in the
center channel (Fig. 4-2B). Next, we held ΔH at 700 µm while varying the height of the center
channel (H) from 300 to 900 µm at a 150 µm step (Fig. 4-2C). Our test results showed that the
design was able to support liquid pinning in a wide range of center channel heights up to 750 µm
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(Fig. 4-2C). We further examined whether the width of the center channel also plays any role in
liquid pinning. With fixed H at 300 µm and ΔH at 700 µm, we found that all the tested width (from
0.8 mm to 2.0 mm) successfully achieved liquid pinning (Fig. 4-2D). These results suggest that
liquid pinning in the hydrogel channel is dependent on and sensitive to the height and height
difference but not the width of the channels in our device design.
Another crucial factor in liquid pinning is the hydrophobicity of the microchannel surfaces,
which determines the surface tension that enables microchannel wetting and retention of the
aqueous solution in the center channel during the pinning process. Plasma treatment is a common
step in microfluidic device fabrication, which covalently bounds PDMS to glass while reducing
the hydrophobicity of the internal surfaces, particularly for those of PDMS [383]. While all the
devices tested so far had been treated with oxygen plasma (under atmospheric condition), we next
specifically examined the impact of such treatment on the ability of achieving liquid pinning in
the device (Fig. 4-2E). Using a design of H at 300 µm and ΔH at 700 µm, we evaluated the liquid
pinning in the devices with plasma treatment on neither, either, or both PDMS and glass surfaces
before device assembly (Fig. 4-2E, bottom). We found that liquid pinning was successful only
when both PDMS and the glass coverslip were treated. Therefore, plasma treatment is necessary
not only for device assembly but also for liquid pinning.
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Figure 4-2. Dependence of liquid pinning on device parameters and surface treatment. (A) Illustration of key
geometric parameters for liquid pinning. (B) Varying the height difference between the center and side channel
with the height of the center channel kept constant (300 µm). (C) Varying the height of the center channel with
the height difference kept constant (700 µm) . (D) Varying the width of center channel with constant height of
the center channel (300 µm) and the height difference (700 µm). (E) Effect of plasma treatment of the PDMS
device and glass substrate on liquid pinning.
4.3.3 Coating hydrogel channel is necessary to prevent leakage of soluble factors
The key design of the device lies in the proper functions of hydrogel barrier
channels. Ideally, a hydrogel barrier should form a good seal around the PDMS/glass interfaces,
allowing for chemoattractant diffusion through the barrier while preventing cells from escaping
the bilayer/cell culture channel. It should also maintain shape within the channel throughout the
111
whole process of lipid bilayer formation, protein tethering, cell seeding and live imaging (Fig. 4-
1B,C).
After an initial screening, we narrowed down to two hydrogel candidates, collagen and
agarose, which maintain their shape in the hydrogel barrier channels (Fig. 4-3A,B). The hydrogel
solution of selection (2.5 mg/mL collagen solution or a 0.8% agarose solution) was injected into
corresponding channels, allowed to be cured or solidified. To test the diffusivity and integrity of
the gel barriers, we injected aqueous solutions with blue and red food dyes one or both reservoir
(source and sink) channels, and monitored the distribution of the colors across the hydrogel barrier
for 2 hours. We found that the blue and red food dyes immediately appeared across the hydrogel
barriers and in the center channel in an uncontrollable manner (Fig. 4-3A,B), suggesting leakages
at the gel-PDMS or gel-glass interfaces.
Poly-lysine is a positively charged synthetic polymer of the amino acid(s) L-lysine or D-
lysine. It has been widely used as an enhancer of electrostatic interactions for surface coating [384].
On the other hand, the agarose polymer contains negatively charged residues, namely pyruvate
and sulfate [385]. We thus hypothesized that pre-coating the channel surfaces with poly-lysine
electrostatically seal the gel-PDMS and gel-glass interfaces. We coated the hydrogel channel with
poly-d-lysine (PDL) prior to injecting and solidifying a 0.8% agarose hydrogel, followed by
infusion of aqueous food dye solutions to the reservoir channels. We indeed observed a steady
retention of the dye solutions in the side channels, which uniformly diffused across the agarose
hydrogel barrier during the 2-hr incubation period (Fig. 4-3C). Considering the superior
performance of the agarose hydrogel and the concern of collagen as an adhesion substrate, we
decided to use agarose gel with PDL precoating for the following chemotaxis studies.
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Figure 4-3. Effect of hydrogel type and poly-D-lysine precoating. Leakage of dye solution into the center channel
when the hydrogel barriers were formed with (A) collagen and (B) agarose hydrogel without precoating in the
device. (C) Illustration of poly-D-lysine (PDL) precoating in the gel channels. (D) PDL precoating prevented
leakage in the agarose hydrogel barrier.
4.3.4 Gradient profiles within the device can be optimized through COMSOL simulation
Upon confirming the requirements of design parameters and feasibility of the hydrogel
barrier, we next utilized COMSOL Multiphysics simulation to determine the optimal device
parameters for the chemotaxis studies (Fig. 4-4A-C). The criteria for an ideal device include a
steep chemokine gradient and a wide migration space in the cell culture channel, and compatibility
with the milling- and liquid pinning-based fabrication/assembly processes. specifically, we used
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the simulated concentration profile across the width of the lipid bilayer/cell culture channel at 1 h
as the readout (Fig. 4-4C), as it is where the cells will be seeded and migrate. The first parameter
that we examined was the hydrogel porosity, defined as volume ratio between pores and total bulk,
which varies between 0 (no pores) and 1 (fully liquid). As a reference, the porosity of commonly
used agarose gel of low concentration (<1%) has a porosity close to 1. We found that the
concentration distribution is similar when the porosity varies in the range of 0.1-1 (Fig. 4-4D),
suggesting that gel porosity is not a critical factor for the gradient profile within the device.
Next, we screened the geometric parameters of the hydrogel barrier and central channel
(Fig. 4-4C, red arrows). We fixed the height of the central lipid bilayer/cell culture channel at 1
mm to allow for a sufficient height difference (ΔH) between the hydrogel barrier and central
channel for liquid pinning (Fig. 4-2B, 4-4C). We first examined the impact of the height of
hydrogel barrier on the gradient profile within the central channel, by varying the height of
hydrogel barrier from 100 to 500 µm (thus ΔH from 900 to 500 µm). We found that the gradient
profiles were largely similar under the heights of 300 and 500 µm, while the steepness of the
gradient near the center dropped more significantly under the 100 and 200 µm heights (Fig. 4-4E).
We then evaluated the impact of hydrogel barrier width on the gradient profile, by varying its value
from 0.2 to 3.2 mm. The simulation showed that the gradient was quickly flattened by the increased
width above 0.4 mm (Fig. 4-4F), suggesting a shift of major diffusion resistance from the central
channel to the hydrogel barriers under the increased gel barrier widths. Lastly, we evaluated the
impact of the central channel width on the gradient profile (Fig. 4-4G). While reducing the channel
width from 2 mm to 1 mm slightly improved the steepness of the gradient at the channel center,
increasing it to 4 mm and 8 mm dramatically flattened the chemoattractant gradient (Fig. 4-4G).
Combining these results and the choice of milling tool size (1/32”), to achieve liquid pinning as
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well as a steep gradient with ease of fabrication, we finalized on a design with a 350 µm hydrogel
channel height, a 0.8 mm hydrogel channel width, and a 2 mm central channel width.
With these chosen parameters, we fabricated a microchannel diffusion device and
characterized the spatiotemporal gradient profiles of 10 kDa fluorescence isothiocyanate (FITC)-
dextran, which serves as a surrogate for the chemoattractant CXCL12 [386]. Fluorescence images
were taken every 5 minutes for 1 hour upon the filling of the source channel with FITC-dextran
(Fig. 4-4H). We observed a series of time-dependent lateral concentration gradients within the
central channel (Fig. 4-4I), which was overall consistent with those from COMSOL simulations
(Fig. 4-4J). Overall, we have designed and optimized the device based on COMSOL simulation
and demonstrated the establishment of a concentration gradient within the device for subsequent
chemotaxis studies.
115
116
Figure 4-4. Characterization of diffusion with COMSOL Multiphysics® and FITC-Dextran diffusion. (A)
Isometric, (B) top, and (C) cross-section views of concentration distribution throughout the diffusion
microdevice in COMSOL simulation. (D-G) COMSOL parameter sweep results of the effects of key geometries
on the concentration profiles across the center channel. Default parameters during sweep include width of the
cell chamber (2 mm), with of the hydrogel chamber (0.8 mm) and height of the hydrogel chamber (0.35 mm).
Simulated effect of (D) hydrogel porosity, (E) height of hydrogel barrier, (F) width of hydrogel channel, and (G)
width of the center SLB/cell loading channel. (H) Diffusion of 10 kDa FITC-Dextran in center chamber over
time. (I) Quantification of fluorescence intensity of 10 kDa FITC-Dextran across the width of the center channel
over time. (J) Simulated concentration profiles of 10 kDa molecule across the center channel over time.
3.3.5 Fluorescence recovery after photobleaching confirms lipid bilayer formation and mobility in
the device
Next, we tested whether lipid bilayer can be formed and tethered with membrane-bound
proteins in the microdevice. To avoid excessive shear flow during the loading and washing steps
in the center channel, which may disrupt lipid bilayer/tethered proteins/attached cells, we used the
difference in Laplace pressures generated by the curved liquid-air interface droplets at the inlet
and outlet of the center channel [387] to inject samples (liposome solution, membrane-bound
proteins, and cells) in the device (Fig. 4-5A). We first established the agarose hydrogel barriers in
the flanking channels, before filling the center channel with aqueous solution. We intentionally
overfilled the channel so that it forms a large droplet at the outlet and a small droplet at the inlet.
The formation of the droplets indicates no leakage through the hydrogel barrier. To demonstrate
the sample loading direction and assess the uniformity of loaded solution, we pipetted 50uL of
green food dye solution to the outlet (large droplet) or inlet (small droplet), respectively. Only the
dye solution loaded at the inlet (small drop) flowed through the whole channel with uniform
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distribution across the width of the channel (Fig. 4-5B), confirming the feasibility of the Laplace
pressure-based sample loading.
We then injected an aqueous solution containing small unilamellar vesicles (SUVs)
composed of synthetic lipid 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with 5 %
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel
salt) (DGS-NTA(Ni)) lipid into the center channel of a prepared device to form a supported lipid
bilayer (SLB). We loaded 6-histidine- and Fc-tagged, fluorescently-labeled intercellular adhesion
molecule-1 (ICAM-1) to the channel to tether ICAM-1 to SLB through Ni-chelation. As an
immobilized control, we also coated a channel with Protein A and immobilized the same ICAM-
1 through the capture of Fc domain by the Protein A layer (Fig. 4-5C). Fluorescence recovery after
photo-bleaching (FRAP) technique was used to confirm the lateral mobility (or immobility) of the
captured ICAM-1 on both surfaces, in which a small region within the channel was photobleached
and monitored for fluorescence recovery for 20 minutes (Fig. 4-5C). The fluorescence recovery
was observed immediately upon photobleaching on the SLB (Fig. 4-5C, top row, mb-ICAM-1;
Fig. 4-5D), while in contrast, no recovery was observed with the immobilized ICAM-1 (im-ICAM-
1) (Fig. 4-5C, bottom row; Fig. 4-5E). Therefore, our device is capable of forming substrates with
membrane bound factors and immobilized factors.
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Figure 4-5 Lipid bilayer formation and confirmation of lateral mobility of the membrane-bound ICAM-1. (A)
Schematic of filling the center channel with Laplace pressure. R and r are the radii of the larger and smaller
droplets, respectively. The difference in pressure generated by the surface tension of each droplet drives liquid
movement in the microchannel. (B) Dye loading test showing the filling direction. (C) Evaluating lateral mobility
of membrane bound (mb-) and immobilized (im-) ICAM-1 with fluorescent recovery after photobleaching
119
(FRAP). (D-E) Line scan of mb-ICAM-1 and im-ICAM-1 fluorescence profiles right after vs. 20 min after
photobleaching.
