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Regional localization and regulation of hematopoietic stem cells in the bone marrow stem cell niche
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Regional localization and regulation of hematopoietic stem cells in the bone marrow stem cell niche
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Content
REGIONAL LOCALIZATION AND REGULATION OF HEMATOPOIETIC STEM
CELLS IN THE BONE MARROW STEM CELL NICHE
by
Narges Rashidi
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment for the Degree
DOCTOR OF PHILOSOPHY
(SYSTEMS BIOLOGY AND DISEASE)
December 2011
Copyright 2011 Narges Rashidi
Dedication
To my mother …
For all her love and support. I wish she were here for yet another beautiful memory.
Acknowledgements
I sincerely would like to thank my advisor, Dr. Gregor B. Adams, PhD, for an amazing
five-year journey. As I have said many times, I have been the luckiest graduate student
there could ever be, and it is all because of his guidance and support. Gregor has been the
best mentor a student can ever have. He has taught me how to think independently and
critically. He not only taught me how to conduct science, but more importantly, how to
present science. It was because of his constant insistence on me giving talks, even on
crutches (!), that I am the confident speaker that I am today. I still have a long way to go
to be anywhere close to my own role model, Gregor. Not only has he been the best
“Boss”, Gregor has been a valuable friend for me helping and supporting me at every step
of my eventful life in the past few years. Even in the darkest days, it would only take a
pep talk from him to make me believe in myself and keep me going.
I am also grateful to the members of my committee: Dr. Baruch Frenkel, DMD, PhD, and
Dr. Krzysztof Kobielak, MD, PhD, for all their thoughtful comments and suggestions. I
realize it takes a lot of time and effort to be on a graduate student’s committee and I am
grateful for that. I especially want to thank Dr. Frenkel for his valuable help with the
osteoclast project and my paper revisions. It meant a lot to have such an expert by my
side helping me with my project. Also, I want to thank Dr. Frenkel’s former post-doc, Dr.
Yankel Gabet, PhD, who offered his expertise with macrophage isolation and
osteoclastogenesis.
I want to thank the program director of Systems Biology and Disease (SBD), Dr. Alicia
McDonough, PhD. Alicia has developed a scientifically rich program based on support
and guidance. It is amazing how involved she is with everybody’s projects and how
sincerely she shares all her resources with the students. I don’t know how she keeps track
of everyone’s progress in the program, while running a successful lab and having a
wonderful family at the same time, but she does it all. Alicia is a true inspiration and is a
model of the graceful, compassionate woman that I strive to be when I grow up!
Also, I would like to thank all the SBD administration staff: Marisela Zuniga, Raquel
Gallardo and especially Dawn Burke for being the great help that they were in these
years.
Of course, none of this work would have been possible without the help of the people in
the Adams lab. First and foremost, I want to thank Ben Lam, my fellow graduate student.
Ben has been a wonderful friend for the past four years and will be for the rest of my life.
He has helped me a lot in the lab, especially with weekend sorting appointments! But
more importantly, he was my “wingman” when looking for post-doc positions at the
Keystone Symposium, and the most influential person when deciding to take the job in
London. I just cannot wait to have him move out there too! I also want to thank Dilani
Rosa for being the “Queen of Histology” that she was. Sitting at the microtome, cutting
sections, and then getting all the histochemistry stainings to work is an exhausting job,
and she did it impeccably. And of course, I am very thankful for all her hard work
keeping the lab up and running as our lab manager. I would especially like to thank Lora
Barsky for teaching me all that I know about flow cytometry and cell sorting. More than
half of the experiments in this dissertation involve flow cytometry, none of which would
have been possible without Lora’s help. I would like to thank Dr. Xiaoying Zhou MD,
PhD, our post-doc, for teaching me all the nuts and bolts of gene expression assays and
helping me with lots of experiments when I was away for my interviews. Also, I should
thank Xiaoying for her timely and insightful questions, which always compelled me to
look further and learn more about my projects. I want to especially express my gratitude
to Tassja Spindler, our fantastic CIRM Bridges intern, for helping me out when I needed
it the most; with many of the last minute experiments for my paper submission and
revision. Tassja is extremely smart and reliable and a delight to work with. And of
course, she is the go-to person if you want to go out, have fun, or find an interesting place
to eat! Thanks for all the tips and all the memories. Also, I want to thank Sapna Shah, the
recent addition to the Adams lab and someone who became a very dear friend for me in
such short time. I am so lucky to have such a caring person in my life and am very sad
that I did not get the chance to spend more time with her. Thanks Sapna, for all the
inspiring and encouraging conversations. I also want to acknowledge and thank the
“future doctors” of the lab. Alan Tseng, a very dear friend, for all his help around the lab
(especially with lots and lots of injections), and more importantly for all the ice-cream-
therapy sessions. And of course, many thanks go to Tigue Tozer, for all his help with the
imaging of my sections, and his contributions to the calvaria project. How could he be the
biggest pain yet the most fun person to work next to, I’ll never know. I want to
acknowledge Sali Liu, our great technician with the cutest British accent, for starting up
the calvaria project. Finally, I would like to thank everybody in the Kobielak lab for
making these years filled with lots of memories at the birthdays and retreats and happy
hours. Being part of this lab and this department had made life just so much more
exciting and fun.
I am especially grateful to my collaborators at the California Institute of Technology for
all their help with the Imaging chapter. First, I want to thank Dr. Periklis Pantazis, PhD,
who developed the imaging modality, for all the valuable insights and discussions about
this project. I especially want to thank William P. Dempsey. Bill taught me lots of the
technical stuff I know about imaging, and helped greatly with all the work in the imaging
chapter. Setting up the collaboration, keeping people at different institutes in the loop,
and getting the experiments done at two facilities was not an easy job, and would have
been impossible without Bill. I would also like to thank Dr. Scott Fraser, PhD, for
making this collaboration possible.
During the past five years I have met many people who have influenced my life a lot and
have also contributed to this work with their support. I would like to use this opportunity
to reflect on some of those people. Marzieh Vali and Nima Shojaee have been the
kindest, most supportive couple ever. They were there to help me and get me through
some of the hardest days of my life. I want to thank them for all they have done for me,
for all the chats over many, many lunches and dinners and teas, and for all their generous
help during the writing of this thesis. I hope having had a 29-year-old daughter for 9
months has not changed their mind about having kids! I also want to thank Niki Bayat
and Amjad Askari for all their support while writing my thesis and on the day of my
defense.
I want to acknowledge a couple of my PIBBS classmates. First, my great friend, Anil
Sindhurakar, for many lunch breaks, coffee breaks, and dinners. Without his pep talks
and sanity checks, getting through this program would have been just so much harder.
Also, I want to thank Reza Kalhor for being an invaluable source of support. listening to
me venting about almost everything these past few years. I am so lucky to have him in
my life.
I want to thank my loving friends, Rana Amini, Mahnaz Sadoughi and Mona Radkani for
all their love throughout my life and especially in the past few months. It was their
constant support through long hours of phone calls that got me through many hurdles of
my life, one of which was writing up my dissertation. It is during times of hardship that
you realize what great friends you have, and I have definitely experienced that first-hand.
I also would like to write a couple of words about the guys in my life who have
influenced me in so many ways and have made me a better person. First, I want to thank
a very dear friend, Kaveh Shoorideh. Kaveh has been a valuable friend and a supportive
person in my life ever since I met him. He unconditionally loved me and was there for me
no matter what it took. It was a sleepless night of his hard work and his meticulousness
and patience that brought this dissertation into its current format. I am so thankful to him
for being so caring and so supportive and for all the great memories throughout these
years.
Also, I would like to thank Ali Vafai for being one of the most influential people in my
life. Ali always believed in me and it was his encouraging words that inspired me,
especially when I first started my PhD. Without his help, his friendship and his constant
support finishing up my PhD would have been much harder. I also owe a special note of
gratitude to the Vafai family who were a big part of my life in the past five years. I have
been so blessed and so honored to know them and I am forever in debt for all their love. I
also want to especially thank Dr. Hassan Vafai, PhD, for all the encouraging
conversations during the most stressful times of writing and defending my dissertation.
Finally and most importantly, I want to thank my family. My brothers, Alireza and Ala,
for making life fun! My lovely sister-in-law, Shiva, for being the sweetest person in the
world, and for all her encouragements from miles away. I should thank my grandparents
for all their love in these years. In the most desperate moments, a few words over the
phone from my grandpa, saying “Everything will be alright!” was enough to calm me
down. And of course my Uncle and Aunt, Hossein and Maryam Shoraka, for being so
caring and helpful during my studies. Visiting them and spending time with them in San
Francisco has always been my favorite relaxing method! And finally, I want to thank my
Mum and Dad for supporting me throughout all these years of growing and learning, and
for being so loving and generous and encouraging. Without them, none of my
achievements would have been possible.
Table of Contents
Dedication ii
Acknowledgements iii
List of Tables xii
List of Figures xiii
Abbreviations xvi
Abstract xxii
Chapter 1: Introduction 1 1.1 What is a Hematopoietic Stem Cell? 1 1.2 Hematopoietic Stem Cell: A historical overview 1 1.3 Identification of mouse HSCs 2 1.4 Isolation of HSCs using cell surface markers 6 1.5 HSC Journey During Development 14 1.6 Stem Cell Niche 18 1.7 Intrinsic Regulators of HSC function 46 1.8 Visualizing HSCs 51
Chapter 2: Materials and Methods 56 2.1 Subjects 56 2.2 Bone Harvesting 56 2.3 Cell Cultures 57 2.4 Colony Forming Unit Cell (CFU-C) Assay 57 2.5 Cobblestone Area Forming Cell (CAFC) Assay 58 2.6 Stromal TRACP & ALP Staining 60 2.7 Flow Cytometry 61 2.8 Competitive Repopulation Assay 66 2.9 Histological Analysis 68
Chapter 3: Alternative Methods of Activation of the Hematopoietic
Stem Cell Niche Reveal a Spatial Localization of
Primitive Cell Subsets in the Adult Bone Marrow 70 3.1 Abstract 70 3.2 Introduction 71 3.3 Materials and Methods 74
3.4 Results 81 3.5 Discussion 98
Chapter 4: Spatially Distinct Niche Regulation of a Stem Cell
Population 102
4.1 Abstract 102 4.2 Introduction 104 4.3 Materials and Methods 107 4.4 Results 116 4.5 Discussion 124
Chapter 5: A Novel Method to Image Hematopoietic Stem Cells in
the Adult Bone Marrow Stem Cell Niche 126 5.1 Abstract 126 5.2 Introduction 127 5.3 Materials and Methods 131 5.4 Results 137 5.5 Discussion 147
Chapter 6: Concluding Remarks and Future Directions 150
Bibliography 158
List of Tables
Table 2.1 Antibody List 63
Table 3.1 Summary of in vivo results 94
List of Figures
Figure 1.1 Overview of hematopoietic development indicating
intermediate cells in the hierarchy of hematopoietic
differentiation 13
Figure 1.2 Overview of migratory and circulatory routes connecting
fetal hematopoietic sites 18
Figure 1.3 Molecular cross-talk between HSC and the endosteal niche 36
Figure 2.1 Colony forming unit cell (CFU-C) assay 58
Figure 2.2 Cobblestone area forming cell (CAFC) assay 60
Figure 2.3 Flow cytometric analysis of hematopoietic cells using Lin,
c-Kit, Sca-1 and Flk2 markers 65
Figure 2.4 Flow cytometric analysis of hematopoietic cells using
SLAM markers 65
Figure 2.5 Competitive repopulation assay (CRA) 67
Figure 3.1 In vivo treatment plan 76
Figure 3.2 Histostaining
of osteoclasts and osteoblasts 80
Figure 3.3 TRACP and ALP activity of treated BM-MNC cultures 82
Figure 3.4 Quantification of TRACP
+
and ALP
+
Cells in Treated BM-
NC Cultures 82
Figure 3.5 Effects of in vitro treatments on growth and functional
potential of BM-MNCs 83
Figure 3.6 Splenocyte-derived osteoclasts enhance hematopoietic
support of BM-MNC cultures 84
Figure 3.7 Effects of in vivo RANK-L and PTH treatments on bone
structure 85
Figure 3.8 Effects of in vivo RANK-L and PTH treatments on BM
stromal cell composition in vivo 87
Figure 3.9 Effects of in vivo RANK-L and PTH treatments on
hematopoietic stem and progenitor cell populations 89
Figure 3.10 Effects of in vivo RANK-L and PTH treatments on
committed progenitor cell populations 90
Figure 3.11 Effects of in vivo RANK-L and PTH treatments on
primitive hematopoietic cell function 91
Figure 3.12 Multi-lineage engraftment of BM-MNCs isolated from
RANK-L and PTH treated mice 92
Figure 3.13 Effects of in vivo RANK-L and PTH treatments on
primitive hematopoietic cell function 93
Figure 3.14 Separation of metaphysis and diaphysis sections of the long
bones 95
Figure 3.15 Effects of in vivo RANK-L and PTH treatments on
hematopoietic cell population in distinct bone regions 97
Figure 3.16 Hypothesized model of localization of the adult HSC in the
endosteal niche 100
Figure 4.1 Cell cycle analysis of hematopoietic cells 115
Figure 4.2 Comparison of hematopoietic stem and progenitor cell
population frequencies between cells of different BM
origin 117
Figure 4.3 Comparison of primitive hematopoietic cell function
between cells of different BM origin 118
Figure 4.4 Comparison of cell cycle profile of BM-MNCs of different
BM origin 118
Figure 4.5 Comparison of gene expression profile of BM-MNCs of
different BM origin 119
Figure 4.6 Comparison of gene expression profile of stromal cells of
different BM origin 120
Figure 4.7 N-cadherin immunohistostaining of bone sections and BM
stromal cultures of different BM origin 121
Figure 4.8 ALP and TRACP activity of BM stromal cultures of
different BM origin 122
Figure 4.9 Comparison of in vitro functional potential of BM stromal
cultures of different BM origin 123
Figure 5.1 Spatiotemporal resolution of imaging modalities used for
HSC visualizing 128
Figure 5.2 Two-photon excited fluorescence versus SHG 129
Figure 5.3 An overview of SHG-nanoprobe labeling 132
Figure 5.4 Ficoll separation of BM-MNCs 132
Figure 5.5 SHG nanoprobe cell labeling 138
Figure 5.6 Effects of BaTiO
3
labeling on growth and functional
potential of hematopoietic cells 138
Figure 5.7 Effects of BaTiO
3
labeling on cellular death 139
Figure 5.8 Bone section imaging and image processing 142
Figure 5.9 Overlaying wide-field histological data with SHG tiled
scan for topological characterization of niche-dependent
HSC localization 143
Figure 5.10 BABB bone clearing 145
Figure 5.11 Characterization of a long bone HSC niche in optical cross
section and optical transverse section 146
Figure 6.1 SHG microendoscopy within a fixed, whole calvarium 156
Abbreviations
7-AAD 7-amino-actinomycin D
-MEM minimum essential medium alpha
AGM aorta-gonadal-mesonephrons
ALP alkaline phosphatase
ANG angiopoietin 1
APC allophycocyanin
BaTiO
3
barium titanate
BABB benzyl alcohol: benzyl benzoate
BFU-E burst-forming unit-erythroid
BM bone marrow
BM-MNC bone marrow mononuclear cells
BMP bone morphogenic protein
BMPRIA bone morphogenic protein receptor IA
BrdU 5-bromo-2’-deoxyuridne
CAFC cobblestone area forming cell
CAR CXCL12-abundant reticular
CD cluster of differentiation
CDK cyclin-dependent kinase
CDKIs cyclin-dependent kinase inhibitors
cDNA complementary DNA
CFU-C colony forming unit cell
CFU-GM granulocyte-macrophage colony-forming units
CFU-S colony-forming units spleen
cKit cellular Kit
CP cysteine proteinases
CRA competitive repopulation assay
CRD cysteine-rich domain
CRU competitive repopulation unit
CXCL12 chemokine (C-X-C motif) ligand 12
CXCR4 chemokine (C-X-C motif) receptor 4
Ddh dopamine hydroxylase
Dkk1 dickkopf1
EC endothelial cell
ECM extracellular matrix
EDTA Ethylenediaminetetracetic acid
eHPCs early hematopoietic progenitor cells
EPCR endothelial protein C receptor
FACS fluorescence activated cell sorting
FBS fetal bovine serum
FL fetal liver
Flk-2 fetal liver kinase-2
Flt3 fms-like tyrosine kinase-3
FSC forward scatter
G-CSF granulocyte colony-stimulating factor
GCV ganciclovir
Gfi1 growth factor independent 1
GFP green fluorescence protein
Gy gray
H2B Histone 2B
H&E hematoxylin and eosin
HOX homeobox
HSC hematopoietic stem cell
HSPC hematopoietic stem and progenitor cell
HUBEC human brain endothelial cells
IACUC institutional animal care and use committee
IHC immunohistochemistry
IL-6 interleukin-6
Jag-1 jagged-1
LEC liver endothelial cell
LEF-1/TCF lymphoid enhancer-binding factor-1/T-cell factor
Lin lineage
LRC label retaining cell
LSK Lin
-
Sca-1
+
c-Kit
+
LSM lymphocyte separation medium
LT-HSC long-term hematopoietic stem cell
LTC-IC long-term culture-initiating cells
mAbs monoclonal antibodies
Mac-1 macrophage-1
MAPK mitogen-activated protein kinase
Mdr multi-drug resistance protein
MPL myeloproliferative leukemia virus oncogene
MPP multipotent progenitor cells
MPS myeloproliferative syndromes
MRI magnetic resonance imaging
MSC mesenchymal stem cell
NE norepinephrine
NICD notch intracellular domain
OPN osteopontin
Par-1 protease-activated receptor 1
PBS phosphate buffered saline
PHSC pluripotent hematopoietic stem cell
PIP3 phosphatidylinositol (3,4,5)-triphosphate
PLL poly-L-lysine
PPR parathyroid hormone/parathyroid hormone-related peptide receptor
PPAR peroxisome proliferator-activated receptor gamma
PtdIns phosphatidylinositol
Pten phosphatase and tensin homologue
PTH parathyroid hormone
PTP protein tyrosin phosphatase
RANK-L receptor activator of nuclear factor- B ligand
RAR retinoic acid receptor
RM red marrow
SCF stem cell factor
SDF-1 stromal cell-derived factor 1
SHG second harmonic generation
sKitL soluble Kit ligand
SLAM signaling lymphocyte activation molecule
SNO spindle-shaped N-cadherin
+
osteoblasts
SNS sympathetic nervous system
Sr strontium
SP side population
SSC side scatter
ST-HSC short-term hematopoietic stem cell
TBLA trabecular-bone-like area
THPO thrombopoietin
TK thymidine kinase
tm-SCF trans-membrane stem cell factor
TNF tumor necrosis factor
TNR transgenic notch reporter
TRACP tartarate resistant acid phosphatase
VEGF vascular endothelial growth factor
VLA very late antigen
WGA wheat germ agglutinin
WT wild-type
Abstract
Adult HSCs reside in the BM in a microenvironment known as the stem cell niche. Many
studies have identified key components of the niche required to maintain the HSCs in
their primitive state. However, other cellular components and the exact location of the
HSC niche are yet to be clearly identified. My studies have demonstrated that the
coordinated interaction between the osteoblasts and the osteoclasts is required for
maintaining HSCs in their BM niche. This also highlighted a structural organization of
the localization of primitive hematopoietic cell subsets in distinct regions of the BM. We
further examined this spatial regulation of HSCs in two anatomically distinct sites, flat
bone and long bones. Our data shows that while HSCs isolated from these two different
bones have identical potential, their maintenance may be regulated by different
mechanisms. These data further demonstrate that although flat bones are desirable sites
for visualizing HSC in their niche, the data derived from imaging HSCs in these bones
may not necessarily correlate with functional analysis data that is mainly derived from
cells of the long bones. Therefore, we developed an imaging modality based on SHG
nanoporbes that can potentially capture the exact localization of HSCs in their niche and
their dynamic nature at single cell resolution within all bones. Understanding all of the
interactions between the stem cells and their niche may yield both practical methods for
manipulating stem cells to achieve therapeutic outcomes and also define a model for the
impact of the microenvironment on stem cell biology.
Chapter 1: Introduction
1.1 What is a Hematopoietic Stem Cell?
The word “Hematopoiesis” comes from ancient Greek word “
µ ” meaning blood and
“ ” meaning to make. The dynamic process of hematopoiesis guarantees
replenishment of the relatively short-lived blood cells. On average 7 × 10
9
blood cells per
kg body weight is produced every day (Purton & Scadden, 2008). The production of
these cells is dependent on the presence of Hematopoietic Stem Cells (HSCs). HSCs are
defined as cells that are able to self-renew and also differentiate into an assortment of
blood cells to preserve a stable source of hematopoiesis throughout life.
1.2 Hematopoietic Stem Cell: A historical overview
The hunt for stem cells began from studies aiming to find a suitable cure for exposure to
lethal doses of irradiation. Experimental evidence, first shown by Jacobson and
colleagues, demonstrated that if spleens of mice exposed to total body x-radiation were
lead-shielded, the survival rate increased from 0.8% to 76% (Jacobson et al., Science
1951). The same results were also observed when the entire head or one hind leg was
lead-shielded (Jacobson et al., 1951). In addition, when isologous marrow isolated from
inbred mice were injected to the mice after x-ray exposure, the survival rate of the mice
increased significantly. (Lorenz et al., 1951; Jacobson et al., Science 1951; Rosenthal et
al., 1960). Multiple reports then conclusively demonstrated that the regenerated marrow
was derived from the injected cells and was responsible for recipients’ survival (Nowell
et al., 1956; Ford et al., 1956).
In 1961, Till and McCulloch quantified the radiation sensitivity of bone marrow (BM)
cells in transplantation settings. Injection of BM cells into isologous irradiated recipient
mice led to the formation of colonies of proliferating myeloid and erythroid cells in the
spleens. These cells, called colony-forming units spleen, or CFU-S, directly correlated
with the number of BM cells injected (Till & McCulloch, 1961). They subsequently
showed that if they pre-marked the donor cells by irradiating the donor mice with sub-
lethal doses of irradiation to introduce unique chromosome breaks, all the cells within a
single spleen colony, which contained different types of blood cells, were derived from a
single cell (Becker et al., 1963). In addition, they were able to show that when using the
cells from a single colony for secondary transplantation, the same unique chromosomal
aberration was observed, thus indicating that these colonies had been regenerated from
the same single cell that had originated the first colony (Simminovitch et al., 1963).
These CFU-S were then proposed to be the pluripotent HSCs that could self-renew and
make all blood cell types.
1.3 Identification of mouse HSCs
Multiple studies demonstrated that spleen colonies originating from a single cell varied
greatly in the content of the differentiated cells (Fowler et al., 1967; Lewis et al., 1964;
Curry & Trentin, 1967). More importantly the number of CFU-S originating from a
single animal were quite heterogeneous as well (Simminovitch et al., 1963). These
observations could have been explained either by varying degrees of self-renewal versus
differentiation or by differences between the influence of the local environment on the
spleen colonies (Curry & Trentin, 1967). However, both of these models rely on the
assumption that the starting CFU-S was a homogenous population of cells with identical
differentiation and self-renewal potential. In 1964, Simminovitch et al. showed that not
all of the spleen colonies were able to give rise to sufficient CFU-S to initiate extended
serial passaging, providing indirect evidence for variations in CFU-S potential
(Simminovitch et al., 1964). Based on such observations, Worton and colleagues
attempted to separate various cellular components of CFU-S by using velocity
sedimentation techniques to sub-fractionate populations of primitive hematopoietic cells
(Worton et al., 1969). They showed that CFU-S from the fraction with slowly
sedimenting cells have an increased capacity of self-renewal, concluding that “at least
part of the heterogeneity observed in the CFU content of individual spleen colonies arises
from the composition of the initial cell suspension, probably from intrinsic differences
between stem cells themselves”.
In 1977, Abramson et al. conducted a series of experiments that identified the
relationship between CFU-S and the lymphoid system. They obtained direct evidence of
the existence of a class of pluripotent stem cells which are progenitors of B- and T-
lymphocytes, as well as myeloid cells. Using the same radiation-induced chromosomal
marking strategy as Till and McCulloch, they marked individual primitive hematopoietic
cells which then repopulated the recipient’s hematopoietic system over the course of
several months. They then looked for the presence of the chromosome breaks in various
hematopoietic organs examining the cells’ proliferative capacity. The result of their study
showed that while there was a minority of cells with characteristics of true pluripotent
stem cells, there also existed restricted progenitor cells, which were only able to give rise
to myeloid cells, or a specific type of lymphocytes.
More evidence for the heterogeneity of the CFU-S came from studies of Jones et al. when
they looked at the nature of the cells responsible for serial transplantations (Jones et al.,
1989). A decline in CFU-S numbers was seen when male BM cells were serially
transplanted into female recipients, although the numbers of granulocyte-macrophage
colony-forming units (CFU-GM) in the BM were not affected. However, these CFU-S
numbers still remained at levels that should have resulted in engraftment. A 0% survival
rate upon secondary transplantation clearly suggested that CFU-S were not the cells
responsible for long-term hematopoietic repopulation. Serial transplantation survival
rates are dependant on the size of the graft (how many cells were injected) and the time
interval between transfers (how soon the cells were re-transplanted). The investigators
thus suggested that there were two phases associated with engraftment that could be
assigned to different cellular populations in the transplanted cells. In the first phase,
committed progenitor cells were proposed to be responsible for the early, transient
repopulation that could have been maintained through serial transplantations. While in
the second phase, the pluripotent stem cells were accountable for the sustained long-term
phase that was eventually lost during serial transplantations (Jones et al., 1989).
To further characterize the developmental potential of HSCs, Lemischka et al. took an
alternative approach by introducing new genes into HSCs via transmissible retrovirus
vectors (Lemischka et al., 1986). Cells were then transplanted into irradiated mice and
the fate of these cells was tracked in the recipient’s hematopoietic system. Donor mice
were treated with 5-fluorouracil (5-FU), a drug that eliminates the committed progenitor
cells and selectively spare the more primitive cells, in order to obtain a higher proportion
of replicating cells that is enriched in primitive stem cells, (Van Zant, 1984). Their study
revealed several classes of stem cells from cells whose progeny were capable of
contributing to multi-lineage repopulation to cells whose progeny were specifically
committed to myeloid or lymphoid lineages. Results of secondary transplantation showed
that various clones of stem cells are sequentially being activated during hematopoiesis,
and point out the possibility of the presence of a mechanism for temporal control of HSC
use upon being transferred into irradiated recipients (Lemischka et al., 1986).
However, this idea of a strict clonal succession model of hematopoiesis was later
challenged when Keller and Snodgrass showed that the progeny of transplanted cells with
retroviral integration sites can function for a significant portion of the lifetime of a mouse
(15 months). These cells demonstrated multi-lineage reconstitution potential in the first
irradiated recipient for a minimum of 8 months, and continued to function for a further 7
months when transplanted into a second recipient. These data provided strong evidence
suggesting that the primitive stem cell population could actually clonally expand and
maintain hematopoiesis during animal’s lifelong (Keller & Snodgrass, 1990).
Together these studies confirmed the presence of a stem cell population in the
hematopoietic system with the capacity to generate progeny of all the blood cell lineages,
as well as the capacity to generate cells with a potential similar to their own. The next
evident step was then to isolate these cells for further therapeutic use.
1.4 Isolation of HSCs using cell surface markers
The first enrichment of CFU-S was achieved by Visser et al. using a three-step isolation
method (Visser et al., 1984). Using fluorescence activated cell sorting (FACS) and a
combination of wheat germ agglutinin conjugated with Fluorescein isothiocyanate
(WGA-FITC) and anti-H-2K labeling, WGA
dim
cells with high H-2K density were
isolated. All of the sorted, pluripotent HSC (PHSC) were either in the G
1
or G
o
phase of
the cell cycle. Electron microscopy revealed that the sorted cells consisted primarily of
two cell types, possibly representing G
1
or G
o
cells (Visser et al., 1984).
Bone marrow cells were then sorted based on their size and density using counterflow
centrifugal elutriation. They were able to separate BM cells into three sub-populations:
(1) the largest cells that were enriched for CFU-GM, but gave only transient, early
engraftment, (2) the intermediate fraction of cells enriched in CFU-S but depleted of
CFU-GM and (3) the fraction of lymphocyte-like cells which were able to give sustained
reconstitution of all hematopoietic cells. They concluded that the first two fractions were
classified as the committed progenitor cells while the latter was the distinguishable
source of true stem cells (Jones et al., 1990).