4.3.6 Jurkat cells have different chemotactic profiles on membrane bound vs. immobilized ICAM-
1
As a proof-of-concept, we next evaluated the chemotactic behaviors of Jurkat T cells on
mb-ICAM-1 vs im-ICAM-1 under a gradient of CXCL12 within the device. After forming the
hydrogel barriers, we first loaded the devices with either SLB with tethered ICAM-1, or
immobilized ICAM-1 through a coated Protein A layer in the center channel. Jurkat cells were
then seeded on the two substrates and incubated for 1 hour to allow for initial cell attachment. To
form a chemokine gradient, we loaded the two reservoir channels with media containing 50 ng/mL
CXCL12 or no CXCL12, respectively. Cells were immediately live imaged at an interval of 5 min
for a total period of 1 hour side-by-side on both surfaces (Fig. 4-6A, B). Noticeably, we observed
little or no cell movement in the control devices without CXCL12 gradient, whether on the
membrane-bound or immobilized ICAM-1 (Fig. 4-6C, D, left panels; scale bars: 5µm). In contrast,
CXCL12 gradient induced massive and persistent migration toward the sources of the gradient in
the Jurkat cell population on both substrates (Fig. 4-6C, D, right panels; scale bars: 50 µm). To
assess the migratory behaviors of the Jurkat cells, we tracked the trajectory of the cell movement
on the substrates, and quantified the total movements, directionality, percent of runs, and the
forward migration index along the x axis (Fig. 4-6E). Among those, total movements indicate the
level of migratory activities, while directionality indicates the randomness of migration. The “runs”
are defined as the movement along the overall migration direction, while “tumbles” are those
backward movements. The percentage of runs thus reflects the responsiveness of migration to the
chemoattractant source. The forward migration index (on the x-axis) is a measure of the efficiency
120
of directed migration toward the chemoattractant. We observed that under both surface conditions,
Jurkat cells had significantly higher total movements and directionality under chemoattractant
gradients than those without the gradient (Fig. 4-6F,G). A detailed analysis on the percentage of
runs and forward migration index further showed that those Jurkat cells migrating on mb-ICAM-
1 on SLBs had higher responsiveness and deficiency in their directed migration toward the
chemoattractant source than their counterparts on the im-ICAM-1 (Fig. 4-6H,I). Our results thus
demonstrated the ability of our device to investigate cell chemotaxis and distinguish the differential
behaviors of cell migration on membrane-bound vs. immobilized factors.
121
Figure 4-6. Jurkat cell chemotaxis towards CXCL12 on membrane bound vs immobilized ICAM-1 in the
diffusion microdevice. Representative images of Jurkat cells and migration trajectories in 1 hour with and
without CXCL12 gradient, on (A) mb-ICAM-1 and (B) im-ICAM-1, respectively. The migration trajectories of
Jurkat T cells in 1 hour on (C) mb-ICAM-1 and (D) im-ICAM-1 without and with CXCL12 gradient. (E)
Schematic of the definitions of cell migration parameters. Quantification of (F) total movements, (G)
directionality, (H) migration persistency as assessed by Runs% and (I) forward migration index, as assessed by
122
averaged movement toward chemoattractant gradient over accumulated distance. n = 66 – 71 single cell
trajectory per condition. Not specified: p>0.05; *: p<0.05; ***: p<0.001; ****: p<0.0001 by ANOVA with
Tukey’s test.
4.3.7 mSCF-VCAM-1 regulate HSC motility but not migration
To assess the effect of membrane-bound interactions on HSC motility and migration, we
incubated cells with lipid bilayers (tethered with mSCF, VCAM-1, or mSCF+VCAM-1) for 1 h,
and live imaged and tracked cells for 1h. We found that HSCs showed slightly increased motility
when seeded on SCF and VCAM-1 tethered lipid bilayers (Fig. 4-7 A-C). However, HSCs did not
migrate towards CXCL12 gradient when seeded in the multifactor diffusion device, and cell
protrusions did not show orientation (towards or against) with respect to the CXCL12 (Fig. 4-7D).
Consistent with our previous result, the protrusions are sites of stable anchorage (Fig. 2-5) and do
not direct HSC chemotaxis.
Figure 4-7. mSCF-VCAM-1 promote HSC local motility but not migration. (A) HSC trajectories on mSCF-
VCAM-1 tethered bilayer without CXCL12 gradient for 1 hr. (B) Total distance traveled by HSCs in 1 h on lipid
123
bilayers. (C) Single-cell velocity is plotted as a function of time. (D) Three HSCs seeded on mSCF-VCAM-1
with CXCL12 diffusing from the top before and after 40 min.
4.4 Discussion
We have developed a cleanroom-free, multifactor device which is, to our knowledge, the
first attempt designed to study cell chemotaxis on a cell membrane mimicking lipid bilayer. We
studied the geometry dependence of liquid pinning and the effect on gradient generation within
the device, which can be utilized to guide and inspire microfluidic device design. Admittedly, the
milling strategy also imposed a limit on the smallest features of the microchannel designs, such as
by the size of the drill bits and the spatial resolution (x, y, and z) of the milling platform. As such,
the sharpest gradient indicated in the COMSOL simulation was not achieved. Nevertheless, the
low price of and ease of access/operation to milling platforms will allow for wide adoption of this
platform for small labs with insufficient resources or those with limited access to cleanroom
facilities. On the other hand, we can further improve the sharpness of gradients within the device
using a milling platform with higher resolution/smaller tools by reducing the width of gel barrier,
and improve the uniformity/longevity of the gradients by increasing the size of reservoirs.
Our platform can be easily adapted and extended to different biological contexts since each
channel within the device can be manipulated independently. By coating the center chamber
differently (ECM proteins, immobilized proteins, cultured cells, etc.), cell adhesion and migration
can be studied accordingly. Furthermore, shear flow can be included as another factor by
connecting the center channel to a syringe pump, to study cell migration under a controlled shear
stress. In addition, by changing the soluble molecule content in the two reservoirs, cell responses
to more soluble factors can be studied simultaneously. Since the migration of many cell types is
124
achieved through cell-cell adhesion and chemokines in the microenvironment, our platform can be
widely adapted to different biological contexts, such as membrane-bound migration in cancer,
immune, and stem cells.
125
Chapter 5: Concluding Remarks and Future Directions
Membrane bound interactions between HSCs and niche cells are of great importance to
many aspects of stem cell functions. In this dissertation, we used and a supported lipid bilayer
model that allow visualization of how HSCs interact with membrane bound factors at single-cell
level.
In chapter 2, we discovered a unique synergy between two membrane bound factors, SCF
and VCAM-1, in regulating HSC adhesion and polarization using the supported lipid bilayer model.
We observed active recruitment of mSCF into tight clusters by HSCs, but not VCAM-1. How
VCAM-1 assisted HSCs to form the unique interaction pattern with SCF requires deeper
investigation. The Among all the adhesion molecules that contribute to HSC maintenance in the
body (reviewed in Chapter 1), we assessed VCAM-1 and its role in HSC activities in depth. We
envision the versatile SLB model holds great potential in exciting future studies for the assessment
of the interaction between HSCs and single or multiple membrane bound factors. Furthermore, as
cell-cell interaction usually has bidirectional signaling impacts to both parties, the SLB can be
used in the other way to mimic the membrane of HSCs assess how stromal cells would respond to
membrane bound HSC proteins.
In chapter 3, we explored the potential of applying the gained knowledge of cell adhesion
to SCF-VCAM-1 tethered lipid bilayer to HSC enrichment in a proof-of-concept study. Using LSK
cells as the starting heterogeneous population, we were able to exclude the weakly adhered
compartment using a shear flow of media and keep the strongly adhered cells on the membrane
for later harvest. We found MPPa cells consist of a considerable proportion of strongly adhered
cells similar to FACS-sorted HSCs. We hypothesize this compartment of MPPa cells are probably
cells with engraftment potential.
126
In chapter 4, we added a chemoattractant gradient to a tethered bilayer by developing a
pillar-free diffusion device and examined membrane bound regulation of cell chemotaxis. This
cleanroom-free, multifactor microfluidic tool is, to our knowledge, the first attempt designed to
study cell chemotaxis on a cell membrane mimicking lipid bilayer and can be adapted to different
biological contexts. We did not observe HSC chemotaxis towards CXCL12 on mSCF-VCAM-1
tethered lipid bilayer. It was probably because the dual membrane bound factors together promotes
cell adhesion and anchorage, instead of migratory activities. However, we cannot rule out the
possibility that a preestablished cell polarity would contribute to HSC movement or homing to
bone marrow niches. Therefore, the relationship between membrane-bound induced HSC polarity
and cell migration requires further investigation, including in vitro and in vivo homing assays.
Overall, this dissertation established a synergy of mSCF-VCAM-1 in regulating mouse
HSC behaviors (protrusion, adhesion, proliferation) in the bone marrow niche and provided
mechanistic details in cellular signaling and application in HSC enrichment. Future endeavors on
investigating whether this synergy is true in human hematopoietic stem and progenitor cells, and
on the potential of targeting mSCF-VCAM-1 synergy for interventions of hematopoietic disorders
would greatly benefit the use of HSCs in regenerative medicine and cell therapy.
127
References
1. Lane, S.W., D.A. Williams, and F.M. Watt, Modulating the stem cell niche for tissue
regeneration. Nat Biotechnol, 2014. 32(8): p. 795-803.
2. Watt, F.M. and B.L.M. Hogan, Out of Eden: Stem Cells and Their Niches. Science, 2000.
287(5457): p. 1427-1430.
3. Driessen, R.L., H.M. Johnston, and S.K. Nilsson, Membrane-bound stem cell factor is a
key regulator in the initial lodgment of stem cells within the endosteal marrow region. Exp
Hematol, 2003. 31: p. 1284-1291.
4. Elaine Fuchs, T.T., Geraldine Guasch, Socializing with the Neighbors: Stem Cells and
Their Niche. Cell, 2004. 116: p. 769–778.
5. Dzierzak, E. and A. Bigas, Blood Development: Hematopoietic Stem Cell Dependence and
Independence. Cell Stem Cell, 2018. 22(5): p. 639-651.
6. Xu, M.-j., et al., Stimulation of Mouse and Human Primitive Hematopoiesis by Murine
Embryonic Aorta-Gonad-Mesonephros–Derived Stromal Cell Lines. Blood, 1998. 92(6):
p. 2032-2040.
7. Ohneda, O., et al., Hematopoietic Stem Cell Maintenance and Differentiation Are
Supported by Embryonic Aorta-Gonad-Mesonephros Region–Derived Endothelium. Blood,
1998. 92(3): p. 908-919.
8. Oostendorp, R.A., et al., Long-term maintenance of hematopoietic stem cells does not
require contact with embryo-derived stromal cells in cocultures. Stem Cells, 2005. 23(6):
p. 842-51.
9. Hadland, B.K., et al., Endothelium and NOTCH specify and amplify aorta-gonad-
mesonephros-derived hematopoietic stem cells. J Clin Invest, 2015. 125(5): p. 2032-45.
128
10. Ruiz-Herguido, C., et al., Hematopoietic stem cell development requires transient Wnt/β-
catenin activity. J Exp Med, 2012. 209(8): p. 1457-68.
11. McGarvey, A.C., et al., A molecular roadmap of the AGM region reveals BMPER as a
novel regulator of HSC maturation. J Exp Med, 2017. 214(12): p. 3731-3751.
12. Rybtsov, S., et al., Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-
3-independent CD43(-) embryonic precursor. Stem Cell Reports, 2014. 3(3): p. 489-501.
13. Gao, X., et al., The hematopoietic stem cell niche: from embryo to adult. Development,
2018. 145(2).
14. Martin, M.A. and M. Bhatia, Analysis of the Human Fetal Liver Hematopoietic
Microenvironment. Stem Cells and Development, 2005. 14(5): p. 493-504.
15. Zhao, M., et al., N-Cadherin-Expressing Bone and Marrow Stromal Progenitor Cells
Maintain Reserve Hematopoietic Stem Cells. Cell Reports, 2019: p. 652-669.
16. McCulloch, E.A. and J.E. Till, Perspectives on the properties of stem cells. Nature
Medicine, 2005. 11(10): p. 1026.