To further identify the properties of stem cells, a pure population of the pluripotent
hematopoietic cells was isolated using “differentiation” markers. These markers were
defined as antigens present on the cells in a particular differentiated population or
lineage. Once isolated, the multipotency of the cells was then assayed by various assays
developed to ensure production of various blood cell lineages by these cells (Whitlock &
Witte, 1982; Spangrude et al., 1988b; Till & McCulloch, 1961).
1.4.1 Lineage Selection
The idea of lineage selection first came from a study where it was shown that the clonal
precursors of the B lineage-engrafting cells lack the surface marker B220, present on the
pre-B and B cells [Coffman & Weissman (a,b), 1981]. It was reasoned that if the B-cell
markers were not present on the ealiest B-lineage progenitors, then markers of other
committed blood lineages such as T-cells (CD4 and CD8), granulocytes (Gr-1),
macrophage/monocytes (Mac-1) and erythroids (Ter119) were also unlikely to be present
on their progenitors. This finding was the origin of the lineage (Lin) antibodies that is
currently used for negative selection of HSCs.
1.4.2 Thy-1
Thy-1 or CD90 is a cell surface marker that was originally discovered to be present in
large amounts on thymocytes. Thy-1 was also shown to be present in reduced amounts on
some lymphoid cell populations. In 1978, it was demonstrated that Thy-1 antigen was
present on pluripotent stem cells in rat hematopoietic tissues at various developmental
stages, in adult BM, neonatal spleen, and fetal liver (Goldschneider et al., 1978). Also,
multipotential lympho-HSCs detected by a 12-day spleen colony assay were shown to
uniformly express Thy-1 even though in low levels. The expression level of this antigen
was further decreased and eventually lost as these cells differentiated into progenitor cells
with restricted developmental potentials (Berman & Basch, 1985). Using Thy-1 antibody,
the frequency of colony-forming cells was enriched 500-fold, but this population was still
heterogeneous requiring other antibodies to further isolate HSCs.
1.4.3 Sca-1
In 1988, Aihara and Klein produced a library of monoclonal antibodies against marrow
Thy1
+
cells made into hybridomas with T-cells (Aihara et al.,1986). One of the antibodies
in this library was shown to recognize BM progenitors of thymic lymphocytes as well as
several other hematolymphoid cells. The antigen recognized by this monoclonal
antibody, called Stem cell antigen-1 (Sca-1), was then used by Spangrude to further
subdivide the Lin
-
Thy-1
lo
cells into two populations; Sca-1
+
and Sca-1
-
(Spangrude et al.,
1988a). They then demonstrated that limiting numbers of Lin
-
Thy-1
lo
Sca-1
+
BM cells
were able to repopulate T-cell, B-cell and myeloid lineaged when injected into irradiated
mice. Also, when intravenously transplanted into lethally irradiated mice, the Lin
-
Thy-
1
lo
Sca-1
+
BM cells were capable of rescuing the mice. Based on their observations, these
cells were hypothesized to include all pluripotent HSCs, although further refinement was
still required to isolate pure population of stem cells (Spangrude et al., 1988b).
1.4.4 c-Kit (CD117)
The W locus (dominant spotting [W] loci) encodes the ligand c-kit proto-oncogene, a
member of receptor tyrosine kinase family, while the Sl locus (Steel factor) encodes the
ligand for c-kit (named stem cell factor [SCF]). Using an anti-Kit monoclonal antibody, it
was shown that c-Kit receptor is expressed on the surface of HSCs. However, the c-Kit-
SCF interaction was not essential for the initiation of hematopoiesis and the self-renewal
of fetal HSCs, as evidenced by HSCs’ maintained functionality in Sl/Sl
d
mice that lack
functional SCF (Ikuta & Weissman, 1992). Combining c-Kit with the previously
discussed stem cell markers, it was shown that while Lin
-
Thy-1
lo
Sca-1
+
c-Kit
-
cells did not
have any progenitor activity, Lin
-
Thy-1
lo
Sca-1
+
c-Kit
+
cells were able to result in long-
term reconstitution in irradiated mice (Morrison & Weissman, 1994) and be most
responsive to a combination of hematopoietic factors such as SCF, IL-3 and interleukin-6
(IL-6) (Okada et al., 1992). Once the reconstituting potential of Lin
-
Thy-1
lo
Sca-1
+
c-Kit
+
cells was confirmed in a series of syngeneic and allogeneic transplants (Uchida &
Weissman, 1992; Uchida et al., 1994), these cells were indicated to be the only functional
long-term self-renewing cells in the marrow. This cell population was later subdivided
into long-term reconstituting cells (Lin
-
Thy-1
lo
Sca-1
+
c-Kit
+
Mac-1
-
CD4
-
[referred to as c-
Kit
+
Mac-1
-
CD4
-
]), which will in turn give rise to transiently reconstituting multipotent
progenitors (Lin
-
Thy-1
lo
Sca-1
+
Mac-1
lo
CD4
lo
[referred to as Mac-1
lo
CD4
lo
]) (Morrison et
al., 1997).
1.4.5 Flk-2 (Flt3, CD135)
Another member of receptor tyrosine kinase family that was shown to have
heterogeneous expression on the stem cell pool is fetal liver kinase-2 (Flk-2) also known
as fms-like tyrosine kinase-3 (Flt3) or CD135. Flk-2 is expressed in early stages of
hematopoiesis and its ligand FL has been suggested to be a potent stimulator of murine
and human growth (Lyman & Jacobsen, 1998). To examine the expression of Flk-2/Flt3
receptor tyrosine kinase on HSCs, the primitive Lin
-
Thy-1
lo
Sca-1
+
c-Kit
+
cells were
sorted. It was shown that while low numbers Flk-2
-
cells were able to give rise to long-
term multi-lineage reconstitution, Flk-2
+
cells were only able to reconstitute the irradiated
recipients for a short amount of time (Christensen & Weissman, 2001). Phenotypic and
functional analysis of these subpopulations revealed that loss of Thy
lo
and gain of Flk-2
expression marks the loss of self-renewal in HSC maturation but sustained lymphoid
restricted reconstitution potential (Christensen & Weissman, 2001; Adolfsson et al.,
2001). Therefore, the HSC population can be classified into long-term HSCs (LT-HSCs:
Thy
lo
Flk-2
-
), short-term HSCs (ST-HSC: Thy
lo
Flk-2
+
) and multipotent progenitors (Thy
-
Flk-2
+
) (Christensen & Weissman, 2001).
1.4.6 CD34
Murine CD34 is a hyperphosphorylated glycoprotein that is expressed on the cell surface
as a either full-length or truncated protein. The role of CD34 in HSCs was first
established in humans where hCD34 is a stage-specific antigen that marks stem and
progenitor cells and its expression is declined as the cells go down the differentiation
hierarchy (Bernson et al., 1988; Civin et al., 1984). Murine CD34, on the other hand, had
not been characterized until 1994, when Krause et al. showed that CD34
-
BM cells
include the primitive hematopoietic cells that are capable of forming CFU-S, CFU-
granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) in vitro.
These cells were also able to repopulate hematopoietic system both short-term and long-
term once transplanted into lethally irradiated mice confirming that the CD34
-
cells
contained both functional hematopoietic progenitor and stem cells (Krause et al., 1994).
1.4.7 SLAM Family
Signaling Lymphocyte Activation Molecule (SLAM) family is a group of 10–11 cell
surface receptors that are tandemly arrayed at a single locus on chromosome 1 (Engel et
al., 2003). This family includes members such as CD150, CD244 and CD48 and have
been identified as regulators of the proliferation and activation of lymphocytes (Howie et
al., 2002; Wang et al., 2004). In 2005, in an attempt to identify genes that are associated
with HSC identity, Kiel et al. compared the gene expression profiles of highly enriched
populations of HSCs defined as Lin
-
Thy-1
lo
Sca-1
+
c-Kit
+
Mac-1
-
CD4
-
and transiently
reconstituting multipotent progenitors (MPPs) defined as Lin
-
Thy-1
lo
Sca-1
+
Mac-
1
lo
CD4
lo
. The founding member of SLAM family, CD150, was one of the genes that was
expressed in much higher levels in HSCs compared to MPPs (Kiel et al., 2005b). This
prompted the expression assessment of other SLAM family members which led to the
discovery that based on the differential expression of cell surface receptors of the SLAM
family on functionally distinct cell populations, HSCs were highly purified as
CD150
+
CD244
CD48
cells while MPPs were CD244
+
CD150
CD48
and most
restricted progenitors were CD48
+
CD244
+
CD150
(Kiel et al., 2005b). Identification of
these markers led to identification of the key role of endothelial cells in the HSC
microenvironment in the BM. A schematic overview of hematopoietic development
indicating the surface markers used for the isolation of the stem and progenitor cells is
shown in figure 1.1.
1.4.8 Side Population (SP)
Hoechst 33342 is a fluorescent vital dye, which readily binds to DNA in live cells. The
quantity of Hoechst 33342 fluorescence therefore, can be used as an indicator of cell
cycle since it directly relates to DNA content. In 1996 it was shown that BM cells labeled
with Hoechst 33342 dye demonstrated a unique fluorescence pattern (Goodell et al.,
1996). When the dye fluorescence was observed simultaneously at two emission
wavelengths (red and blue) a small distinct side population (SP) of cells with low staining
pattern existed. This population happened to express markers of HSCs as they were Lin
-
Sca-1
+
cells. To verify the function of these cells, they were sorted and used in
competitive repopulation assays, where it was shown that the multi-lineage reconstituting
cells of mouse BM reside in the SP fraction of cells. It was noteworthy that formation of
SP profile was blocked by verapamil, indicating that the low staining pattern of the SP
cells was due to a multi-drug resistance protein (mdr) or mdr-like mediated efflux of the
dye from HSCs (Goodell et al., 1996). The true stem cell nature of the SP cells was later
confirmed by various studies that looked into the expression of established stem cell
markers by these cells (Goodell et al., 1997; Weksberg et al., 2008).
Figure 1.1 Overview of hematopoietic development indicating intermediate cells in the hierarchy of
hematopoietic differentiation. Surface markers used for isolation are indicated for the stem and
progenitor cells. HSC, hematopoietic stem cell; MPP, multi-potent progenitors; CMP, common myeloid
progenitor; CLP, common lymphoid progenitor; BLP, B-lymphocyte progenitor; ProT, T-cell
progenitor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythroid progenitor;
MkP, megakaryocyte progenitor; EP, erythroid progenitor
1.4.9 CD45
CD45 is a member of the mouse Ly-5 system which is defined based on expression of
members of protein tyrosin phosphatase (PTP) family. Various molecular isoforms of
these transmembrane glycoproteins are expressed by most of hematopoietic cells and
typify them according to their lineage or stage of differentiation from the HSCs (Saga et
al., 1988). Although not a specific marker for HSCs, CD45 is very important in the HSC
field. The major breakthrough in the analysis of HSCs in vivo came from studies where
Boyse and colleagues developed Ly5/CD45 (leukocyte common antigen) congenic
mouse strains on a C57/Bl6 background (Shen et al., 1986). Using these congenic mice
and the monoclonal antibodies (mAbs) developed for them, it was possible to transplant
cells from different strains into the host and distinguish donor cells from host cells.
1.5 HSC Journey During Development
In vertebrates, hematopoiesis occurs from different anatomical sites during development.
The use of multiple hematopoietic niches during development has been observed in many
species (Ciau-Utiz et al., 2000; Tavian & Péault, 2005; Traver & Zon, 2002) thus
allowing for production of differentiated blood cells that would be immediately used
during embryonic development while forming an undifferentiated HSC reservoir and
preserving that for future use. While various sites can have different inductive signals to
maintain HSCs, the compartmentalization of embryonic hematopoiesis also allows
formation of mature blood cells at one site while the “stem-ness” of the cells is preserved
at the other (Mikkola & Orkin, 2006).
Yolk Sac- In mammalians, a subset of embryonic ventral mesoderm cells are committed
to becoming blood cells upon gastrulation (McGarth & Palis, 2005). These cells,
allocated in the yolk sac, form blood islands and initiate primitive hematopoiesis between
embryonic day 7 (E7.0) and E8.25. Their main function is erythrocyte production for
tissue oxygenation. Even though there are studies suggesting that the E12.5 yolk sac
microenvironment is capable of supporting HSC development (Kumaravelu, 2002), the
lack of inductive differentiation signals to generate blood cell lineages force
hematopoietic cells to take home in a new niche.
1.5.1 Aorta-gonadal-mesonephrons (AGM) region
By E8.5, hematopoietic progenitor cells are found in the mouse AGM region of the
developing embryo. The HSCs found there are capable of reconstituting adult recipient
hematopoiesis by E10.5-11.0 (Jaffredo et al., 2005). Although the AGM region is a
source of HSCs, “further maturation process is required to confer on them the
engraftment and self-renewal abilities in the adult microenvironement” (Mikkola &
Orkin, 2006).
1.5.2 Placenta
The placenta is the organ responsible for feto-maternal exchange from midgestation and
production important pregnancy-related hormones and growth factors (Rossant & Cross,
2001). Definitive multi-lineage progenitors and HSCs appear in the placenta by E10.5-
11.0. The placental HSCs have the functional properties of adult HSCs; yet have a very
rapid expansion rate comparable to the cells from yolk sac or AGM (Gekas et al., 2005).
1.5.3 Fetal liver (FL)
Later during development, the FL will be invested by HSCs and remain the main site of
hematopoietic cell production until approximately the second trimester. There is thought
to be two major vascular circuits connecting to the FL. First, the vitelline vessels that
serves as the route for the myeloerythroid progenitors to arrive at the FL from the yolk
sac. Second, the umbilical vein that brings the first HSCs to the FL from the placenta and
AGM (Mikkola & Orkin, 2006). It has been shown that while HSCs in the adult BM
niche remain relatively quiescent, the FL HSCs are actively cycling resulting in their
reconstitution advantage compared to BM HSCs when transplanted into irradiated mice
(Harrison et al., 1997; Morrison et al., 1995; Rebel et al., 1996).
Although the nature of the FL is yet to be fully defined, a recent study investigated the
niche and the molecular mechanism of HSC maintenance in mouse FL using HSCs
expressing endothelial protein C receptor (EPCR) (Iwasaki et al., 2010). EPCR is a type I
transmembrane glycoprotein
expressed mainly in endothelial cells of larger blood vessels,
liver sinusoids, monocytes, leukocytes, and several tumor cells as well as HSCs and is
known for its anti-coagulant and cytoprotective functions. Here, it was shown that only
EPCR
+
Lin
-
Sca-1
+
c-Kit
+
(LSK) cells originating from FL but not EPCR
cells had HSC
characteristics of being able to exhibit long-term reconstitution in irradiated mice. In
order to exert its cytoprotective activity, EPCR requires protease-activated receptor 1
(Par-1) as a cofactor to interact with activated protein C (APC). The antiapoptotic effect
of APC on EPCR
+
HSCs and the expression of Par-1 mRNA in these cells suggested the
involvement of the cytoprotective APC/EPCR/Par-1 pathway in HSC maintenance.
Immunohistochemistry revealed that EPCR
+
cells were localized adjacent to the
sinusoidal network, where APC and extracellular matrix (ECM) are abundant, suggesting
that HSCs in FL were maintained in the APC- and ECM-rich perisinusoidal niche
(Iwasaki et al., 2010).
1.5.4 Bone Marrow (BM)
The functional HSCs start colonizing the BM at around E17.5 in the mouse. The exact
transition process from the FL to the BM still remains to be elucidated, however, the
early fetal BM (E12.5-15.5) microenvironment is unable to attract circulating HSCs and
the presence of osteoblasts and calcification of the bone is required for seeding of the BM
to be successful (Mikkola & Orkin, 2006). Following the cell migration which is
completed shortly after birth, the BM will be the major hematopoietic site and source of
HSCs throughout adulthood.
A schematic overview of the migratory and circulatory routes during fetal hematopoiesis
is shown in figure 1.2.
1.6 Stem Cell Niche
As mentioned previously, the defining characterizations of HSCs are their ability to
undergo self-renewing divisions and multi-lineage differentiation. It is evident that to
maintain the regenerative needs of the hematopoietic system throughout the life span of
the organism these two processes need to be closely balanced. While there are intrinsic
regulators that control entry of the stem cells to the cell cycle and play a key role in stem
cells’ quiescence and physiology, the expression of these molecules is also dependent on
the extrinsic signals from the environment that the stem cells inhabit.
In 1972, Lord & Hendry showed that in the adult BM, the CFU-S are not randomly
distributed. Instead, higher numbers of CFU-S are found in the areas closer to the inner
Figure 1.2 Overview of migratory and circulatory routes connecting fetal hematopoietic sites.
Hemogenic mesoderm cells form blood islands in the yolk sac (yellow) and start blood formation there
and then later at the aorta-gonadmesonephros (AGM) region (green) and the placenta (blue). There are
two main circulatory routes that connect fetal hematopoietic organs. The yolk sac connects to the fetal
liver (red) via the vitelline vein. The umbilical vein connects the placenta to the fetal liver. From the
fetal liver, the bone marrow (orange) is then seeded by HSCs through peripheral circulation right
around birth time. The timing of these events is outlined by the timescale in embryonic days below.
surface of the bone (endosteum) compared to the center of the BM cavity (Lord &
Hendry, 1972). They also observed that the CFU-S closer to the endosteum were more
proliferative compared to those harvested from the axial shaft of the femur. On the other
hand, colony forming unit cells (CFU-Cs), that represents the committed precursor cells
of granulocytic cells, were mainly concentrated at areas further from the bone surface.
This suggested that the endosteum of the bone forms a matrix for the CFU-S that was
required for their maintenance. Once differentiated into CFU-C, the cells move toward
the center of the BM cavity producing the peak of CFU-C closer to the axial shaft of the
femur (Lord et al., 1975). In 1978, Gong separated and studied the loosely adherent cells
of the central BM [the red marrow (RM) cells] and the endosteal marrow (EM). He
showed that although the EM cells had similar morphology to that of lymphocytes, they
were in fact a class of cells that were different from any hematopoietic cells normally
found in the RM. He suggested that these “unidentified endosteal marrow cells” could be
CFU-S (Gong, 1978).
In 1978, based on these studies on hematopoietic cells, Schofield proposed the concept of
stem cell niche (Schofield, 1978). In his hypothesis, a niche is composed of heterologous
subsets of cells and extracellular substrates that house stem cells. The cellular
components and the products of the niche provide the cells with the physical interactions
and the molecular basis for a balance of inhibitory and stimulatory signals that governs
stem cell self-renewal and differentiation (Adams & Scadden, 2006)
.
Schofield
hypothesized that as long as the stem cell is in association with the niche, it essentially
becomes a “fixed tissue cell” that is protected from differentiation and apoptotic stimuli
maintaining its stem-cell state (Schofield, 1978). The existence and nature of such niches
for maintenance of stem cells has been extensively studied and confirmed in lower
organisms such as the gonadal tissue in Drosophila melanogaster and Caenorhabditis
elegans (Xie & Spradling, 2000; Crittenden et al., 2002). However, it has not been until
recently that the structure of the HSC niche and the molecular interactions between them
and their niche components was examined.
In the adult BM niche, HSCs are largely found in a quiescent state. The BM stromal cells
construct the HSC niche structure in the BM. These stromal cells, which have a
mesenchymal stem cell (MSC) origin, are speculated to be responsible for maintenance
of the balance between HSC quiescence, self-renewal, proliferation and differentiation.
The stromal cells whose role in HSC niche has been well studied include osteoblasts,
endothelial cells and adipocytes.
1.6.1 Osteoblasts
Close proximity of the HSCs to the endosteal surface of the bone (Lord et al., 1975;
Gong, 1978) made the bone-lining cells of osteoblastic lineage the natural candidates to
contribute to the HSC niche. Osteoblasts are found at the interface between the bone and
marrow (endosteum). These cells are responsible for the secretion of extracellular BM
matrix as well as the regulation of mineralization (Aguila & Rowe, 2005). They also are
the major regulators of osteoclast differentiation (Martin & Sims, 2005).
Evidence for the contribution of osteoblasts to the HSC niche first came from in vitro
studies. Human osteoblasts were shown to support HSC survival in vitro through
production of Granulocyte Colony-stimulating Factor (G-CSF) (Taichman & Emerson,
1994). These cells were able to maintain long-term culture-initiating cells (LTC-IC) and
resulted in a significant expansion of progenitor cells (CFU-Cs) in vitro further providing
evidence that the presence of HSCs in close proximity to endosteal surfaces in vivo may
be due in part to a requirement for osteoblast-derived products (Taichman et al., 1996).
Additionally, it has been shown that during whole BM transplantations, osteoblastic
progenitors engraft into the recipient BM and give rise to bone lining cells and osteocytes
which function as bone matrix producing cells (Nilsson et al., 1999). When co-
transplanted with HSCs, osteoblasts facilitated engraftment of stem cells in an allogeneic
environment and the transplanted mice demonstrated excellent long-term survival (El-
Badri et al., 1998).
In vivo evidence for contribution of osteoblasts to HSC niche came from studies using
genetically altered animal models to activate or destruct osteoblastic cells. Zhang et al.
showed that mice with conditional inactivation of bone morphogenic protein receptor
type IA (BMPRIA), using a Mx1-Cre system, have ectopic formation of trabecular-bone-
like area (TBLA) in the long-bone region along the endosteal surface that was due to an
increase in osteoblast number and the rate of bone formation (Zhang et al., 2003). The
increased TBLA correlated with an expanded HSC pool size as shown both by
immunophenoypical analysis as well as functional competitive repopulation unit (CRU)
assay. Using this assay, they showed that the functional stem cell number increased 2.2-
fold in the mutant mice compared with controls. They also demonstrated that two
adherens junction molecules, N-cadherin and -catenin, are asymmetrically localized
between the spindle-shaped N-cadherin
+
osteoblast (SNO) cells and the long-term HSCs.
The increase in the HSC frequency was in a non-autonomous, microenvironment-
dependent manner given the expression pattern of BMPRIA that was exclusive to the
osteoblasts. It was therefore concluded that HSCs are attached to an SNO cell subset that
is responsible for regulating the niche size (Zhang et al., 2003).
A complementary study looking into the role of osteoblastic cells in the HSC niche was
published by Calvi et al., who assessed mice that were genetically altered to express a
constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor
(PPR) under the control of the 1(I) collagen promoter, active specifically in osteoblastic
cells (Calvi et al., 2003). These transgenic mice showed an increase in the stem-cell-
enriched LSK subpopulation of bone marrow mononuclear cells (BM-MNC) fraction in
the BM. The increase in LSK cells was accompanied with an enhanced functionality of
these cells as assessed using a quantative LTC-IC assay which is a correlative of in vivo
HSC function. They further confirmed the increase in stem cell population by
competitive transplantation of wild-type (WT) and transgenic cells into irradiated
recipient which indicated a superior engraftment of transgenic cells. To elucidate the
possible mechanism for the effect seen, they demonstrated that the increased number of
the primitive cells was stroma-dependent and was also accompanied by an elevated
expression level of the Notch ligand, Jagged-1 (Jag-1) in the osteoblasts of the transgenic
mouse. Reduction of the supportive capacity of transgenic stroma by administration of -
secretase inhibitor, that blocks Notch cleavage, further supported the proposed model for
the observed effect of PPR activation. In this model, PPR activation in the osteoblastic
population increases cell number and the overall production of Jagged-1. This, in turn,
may activate Notch on primitive hematopoietic cells, resulting in expansion of the stem
cell compartment.
Similar effects were observed using exogenous parathyroid hormone (PTH), which
suggested a possible role for PTH to regulate HSCs. BM stromal cells expanded in the
presence of PTH led to an increased LTC-IC support similar to the results from the
transgenic mice stromal cells. Addition of -secretase inhibitor to the stromal cultures
abrogated the impact of PTH on the stromal support of primitive hematopoietic cells,
which was suggestive of Notch involvement. As a final piece of evidence, they examined
in vivo effects of exogenous administration of PTH. Injecting WT mice with PTH for 4
weeks significantly increased the absolute numbers of LSK cells. Once used in a
competitive transplantation setting, the hematopoietic cells from PTH treated cells
exhibited an enhanced engraftment rate into secondary irradiated recipients compared to
WT cells (Calvi et al., 2003).
Further evidence for the role of osteoblasts in the HSC niche came from studies where
decreased osteoblast number led to a reduction in HSC numbers. In one study, a
transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the
control of a 2.3-kilobase fragment of the rat collagen 1(I) promoter (Col2.3 TK) was
used. When these mice were treated with ganciclovir (GCV) for 16 days, they displayed
extensive destruction of the bone lining cells and also decreased osteoclast number
(Visnjic et al., 2001). These mice provide a reversible model for directly ablating
osteoblasts and studying their effect on the HSCs. In fact, upon GCV treatment, the
progressive bone loss and decrease in BM cellularity was accompanied by decreased BM
and increased extrameduallary (in liver and spleen) hematopoiesis. After withdrawal of
GCV, osteoblasts reappeared in the bone compartment leading to a recovery of medullary
and decrease in extramedullary hematopoiesis (Visnjic et al., 2004).
However, not all the studies that deplete osteoblasts in the BM show a decrease in the
number of HSCs. Kiel et al. used biglycan-deficient mice to test whether depletion of
osteoblasts and osteoblast progenitors would reduce the number of HSCs (Kiel et al.,
2007). Biglycan is an extracellular matrix proteoglycan that is most prominently
expressed by osteoblasts and chondrocytes. biglycan-deficient mice develop an
osteoporosis-like phenotype, with less trabecular bone, fewer osteoblasts, and fewer
osteoblast progenitors. No changes in the hematopoiesis of these deficient mice were
observed compared to their WT littermates and they both had comparable BM cellularity
and HSC frequency as well as similar repopulating potential as measured by competitive
repopulation assays (CRAs). Based on these observations, the investigators suggested
that osteoblasts are not a limiting component of adult BM HSC niches. However,
osteoblasts were never completely eliminated from biglycan-deficient mice as shown by
existence of osteopontin
+
osteoblasts at the endosteal surface of the femurs, therefore this
study could not suggest that these cells are not involved in HSC maintenance.
Yet, assuming osteoblasts are the only regulators of the HSC niche would be naïve. In
fact, it has been shown that global osteoblastic expansion by itself is not sufficient to
expand HSCs (Lymperi et al., 2008). In this study osteoblastic function was enhanced
using strontium (Sr), an anabolic agent that also prevents osteoclastic function. While
morphometric analysis of the bones from the mice treated with Sr showed increased
osteoblast number, bone volume, and trabecular thickness, no effects were seen in the
primitive HSC population and the reconstitution ability of these cells were also impeded.
Correlating with this was the observation that Sr-treated osteoblastic cells did not display
an increase either in RANK-L expression or in N-cadherin
+
osteoblasts and N-cadherin
transcripts, whereas PTH treatment (which increases HSC number in the niche) did.
These data suggest that increasing the overall number and function of osteoblasts without
increasing N-cadherin
+
cells and receptor activator of nuclear factor- B ligand (RANK-
L) expression is not sufficient to enhance HSC quantity and function. This suggests a
potential role for cells of osteoclastic lineage in the HSC niche (Lymperi et al., 2008).
1.6.2 Molecular Crosstalk at the Endosteal Niche
While the role of osteoblasts in the HSC niche has been extensively studied, the exact
mechanism through which these cells exert their effect is yet to be clearly deciphered.
However various products of these cells as well as direct cell-cell interactions have been
shown to act as regulators of HSC physiology in the BM niche:
Angiopoietin 1 (Ang1)- Ang1 is an angiogenic factor and a ligand for Tie2, a receptor
tyrosine kinase expressed on endothelial cells (ECs) and HSCs (Dumont et al., 1992;
Iwama et al., 1993). In 2004, Arai and collegues showed that osteoblasts express Ang1
and maintain HSCs in vitro (Arai et al., 2004). They also demonstrated that Tie2
+
LSK
cells were quiescent and anti-apoptotic as confirmed by their insensitivity to
myelosuppressive 5-FU treatment. Tie2
+
HSCs were shown to co-express c-Kit and
adhere to Ang1-expressing osteoblasts at the endosteal surface. The BM-derived
osteoblasts were able to support Tie2
+
LSK cells in vitro and disruption of Tie2/Ang1
interaction by adding Tie2-F
c
(a soluble form of Tie2 receptor) inhibited cobblestone
formation in cobblestone area forming cell (CAFC) assays. Interestingly, exogenous
Ang1 (expressed through transplanting BM cells transduced with an Ang-1 expressing
retrovirus) increased the number of quiescent HSCs by 1.5 fold. They also demonstrated
that Tie2/Ang1 signaling protects HSCs from myelosuppressive treatements in vivo (Arai
et al., 2004).