17. Bonnet, D., Haematopoietic stem cells. Journal of Pathology, 2002. 197: p. 430–440.
18. Wilkinson, A.C., et al., Long-term ex vivo expansion of mouse hematopoietic stem cells.
Nat Protoc, 2020. 15(2): p. 628-648.
19. Osawa, M., et al., Long-term lymphohematopoietic reconstitution by a single CD34-
low/negative hematopoietic stem cell. Science, 1996. 273(5272): p. 242.
20. Wilkinson, A.C., et al., Long-term ex vivo haematopoietic-stem-cell expansion allows
nonconditioned transplantation. Nature, 2019.
21. Copelan, E.A., Hematopoietic Stem-Cell Transplantation. New England Journal of
Medicine, 2006. 354: p. 1813-26.
129
22. Shouval, R., et al., Outcomes of allogeneic haematopoietic stem cell transplantation from
HLA-matched and alternative donors: a European Society for Blood and Marrow
Transplantation registry retrospective analysis. The Lancet Haematology, 2019. 6(11): p.
e573-e584.
23. Liso, A., et al., Hematopoietic Stem Cell Transplantation: A Bioethical Lens. Stem Cells
Int, 2017. 2017: p. 1286246.
24. Schriber, J., et al., Second unrelated donor hematopoietic cell transplantation for primary
graft failure. Biol Blood Marrow Transplant, 2010. 16(8): p. 1099-106.
25. Remberger, M., et al., Second allogeneic hematopoietic stem cell transplantation: a
treatment for graft failure. Clin Transplant, 2011. 25(1): p. E68-76.
26. Arfons, L.M., et al., Second hematopoietic stem cell transplantation in myeloid
malignancies. Curr Opin Hematol, 2009. 16(2): p. 112-23.
27. Eaves, C.J., Hematopoietic stem cells: concepts, definitions, and the new reality. Blood,
2015. 125(17): p. 2605-13.
28. Seita, J. and I.L. Weissman, Hematopoietic stem cell: self-renewal versus differentiation.
Wiley Interdiscip Rev Syst Biol Med, 2010. 2(6): p. 640-53.
29. Orkin, S.H. and L.I. Zon, Hematopoiesis: an evolving paradigm for stem cell biology. Cell,
2008. 132(4): p. 631-44.
30. Yamamoto, R., A.C. Wilkinson, and H. Nakauchi1, Changing concepts in hematopoietic
stem cells. Science, 2018. 362: p. 895–896.
31. Dahlberg, A., C. Delaney, and I.D. Bernstein, Ex vivo expansion of human hematopoietic
stem and progenitor cells. Blood, 2011. 117: p. 6083-6090.
130
32. Kumar, S. and H. Geiger, HSC Niche Biology and HSC Expansion Ex Vivo. Trends in
Molecular Medicine, 2017. 23: p. 799-819.
33. Robin, C. and C. Durand, The roles of BMP and IL-3 signaling pathways in the control of
hematopoietic stem cells in the mouse embryo. Int J Dev Biol, 2010. 54(6-7): p. 1189-200.
34. Kumar, A., S.S. D'Souza, and A.S. Thakur, Understanding the Journey of Human
Hematopoietic Stem Cell Development. Stem Cells Int, 2019. 2019: p. 2141475.
35. Cao, H., A. Oteiza, and S.K. Nilsson, Understanding the role of the microenvironment
during definitive hemopoietic development. Exp Hematol, 2013. 41: p. 761-768.
36. Gekas, C., et al., Hematopoietic stem cell development in the placenta. Int J Dev Biol, 2010.
54(6-7): p. 1089-98.
37. Ottersbach, K. and E. Dzierzak, The placenta as a haematopoietic organ. Int J Dev Biol,
2010. 54(6-7): p. 1099-106.
38. Rybtsov, S.A. and M.A. Lagarkova, Development of Hematopoietic Stem Cells in the Early
Mammalian Embryo. Biochemistry (Mosc), 2019. 84(3): p. 190-204.
39. Cheng, M., et al., CXCR4-mediated bone marrow progenitor cell maintenance and
mobilization are modulated by c-kit activity. Circulation Research, 2010. 107: p. 1083-
1093.
40. Singh, P., K.S. Mohammad, and L.M. Pelus, CXCR4 expression in the bone marrow
microenvironment is required for hematopoietic stem and progenitor cell maintenance and
early hematopoietic regeneration after myeloablation. Stem Cells, 2020.
41. Wei, Q. and P.S. Frenette, Niches for Hematopoietic Stem Cells and Their Progeny.
Immunity, 2018. 48: p. 632-648.
131
42. Morrison, S.J. and D.T. Scadden, The bone marrow niche for haematopoietic stem cells.
Nature, 2014. 505: p. 327-34.
43. Pinho, S. and P.S. Frenette, Haematopoietic stem cell activity and interactions with the
niche. Nature Reviews Molecular Cell Biology, 2019.
44. Lee, D., D.W. Kim, and J.Y. Cho, Role of growth factors in hematopoietic stem cell niche.
Cell Biol Toxicol, 2020. 36(2): p. 131-144.
45. Zhang, P., et al., The physical microenvironment of hematopoietic stem cells and its
emerging roles in engineering applications. Stem Cell Res Ther, 2019. 10(1): p. 327.
46. Jhala, D. and R. Vasita, A Review on Extracellular Matrix Mimicking Strategies for an
Artificial Stem Cell Niche. Polymer Reviews, 2015. 55(4): p. 561-595.
47. Calvi, L.M., et al., Osteoblastic cells regulate the haematopoietic stem cell niche. Nature,
2003. 425(6960): p. 841-46.
48. Zhang, J., et al., Identification of the haematopoietic stem cell niche and control of the
niche size. Nature, 2003. 425(6960): p. 836-41.
49. Guerrouahen, B.S., I. Al-Hijji, and A.R. Tabrizi, Osteoblastic and vascular endothelial
niches, their control on normal hematopoietic stem cells, and their consequences on the
development of leukemia. Stem Cells Int, 2011. 2011: p. 375857.
50. Crane, G.M., E. Jeffery, and S.J. Morrison, Adult haematopoietic stem cell niches. Nature
Reviews Immunology, 2017.
51. Kunisaki, Y., et al., Arteriolar niches maintain haematopoietic stem cell quiescence.
Nature, 2013. 502: p. 637-643.
52. He, N., et al., Bone Marrow Vascular Niche: Home for Hematopoietic Stem Cells. Bone
Marrow Research, 2014. 2014: p. 1-8.
132
53. Doan, P.L. and J.P. Chute, The vascular niche: home for normal and malignant
hematopoietic stem cells. Leukemia, 2012. 26(1): p. 54-62.
54. Tormin, A., et al., Human Primary CD271+/CD45−/CD146−/Low and
CD271+/CD45−/CD146+ Bone Marrow Cells Are Developmentally Closely-Related
Stroma Stem Cells with Similar Functional Properties but Different In-Situ Localization.
2010, American Society of Hematology.
55. Matsuoka, Y., et al., Prospectively Isolated Human Bone Marrow Cell-Derived MSCs
Support Primitive Human CD34-Negative Hematopoietic Stem Cells. Stem Cells, 2015.
33(5): p. 1554-65.
56. Harkness, L., et al., CD146/MCAM defines functionality of human bone marrow stromal
stem cell populations. Stem Cell Res Ther, 2016. 7: p. 4.
57. Acar, M., et al., Deep imaging of bone marrow shows non-dividing stem cells are mainly
perisinusoidal. Nature, 2015. 526: p. 126-130.
58. Pinho, S., et al., PDGFRα and CD51 mark human Nestin + sphere-forming
mesenchymal stem cells capable of hematopoietic progenitor cell expansion. The Journal
of experimental medicine, 2013. 210: p. 1351-1367.
59. Méndez-Ferrer, S., et al., Mesenchymal and haematopoietic stem cells form a unique bone
marrow niche. Nature, 2010. 466: p. 829-834.
60. Ding, L., et al., Endothelial and perivascular cells maintain haematopoietic stem cells.
Nature, 2012. 481: p. 457-462.
61. Li, H., et al., Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord
Blood Hematopoietic Stem and Progenitor Cells. Int J Mol Sci, 2019. 20(8).
133
62. Pinho, S., et al., Lineage-Biased Hematopoietic Stem Cells Are Regulated by Distinct
Niches. Developmental Cell, 2018. 44: p. 634-641.e4.
63. Boulais, P. and P. Frenette, Making sense of hematopoietic stem cell niches. Blood, 2015.
125: p. 2621-2630.
64. Cheng C. Zhang, H.F.L., Cytokines regulating hematopoietic stem cell function. Current
Opinion in Hematology, 2008. 15: p. 307– 311.
65. Zhang, C.C. and H.F. Lodish, Cytokines regulating hematopoietic stem cell function.
Current opinion in hematology, 2008. 15: p. 307– 311.
66. Liu, H., et al., Structural basis for stem cell factor-KIT signaling and activation of class III
receptor tyrosine kinases. EMBO Journal, 2007. 26: p. 891-901.
67. Lennartsson, J. and L. Ronnstrand, Stem Cell Factor Receptor/c-Kit: From Basic Science
to Clinical Implications. Physiological Reviews, 2012. 92: p. 1619-1649.
68. Arai, F., et al., Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence
in the bone marrow niche. Cell, 2004. 118: p. 149-161.
69. Arai, F., et al., Role of N-cadherin in the regulation of hematopoietic stem cells in the bone
marrow niche. Annals of the New York Academy of Sciences, 2012. 1266: p. 72-77.
70. Avecilla, S.T., et al., Chemokine-mediated interaction of hematopoietic progenitors with
the bone marrow vascular niche is required for thrombopoiesis. Nat Med, 2004. 10(1): p.
64-71.
71. Kakeda, M., et al., Role of the thrombopoietin (TPO)/Mpl system: c-Mpl-like
molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis. Develop.
Growth Differ., 2002. 44: p. 63–75.
134
72. Yoshihara, H., et al., Thrombopoietin/MPL signaling regulates hematopoietic stem cell
quiescence and interaction with the osteoblastic niche. Cell Stem Cell, 2007. 1(6): p. 685-
97.
73. Qian, H., et al., Critical role of thrombopoietin in maintaining adult quiescent
hematopoietic stem cells. Cell Stem Cell, 2007. 1(6): p. 671-84.
74. Ueda, T., et al., Expansion of human NOD/SCID-repopulating cells by stem cell factor,
Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest, 2000.
105(7): p. 1013-21.
75. Levis, M., FLt3 dancing on the stem cell. Journal of Experimental Biology, 2017. 214(7):
p. 1857.
76. Ding, L. and S.J. Morrison, Haematopoietic stem cells and early lymphoid progenitors
occupy distinct bone marrow niches. Nature, 2013. 495: p. 231-235.
77. Linnekin, D., Early signaling pathways activated by c-Kit in hematopoietic cells.
International Journal of Biochemistry and Cell Biology, 1999. 31: p. 1053-1074.
78. Rönnstrand, L., Signal transduction via the stem cell factor receptor/c-Kit. Cellular and
Molecular Life Sciences, 2004. 61: p. 2535-2548.
79. Papayannopoulou, T., G.V. Priestley, and B. Nakamoto, Anti-VLA4/VCAM-1-induced
mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood,
1998. 91: p. 2231-2239.
80. Tay, J., J.P. Levesque, and I.G. Winkler, Cellular players of hematopoietic stem cell
mobilization in the bone marrow niche. Int J Hematol, 2017. 105(2): p. 129-140.
81. Asada, N., S. Takeishi, and P.S. Frenette, Complexity of bone marrow hematopoietic stem
cell niche. International Journal of Hematology, 2017. 106: p. 45-54.
135
82. Asada, N., et al., Differential cytokine contributions of perivascular haematopoietic stem
cell niches. Nature Cell Biology, 2017. 19: p. 214-223.
83. Nakamura-Ishizu, A., H. Takizawa, and T. Suda, The analysis, roles and regulation of
quiescence in hematopoietic stem cells. Development, 2014. 141: p. 4656-4666.
84. Lévesque, J.-P., et al., Disruption of the CXCR4/CXCL12 chemotactic interaction during
hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. Journal of
Clinical Investigation, 2003. 111(2): p. 187-196.