Thrombopoietin (THPO)- THPO is a highly glycosylated protein which has an
established role in megakaryocytogenesis and the regulation of platelet production
(Kaushansky K, 2006). However, the interaction between THPO and its receptor Mpl
was also shown to be important in the regulation of the HSC compartment (Kimura et al.,
1998). Here it was demonstrated that BM cells from mpl
-/-
mice had 8- to 10-fold fewer
spleen colonies than WT marrow, an effect that was intrinsic to the cells since the mpl
-/-
niche was capable of supporting WT cells. The mpl
-/-
cells also failed to reconstitute the
hematopoietic system in CRA and serial transplantation showed an impaired self-renewal
of these cells.
Later it was shown that Mpl is expressed on LSKCD34
FLT3
Tie2
+
and
LSKCD48
CD150
+
HSCs (Yoshihara et al., 2007). The Mpl
+
cells were resistant to 5-
FU-induced myelosupression further showing that Mpl is expressed on the quiescent
long-term HSCs. These 5-FU-resistant Tie2
+
Mpl
+
cells were localized to the endosteal
surface where they attached to THPO-expressing osteoblasts. Mpl
+
cells showed
functional advantage when compared to Mpl
-
cells in CRA and serial transplantation
assays. Quantitative real-time PCR showed that THPO/Mpl signaling upregulated 1-
integrin and cyclin-dependent kinase inhibitor, p57
Kip2
, in HSCs. Furthermore, inhibition
of this pathway by anti-Mpl neutralizing antibody, AMM2, reduced the number of
quiescent long-term HSCs (LT-HSCs) and allowed exogenous HSC engraftment without
irradiation. By contrast, exogenous THPO stimulation transiently increased quiescent
HSC population and subsequently induced HSC proliferation in vivo. Together these data
suggested a critical role for the THPO/Mpl signaling pathway in regulation of HSC in
their osteoblastic niche (Yoshihara et al., 2007).
Notch Pathway- The notch signaling pathway is an evolutionary preserved pathway that
has been shown to have a critical role in various cell-fate decisions. A role of Notch was
first implicated in hematopoiesis field when it was shown to be expressed on human
CD34
+
hematopoietic precursors (Milner et al., 1994). Later, it was shown that Notch and
its ligand, Jag-1, was expressed by both primitive and mature human hematopoietic cells
as well as BM stromal cells indicating that both these cells are capable of inducing, and
also responding, to Notch signaling (Karanu et al., 2000). CD34
+
CD38
Lin
human stem
cells were then stimulated by soluble human-Jag-1 [(h)Jag-1] both in vitro and in vivo.
While only a modest expanding effect was observed following (h)Jag-1 treatment,
intravenous transplantation of the treated cultured cells into immunodeficient mice
revealed that (h)Jag-1 induces the survival and expansion of functional human stem cells
(Karanu et al., 2000). The supportive role of Notch ligand families, Delta and Jagged,
were then investigated in both murine and human system confirming their ability to
expand hematopoietic progenitor populations in vitro (Karanu et al., 2001; Vas et al.,
2004; Delaney et al., 2005).
Later, to determine whether activation of Notch influences stem cell function, Stier et al.
studied the influence of activated Notch in V(D)J recombination activation gene-1
(RAG1)-deficient BM stem cells (Stier et al., 2002). Since RAG-1 is essential for
lymphocyte development, RAG-1-deficient mice have no mature B- and T-lymphocytes.
Also, RAG-1
/ Sca1
+
Lin
cells transduced with activated Notch1 show a higher LTC-IC
frequency in vitro than control vector-transduced cells. In vivo, activated Notch1
expanded primitive RAG-1
/
hematopoietic cells and enhanced the self-renewal of RAG-
1
/ HSCs as measured by serial BM transplantation. It was noteworthy that preferential
lymphoid over myeloid lineage commitment was observed upon differentiation (Stier et
al., 2002). These data suggested Notch1 as a target for stem cell manipulation strategies,
an idea that was further corroborated with findings of Calvi et al. when they
demonstrated how constitutively active PPR expanded HSC pool size through increasing
levels of Notch intracellular domain (NICD) (Calvi et al., 2003).
Finally, using a Transgenic Notch Reporter (TNR) mouse, Duncan et al. showed that
Notch signaling was active in HSCs in trabecular bone in vivo and down-regulated as
HSCs differentiated. Inhibition of Notch signaling, by -secretase inhibitor, led to
accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo (Duncan et al.,
2005).
Together these data suggest an important role of Jagged/Notch signaling in the
extracellular regulation of HSCs. However, whether this pathway is necessary for HSC
maintenance is still in question as reports from Mancini et al. showed that an inducible
Cre-loxP–mediated inactivation of the Jag-1 gene in BM cells does not impair HSC self-
renewal or differentiation. Also, simultaneous inactivation of Jag-1 in the BM and
Notch1 in HSCs does not affect their self-renewal potential reconstitution potential
(Mancini et al., 2005). These observations could be due to compensatory mechanisms
mediated by Notch receptors and ligands. Wnt Pathway- The wnt gene family is known to regulate the cell fate and cell-cell
interactions of multipotential cells in a variety of tissues. Wnt signaling pathway was first
examined in fetal hematopoiesis where it was shown that treatment of HSC populations
in culture with soluble Wnt proteins stimulated expansion of cells by enhancing self-
renewal and proliferation of HSCs (Austin et al., 1997). In humans, Wnt is expressed in
both adult and fetal BM stroma as well as Lin
-
CD34
+
primitive cells and has a positive
effect on their proliferation (Van den Berg et al., 1998). This effect was further
confirmed when soluble Wnt binding antagonists (a soluble form of frizzled cysteine-rich
domain (CRD) that inhibits the binding of Wnt proteins to the frizzled receptor) were
shown to reduce the proliferative capacity of HSCs in vitro (Reya et al., 2003). In this
study, it was shown that the canonical Wnt signaling through -catenin is responsible for
the effects seen, as constitutively active -catenin expression in c-Kit
+
Thy-1.1
lo
Lin
-/lo
Sca-
1
+
HSCs led a self-renewal of these cells and their reconstitution advantage. Confirming
this observation, it was shown that HSCs signal through lymphoid enhancer-binding
factor-1/T-cell factor (LEF-1/TCF) complex and self-renewal stimulating genes such as
Notch1 and HoxB4 are expressed in them (Reya et al., 2003).
However, contradictory to Reya et al. were observations of experiments in which -
catenin was conditionally expressed in vivo under control of the ROSA26 locus
(Kirstetter et al., 2006). Here, not only was no increase in self-renewal observed, but it
also caused a multilineage differentiation block and compromised HSC maintenance. It
was also shown that inducible Cre-loxP-mediated inactivation of the -catenin gene in
BM progenitor cells did not result in any in vivo phenotype (Cobas et al., 2004).
Finally, more discrepancies were introduced to the field when Wnt pathway was inhibited
in the HSCs osteoblastic niche in vivo (Fleming et al., 2008). Here, they used transgenic
mice that had an osteoblast-specific promoter driving expression of the inhibitor of
canonical Wnt signaling, Dickkopf1 (Dkk1). While osteoblast-specific expression of
Dkk1 did not affect blood or marrow primitive hematopoietic cell populations at steady
state, when transplanted into irradiated recipients, the frequency of repopulating cells
present in BM isolated from individual Dkk1-expressing animals revealed a 2-fold
elevation in the number of functional reconstituting HSCs. These transplant results
indicate that cells isolated from the Dkk1-expressing niche are capable of reconstituting
irradiated recipients and appear to be present at a higher frequency when Wnt has been
inhibited in this location. A progressive decline in regenerative function of these HSCs
was also observed after serial transplantations. The effects were microenvironment
determined, but irreversible if the cells were transferred to a normal host (Fleming et al.,
2008).
These discrepancies further confirm that the exact role of Wnt signaling in the HSC niche
is yet to be discovered however, this pathway can be a potential therapeutic target for
various hematopoietic diseases.
Osteopontin (OPN)- OPN is an acidic glycoprotein, which is a product of osteoblasts
and secreted into the bone ECM. OPN can exert its effect on the cells through either its
interaction with integrins ( 5
3
intergrin) or with CD44, activating multiple signaling
pathways (Denhardt & Guo, 1993). In the bone, OPN is expressed by cells of the
osteoblastic lineage mainly at the site of bone-remodeling. However its absence does not
affect bone morphology trabecular spaces associated with stem cell localization, or
osteoblasts under homeostatic conditions (McKee et al., 1993; Rittling et al., 1998).
It has been shown that OPN-deficient mice had an increase in HSC numbers.
Transplanting WT HSC into opn
-/-
recipient resulted in an increase in HSC numbers,
indicating that OPN acts through a non-cell autonomous, niche-dependent manner. The
HSCs from OPN-deficient mice had a comparable cell-cycle profile to that of WT mice.
Sstem cell expansion in these mice occurred without increased proliferation through
Notch1 activation where stem cell self-renewal was favored over differentiation.
Activation of Notch1 on primitive hematopoietic cells in vivo as a result of OPN absence
resulted in an increase in the number of primitive cells, but reduced number of progenitor
cells. On the other hand, increase in apoptosis of hematopoietic cells seemed to also be
responsible for the limiting effect of OPN on the HSC pool (Stier et al., 2005).
It was also shown that primitive hematopoietic cells specifically attached to OPN in vitro
via 1
integrin. Furthermore, treating the cells with exogenous OPN potently suppressed
the proliferation of primitive progenitor cells in vitro further confirming the negative
regulatory effects of OPN in the HSC niche (Nilsson et al., 2005).
Stem Cell Factor (SCF)- The steel (Sl) locus encodes both secreted and trans-membrane
SCF (tm-SCF). SCF signals by ligand-mediated dimerization of its receptor c-Kit, which
is expressed at high levels by HSCs and other stem cells. tm-SCF, which is expressed by
osteoblasts has been shown to result in a more continuous activation of the c-Kit receptor
than the soluble isoform (Miyazawa et al., 1995) and is crucial for long-term
maintenance and self-renewal of HSCs through promoting their adhesion to stromal cells
via activation of Very Late Antigen 4 (VLA4: 4
1
Integrin) and Very Late Antigen 5
(VLA5: 5
1
Integrin) (Kinashi & Springer, 1994). These effects were recapitulated when
in vivo administration of SCF was shown to expand the absolute number of HSC per
mouse, up to threefold as shown by limiting dilution repopulating assay (Bodine et al.,
1993).
It was also shown that transplanting normal HSCs into Sl/Sl
d
mice, who had a
microenvironment devoid of tm-SCF, resulted in normal homing but impaired lodging
and engraftment
of the cells (Driessen et al., 2003). The niche-dependent effect of tm-
SCF was further investigated when BM-MNCs from young Sl/Sl
d
mice were shown to
have normal long-term repopulating (LTR) potential (Barker, 1994) but, BM cells from
old Sl/Sl
d
mice had reduced LTR activity suggesting that the stem cells, when retained in
the mutant environment into adulthood, are either reduced in number or phenotypically
altered by lack of the tm-SCF (Barker, 1997). Together, these data emphasize the role of
tm-SCF in maintaining the long-term HSC activity in the adult BM niche.
Stromal cell-derived factor 1 (SDF-1) & CXCR4- SDF-1 [also known as Chemokine
(C-X-C motif) ligand 12 (CXCL12)] is a chemokine that is expressed by several BM
stromal cells including osteoblasts and vascular endothelial cells (Wilson & Trumpp,
2006). The role of SDF-1 during ontogeny is well documented and it has been shown that
its interaction with its receptor, CXCR4, is required in colonization of the BM by HSCs
and myeloid cells (Nagasawa et al., 1996; Ara et al., 2003). SDF-1 has also been shown
to synergistically work with steel factor to exert their chemoattraction effects on fetal
liver HSCs when homing to and seeding BM and spleen during late fetal development
(Christensen et al., 2004). Also, in the BM, CXCR-4 has been shown to be important in
retention of stem cells in their niche as shown in studies where CXCR4-deficient fetal
liver cells were transplanted into wild-type recipients (Ma et al. 1999). In these mice,
high circulating levels of myeloid and B-lymphoid precursor cells were observed in the
blood at the expense of low cell numbers in the BM (Ma et al. 1999).
In adult hematopoietic system, HSCs were shown to specifically migrate towards SDF-1
in vitro but not towards any other single chemokine (Wright et al., 2002). Recent studies
investigating the mechanisms of the CXCL12-CXCR-4 pathway revealed the
involvement of FAK in mediating the chemotactic effect of CXCL12 in hematopoietic
progenitor cells (Glodek et al., 2007). It was indicated that deletion of FAK via a Cre-
Flox system resulted in impaired CXCL12-induced migration suggesting a potential role
for FAK as an intermediary in signaling pathways controlling hematopoietic cell
lodgment and lineage development (Glodek et al., 2007).
A schematic overview of the molecular cross-talk between HSC and the osteoblast cell
including the ligand-receptor interaction and the adhesion molecules discussed above is
shown in figure 1.3.
1.6.3 Endothelial Cells
The BM vasculature is made of a network of thin-walled and fenestrated sinusoidal
vessels. These vessels are lined by endothelial cells; cells that just like hematopoietic
cells are derived from hemangioblasts during development (Kopp et al., 2005). Indeed,
from a developmental point of view, hematopoietic and endothelial cells have been
shown to be interconnected at various stages of ontogeny from yolk sac (Shalaby et al.,
1995; Lu et al., 1996) to AGM (Tavian et al., 1996), to FL (Oberlin et al., 2002).
Hematopoietic cells then need to colonize the BM, a process in which, as mentioned
Figure 1.3 Molecular cross-talk between HSC and the endosteal niche. Schematic diagram of the
endosteal niche–stem-cell interaction showing putative ligand–receptor interactions and adhesion
molecules. ANG1, angiopoietin-1; BMP, bone morphogenetic protein; BMPR1A, BMP receptor 1A;
SDF1, stromal cell-derived factor 1; CXCR4, CXC-chemokine receptor 4; HSC, hematopoietic stem
cell; LRP, low-density-lipoprotein-receptor-related protein; MPL, myeloproliferative leukemia
receptor; OPN, osteopontin; PPR, PTH/PTH-related protein receptor; PTH, parathyroid hormone;
SCF, stem-cell factor; THPO, thrombopoietin; TIE2, tyrosine kinase receptor 2.
earlier, the role of SDF-1 has been proven. It has been shown that mutant mice with a
targeted ablation of sdf-1 die perinatally and have a disrupted B-cell lymphopoiesis and
BM myelopoiesis (Nagasawa et al., 1996). The BM colonization defect in these mice can
be rescued by enforced expression of SDF-1 under the control of vascular-specific Tie-2
regulatory sequences in the vascular endothelial cells (Ara et al., 2003), suggesting the
essential role of endothelial cells for colonization of the fetal BM by HSCs in the
presence of SDF-1.
In addition to the large body of evidence highlighting the role of endothelial cells in
hematopoiesis in vivo, it has been demonstrated that endothelial cells derived from
various tissues can support HSCs and progenitor cells in culture systems. BM endothelial
cell monolayers were shown to support proliferation and differentiation of myeloid and
megakaryocytic progenitors in culture, via production of IL-6, Kit-ligand, G-CSF, and
granulocyte macrophage colony-stimulating factor (GM-CSF) (Rafii et al., 1995). Also,
Lin
-
Sca1
+
cells isolated from 5-FU treated mice were cultured on endothelial cell cultures
derived from adult murine liver [liver endothelial cell (LEC)] and were capable of
forming cobblestone areas as well as hematopoietic progenitors that are able to form
colonies (Cardier and Barberá-Guillem, 1997). Another hematopoietically active site
whose endothelial cells have been shown to support HSCs in vivo is the AGM. Analysis
of two different AGM-derived CD34
+
cell lines revealed that one was able to efficiently
induce FL HSCs to differentiate down erythroid, myeloid, and B-lymphoid pathways
while the other was able to stimulate expansion of Lin
-
CD34
+
Sca-1
+
c-Kit
+
cells could
competitively repopulate the BM of lethally irradiated mice (Ohneda et al., 1998).
The first non-hematopoietic site that was studied as a potential culture system for
supporting HSCs was brain endothelial cells (Chute et al., 2002). In these studies adult
BM CD34
+
cells were co-cultured with human brain endothelial cells (HUBECs) for 7
days. This system supported a 5.4-fold increase in CD34
+
cells and produced progeny
that engrafted NOD/SCID mice at significantly higher rates than fresh adult BM CD34
+
cells. The recipients showed lymphoid and myeloid differentiation upon hematopoietic
reconstitution, indicating that a primitive hematopoietic cell was preserved during culture
(Chute et al., 2002). Later, in a comprehensive study, Li et al. isolated primary EC
populations from several non-hematopoietic organs and co-cultured BM Lin
-
Sca1
+
c-Kit
+
cells with them for 7 days. They demonstrated that while brain and heart endothelial cell
monolayers significantly increased the number of CFU-S day-8 colonies, lung and liver
endothelial cell monolayers maintained, and kidney endothelial cell monolayer markedly
decreased these colonies after 7 days of coculture. Also, HSC competitive repopulating
potential was maintained during the heart and liver endothelial cell 7-day cocultures but
was lost in the kidney coculture (Li et al., 2004).
In vivo, endothelial cells form the barrier between the developing hematopoietic cells and
peripheral circulation. Therefore, they are the initial gate of entrance through which all
blood cells enter circulation (for example during mobilization) and also the final site
whereby the cells leave the blood and enter the BM (for example during homing)
(Avecilla et al., 2004; Chute JP,2006; Laird et al., 2008; Lapidot et al., 2005; Winkler
and Lévesque, 2006; Wright et al., 2001). Direct evidence for the role of endothelial cells
in maintaining the HSC niche in vivo came from two studies where hematopoietic stem
and progenitor cells where shown to reside in proximity of endothelial cells. In one study,
Sipkins et al. used intravital imaging of calvarium BM to show that fluorescently labeled
primitive hematopoietic cells injected into mice adhere to BM microvasculature at
specialized domains (Sipkins et al., 2005). At these sites, the vasculature was shown to
express the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete,
discontinuous areas. The hematopoietic stem and progenitor cells were observed at these
sites as early as 2 hours after intravenous injection and persisted or increased in number
over a 70 day interval (Sipkins et al., 2005). Although these data suggest the existence of
a potential perivascular niche the cell populations injected were not highly purified for
HSCs (the cells used were a pool of Lin
-
Sca1
+
c-Kit
+
cells along with and Lin
-
Sca1
+
and
Lin
-
c-Kit
+
cells). However, the discovery of SLAM antigens as markers of HSCs enabled
Kiel et al. to look into the localization of HSCs in the BM where they showed that the
majority of CD150
+
CD48
CD244
CD41
Lin
HSCs reside in the perivascular region
with a minority of them at the periendostal sites (Kiel et al., 2005b). Later, it was shown
that these sinusoidsal endothelial cells are surrounded by CXCL12-abundant reticular
(CAR) cells that are also found at the endosteum (Sugiyama et al., 2006). However, it
was not until 2010 that endothelial cells were shown to be indispensable for HSC self-
renewal (Butler et al., 2010). In this study an endothelial cell line that is capable of
growing in culture in a serum- and cytokine-free conditions (E4ORF1
+
endothelial cell
line) were used to support HSCs in vitro. Lin
-
Sca1
+
c-Kit
+
cells cocultured with this cell
line in presense of soluble Kit ligand (sKitL) showed a dramatic 15-fold hematopoietic
cell expansion for more than 21 days. These expanded hematopoietic cell population
showed an increase in progenitor cell numbers as determined by a CFU-C assay. These
cultured Lin
-
Sca1
+
c-Kit
+
cells were able to support long-term multi-lineage engraftment
of irradiated mice. Angiocrine expression of Notch ligands by endothelial cells were
proposed to be responsible for the effect seen and LT-HSCs were detected in cellular
contact with sinusoidal endothelial cells in transgenic notch-reporter (TNR.Gfp) mice
(Butler et al., 2010).
Finding HSCs at the BM microvasculature region is formally possible since it serves as a
barrier for stem cell trafficking into and out of the circulation. While these data propose a
regulatory role for endothelial cells, further evidence is required for suggesting the
presence of a vascular “niche” for HSCs that is capable of supporting self-renewal and
multi-lineage differentiation of stem cells. Also, the recent studies that suggest HSCs
reside in a hypoxic microenvironment (Parmar et al., 2007; Kubota et al., 2008) further
propose the endosteal surface as a more plausible site for slow-cycling HSCs to reside as
opposed to the BM vascular system where they are readily accessible to diffusible agents
(Adams, 2008).
1.6.4 Adipocytes
Adipocytes, the main cellular component of adipose tissue, are generated from
mesenchymal progenitor cells of mesodermal origin (Majka et al., 2011) where
peroxisome proliferator-activated receptor gamma (PPAR ) is known as the established
regulator of adipocyte differentiation (Lazar, 2005; Farmer, 2006). Once the regression of
hematopoiesis from the appendicular skeleton to the axial skeleton occurs during the
adult human lifespan, the rest of the BM undergoes adipose conversion (ie, “red” marrow
turns into “yellow”marrow) (Bianco, 2009). While the common origin of adipocytes with
osteoblastic lineage put forward the possibility that just like osteoblasts, they also play a
role in the HSC niche, their effect is still quite controversial.
Several reports have suggested that adipocytes and adipose tissue are capable of
regulating hematopoiesis (Corre et al., 2004; Cousin et al., 2003; Hangoc et al., 1993).
Two adipocyte-derived factors that have been suggested to be responsible for the effects
are Leptin and Adiponectin. Leptin has been shown to have a proliferative effect on
multilineage progenitor, as shown by increased myelopoiesis, erythropoiesis and
especially lymphopoiesis (Bennet et al., 1996; Gimble et al., 1990; Umemoto et al.,
1997). Analysis of db/db mice, that have a truncated leptin receptor, revealed a defective
lymphopoiesis at the homeostatic steady-state conditions as well as post-irradiation insult
(Bennet et al., 1996). Adiponectin, another protein expressed by adipocytes, however has
also been described as being a negative regulator of myelomocytic cells (Yokota et al.,
2000). In vitro, adiponectin suppressed colony formation from CFU-GM and also
inhibited mature macrophage functions, while it did not have any effect on BFU-E
colonies (Yokota et al., 2000). It was later proposed that adiponectin also has negative
effects on B-lymphpoiesis through activation of the cyclooxygenase-prostaglandin
pathway in stromal cells (Yokota et al., 2003).
HSCs also were originally suggested to be negatively regulated by adipocytes. Using the
spine of adult mice as a natural manifestation of a BM adipocyte gradient, where thoracic
vertebrae are virtually adipocyte-free and tail vertebrae being highly adipocytic, Naveiras
and colleagues showed that HSC numbers are reduced in adipocyte-rich BM (Naveiras et
al., 2009). They used A-ZIP/F1 fatless mice to look into the functional properties of
adipocytes and showed that the lack of adipocytes in these mice enhanced hematopoietic
recovery post transplantation. This enhancement was then reproduced pharmacologically
in WT mice by inhibiting adipocyte formation through of PPAR- inhibition (Naveiras et
al., 2009) establishing the role of adipocytes as negative regulators of HSCs. The
negative effect of adipocyte were further explored in vitro when Lin
-
Sca-1
+
c-Kit
+
cells
were cultured on GZL stromal cell line which has a high adipocyte content (Chitteti et
al., 2010). This stromal cell line were not able to efficiently support CFU-C expansion
and the Lin
-
Sca-1
+
c-Kit
+
cells had lower repopulating potential when compared to cells
grown on osteoblastic stromal layers. This suppressive effect of adipocytes was thought
to be due to an up-regulation of neuropilin-1 and adiponectin expression (Chitteti et al.,
2010).
Conversely, adipocytes have also been found to support HSCs, reappearing at day 7 after
irradiation injury, corresponding to the initiation of hematopoietic proliferation
(Yamazaki & Allen, 1991). Also, contrary to its suppressive effects on myeloid and
lymphoid cells, adiponectin was demonstrated to enhance HSC proliferation in vitro
expanding Lin
-
Sca-1
+
c-Kit
+
Thy-1
lo
cells that had a more efficient reconstitution ability
compared to non-treated HSCs (DiMascio et al., 2007). The adiponectin effect on HSCs
was mediated by signaling through its receptor, AdipoR1, which is expressed on HSCs
(DiMascio et al., 2007). Also, it is noteworthy to mention that the CAR cells whose
supportive role in the HSC niche is well established has an adipo-osteogenic origin
(Omatsu et al., 2010).
It has been shown that adipose tissue can support aspects of hematopoiesis in states of
stress, as was observed in retinoic acid receptor (RAR )-deficient animals who
developed myeloproliferative syndromes (MPS) (Walkley et al., 2007). These mice had
significantly increased granulocyte/macrophage progenitors and granulocytes in BM,
peripheral blood, and spleen, a phenotype that was more pronounced in older mice. These
older mice also had a strikingly high number of immature and maturing hematopoietic
cells in their adipose tissue. The MPS phenotype was suggested to be partially due to a
raised tumor necrosis factor (TNF ) expression in RAR -deficient animals.
Interestingly, TNF has been shown to increase leptin and reduce adiponectin expression
in adipose tissue, hence it is possible that increased TNF signaling directly enhanced the
hematopoietic support potential of adipose tissue in the older RAR null mice (Walkley
et al., 2007).
1.6.5 Nervous System
BM has been shown to be a highly innervated site with both myelinated and
nonmyelinated nerve fibers connected to stromal layers via gap junctions forming
potential functional units for signal transduction called the "neuro-reticular complex"
(Yamazaki and Allen 1990). The nervous system had been shown to have an effect on
BM composition in a study where surgical severance of femoral nerve resulted in
decreased BM cellularity and significant mobilization of progenitor cells. This effect was
replicated with administration of 6-hydroxydopamine that blocked neurotransmitter
synthesis in adrenergic and dopaminergic nerve fibers (Afan et al., 1997).
Further evidence that nervous system might affect the HSC niche actually, came from
studying the role of sympathetic nervous system (SNS) on the osteoblasts (Takeda et al.,
2002). Here, it was shown that osteoblasts express 2
-adrenergic receptors that can
respond to catecholamine ligands for adrenergic receptors, norepinephrine (NE) and
epinephrine, secreted from hypothalamic nerve ends resulting in increased bone
formation (Takeda et al., 2002). Correlating with these data, it was shown that NE
controls the suppression of osteoblasts and down-regulation of CXCL12 expression in
bone cells that occurs in response to G-CSF treatment (Katayama et al., 2006). In this
study, a mouse model with disrupted SNS was used [UDP-galactose:ceramide
galactosyltransferase (Cgt)-deficient mice] that displayed a defect in nerve conduction
due to a lack of myelin. No mobilization occurred in these mice in response to G-CSF
treatment. The inability of these mice to mobilize was not mediated by hematopoietic
self-autonomous mechanisms, but was accredited to low but sustained levels of CXCL12
in the bone tissue and compromised osteoblast function. It was proposed that adrenergic
receptors are required for mobilization of stem cells induced by G-CSF, since dopamine
hydroxylase [Ddh (responsible for the conversion of dopamine into norepinephrine)] null
mice also showed a defective mobilization in response to G-CSF which was rescued with
administration of a 2-adrenergic agonist (Katayama et al., 2006).
A more recent study also revealed that trafficking of hematopoietic stem and progenitor
cells in and out of the blood stream exhibit robust circadian fluctuations that were due to
a cyclical expression of CXCL12. It was shown that circadian secretion of NE by the
SNS was the reason for the effects seen. (Méndez-Ferrer et al., 2008).
It should be noted that immature human CD34
+
cells also have been shown to express
both forms of catecholamine receptors that gets over-expressed in response to G-CSF and
GM-CSF cytokines (Spiegel et al., 2007). NE activation of these receptors resulted in
increased motility, proliferation and colony formation of human progenitor cells. Also
NE treatment enhanced human CD34
+
cell engraftment of NOD-SCID mice through Wnt
signaling activation and increased cell mobilization and BM Lin
-
Sca-1
+
c-Kit
+
cell
numbers (Spiegel et al., 2007).
Summary
Together these data suggests that HSC niche is a lot more complex than a single cell type
and there are many other cellular components interacting with each other through direct
cell-cell contact and soluble factors to keep the HSCs in place.