85. Decker, M., et al., Hepatic thrombopoietin is required for bone marrow hematopoietic stem
cell maintenance. Science (New York, N.Y.), 2018. 360: p. 106-110.
86. Puri, M.C. and A. Bernstein, Requirement for the TIE family of receptor tyrosine kinases
in adult but not fetal hematopoiesis. Proc Natl Acad Sci U S A, 2003. 100(22): p. 12753–
12758.
87. Zhou, B.O., L. Ding, and S.J. Morrison, Hematopoietic stem and progenitor cells regulate
the regeneration of their niche by secreting Angiopoietin-1. eLife, 2015. 4: p. e05521.
88. Martin, I., et al., Fibroblast Growth Factor-2 Supports ex Vivo Expansion and
Maintenance of Osteogenic Precursors from Human Bone Marrow. Endocrinology, 1997.
138(10): p. 4456-4462.
89. Haan, G.d., et al., In Vitro Generation of Long-Term Repopulating Hematopoietic Stem
Cells by Fibroblast Growth Factor-1. Developmental Cell, 2003. 4: p. 241–251.
90. Itkin, T., et al., FGF-2 expands murine hematopoietic stem and progenitor cells via
proliferation of stromal cells, c-Kit activation, and CXCL12 down-regulation. Blood, 2012.
120(9): p. 1843-55.
136
91. Zhao, M., et al., Megakaryocytes maintain homeostatic quiescence and promote post-
injury regeneration of hematopoietic stem cells. Nat Med, 2014. 20(11): p. 1321-6.
92. Yoon, K.A., et al., Fibroblast growth factor 2 supports osteoblastic niche cells during
hematopoietic homeostasis recovery after bone marrow suppression. Cell Commun Signal,
2017. 15(1): p. 25.
93. Blank, U. and S. Karlsson, The role of Smad signaling in hematopoiesis and translational
hematology. Leukemia, 2011. 25(9): p. 1379-88.
94. Nilsson, S.K., et al., Osteopontin, a key component of the hematopoietic stem cell niche
and regulator of primitive hematopoietic progenitor cells. Blood, 2005. 106(4): p. 1232-9.
95. Canaani, J., O. Kollet, and T. Lapidot, Neural regulation of bone, marrow, and the
microenvironment. Frontiers in Bioscience, 2011. 3: p. 1021-1031.
96. Bendall, L.J. and K.F. Bradstock, G-CSF: From granulopoietic stimulant to bone marrow
stem cell mobilizing agent. Cytokine Growth Factor Rev, 2014. 25(4): p. 355-67.
97. Zhang, W., et al., Effects of insulin and insulin-like growth factor 1 on osteoblast
proliferation and differentiation: differential signalling via Akt and ERK. Cell Biochem
Funct, 2012. 30(4): p. 297-302.
98. Caselli, A., et al., IGF-1-mediated osteoblastic niche expansion enhances long-term
hematopoietic stem cell engraftment after murine bone marrow transplantation. Stem
Cells, 2013. 31(10): p. 2193-204.
99. Graddis, T.J., et al., Structure-function analysis of FLT3 ligand-FLT3 receptor interactions
using a rapid functional screen. Journal of Biological Chemistry, 1998. 273: p. 17626-
17633.
137
100. Mehrasa, R., et al., Mesenchymal stem cells as a feeder layer can prevent apoptosis of
expanded hematopoietic stem cells derived from cord blood. International journal of
molecular and cellular medicine, 2014. 3: p. 1-10.
101. Nitsche, A., et al., Interleukin-3 Promotes Proliferation and Differentiation of Human
Hematopoietic Stem Cells but Reduces Their Repopulation Potential in NOD/SCID Mice.
Stem Cells, 2003. 21(2): p. 236-244.
102. Bernad, A., et al., Interleukin-6 Is Required In Vivo for the Regulation of Stem Cells and
Committed Progenitors of the Hematopoietic System. Immunity, 1994. 1: p. 725-731.
103. Chabot, B., et al., The proto-oncogene c-kit encoding a transmembrane tyrosine kinase
receptor maps to the mouse W locus. Nature, 1988. 335(6185): p. 88-89.
104. Zsebo, K.M., et al., Stem cell factor is encoded at the SI locus of the mouse and is the ligand
for the c-kit tyrosine kinase receptor. Cell, 1990. 63(1): p. 213-224.
105. Li, C.L. and G.R. Johnson, Stem Cell Factor Enhances the Survival But Not the Self-
Renewal of Murine Hematopoietic Long-Term Repopulating Cells. Blood, 1994. 84(2): p.
408-414.
106. Miller, C.L., et al., Impaired Steel Factor Responsiveness Differentially Affects the
Detection and Long-Term Maintenance of Fetal Liver Hematopoietic Stem Cells In Vivo.
Blood, 1997. 89(4): p. 1214-1223.
107. Eaves, C., et al., Hematopoietic stem cells: Inferences from in vivo assays. STEM CELLS,
1997. 15(S2): p. 1-5.
108. Holyoake, T.L., et al., Ex Vivo Expansion With Stem Cell Factor and Interleukin-11
Augments Both Short-Term Recovery Posttransplant and the Ability to Serially Transplant
Marrow. Blood, 1996. 87(11): p. 4589-4595.
138
109. Broudy, V.C., Stem Cell Factor and Hematopoiesis. Blood, 1997.
110. Bearman, S.I., Use of stem cell factor to mobilize hematopoietic progenitors. Current
opinion in hematology, 1997. 4: p. 157-162.
111. Nakamura, Y., et al., Soluble c-kit receptor mobilizes hematopoietic stem cells to
peripheral blood in mice. Exp Hematol, 2004. 32: p. 390-396.
112. Frimberger, A.E., et al., The fleet feet of haematopoietic stem cells: rapid motility,
interaction and proteopodia. British journal of haematology, 2001. 112: p. 644-654.
113. Reber, L., C.A. Da Silva, and N. Frossard, Stem cell factor and its receptor c-Kit as targets
for inflammatory diseases. Eur J Pharmacol, 2006. 533(1-3): p. 327-40.
114. Flanagan, J.G., D.C. Chan, and P. Leder, Transmembrane form of the kit ligand growth
factor is determined by alternative splicing and is missing in the SId mutant. Cell, 1991.
64(5): p. 1025-1035.
115. Barker, J.E., Sl/Sld hematopoietic progenitors are deficient in situ. Exp Hematol, 1994.
22(2): p. 174-177.
116. Heissig, B., et al., Recruitment of stem and progenitor cells from the bone marrow niche
requires MMP-9 mediated release of Kit-ligand. Cell, 2002. 109: p. 625-637.
117. Friel, J., et al., Hierarchy of Stroma-derived Factors in Supporting Growth of Stroma-
dependent Hemopoietic Cells : Membrane-bound SCF is Sufficient to Confer Stroma
Competence to Epithelial Cells Growth Factors, 2009. 20(1): p. 35–51.
118. Ajami, M., et al., Comparison of cord blood CD34 + stem cell expansion in coculture with
mesenchymal stem cells overexpressing SDF-1 and soluble /membrane isoforms of SCF. J
Cell Biochem, 2019. 120(9): p. 15297-15309.
139
119. Takagi, S., et al., Membrane-bound human SCF/KL promotes in vivo human hematopoietic
engraftment and myeloid differentiation. Blood, 2012. 119(12): p. 2768-77.
120. Liu, J., et al., Chapter Twelve - Notch Signaling in the Regulation of Stem Cell Self-
Renewal and Differentiation, in Current Topics in Developmental Biology, R. Kopan,
Editor. 2010, Academic Press. p. 367-409.
121. Lampreia, F.P., J.G. Carmelo, and F. Anjos-Afonso, Notch Signaling in the Regulation of
Hematopoietic Stem Cell. Curr Stem Cell Rep, 2017. 3(3): p. 202-209.
122. Kumano, K., et al., Notch1 but not Notch2 is essential for generating hematopoietic stem
cells from endothelial cells. Immunity, 2003. 18(5): p. 699-711.
123. Duncan, A.W., et al., Integration of Notch and Wnt signaling in hematopoietic stem cell
maintenance. Nat Immunol, 2005. 6(3): p. 314-22.
124. Butler, J.M., et al., Endothelial cells are essential for the self-renewal and repopulation of
Notch-dependent hematopoietic stem cells. Cell Stem Cell, 2010. 6(3): p. 251-64.
125. Varnum-Finney, B., C. Brashem-Stein, and I.D. Bernstein, Combined effects of Notch
signaling and cytokines induce a multiple log increase in precursors with lymphoid and
myeloid reconstituting ability. Blood, 2003. 101: p. 1784-1789.
126. Karanu, F.N., et al., The Notch Ligand Jagged-1 Represents a Novel Growth Factor of
Human Hematopoietic Stem Cells. J Exp Med, 2000. 192(9): p. 1365–1372.
127. Mancini, S.J., et al., Jagged1-dependent Notch signaling is dispensable for hematopoietic
stem cell self-renewal and differentiation. Blood, 2005. 105(6): p. 2340-2.
128. Guo, P., et al., Endothelial jagged-2 sustains hematopoietic stem and progenitor
reconstitution after myelosuppression. J Clin Invest, 2017. 127(12): p. 4242-4256.
140
129. Sacchetti, B., et al., Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can
Organize a Hematopoietic Microenvironment. Cell, 2007. 131(2): p. 324-336.
130. Ehninger, A. and A. Trumpp, The bone marrow stem cell niche grows up: mesenchymal
stem cells and macrophages move in. The Journal of Experimental Medicine, 2011. 208(3):
p. 421-428.
131. Shi, Y., D.J. Riese, 2nd, and J. Shen, The Role of the CXCL12/CXCR4/CXCR7 Chemokine
Axis in Cancer. Front Pharmacol, 2020. 11: p. 574667.
132. Broxmeyer , H.E., et al., Rapid mobilization of murine and human hematopoietic stem and
progenitor cells with AMD3100, a CXCR4 antagonist. Journal of Experimental Medicine,
2005. 201(8): p. 1307-1318.
133. Sugiyama, T., et al., Maintenance of the Hematopoietic Stem Cell Pool by CXCL12-CXCR4
Chemokine Signaling in Bone Marrow Stromal Cell Niches. Immunity, 2006. 25: p. 977-
988.
134. Wright, D.E., et al., Hematopoietic Stem Cells Are Uniquely Selective in Their Migratory
Response to Chemokines. The Journal of experimental medicine, 2002. 195(9): p. 1145-
1154.
135. Yu, L., et al., Identification and expression of novel isoforms of human stromal cell-derived
factor 1. Gene, 2006. 374: p. 174-9.
136. De La Luz Sierra, M., et al., Differential processing of stromal-derived factor-1α and
stromal-derived factor-1β explains functional diversity. Blood, 2004. 103(7): p. 2452-2459.
137. Rueda, P., et al., The CXCL12gamma chemokine displays unprecedented structural and
functional properties that make it a paradigm of chemoattractant proteins. PLoS One,
2008. 3(7): p. e2543.
141
138. Kollet, O., et al., Osteoclasts degrade endosteal components and promote mobilization of
hematopoietic progenitor cells. Nat Med, 2006. 12(6): p. 657-64.
139. Mooney, C., et al., Selective Expression of Flt3 within the Mouse Hematopoietic Stem Cell
Compartment. International Journal of Molecular Sciences, 2017. 18: p. 1037.
140. Jacobsen, S.E., et al., The FLT3 ligand potently and directly stimulates the growth and
expansion of primitive murine bone marrow progenitor cells in vitro: synergistic
interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors. The
Journal of experimental medicine, 1995. 181(4): p. 1357-1363.
141. Hudak, S., et al., FLT3/FLK2 ligand promotes the growth of murine stem cells and the
expansion of colony-forming cells and spleen colony-forming units. Blood, 1995. 85(10):
p. 2747-2755.
142. Hirayama, F., et al., The flt3 ligand supports proliferation of lymphohematopoietic
progenitors and early B-lymphoid progenitors. Blood, 1995. 85(7): p. 1762-1768.
143. Gabbianelli, M., et al., Multi-level effects of flt3 ligand on human hematopoiesis: expansion
of putative stem cells and proliferation of granulomonocytic progenitors/monocytic
precursors. Blood, 1995. 86(5): p. 1661-1670.
144. Banu, N., et al., Modulation of haematopoietic progenitor development by FLT-3 ligand.
Cytokine Growth Factor Rev, 1999. 11(9): p. 679-688.
145. McKenna, H.J., et al., Mice lacking flt3 ligand have deficient hematopoiesis affecting
hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood, 2000.
95(11): p. 3489-97.
146. Savvides, S.N., T. Boone, and P. Andrew Karplus, Flt3 ligand structure and unexpected
commonalities of helical bundles and cystine knots. Nat Struct Biol, 2000. 7(6): p. 486-91.
142
147. Lyman, S.D., et al., Identification of soluble and membrane-bound isoforms of the murine
flt3 ligand generated by alternative splicing of mRNAs. Oncogene, 1995. 10(1): p. 149-57.
148. Chen, S., M. Lewallen, and T. Xie, Adhesion in the stem cell niche: biological roles and
regulation. Development, 2013. 140: p. 255-265.
149. Jeannet, R., et al., Alcam regulates long-term hematopoietic stem cell engraftment and self-
renewal. Stem Cells, 2013. 31(3): p. 560-71.
150. Papayannopoulou, T. and B. Nakamoto, Peripheralization of hemopoietic progenitors in
primates treated with anti-VLA4 integrin. Proceedings of the National Academy of
Sciences, 1993. 90(20): p. 9374-8.
151. Vermeulen, M., et al., Role of Adhesion Molecules in the Homing and Mobilization of
Murine Hematopoietic Stem and Progenitor Cells. Blood, 1998. 92(3): p. 894-900.
152. Wilson, A. and A. Trumpp, Bone-marrow haematopoietic-stem-cell niches. Nature
Reviews Immunology, 2006. 6(2): p. 93-106.
153. Kulkarni, R. and V. Kale, Physiological Cues Involved in the Regulation of Adhesion
Mechanisms in Hematopoietic Stem Cell Fate Decision. Frontiers in Cell and
Developmental Biology, 2020. 8.
154. Zhang, J., et al., Identification of the haematopoietic stem cell niche and control of the
niche size. Nature, 2003. 425(6960): p. 836-41.
155. Nakamura, Y., et al., Isolation and characterization of endosteal niche cell populations
that regulate hematopoietic stem cells. Blood, 2010. 116(9): p. 1422-32.
156. Hosokawa, K., et al., Cadherin-based adhesion is a potential target for niche manipulation
to protect hematopoietic stem cells in adult bone marrow. Cell Stem Cell, 2010. 6(3): p.
194-8.
143
157. Hosokawa, K., et al., Knockdown of N-cadherin suppresses the long-term engraftment of
hematopoietic stem cells. Blood, 2010. 116(4): p. 554-63.
158. Kiel, M.J., G.L. Radice, and S.J. Morrison, Lack of evidence that hematopoietic stem cells
depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell Stem
Cell, 2007. 1(2): p. 204-17.
159. Bromberg, O., et al., Osteoblastic N-cadherin is not required for microenvironmental
support and regulation of hematopoietic stem and progenitor cells. Blood, 2012. 120(2):
p. 303-13.
160. Greenbaum, A.M., et al., N-cadherin in osteolineage cells is not required for maintenance
of hematopoietic stem cells. Blood, 2012. 120(2): p. 295-302.
161. Kiel, M.J., et al., Hematopoietic Stem Cells Do Not Depend on N-Cadherin to Regulate
Their Maintenance. Cell Stem Cell, 2009. 4: p. 170-179.
162. Haug, J.S., et al., N-cadherin expression level distinguishes reserved versus primed states
of hematopoietic stem cells. Cell Stem Cell, 2008. 2(4): p. 367-79.
163. Calderwood, D.A., S.J. Shattil, and M.H. Ginsberg, Integrins and actin filaments:
Reciprocal regulation of cell adhesion and signaling. Journal of Biological Chemistry,
2000. 275: p. 22607-22610.
164. Shen, B., M.K. Delaney, and X. Du, Inside-out, outside-in, and inside-outside-in: G protein
signaling in integrin-mediated cell adhesion, spreading, and retraction. Curr Opin Cell
Biol, 2012. 24(5): p. 600-6.
165. Luo, B.H., C.V. Carman, and T.A. Springer, Structural basis of integrin regulation and
signaling. Annu Rev Immunol, 2007. 25: p. 619-47.
144
166. Mahabeleshwar, G.H., et al., Mechanisms of integrin-vascular endothelial growth factor
receptor cross-activation in angiogenesis. Circ Res, 2007. 101(6): p. 570-80.
167. Petruzzelli, L., M. Takami, and H.D. Humes., Structure and function of cell adhesion
molecules. The American journal of medicine, 1999. 106(4): p. 467-476.
168. Li, D., et al., VCAM-1+ macrophages guide the homing of HSPCs to a vascular niche.
Nature, 2018: p. 1.
169. Papayannopoulou, T., Craddock, C., Nakamoto, B., Priestley, G V, Wolf, N S., The
VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of
transplanted murine hemopoietic progenitors between bone marrow and spleen.
Proceedings of the National Academy of Sciences of the United States of America, 1995.
92: p. 9647-9651.
170. Oostendorp, R.A. and P. Dormer, VLA-4-mediated interactions between normal human
hematopoietic progenitors and stromal cells. Leuk Lymphoma, 1997. 24(5-6): p. 423-35.
171. Qin, G., et al., Functional disruption of α4 integrin mobilizes bone marrow–derived
endothelial progenitors and augments ischemic neovascularization. The Journal of
experimental medicine, 2006. 203: p. 153-163.
172. Potocnik, A.J., C. Brakebusch, and R. Fässler, Fetal and adult hematopoietic stem cells
require β1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity
2000. 12(6): p. 653-663.
173. Scott, L.M., G.V. Priestley, and T. Papayannopoulou, Deletion of α 4 Integrins from Adult
Hematopoietic Cells Reveals Roles in Homeostasis , Regeneration , and Homing.
Molecular and cellular biology, 2003. 23: p. 9349-9360.
145
174. Huygen, S., et al., Adhesion ofs synchronized human hematopoietic progenitor cells to
fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle
transit in ex vivo culture. Blood, 2002. 100(6): p. 2744-2752.
175. Lubkova, O., et al., VCAM-1 expression on bone marrow stromal cells from patients with
myelodysplastic syndromes. Bulletin of experimental biology and medicine, 2011. 151(1):
p. 13-15.
176. Reina, M. and E. Espel, Role of LFA-1 and ICAM-1 in Cancer. Cancers (Basel), 2017.
9(11).
177. Liu, W., et al., Rational identification of a Cdc42 inhibitor presents a new regimen for
long-term hematopoietic stem cell mobilization. Leukemia, 2018.
178. Peled, A., et al., The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5
on immature human CD34+ cells: role in transendothelial/stromal migration and
engraftment of NOD/SCID mice. Blood, 2000. 95(11): p. 3289-3296.
179. Cao, B., et al., Junctional Adhesion Molecule a (Jam-a) Is Critical for Hsc Homing and
Maintenance of Stem Cell Quiescence in the Endosteal Niche. Experimental Hematology,
2019. 76.
180. Arcangeli, M.L., et al., JAM-B regulates maintenance of hematopoietic stem cells in the
bone marrow. Blood, 2011. 118(17): p. 4609-19.
181. Praetor, A., et al., Genetic deletion of JAM-C reveals a role in myeloid progenitor
generation. Blood, 2009. 113(9): p. 1919-28.
182. Arcangeli, M.L., et al., Function of Jam-B/Jam-C interaction in homing and mobilization
of human and mouse hematopoietic stem and progenitor cells. Stem Cells, 2014. 32(4): p.
1043-54.
146
183. Chitteti, B.R., et al., CD166 regulates human and murine hematopoietic stem cells and the
hematopoietic niche. Blood, 2014. 124(4): p. 519-29.
184. Yokota, T., et al., The endothelial antigen ESAM marks primitive hematopoietic
progenitors throughout life in mice. Blood, 2009. 113(13): p. 2914-23.
185. Ooi, A.G., et al., The adhesion molecule esam1 is a novel hematopoietic stem cell marker.
Stem Cells, 2009. 27(3): p. 653-61.
186. Sugano, Y., et al., Junctional adhesion molecule-A, JAM-A, is a novel cell-surface marker
for long-term repopulating hematopoietic stem cells. Blood, 2008. 111(3): p. 1167-72.
187. Henry, E., et al., JAM-C/Jam-C Expression Is Primarily Expressed in Mouse
Hematopoietic Stem Cells. Hemasphere, 2021. 5(7): p. e594.
188. Forsberg, E.C., et al., Differential expression of novel potential regulators in hematopoietic
stem cells. PLoS Genet, 2005. 1(3): p. e28.
189. Nagamatsu, G., et al., A CTX family cell adhesion molecule, JAM4, is expressed in stem
cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages.
Mol Cell Biol, 2006. 26(22): p. 8498-506.
190. Chang, C.H., et al., Junctional Adhesion Molecule-A Is Highly Expressed on Human
Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine
Receptor CXCR4. Stem Cells, 2016. 34(6): p. 1664-78.
191. Kinashi, T. and T.A. Springer, Steel factor and c-kit regulate cell-matrix adhesion. Blood,
1994. 83: p. 1033-8.
192. Schumann, K., et al., Immobilized chemokine fields and soluble chemokine gradients
cooperatively shape migration patterns of dendritic cells. Immunity, 2010. 32(5): p. 703-
13.
147
193. Tabone-Eglinger, S., et al., Membrane-bound Kit ligand regulates melanocyte adhesion
and survival, providing physical interaction with an intraepithelial niche. The FASEB
Journal, 2012. 26: p. 3738-3753.
194. Jonathan Hoggatt, L.M.P., Mobilization of hematopoietic stem cells from the bone marrow
niche to the blood compartment. Stem Cell Research & Therapy, 2011. 2(13).
195. Ridley, A.J., et al., Cell Migration: Integrating Signals from Front to Back. Science, 2003.
302: p. 1704-1709.
196. Francis, K., et al., Two new pseudopod morphologies displayed by the human
hematopoietic KG1a progenitor cell line and by primary human CD34(+) cells. Blood,
1998. 92: p. 3616-23.
197. Fruehauf, S., et al., Functional characterization of podia formation in normal and
malignant hematopoietic cells. Journal of Leukocyte Biology, 2002. 71: p. 425-432.
198. Rettig, M.P., G. Ansstas, and J.F. DiPersio, Mobilization of hematopoietic stem and
progenitor cells using inhibitors of CXCR4 and VLA-4. Leukemia, 2012. 26(1): p. 34-53.
199. Kovach, N.L., et al., Stem cell factor modulates avidity of alpha 4 beta 1 and alpha 5 beta
1 integrins expressed on hematopoietic cell lines. Blood, 1995. 85(1): p. 159-167.
200. Moll, N.M. and R.M. Ransohoff, CXCL12 and CXCR4 in bone marrow physiology. Expert
Review of Hematology, 2010. 3: p. 315-322.
201. Pham, K., F. Sacirbegovic, and S.M. Russell, Polarized cells, polarized views: asymmetric
cell division in hematopoietic cells. Front Immunol, 2014. 5: p. 26.
202. Knoblich, J.A., Asymmetric cell division: recent developments and their implications for
tumour biology. Nat Rev Mol Cell Biol, 2010. 11(12): p. 849-60.
148
203. Zhang, J. and L. Li, BMP signaling and stem cell regulation. Dev Biol, 2005. 284(1): p. 1-
11.
204. Knoblich, J.A., Mechanisms of asymmetric stem cell division. Cell, 2008. 132(4): p. 583-
97.
205. Wu, M., et al., Imaging hematopoietic precursor division in real time. Cell Stem Cell, 2007.
1(5): p. 541-54.
206. Schroeder, T., Asymmetric cell division in normal and malignant hematopoietic precursor
cells. Cell Stem Cell, 2007. 1(5): p. 479-81.
207. Yassin, M. and S.M. Russell, Polarity and asymmetric cell division in the control of
lymphocyte fate decisions and function. Curr Opin Immunol, 2016. 39: p. 143-9.