1.7 Intrinsic Regulators of HSC function
Regulation of self-renewal, specifically, has been shown to be of critical importance since
inadequate self-renewal can potentially cause tissue failure, and is probably one
component of the aging process (Janzen et al., 2006; Rando, 2006; Gomez et al., 2005;
Sudo et al., 2000). On the other hand, if not properly restrained, production of large
numbers of undifferentiated stem cells will provide the necessary substrate for malignant
transformation and pathogenic conditions (Domen & Weissman, 2000; Taipale &
Beachy, 2001). Self-renewing stem cells will necessarily undergo cell division that will
give rise to daughter cells with identical potential as the original one. Therefore, the
regulation of cell-cycle progression in these cells is of critical importance.
Contrary to embryonic stem cells that have a short cell cycle and extraordinary rapid
proliferation potential, adult stem cells are characterized by their relative proliferative
quiescence. Specifically, HSCs are largely found in the G
0
or G
1
phase of the cell cycle.
Using 5-bromo-2’-deoxyuridine (BrdU; a synthetic analogue of thymidine that can be
incorporated into newly synthesized DNA and identified with specific antibodies)
incorporation to study cell-cycle kinetics of HSCs, it was shown that while only 8% of
LT-HSCs (defined as c-Kit
hi
Thy1.1
lo
Sca
+
Lin
-
cells) asynchronously enter cell cycle per
day, by 6 months, 99% of LT-HSCs have divided (Cheshier et al., 1999). According to
this study, 75% of LT-HSCs are quiescent in G
0
at any one time. This quiescent state has
important functional roles in the physiology of HSCs to maintain the stem cell population
and preserve them against toxic injuries. In addition, HSCs have functional limitations
that are usually hastened by proliferative stress. This is most obvious during serial
transplantation experiments when the reconstituting ability of HSCs will gradually
decline and eventually exhaust, as evidenced by studies of Harrison et al. where they
showed that even upon a single transplantation the growth potential of the transplanted
cells in spleen declined 1.5- to 2.5-fold (Harrison et al., 1978). To further examine the
relationship between cell-cycle entrance and HSC self-renewal potential, mouse strains
with different life-spans were studied. It was observed that the pool size of the most
primitive HSC was bigger in the long-lived C57/Bl6 strain compared to that of the short-
lived DBA/2 strain. HSCs from the short-lived DBA/2 strain also had much higher
proliferation potential compared to the C57/Bl6 mice (Phillips et al., 1992). Later, the
same group showed that not only is longer life-span correlated with more stem cells, but
HSCs derived from faster cycling animals (usually short-lived strains) functionally
exhaust more rapidly (de Haan, 1997). The reverse scenario was observed when cells
from older mice from these two strains were compared to each other.
As witnessed by these data, it is clear that the cell cycle entry of these stem cells be
closely regulated to maintain their population. Various genetic mouse models, as listed
below, have been developed to investigate the effect of increasing and/or decreasing cell
cycle entry on stem cell self-renewal and function.
1.7.1 Cyclin-dependent kinase inhibitors (CDKIs)
CDKIs interact with cyclin-CDK complexes and block the kinase activity of the CDKs.
There are two major groups of CDKIs: the INK4 family and the CIP/KIP family. Over-
expression of CIP/KIP inhibitors causes G
1
cell cycle arrest, suggesting their preferential
targeting of the G
1
cyclin-CDK complexes (Lee &Yang, 2001). P21
CIP/WAF1
(P21) is one
member of the CIP/KIP family that inhibits kinase activity of cyclinE/CDK2. The role of
P21 in HSC quiescence was first studied in mice whose cell cycle entrance has been
hastened by knocking out the p21 gene (Cheng et al., 2000). These mice had an increased
cellularity and proliferation in normal homeostatic conditions. However, these mice
showed an increased sensitivity of primitive cells to cell-cycle specific myelotoxic
injuries such as 5-FU treatment. They also showed a survival disadvantage during serial
transplantation experiments. Interestingly, it was later shown that p21
-/-
C57/Bl6 mice did
not have such drastic changes in their HSC phenotypes (van Os et al., 2007), which could
be a result of intrinsic cell-cycle differences between various strains, as discussed before.
1.7.2 Growth factor independent 1 (Gfi1)
The zinc-finger repressor Gfi-1 is a cooperating oncogene in lymphoid cells. The Gfi-
1 locus is among the most frequent sites of retroviral integration contributing to the
development of lymphoid tumors in mice (van Lohuizen et al., 1991). It was shown that
in young mice lacking GFI-1, the LSK Flt3
+
HSCs are increased in numbers. These cells
were also more proliferative as shown by BrdU incorporation and cell cycle analysis.
When compared to their WT counterparts, Gfi-1
-/-
HSCs are functionally compromised in
CRAs and serial transplantation assays. The low level of p21 expression was a plausible
explanation of the defects observed in the Gfi-1
-/-
HSCs (Hock et al., 2004).
1.7.3 Phosphatase and tensin homologue (Pten)
PTEN is a dual-specific phosphatase that has activity against lipid and protein substrates.
PTEN is a negative regulator of phosphatidylinositol 3-kinase (PI3K) by targeting
phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3, abbreviated PIP3] and
converting it back to PIP2. Since the PI3K pathway regulates various cellular processes,
such as proliferation, growth, apoptosis and cytoskeletal rearrangement, its inhibitors
including PTEN are usually tumor suppressors. In fact, Pten is commonly deleted or
otherwise inactivated in diverse cancers, including hematopoietic malignancies. To
identify potential therapies to selectively target cancer stem cells, Yilmaz et al.
conditionally knocked out Pten in mice (Yilmaz et al., 2006). These mice showed a
transient increase in HSC numbers followed by a depletion of immunophenotypic HSCs.
The long-term loss of HSCs and engraftment disadvantage in CRAs compared to WT
littermates, were paradoxically accompanied with a myeloproliferative disease and
eventual leukemia in the animals (Zhang et al., 2006). These observations provide
evidence that PTEN is necessary for maintaining HSCs in a quiescent state. Once deleted,
HSCs enter cell cycle leading to a transient increase in the cell numbers but would
eventually exhaust the stem cell pool. However, mutations in a small subset of Pten
-/-
HSCs that have avoided exhastion could cause uncontrolled proliferation and leukemia.
1.7.4 Hoxb4
The Homeobox (HOX) genes encode DNA binding proteins that play a major role in cell
fate decisions. In vitro overexpression of Hoxb4 in cell cultures before transplanting them
into mice led to expansion of these cells in vivo post-transplantation compared to WT
cells and also their competitive advantage in CRAs (Sauvageau et al., 1994;
Thorsteinsdotir et al., 1999). This enhanced functional advantage was maintained through
secondary transplants (Sauvageau et al., 1995; Thorsteinsdotir et al., 1999). Further
investigation of the kinetics of Hoxb4-transduced HSCs revealed that although the
activity of HOXB4 appeared to be cell autonomous and non-transferrable to non-induced
cells, it seems that HOXB4 might require an environment that is only present during the
first few weeks following irradiation and transplantation leading to a fast expansion of
the cells, which is followed by a mild expansion in the next 10 weeks and finally
reaching a maintenance plateau (Antonchuk et al., 2002). Once again, cell-cycle
regulatory genes seem to be the targets of HOX family especially those that are
responsible for transition through G
1
(Del Bene & Wittbrodt, 2005). Specifically, it was
shown that expression of cyclin D2, cyclin D3, and cyclin E, was upregulated during
HOXB4-induced self-renewal of HSCs resulting in a facilitated transition through the
restriction point toward late G
1
phase and therefore a short cell-cycle (Satoh et al., 2004).
Based on the above evidence, it seems that early G
1
phase during cell cycle is a critical
period and the amount of time spent in that phase affect the exposure of the cells to
exhaustion-inducing stimuli present in the cell. In particular, it has been shown that
prolonged signaling through mitogen-activated protein kinase (MAPK) pathway present
in G
1
phase might affect the cell signaling decision and result in differentiation,
senescence or apoptosis, rather than stem cell self-renewal (Marshall, 1995). Therefore,
most genes (p21,Pten and Gfi1) that prevent the entry of quiescent HSCs into cell cycle
preserve their function by limiting their exposure to such exhaustive stimuli in G
1
phase.
At the same time, if the transition period through the early and late G
1
phase is shortened
(as observed in overexpression of Hoxb4 gene), the cell-fate decision is altered and
shifted to more self-renewal because of the limited exposure to exhaustion-inducing
signals (Orford & Scadden, 2008).
1.8 Visualizing HSCs
Revealing how the HSC niche coordinates the signaling pathways that modulate the stem
cells decision to self-renew or differentiate requires dynamic visualization of the stem
cells within their niche in vivo during these processes. However, as mentioned before,
adult HSCs reside within the cancellous bone that is notoriously difficult to image
through. HSCs are also highly motile, trafficking through the peripheral blood (Wright et
al., 2001) and therefore are highly refractory to imaging studies.
Therefore, studies attempting to look into the localization of stem cells and their
interaction with their niche have mostly relied on examining the bones post-mortem via
immunohistochemical analysis. In most of these studies, fluorescently-labeled HSCs
were transplanted into non-ablated WT recipient and were shown to selectively localize
to the endosteal region of the bone 5 hours post-transplantation (Nilsson et al., 2001).
Histone 2B-GFP (H2B-GFP) transgenic mice have also been used in order to examine the
localization of the HSCs (Challen & Goodell, 2008). However, the leaky expression of
GFP in the HSCs prohibits the use of this model to study these cell types in vivo.
Since, HSCs are thought to be in a state of quiescence, BrdU has been used to identify the
label retaining cells (LRCs) that would contain the stem cell populations. These LRCs are
found to be located next to N-cadherin expressing spindle-shaped osteoblasts (Zhang et
al., 2003). But, the nature of these LRCs and whether they actually are HSCs is in
question (Kiel et al., 2007), especially since they fail to express c-Kit (Kubota et al.,
2008). On the other hand, immunohistostaining of the SLAM family markers (CD150
and CD48), which were shown to be reliable markers of HSCs, revealed that most of the
CD150
+
CD48
-
HSCs were associated with the endothelial cells in the diaphysis area of
the bone (Kiel et al., 2005b). As informative as the immunohistochemical analysis is in
studies of the HSC microenvironment, it is very invasive and does not allow dynamic
imaging of the endogenous HSCs that are resident in the niche.
To have a clear picture of the HSC niche and the interactions among the different
components, in vivo imaging of the niche has been sought out. However, there are two
major physical barriers to imaging HSCs in the BM: the bone itself and the muscle and
the skin surrounding the bone. To circumvent these barriers some ex vivo imaging
methods have been developed, where visualization of the HSCs has been attempted in the
bones dissected from the recipients shortly after transplantation has occurred. In one
study, GFP
+
HSCs were transplanted into myeoablated and non-myeloablated mice and
the reconstitution process of these cells were then observed at various time points post-
transplantation in the dissected bones using a fluorescent stereomicroscope (Yoshimoto et
al., 2003). In a similar study, an ex vivo real-time imaging technique was developed to
trace homing of GFP
+
HSCs in the femur four hours after transplantation (Xie et al.,
2009). These studies demonstrated that BM-MNCs preferentially engraft in clusters to the
epiphysis of the bone. Also, HSCs tend to home to the endosteal region of the trabecular
area and interact with osteoblasts in this niche. However, since the bones have been
dissected out of the animal, dynamic imaging of the niche in live animal could not be
achieved.
In vivo imaging can be achieved via two different techniques. In the first method, an
optical window is placed over the distal epiphyses of the femur following detachment of
the skin and quadriceps muscle (Askenasy & Farkas, 2002; Askenasy et al., 2002). Here,
it was confirmed that hematopoietic stem and progenitor cells (HSPCs) engraft in clusters
to the endosteal surface of the epiphyses as early as 24 hours. A similar approach to
image through the bone was taken by removing the muscle and shaving the bone to a
thickness of 30-50 µm that allows better light penetration (Köhler et al., 2009). Using this
method it was reported that aged early hematopoietic progenitor cells (eHPCs) are
present with increased cell protrusion movement in vivo and localize more distantly to the
endosteum compared with young eHPCs. While, these methods provide useful real-time
information about the localization of the stem cells in the niche, the imaging is restricted
to the site of the window and the surgical procedure may contribute to the effects
observed. The second in vivo imaging technique involves imaging the cells in the
calvaria. Here, the bone is so thin that the BM can be macroscopically identified using
two-photon microscopy (Halin et al., 2005; Junt et al., 2007). This imaging technique is
widely used to image live cells in vivo for longer periods of time. Using this technique,
various selectin molecules have been identified to contribute to trafficking of
hematopoietic progenitor cells across the blood vessels (Mazo et al., 1998; Mazo et al.,
2002). Similarly, endothelial microdomains enriched in E-selectin and stromal-cell-
derived factor-1 (SDF-1) were shown to facilitate engraftment of various tumor cell lines
to the BM (Sipkins et al., 2005). Finally, high-resolution confocal microscopy and two-
photon video imaging were used to visualize how individual HSCs interact with niche
constituent cells in the living mice over time (Lo Celso et al., 2009). Here, it was
demonstrated that the HSCs are located in close proximity of osteoblasts and endothelial
cells. Yet, these two niches were indistinguishable in the calvarium BM. However, the
question that remains unanswered in all these studies is how relevant the calvarium BM is
to the BM in other bones. The calvaria is a spatially and anatomically different bone from
the tibia and femur, and while all functional analysis of the HSC niche is performed using
the cells from these long bones, the exact nature of the HSC niche in the calvarium is yet
to be clarified. On the other hand, two-photon microscopy lacks the required tissue-
penetration to image through long bones and the auto-fluorescence from the surrounding
tissue further restricts the use of this method to a depth of 150µm, which means that only
HSCs 60µm from the endosteal surface can be reliably visualized (Lo Celso et al., 2009).
Other methods that will yield the required depth to image into the BM of live animal
without surgical interventions include bioluminescence animals and magnetic resonance
imaging (MRI). However, these methods lack the resolution that is reliable enough to
draw any conclusion about the cell-cell interactions within the niche (Daldrup-Link et al.,
2005; Partlow et al., 2007; Schroeder, 2008). Also, the long imaging time prohibits
dynamic imaging required to visualize highly motile HSCs (Maxwell et al., 2008).