208. Zhang, X., et al., Bioengineering tools for probing intracellular events in T lymphocytes.
Wiley Interdiscip Rev Syst Biol Med, 2020: p. e1510.
209. The Immunological Synapse: A Molecular Machine Controlling T Cell Activation.
210. Ting, S.B., et al., Asymmetric segregation and self-renewal of hematopoietic stem and
progenitor cells with endocytic Ap2a2. Blood, 2012. 119(11): p. 2510-22.
211. Roth, T.M., Y.M. Yamashita, and J. Cheng, Asymmetric Centrosome Behavior in Stem Cell
Divisions, in The Centrosome. 2012. p. 99-110.
212. Mendelson, A. and P.S. Frenette, Hematopoietic stem cell niche maintenance during
homeostasis and regeneration. Nature Medicine, 2014. 20: p. 833-846.
213. Isern, J., et al., The neural crest is a source of mesenchymal stem cells with specialized
hematopoietic stem cell niche function. Elife, 2014. 3: p. e03696.
214. Greenbaum, A., et al., CXCL12 in early mesenchymal progenitors is required for
haematopoietic stem-cell maintenance. Nature, 2013. 495: p. 227-230.
149
215. Coskun, S., et al., Development of the fetal bone marrow niche and regulation of HSC
quiescence and homing ability by emerging osteolineage cells. Cell Rep, 2014. 9(2): p.
581-90.
216. Wu, J.Y., et al., Osteoblastic regulation of B lymphopoiesis is mediated by Gsα-dependent
signaling pathways. Proceedings of the National Academy of Sciences, 2008. 105(44): p.
16976-16981.
217. Park, D., et al., Endogenous bone marrow MSCs are dynamic, fate-restricted participants
in bone maintenance and regeneration. Cell Stem Cell, 2012. 10(3): p. 259-72.
218. Fujita, J., et al., In vitro duplication and in vivo cure of mast-cell deficiency of Sl/Sld mutant
mice by cloned 3T3 fibroblasts. Proceedings of the National Academy of Sciences, 1989.
86(8): p. 2888-2891.
219. Nocka, K., et al., Expression of c-kit gene products in known cellular targets of W
mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant
mice. Genes & development, 1989. 3(6): p. 816-826.
220. Sharma, Y., C.M. Astle, and D.E. Harrison, Heterozygous kit mutants with little or no
apparent anemia exhibit large defects in overall hematopoietic stem cell function. Exp
Hematol, 2007. 35(2): p. 214-220.
221. Wattrus, S.J. and L.I. Zon, Stem cell safe harbor: the hematopoietic stem cell niche in
zebrafish. Blood Adv, 2018. 2(21): p. 3063-3069.
222. Tamplin, O.J., et al., Hematopoietic stem cell arrival triggers dynamic remodeling of the
perivascular niche. Cell, 2015. 160: p. 241-252.
223. Mahony, C.B., et al., Tfec controls the hematopoietic stem cell vascular niche during
zebrafish embryogenesis. Blood, 2016. 128(10): p. 1336-45.
150
224. Mahony, C.B., C. Pasche, and J.Y. Bertrand, Oncostatin M and Kit-Ligand Control
Hematopoietic Stem Cell Fate during Zebrafish Embryogenesis. Stem Cell Reports, 2018.
10(6): p. 1920-1934.
225. Theodore, L.N., et al., Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic
Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization. Stem Cell
Reports, 2017. 8(5): p. 1226-1241.
226. Dimitroff, C.J., et al., CD44 Is a Major E-Selectin Ligand on Human Hematopoietic
Progenitor Cells. The Journal of Cell Biology, 2001. 153(6): p. 1277–1286.
227. Mohle, R., S. Rafii, and M.A.S. Moore, The role of endothelium in the regulation of
hematopoietic stem cell migration. Stem cells, 1998. 16: p. 159-165.
228. Petit, I., D. Jin, and S. Rafii, The SDF-1-CXCR4 signaling pathway: a molecular hub
modulating neo-angiogenesis. Trends Immunol, 2007. 28(7): p. 299-307.
229. Kissa, K. and P. Herbomel, Blood stem cells emerge from aortic endothelium by a novel
type of cell transition. Nature, 2010. 464(7285): p. 112-5.
230. Bertrand, J.Y., et al., Haematopoietic stem cells derive directly from aortic endothelium
during development. Nature, 2010. 464(7285): p. 108-11.
231. Boisset, J.C., et al., In vivo imaging of haematopoietic cells emerging from the mouse aortic
endothelium. Nature, 2010. 464(7285): p. 116-20.
232. Katayama, Y., et al., Signals from the sympathetic nervous system regulate hematopoietic
stem cell egress from bone marrow. Cell, 2006. 124(2): p. 407-21.
233. Ferrell, P.I., et al., Functional assessment of hematopoietic niche cells derived from human
embryonic stem cells. Stem Cells Dev, 2014. 23(12): p. 1355-63.
151
234. Punzel, M., et al., The symmetry of initial divisions of human hematopoietic progenitors is
altered only by the cellular microenvironment. Exp Hematol, 2003. 31: p. 339-347.
235. Dexter, T.M., T.D. Allen, and L.G. Lajtha, Conditions Controlling the Proliferation of
Haemopoietic Stem Cells In Vitro. Journal of cellular physiology, 1977. 91(3): p. 335-344.
236. Kadereit, S., et al., Expansion of LTC-ICs and Maintenance of p21 and BCL-2 Expression
in Cord Blood CD34+/CD38– Early Progenitors Cultured over Human MSCs as a Feeder
Layer. Stem Cells, 2002.
237. Wagner, W., et al., Molecular evidence for stem cell function of the slow-dividing fraction
among human hematopoietic progenitor cells by genome-wide analysis. Blood, 2004. 104:
p. 675-686.
238. Herrera, L., et al., OP9 feeder cells are superior to M2-10B4 cells for the generation of
mature and functional natural killer cells from umbilical cord hematopoietic progenitors.
Frontiers in Immunology, 2017. 8: p. 1-10.
239. Rusmini, F., Z. Zhong, and J. Feijen, Protein Immobilization Strategies for Protein
Biochips. Biomacromolecules, 2007. 8: p. 1775-1789.
240. Kam, L.C., K. Shen, and M.L. Dustin, Micro- and Nanoscale Engineering of Cell Signaling.
Annual Review of Biomedical Engineering, 2013. 15: p. 305-326.
241. Wang, X., et al., Discriminating the Independent Influence of Cell Adhesion and Spreading
Area on Stem Cell Fate Determination Using Micropatterned Surfaces. Sci Rep, 2016. 6:
p. 28708.
242. Dirar, Q., et al., Activation and degranulation of CAR-T cells using engineered antigen-
presenting cell surfaces. PLoS One, 2020. 15(9): p. e0238819.
152
243. Celebi, B., D. Mantovani, and N. Pineault, Effects of extracellular matrix proteins on the
growth of haematopoietic progenitor cells. Biomed Mater, 2011. 6(5): p. 055011.
244. Altrock, E., et al., The significance of integrin ligand nanopatterning on lipid raft
clustering in hematopoietic stem cells. Biomaterials, 2012. 33(11): p. 3107-18.
245. Furukawa, K., Artificial Cell Membrane on Patterned Surface––Growth Control and
Microchannel Device Application. NTT Technical Review, 2009. 7(8).
246. Mingeot-Leclercq, M.P., et al., Atomic force microscopy of supported lipid bilayers. Nat
Protoc, 2008. 3(10): p. 1654-9.
247. Crites, T.J., et al., Supported Lipid Bilayer Technology for the Study of Cellular Interfaces.
Curr Protoc Cell Biol, 2015. 68: p. 24 5 1-31.
248. Plant, A.L., et al., Phospholipid/Alkanethiol Bilayers for Cell-Surface Receptor Studies by
Surface Plasmon Resonance. Anal Biochem, 1995. 226: p. 342-348.
249. Pum, D., et al., Patterning of Monolayers of Crystalline S-layer Proteins on a Silicon
Surface by Deep Ultraviolet Radiation. Microelectronic engineering, 1997. 35: p. 297-300.
250. Yang, T., et al., Fabrication of Phospholipid Bilayer-Coated Microchannels for On-Chip
Immunoassays. Anal Chem, 2001. 73: p. 165-169.
251. Yang, T., et al., Investigations of Bivalent Antibody Binding on Fluid-Supported
Phospholipid Membranes: The Effect of Hapten Density. J Am Chem Soc, 2003. 125: p.
4779-4784.
252. Xu, L., et al., Self-Assembly of a Virus-Mimicking Nanostructure System for Efficient
Tumor-Targeted Gene Delivery. Hum Gene Ther, 2002. 13: p. 469–481.
153
253. Ono, A. and E.O. Freed, Plasma membrane rafts play a critical role in HIV-1 assembly
and release. Proceedings of the National Academy of Sciences, 2001. 98(24): p. 13925–
13930.
254. Kasahara, K., et al., Association of GPI-Anchored Protein TAG-1 with Src-Family Kinase
Lyn in Lipid Rafts of Cerebellar Granule Cells. Neurochemical Research, 2002. 27: p. 823–
829.
255. Qi, S.Y., J.T. Groves, and A.K. Chakraborty, Synaptic pattern formation during cellular
recognition. Proceedings of the National Academy of Sciences, 2001. 98(12).
256. Stoddart, A., et al., Lipid Rafts Unite Signaling Cascades with Clathrin to Regulate BCR
Internalization. Immunity, 2002. 17: p. 451–462.
257. Xu, Q., et al., EphA2 receptor activation by monomeric ephrin-A1 on supported
membranes. Biophysical Journal, 2011. 101: p. 2731-2739.
258. Hartman, N.C., J.a. Nye, and J.T. Groves, Cluster size regulates protein sorting in the
immunological synapse. Proceedings of the National Academy of Sciences of the United
States of America, 2009. 106: p. 12729-12734.
259. Manz, B.N. and J.T. Groves, Spatial organization and signal transduction at intercellular
junctions. Nature Reviews Molecular Cell Biology, 2010. 11: p. 342-352.
260. Dustin, M.L., et al., TCR-Mediated Adhesion of T Cell Hybridomas to Planar Bilayers
Containing Purified MHC Class II/Peptide Complexes and Receptor Shedding During
Detachment. J Immunol, 1996. 157: p. 2014-2021.
261. Dustin, M.L., et al., A Novel Adaptor Protein Orchestrates Receptor Patterning and
Cytoskeletal Polarity in T-Cell Contacts. Cell, 1998. 94: p. 667–677.
154
262. Dustin, M.L., Adhesive Bond Dynamics in Contacts between T Lymphocytes and Glass-
supported Planar Bilayers Reconstituted with the Immunoglobulin-related Adhesion
Molecule CD58. Journal of Biological Chemistry, 1997. 272(25): p. 15782–15788.
263. Grakoui, A., et al., The immunological synapse: a molecular machine controlling T cell
activation. Science, 1999. 285: p. 221-227.
264. Milhiet, P.-E., et al., Spontaneous insertion and partitioning of alkaline phosphatase into
model lipid rafts. EMBO reports, 2002. 3(5): p. 485–490.
265. Rinia, H.A., et al., Domain Formation in Phosphatidylcholine Bilayers Containing
Transmembrane Peptides: Specific Effects of Flanking Residues. Biochemistry, 2002. 41:
p. 2814-2824.
266. Montero, M.T., et al., Interfacial Membrane Effects of Fluoroquinolones as Revealed by a
Combination of Fluorescence Binding Experiments and Atomic Force Microscopy
Observations. Langmuir, 2006. 22: p. 7574-7578.
267. Giocondi, M.-C., et al., Phase Topology and Growth of Single Domains in Lipid Bilayers.
Langmuir, 2001. 17: p. 1653-1659.
268. Kaizuka, Y., et al., Mechanisms for segregating T cell receptor and adhesion molecules
during immunological synapse formation in Jurkat T cells. Proc Natl Acad Sci U S A, 2007.
104(51): p. 20296–20301.
269. Ferraro, F., C.L. Celso, and D. Scadden, Adult stem cells and their niches. Advances in
experimental medicine and biology, 2010. 695: p. 155-168.
270. Lane, S.W., D.A. Williams, and F.M. Watt, Modulating the stem cell niche for tissue
regeneration. Nature biotechnology, 2014. 32(8): p. 795-803.
155
271. Caplan, A.I., Are All Adult Stem Cells The Same? Regenerative Engineering and
Translational Medicine, 2015. 1(1): p. 4-10.
272. Barker, J.E., SL/SL(D) hematopoietic progenitors are deficient in-situ. Experimental
Hematology, 1994. 22(2): p. 174-177.
273. Driessen, R.L., H.M. Johnston, and S.K. Nilsson, Membrane-bound stem cell factor is a
key regulator in the initial lodgment of stem cells within the endosteal marrow region.
Experimental Hematology, 2003. 31: p. 1284-1291.
274. Kollet, O., et al., Osteoclasts degrade endosteal components and promote mobilization of
hematopoietic progenitor cells. Nature Medicine, 2006. 12(6): p. 657-664.
275. Jacobsen, K., et al., Adhesion receptors on bone marrow stromal cells: in vivo expression
of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in
normal and gamma-irradiated mice. Blood, 1996. 87(1): p. 73-82.
276. Walasek, M.A., R. van Os, and G. de Haan, Hematopoietic stem cell expansion: challenges
and opportunities. Annals of the New York Academy of Sciences, 2012. 1266(1): p. 138-
150.
277. Mahadik, B.P., et al., The use of covalently immobilized stem cell factor to selectively affect
hematopoietic stem cell activity within a gelatin hydrogel. Biomaterials, 2015. 67: p. 297-
307.
278. Ajami, M., et al., Comparison of cord blood CD34 + stem cell expansion in coculture with
mesenchymal stem cells overexpressing SDF-1 and soluble /membrane isoforms of SCF.
Journal of Cellular Biochemistry, 2019. 120(9): p. 15297-15309.
156
279. Feng, Q., et al., Expansion of engrafting human hematopoietic stem/progenitor cells in
three-dimensional scaffolds with surface-immobilized fibronectin. J Biomed Mater Res A,
2006. 78(4): p. 781-91.
280. Acar, M., et al., Deep imaging of bone marrow shows non-dividing stem cells are mainly
perisinusoidal. Nature, 2015. 526: p. 126.
281. Morrison, S.J. and D.T. Scadden, The bone marrow niche for haematopoietic stem cells.
Nature, 2014. 505(7483): p. 327-334.
282. Fonseca, A.V., et al., Polarization and migration of hematopoietic stem and progenitor
cells rely on the RhoA/ROCK I pathway and an active reorganization of the microtubule
network. J Biol Chem, 2010. 285(41): p. 31661-71.
283. Jing, D., et al., Hematopoietic stem cells in co-culture with mesenchymal stromal cells -
modeling the niche compartments in vitro. Haematologica, 2010. 95: p. 542-550.
284. Cuchiara, M.L., et al., Covalent immobilization of stem cell factor and stromal derived
factor 1α for in vitro culture of hematopoietic progenitor cells. Acta Biomaterialia, 2013.
9: p. 9258-9269.
285. Grakoui, A., et al., The immunological synapse: a molecular machine controlling T cell
activation. Science, 1999. 285: p. 221-227.
286. Manz, B.N. and J.T. Groves, Spatial organization and signal transduction at intercellular
junctions. Nat Rev Mol Cell Biol, 2010. 11(5): p. 342-52.
287. Kumar, K., et al., Formation of supported lipid bilayers on indium tin oxide for
dynamically-patterned membrane-functionalized microelectrode arrays. Lab Chip, 2009.
9(5): p. 718-25.
157
288. Shen, K., et al., Supported Lipid Bilayers for Patterning the Cell- Substrate Interface.
Journal of the American Chemical Society, 2009. 131: p. 13204-13205.
289. Barr, V.A. and S.C. Bunnell, Interference Reflectance Microscopy. Curr Protoc Cell Biol,
2009: p. 4.23.1-4.23.19.
290. Yen, D.P., Y. Ando, and K. Shen, A cost-effective micromilling platform for rapid
prototyping of microdevices. TECHNOLOGY, 2016. 04(04): p. 234-239.
291. Axel Ullrich, J.S., Signal Transduction by Receptors with Tyrosine Kinase Activity. Cell,
1990. 61: p. 203-212.
292. Edidin, M., Lipids on the frontier: a century of cell-membrane bilayers. Nat Rev Mol Cell
Biol, 2003. 4(5): p. 414.
293. Horiuchi, K., et al., Ectodomain shedding of FLT3 ligand is mediated by TNF-alpha
converting enzyme. Journal of immunology (Baltimore, Md. : 1950), 2009. 182(12): p.
7408-7414.
294. Decker, M., et al., Hepatic thrombopoietin is required for bone marrow hematopoietic stem
cell maintenance. Science, 2018. 360(6384): p. 106-110.
295. Zhou, B.O., et al., Leptin Receptor-expressing mesenchymal stromal cells represent the
main source of bone formed by adult bone marrow. Cell stem cell, 2014. 15: p. 154-168.
296. Zhou, B.O., et al., Leptin-receptor-expressing mesenchymal stromal cells represent the
main source of bone formed by adult bone marrow. Cell Stem Cell, 2014. 15: p. 154-168.
297. Shin, J.Y., et al., High c-Kit expression identifies hematopoietic stem cells with impaired
self-renewal and megakaryocytic bias. The Journal of Experimental Medicine, 2014. 211:
p. 217-231.
158
298. Chen, S., M. Lewallen, and T. Xie, Adhesion in the stem cell niche: biological roles and
regulation. Development (Cambridge, England), 2013. 140(2): p. 255-265.
299. Dustin, M.L., Cell adhesion molecules and actin cytoskeleton at immune synapses and
kinapses. Current Opinion in Cell Biology, 2007. 19: p. 529-533.
300. Shin, J.-W., et al., Contractile Forces Sustain and Polarize Hematopoiesis from Stem and
Progenitor Cells. Cell Stem Cell, 2014. 14(1): p. 81-93.
301. Alexeev, V. and K. Yoon, Distinctive Role of the cKit Receptor Tyrosine Kinase Signaling
in Mammalian Melanocytes. Journal of Investigative Dermatology, 2006. 126(5): p. 1102-
1110.
302. Matsunaga, T., et al., Interaction between leukemic-cell VLA-4 and stromal fibronectin is
a decisive factor for minimal residual disease of acute myelogenous leukemia. Nature
Medicine, 2003. 9(9): p. 1158-1165.
303. Dittmann, A., et al., The Commonly Used PI3-Kinase Probe LY294002 Is an Inhibitor of
BET Bromodomains. ACS Chemical Biology, 2014. 9(2): p. 495-502.
304. Bilanges, B., Y. Posor, and B. Vanhaesebroeck, PI3K isoforms in cell signalling
and vesicle trafficking. Nature Reviews Molecular Cell Biology, 2019. 20(9): p. 515-534.
305. Hemmati, S., et al., PI3 kinase alpha and delta promote hematopoietic stem cell activation.
JCI insight, 2019. 5(13): p. e125832.
306. Mazzoldi, E.L., et al., A juxtacrine/paracrine loop between C-Kit and stem cell factor
promotes cancer stem cell survival in epithelial ovarian cancer. Cell Death Dis, 2019.
10(6): p. 412.
307. Miyamoto, K., et al., Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell
Pool. Cell Stem Cell, 2007. 1: p. 101-112.
159
308. Crane, G.M., E. Jeffery, and S.J. Morrison, Adult haematopoietic stem cell niches. Nature
reviews. Immunology, 2017.
309. Varnum-Finney, B., et al., Notch2 governs the rate of generation of mouse long- and short-
term repopulating stem cells. The Journal of Clinical Investigation, 2011. 121(3): p. 1207-
1216.
310. Carrasco, Y.R., et al., LFA-1/ICAM-1 interaction lowers the threshold of B cell activation
by facilitating B cell adhesion and synapse formation. Immunity, 2004. 20(5): p. 589-99.
311. Christodoulou, C., et al., Live-animal imaging of native haematopoietic stem and
progenitor cells. Nature, 2020. 578(7794): p. 278-283.
312. Zhang, J., et al., In situ mapping identifies distinct vascular niches for myelopoiesis. Nature,
2021. 590(7846): p. 457-462.
313. Lennartsson, J. and L. Rönnstrand, Stem Cell Factor Receptor/c-Kit: From Basic Science
to Clinical Implications. Physiological Reviews, 2012. 92(4): p. 1619-1649.
314. Thoren, L.A., et al., Kit Regulates Maintenance of Quiescent Hematopoietic Stem Cells.
The Journal of Immunology, 2008. 180(4): p. 2045-2053.
315. Francis, K., et al., Two new pseudopod morphologies displayed by the human
hematopoietic KG1a progenitor cell line and by primary human CD34(+) cells. Blood,
1998. 92: p. 3616-23.
316. Wagner, W., et al., Hematopoietic Progenitor Cells and Cellular Microenvironment:
Behavioral and Molecular Changes upon Interaction. Stem Cells, 2005. 23: p. 1180-1191.
317. Wagner, W., et al., Adhesion of hematopoietic progenitor cells to human mesenchymal
stem cells as a model for cell-cell interaction. Experimental Hematology, 2007. 35: p. 314-
325.
160
318. Sims, T.N., et al., Opposing effects of PKCtheta and WASp on symmetry breaking and
relocation of the immunological synapse. Cell, 2007. 129(4): p. 773-85.
319. Hind, L.E., W.J.B. Vincent, and A. Huttenlocher, Leading from the Back: The Role of the
Uropod in Neutrophil Polarization and Migration. Developmental cell, 2016. 38(2): p.
161-169.
320. Florian, Maria C., et al., Cdc42 Activity Regulates Hematopoietic Stem Cell Aging and
Rejuvenation. Cell Stem Cell, 2012. 10(5): p. 520-530.
321. Zhou, H., et al., Non-invasive Optical Biomarkers Distinguish and Track the Metabolic
Status of Single Hematopoietic Stem Cells. iScience, 2020. 23(2).
322. Xu, J., et al., Divergent Signals and Cytoskeletal Assemblies Regulate Self-Organizing
Polarity in Neutrophils. Cell, 2003. 114(2): p. 201-214.
323. Sánchez-Madrid, F. and J.M. Serrador, Bringing up the rear: defining the roles of the
uropod. Nature Reviews Molecular Cell Biology, 2009. 10(5): p. 353-359.
324. Husson, J., et al., Force Generation upon T Cell Receptor Engagement. PLOS ONE, 2011.
6(5): p. e19680.
325. Hyun, Y.M., et al., Uropod elongation is a common final step in leukocyte extravasation
through inflamed vessels. J Exp Med, 2012. 209(7): p. 1349-62.
326. Carrasco, Y.R. and F.D. Batista, B-cell activation by membrane-bound antigens is
facilitated by the interaction of VLA-4 with VCAM-1. The EMBO Journal, 2006. 25(4): p.
889-899.
327. Scott, L.M., G.V. Priestley, and T. Papayannopoulou, Deletion of α 4 Integrins from Adult
Hematopoietic Cells Reveals Roles in Homeostasis , Regeneration , and Homing. 2003. 23:
p. 9349-9360.
161
328. Buitenhuis, M., The role of PI3K/protein kinase B (PKB/c-akt) in migration and homing
of hematopoietic stem and progenitor cells. Curr Opin Hematol, 2011. 18(4): p. 226-30.
329. Yamazaki, S., et al., Cytokine signals modulated via lipid rafts mimic niche signals and
induce hibernation in hematopoietic stem cells. 2006. 25: p. 3515-3523.
330. Hu, H., et al., Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of
Aldolase from the Actin Cytoskeleton. Cell, 2016. 164(3): p. 433-46.
331. Takubo, K., et al., Regulation of glycolysis by Pdk functions as a metabolic checkpoint for
cell cycle quiescence in hematopoietic stem cells. Cell Stem Cell, 2013. 12(1): p. 49-61.
332. Gritsman, K., et al., Hematopoiesis and RAS-driven myeloid leukemia differentially require
PI3K isoform p110α. The Journal of clinical investigation, 2014. 124(4): p. 1794-1809.
333. Brown, J.R., The PI3K pathway: clinical inhibition in chronic lymphocytic leukemia.
Semin Oncol, 2016. 43(2): p. 260-4.
334. Kalaitzidis, D., et al., mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-
evoked leukemogenesis. Cell Stem Cell, 2012. 11(3): p. 429-39.
335. Metcalf, D., et al., Multipotential hematopoietic blast colony-forming cells exhibit delays
in self-generation and lineage commitment. Proceedings of the National Academy of
Sciences, 2010. 107(37): p. 16257-16261.
336. Cheshier, S.H., et al., In vivo proliferation and cell cycle kinetics of long-term self-
renewing hematopoietic stem cells. Proceedings of the National Academy of Sciences,
1999. 96(6): p. 3120-3125.
337. Ikuta, K. and I.L. Weissman, Evidence that hematopoietic stem cells express mouse c-kit
but do not depend on steel factor for their generation. Proceedings of the National
Academy of Sciences, 1992. 89(4): p. 1502-1506.
162
338. Kato, K. and A. Radbruch, Isolation and characterization of CD34+ hematopoietic stem
cells from human peripheral blood by high ‐gradient magnetic cell sorting. Cytometry: The
Journal of the International Society for Analytical Cytology, 1993. 14(4): p. 384-392.
339. Morcos, M.N.F., et al., SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem
and Progenitor Cells. Stem Cell Reports, 2017. 8(6): p. 1472-1478.
340. Oguro, H., L. Ding, and S.J. Morrison, SLAM family markers resolve functionally distinct
subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell,
2013. 13: p. 102-116.
341. Chen, J.Y., et al., Hoxb5 marks long-term haematopoietic stem cells and reveals a
homogenous perivascular niche. Nature, 2016. 530: p. 223-227.
342. Golebiewska, A., et al., Critical appraisal of the side population assay in stem cell and
cancer stem cell research. Cell stem cell, 2011. 8(2): p. 136-147.
343. Margaret A. Goodell, et al., Isolation and Functional Properties of Murine Hematopoietic
Stem Cells that are Replicating In Vivo. J. Exp. Med., 1996. 183: p. 1797-1806.
344. van Buul, J.D. and P.L. Hordijk, Signaling in leukocyte transendothelial migration.
Arterioscler Thromb Vasc Biol, 2004. 24(5): p. 824-33.
345. Kawabata, K., et al., A cell-autonomous requirement for CXCR4 in long-term lymphoid
and myeloid reconstitution. Proceedings of the National Academy of Sciences, 1999. 96:
p. 5663–5667.
346. Ma, Q., D. Jones, and T.A. Springer, The Chemokine Receptor CXCR4 Is Required for the
Retention of B Lineage and Granulocytic Precursors within the Bone Marrow
Microenvironment. Immunity, 1999. 10: p. 463–471.
163
347. Foudi, A., et al., Reduced retention of radioprotective hematopoietic cells within the bone
marrow microenvironment in CXCR4-/- chimeric mice. Blood, 2006. 107(6): p. 2243-51.
348. Hao, J., et al., Membrane-bound SCF and VCAM-1 synergistically regulate the
morphology of hematopoietic stem cells. J Cell Biol, 2021. 220(10).
349. Zaro, B.W., et al., Proteomic analysis of young and old mouse hematopoietic stem cells
and their progenitors reveals post-transcriptional regulation in stem cells. Elife, 2020. 9.
350. Fleming, W.H., et al., Functional heterogeneity is associated with the cell cycle status of
murine hematopoietic stem cells. The Journal of cell biology, 1993. 122(4): p. 897-902.
351. Passegue, E., et al., Global analysis of proliferation and cell cycle gene expression in the
regulation of hematopoietic stem and progenitor cell fates. J Exp Med, 2005. 202(11): p.
1599-611.
352. Wilson, A., E. Laurenti, and A. Trumpp, Balancing dormant and self-renewing
hematopoietic stem cells. Curr Opin Genet Dev, 2009. 19(5): p. 461-8.
353. Sarma, N.J., A. Takeda, and N.R. Yaseen, Colony forming cell (CFC) assay for human
hematopoietic cells. JoVE (Journal of Visualized Experiments), 2010(46): p. e2195.
354. Liu, L., et al., Homing and long-term engraftment of long- and short-term renewal
hematopoietic stem cells. PLoS One, 2012. 7(2): p. e31300.
355. Fu, X., et al., Mesenchymal Stem Cell Migration and Tissue Repair. Cells, 2019. 8(8).
356. Cornwell, K.G., B.R. Downing, and G.D. Pins, Characterizing fibroblast migration on
discrete collagen threads for applications in tissue regeneration. J Biomed Mater Res A,
2004. 71(1): p. 55-62.
164
357. Simula, L., et al., Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T
Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming. Cell Rep,
2018. 25(11): p. 3059-3073 e10.
358. Rodero, M.P., et al., Immune surveillance of the lung by migrating tissue monocytes. Elife,
2015. 4: p. e07847.
359. Jones, D.H., et al., Regulation of cancer cell migration and bone metastasis by RANKL.
Nature, 2006. 440(7084): p. 692-6.
360. Zhao, J., et al., Mitochondrial dynamics regulates migration and invasion of breast cancer
cells. Oncogene, 2013. 32(40): p. 4814-24.
361. Sribenja, S., et al., Roles and mechanisms of beta-thymosins in cell migration and cancer
metastasis: an update. Cancer Invest, 2013. 31(2): p. 103-10.
362. Hunter, M.C., A. Teijeira, and C. Halin, T Cell Trafficking through Lymphatic Vessels.
Front Immunol, 2016. 7: p. 613.
363. Krummel, M.F., F. Bartumeus, and A. Gerard, T cell migration, search strategies and
mechanisms. Nat Rev Immunol, 2016. 16(3): p. 193-201.
364. Mierke, C.T., Role of the endothelium during tumor cell metastasis: is the endothelium a
barrier or a promoter for cell invasion and metastasis? J Biophys, 2008. 2008: p. 183516.
365. Orr, F., et al., Interactions between cancer cells and the endothelium in metastasis. The
Journal of pathology, 2000. 190(3): p. 310-29.
366. Kao, W.-T., et al., Investigation of MMP-2 and-9 in a highly invasive A431 tumor cell sub-
line selected from a Boyden chamber assay. Anticancer research, 2008. 28(4B): p. 2109-
2120.
165
367. Brown, N.S., & Bicknell, R., Cell migration and the boyden chamber. In Metastasis
Research Protocols. Humana Press, Totowa, NJ., 2001: p. 47-54.
368. Lomakina, E.B.a.W., R.E., Micromechanical tests of adhesion dynamics between
neutrophils and immobilized ICAM-1. Biophysical journal, 2004. 86(2): p. 1223-1233.
369. Feigelson, S.W., et al., Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1
is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open
headpiece high-affinity state. J Immunol, 2010. 185(12): p. 7394-404.
370. Jacobson, K., P. Liu, and B.C. Lagerholm, The Lateral Organization and Mobility of
Plasma Membrane Components. Cell, 2019. 177(4): p. 806-819.
371. Ostrowski, M.A., et al., Microvascular endothelial cells migrate upstream and align
against the shear stress field created by impinging flow. Biophys J, 2014. 106(2): p. 366-
74.
372. Bourget, J.M., et al., Patterning of Endothelial Cells and Mesenchymal Stem Cells by
Laser-Assisted Bioprinting to Study Cell Migration. Biomed Res Int, 2016. 2016: p.
3569843.
373. Wong, B.S., et al., A microfluidic cell-migration assay for the prediction of progression-
free survival and recurrence time of patients with glioblastoma. Nat Biomed Eng, 2020.
374. Pavesi, A., et al., A 3D microfluidic model for preclinical evaluation of TCR-engineered T
cells against solid tumors. JCI Insight, 2017. 2.
375. Carrasco, Y.R. and F.D. Batista, B-cell activation by membrane-bound antigens is
facilitated by the interaction of VLA-4 with VCAM-1. EMBO J, 2006. 25(4): p. 889-99.
376. Torres, A.J., et al., Functional single-cell analysis of T-cell activation by supported lipid
bilayer-tethered ligands on arrays of nanowells. 2014. 13: p. 90-99.
166
377. Yen, D.P., Y. Ando, and K. Shen, A cost-effective micromilling platform for rapid
prototyping of microdevices. Technology (Singap World Sci), 2016. 4(4): p. 234-239.
378. Truong, D., et al., Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma
Microenvironment. Sci Rep, 2016. 6: p. 34094.
379. Haessler, U., et al., Migration dynamics of breast cancer cells in a tunable 3D interstitial
flow chamber. Integr Biol (Camb), 2012. 4(4): p. 401-9.
380. Polacheck, W.J., et al., Mechanotransduction of fluid stresses governs 3D cell migration.
Proc Natl Acad Sci U S A, 2014. 111(7): p. 2447-52.
381. Vickerman, V. and R.D. Kamm, Mechanism of a flow-gated angiogenesis switch: early
signaling events at cell-matrix and cell-cell junctions. Integr Biol (Camb), 2012. 4(8): p.
863-74.
382. Tung, C.K., et al., A contact line pinning based microfluidic platform for modelling
physiological flows. Lab Chip, 2013. 13(19): p. 3876-85.
383. Bhattacharya, S., et al., Studies on surface wettability of poly(dimethyl) siloxane (PDMS)
and glass under oxygen-plasma treatment and correlation with bond strength. Journal of
Microelectromechanical Systems, 2005. 14(3): p. 590-597.
384. Mazia, D., G. Schatten, and W. Sale, Adhesion of cells to surfaces coated with polylysine.
Applications to electron microscopy. The Journal of cell biology, 1975. 66(1): p. 198-200.
385. DW., R., Agar and agarose: indispensable partners in biotechnology. Industrial &
engineering chemistry product research and development, 1984. 23(1): p. 17-21.
386. Davidson, S.M., et al., Remote ischaemic preconditioning involves signalling through the
SDF-1alpha/CXCR4 signalling axis. Basic Res Cardiol, 2013. 108(5): p. 377.
167
387. Ju, J., et al., Backward flow in a surface tension driven micropump. Journal of
Micromechanics and Microengineering, 2008. 18(8).
Abstract (if available)
Abstract
In vivo, adult stem cells reside in specific tissue locations known as their niches, which contain certain combinations of factors that maintain and regulate the stem cell functions. Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells. Yet little is known about the underlying mechanisms. Elucidating those factors and their underlying mechanisms is thus instrumental to stem cell biology and regenerative medicine. In this dissertation, we used a tethered supported lipid bilayer (SLB) model to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells, and investigated the effects of membrane-bound factors on HSC morphology and adhesion, using mouse cells as a model. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with vascular cell adhesion molecule 1 (VCAM-1) on the bilayer, which drastically enhances HSC adhesion, instead of migration, as assessed in a microfluidic model. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF-VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC quiescence, and minimizes its loss induced by soluble SCF. As a proof-of-concept, we further demonstrated the feasibility of applying the observed synergy to HSC enrichment from a pool of progenitors. Our work thus established a novel mechanism of mSCF and VCAM-1 in synergistically regulating HSC behaviors (protrusion, adhesion, proliferation) in the bone marrow niche, which can potentially be translated to human HSCs and benefit basic/clinical research and applications.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Hao, Jia
(author)
Core Title
Membrane-bound regulation of hematopoietic stem cells
School
Viterbi School of Engineering
Degree
Doctor of Philosophy
Degree Program
Biomedical Engineering
Degree Conferral Date
2022-08
Publication Date
07/23/2024
Defense Date
06/09/2022
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
hematopoietic stem cells,membrane-bound factors,OAI-PMH Harvest,stem cell factor,stem cell morphology,stem cell niche,VCAM-1
Format
application/pdf
(imt)
Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Shen, Keyue (
committee chair
), Finley, Stacey (
committee member
), Kim, Yong-Mi (
committee member
), Lu, Rong (
committee member
), McCain, Megan (
committee member
)
Creator Email
jh2499@cornell.edu,jiahao@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC111375292
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UC111375292
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etd-HaoJia-10947
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Hao, Jia
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texts
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20220728-usctheses-batch-962
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
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Tags
hematopoietic stem cells
membrane-bound factors
stem cell factor
stem cell morphology
stem cell niche
VCAM-1