Chapter 2: Materials and Methods
2.1 Subjects
Six- to eight-week-old WT male C57Bl/6 and B6.SJL mice (Taconic, Oxnard, CA) were
obtained and used in accordance with the University of Southern California Institutional
Animal Care and Use Committee (IACUC) guidelines. Mice were housed in sterilized
microisolator cages (5 mice per cage) and received autoclaved food and water ad libitum.
To sacrifice the mice, they were placed in a standard CO
2
chamber attached a pressurized
CO
2
tank. The mice were exposed to the gas for 5 minutes to attain complete asphyxia.
Inhalation of CO
2
was followed by cervical dislocation to ensure sacrifice of the animals.
2.2 Bone Harvesting
Sacrificed mice were pinned down to the dissection board and doused in 70% ethanol to
sterilize. All the surgical tools were sterilized using 70% ethanol. Fur and skin from legs
were removed by lifting skin at the base of each leg with tweezers and cutting away skin
across thigh and down to ankle. The skin was peeled down the leg and over foot and
firmly tugged until it is removed. The muscles were removed from the entire leg so that
the bones are completely exposed. The entire leg was removed by cutting above the hip
joint ensuring to keep the top of the femur. The bones were cleaned off the remaining
muscle and placed in Myelocult
®
M5300 culture medium (StemCell Technologies,
Vancouver, BC) containing 10% Penicilin/Streptomycin (Cellgro
®
by Mediatech, Inc.,
Manassas, VA). The either ends of the tibia and femur were cut using a No.10 scalpel
(Becton Dickinson, Franklin Lakes, NJ) and the BM was flushed out using a 1ml syringe
attached with a 25-guage needle (Becton Dickinson). To remove any remaining bone
fragments or hair, the BM solution was filtered using a 70 µm cell strainer (Becton
Dickinson).
2.3 Cell Cultures
BM cells flushed from femurs and tibias were cultured at a concentration of 5×10
6
cells/ml
at 37
o
C/5% CO
2
in a humidified atmosphere in T-25 flasks (Becton Dickinson)
using Myelocult
®
M5300 culture medium (Stem Cell Technologies) containing 10%
penicillin/streptomycin (Cellgro
®
by Mediatech Inc.). The medium was changed after
three days, and the adherent layer of BM-MNCs was cultured for a further 10 days,
before future experiments were performed using them.
2.4 Colony Forming Unit Cell (CFU-C) Assay
MethoCult
®
GF M3434 (Stem Cell Technologies) medium containing fetal bovine serum,
bovine serum albumin, recombinant human (rh)-insulin, human transferrin (iron-
saturated), 2-mercaptoethanol, recombinant murine (rm)-stem cell factor, rm IL-3, rh
IL-6, rh erythropoietin was defrosted at room temperature and vortexed well. Murine
BM-MNCs were re-suspended in Myelocult
®
M5300 culture medium and were added to
one tube of medium in a total volume of 0.3 ml to yield a final cell concentration 20,000
cells/1.1 ml. The tubes were then vortexed rigorously and let stand until all bubbles had
risen to the top (approximately 10 minutes). A 3ml syringe attached to 18-guage needle
(Becton Dickinson) was then used to plate out the cells (in duplicate) in a volume of
1.1ml per well in a 6 well plate (VWR). Distilled water was added to the empty wells.
The plates were incubated at 37
o
C/5% CO
2
in a humidified atmosphere for 7 days before
they were scored for the number of colonies formed in each well (Figure 2.1).
2.5 Cobblestone Area Forming Cell (CAFC) Assay
The BM stromal cell cultures were trypsinized from the T-25 flasks. To trypsinize the
cells, the medium was first aspirated off the T-25 flask. The cells were washed once with
PBS and then enough trypsin (Cellgro
®
by Mediatech Inc.) was added to the cells to
cover the whole surface of the flask (1.5 ml for T-25 flask or 3 ml for T-75 flask). The
cells were incubated with trypsin for five minutes. The process was then halted by adding
Minimum Essential Medium (MEM) alpha medium (Cellgro
®
by Mediatech Inc.)
Figure 2.1 Colony forming unit cell (CFU-C) assay. CFU-C assay is used to measure hematopoietic
progenitor cell number or activity in vitro.
supplemented with 10% Fetal Bovine Serum [FBS (Cellgro
®
by Mediatech Inc.)] and
10% Penicillin/Streptomycin (this medium will be refered to as 10% -MEM from now
on) in a quantity three times larger than the initial trypsin volume (4.5 ml for T-25 flask
or 9 ml for T-75 flask). The cells were then irradiated at 15 Gray (Gy) using a cesium-
based irradiator to serve as stromal layers for the CAFC assay. The cells were then plated
at a concentration of 2.5x10
4
cells/well in flat bottom 96-well plates. BM-MNCs obtained
from tibias of C57Bl/6 mice were then seeded in two-fold serial dilutions on top of these
stromal layers starting with 10
5
cell/well as the highest concentration in 10% -MEM.
The cells were cultured in a humidified atmosphere at 33°C/5% CO
2
for five weeks. The
presence of cobblestone areas was scored on week 5, and the frequency of CAFCs were
calculated using the L-Calc software (StemCell Technologies) (Figure 2.2).
2.6 Stromal TRACP & ALP Staining
BM-MNCs were trypsinized and plated at a concentration of 100,000 cells/ well in a cell
culture 48-well plate. The Tartarate Resistant Acid Phosphatase (TRACP) and Alkaline
Phosphatase (ALP) staining (TAKARA BIO INC, Japan) were performed as described.
The culture supernatant was discarded and the cells were washed once with sterilized
phosphate buffered saline (PBS). Cells were fixed with 100 µl of fixation solution (the
reagent is ready to be used; no treatment is needed) per well at room temperature for 5
minutes. The fixation solution was then diluted and washed twice, using 1 ml of sterilized
distilled water. The appropriate substrate solutions for acid phosphatase and alkaline
Figure 2.2 Cobblestone area forming cell (CAFC) assay. CAFC assay is used to measure
hematopoietic primitive cell number or activity in vitro.
phosphatase were then prepared according to the instruction. For ALP staining, one tablet
of ALP substrate was dissolved in 10 ml of PBS. The substrate solution is made at least
20 minutes before use to give enough time for complete dissolution. The principle for
staining of alkaline phosphatase is as follows:
For TRACP staining, the premixed substrate in a vial is dissolved in 10 ml of PBS. For
detection of tartarate-resistant enzyme, 0.1 volume of sodium tartarate was added to the
substrate solution. The principle for staining of acid phosphatase is as follows:
The substrate solutions were then added to the fixed cells (100 µl/well). The plates were
then covered and incubated at 37 ºC for 1 hour for reaction. The solution was then
discarded and the cells were washed three times with sterilized distilled water to
terminate reaction. The samples were examined with Nikon Eclipse 50i upright
microscope (Nikon, Melville, NY).
2.7 Flow Cytometry
For flow cytometric analysis of cells, BM-MNCs were stained in PBS (Mediatech Inc.)
with monoclonal fluorescent labeled antibodies at a concentration of 3:200. Following
primary antibody staining, cells were washed and then stained with appropriate
fluorescent labeled streptavidin (Becton Dickinson, Franklin Lakes, NJ) as the secondary
label (if needed) at a concentration of 5:200. All the antibodies used in various
experiments are enlisted in Table 2.1.
At each step, the cells were stained for 15 minutes on ice in the dark. Red cells were then
lysed in 1X FACS lysing solution (Becton Dickinson) at room temperature. The labeled
cells were then fixed in 10% buffered formalin (Harleco, Darmstadt, Germany) and
analyzed by flow cytometry using a LSR II cell flow cytometer (Becton Dickinson). In
order to sort different populations of cells for transplantation or RNA extraction
purposes, once stained hematopoietic stem and progenitor cells were sorted (without
being lysed and fixed) using a FACSAria flow cytometer (Becton Dickinson) based on
established cell surface markers as detailed below.
Antibody Antigen Recognized Clone Attached to
B220
B-Cell Specific
CD45R Isoform
RA3-6B2
CD3e
T-Cell Receptor
(TCR)
145-2C11
CD11b
Macrophage-1 (Mac-
1) Antigen
M1/70
Gr-1
Myeloid
Differentiation
Antigen
RB6-8C5
TER119 Ter-119 Antigen TER-119
Biotin
(Biotinylated
Lineage Panel)
CD117 (c-Kit) Stem Cell Factor 2B8
Allophycocyanin
[APC]
CD135 (Flk2) Fetal Liver Kinase 2 A2F10.1 Phycoerythrin [PE]
CD150
CD150 Antigen:
Signaling
Lymphocyte
Activation Molecule
(SLAM)
TC15-12F12.2
Phycoerythrin-Cy7
[PE-Cy7]
CD41 Fibrinogen Receptor MWReg30 Biotinylated
CD45 Fetal Liver Kinase 2 A2F10.1 Phycoerythrin [PE]
Table 2.1 Antibody list. All the antibodies used in the experiments and the most commonly used fluorescent
colors attached to them are listed above
For flow cytometry analysis, using Lin, c-Kit, Sca-1 and Flk2, one million events were
recorded for each sample. The consecutive gating of the samples is shown in the figure
2.3. Briefly, the BM-MNCs were chosen based on the forward and side scatter (FSC and
SSC) plots, excluding the debris that had low forward and side scatter. Those cells were
then plotted based on expression of c-Kit and lineage surface markers. The Lin
-
c-Kit
+
cells, which include hematopoietic stem and progenitor cells were then chosen and
further plotted based on Sca-1 and Flk-2 expression.
Antibody Antigen Recognized Clone Attached to
CD45
Leukocyte Common
Antigen (LCA)
30-F11
Allophycocyanin-
Cy7 [APC-Cy7]
CD45.1/Ly5.1
LCA expressed by
SJL/J strain
A20 Phycoerythrin [PE]
CD45.2/Ly5.2
LCA expressed by
C57BL/6 strain
104
Fluorescein
isothiocyanate
[FITC]
CD48
CD48 Antigen
(BCM1)
HM48-1
Fluorescein
isothiocyanate
[FITC]
Sca1 Stem Cell Antigen 1 E13-161.7 Phycoerythrin [PE]
Streptavidin Primary Antibodies
N/A
[PE-Cy5]
Table 2.1 Antibody list, continued. All the antibodies used in the experiments and the most
commonly used fluorescent colors attached to them are listed above.
For another series of experiments, using the SLAM markers to isolate HSCs, the same
number of events was collected and various hematopoietic stem and progenitor cell
populations were identified based on differential expression of SLAM cell surface
markers. The gating strategy is outlined in figure 2.4.
Figure 2.4 Flow cytometric analysis of hematopoietic cells using SLAM markers. CD45, Lineage, c-Kit,
Sca-1 and SLAM markers were used for immuno-phenotypic analysis of hematopoietic cells.
Figure 2.3 Flow cytometric analysis of hematopoietic cells using Lin, c-Kit, Sca-1 and Flk2 markers.
Lineage, c-Kit, Sca-1 and Flk2 markers were used for immuno-phenotypic analysis of hematopoietic cells.
2.8 Competitive Repopulation Assay
BM-MNCs from the bones of interest were obtained from the donor C57Bl/6 (CD45.2)
mice and the competitor B6.SJL (CD45.1) mice. 250,000 BM-MNCs or 500 purified
HSCs from the donor were mixed with 500,000 BM-MNCs from the competitor mice and
a total volume of 0.2 ml was injected into the tail vein of B6.SJL recipient mice that were
irradiated at 9 Gy less than 24 hours prior transplantation (Figure 2.5). For the tail vein
injections, the recipient mouse was heated up using a heating lamp for 5 minutes to
ensure vasodilation. The mouse was then placed in a restrainer with their tail accessible.
The veins on the lateral aspect of the mouse's tail are an excellent site for intravenous
injections since the principal function of these veins is for thermoregulation and they will
dilate when the mouse’s body temperature rises to disseminate heat. 27-gauge needles
were used to do the injections. Once the vein was located, the needle was inserted slowly,
visualizing the needle as it entered the vein. Once the vein's wall was penetrated, the
needle was directed cranially approximately 5 mm. The injection was then performed
slowly to avoid vascular overload or rupture of the vein from excess pressure. Pressure
was applied over the injection site by gently holding an alcohol wipe over the injection
site for approximately 30 seconds to prevent hematoma formation.
Engraftment levels and multilineage reconstitution were measured by monitoring the
levels of donor cell contribution to host hematopoiesis. This was achieved by obtaining
peripheral blood samples from the tails of recipient mice starting at week 4, for a total of
24 weeks. PE Anti-mouse CD45.1, FITC anti-mouse CD45.2, APC anti-mouse CD3e,
APC-Cy7 anti-mouse CD11b, and biotin anti-mouse B220 antibodies (all from Becton
Dickinson) were used to stain the peripheral blood samples in 1X PBS, at a concentration
of 3:200. PE-Cy5 streptavidin was used as secondary antibody staining, as described
previously in section 2.5. Cells were then incubated in 1X FACS lysing solution (Becton
Dickinson) for 1 minute at room temperature and re-suspended in 10% buffered formalin.
The levels of donor cell contribution were measured by flow cytometry using an LSR II
flow cytometer.
Figure 2.5 Competitive repopulation assay (CRA). CRA is used to measure primitive cell activity in vivo.
2.9 Histological Analysis
After removal of the skin tissue, whole legs from treated mice were fixed overnight in
10% Formalin at 4
o
C. Muscle tissue was then dissected away from the tibia and the bone
was fixed again overnight in 10% Formalin at 4
o
C. Decalcification followed by either
20% Ethylenediaminetetracetic acid (EDTA) in PBS, changed every other day, over 1
two-week period or Immunocal (Decal Corporation Group, Tallman, NY) over a three-
day period. The samples were then processed using the Tissue-Tec Vip-6 (Sakura Finetek
USA Inc., Torance, CA). Tibias were then embedded frontal ridge side down in paraffin.
Whole bone sections were then cut via microtome at 5µm and were used for further
staining.
2.9.1 Hematoxylin and Eosin (H&E) Staining
H&E staining was performed according to standard Harris Hematoxylin & Eosin Y
progressive staining method. Briefly, the slides were warmed up overnight at 47
o
C and
de-paraffinized by Xylene (3 x 2 mins) and 100% Ethanol (2 x 1 min), then rehydrated in
95% Ethanol (1 min) and distilled Water (2 x 1 min). Slides were then stained in Harris
Hematoxylin (Harleco) for 1.25 minutes, which was followed by running tap water (1
min) and distilled water (2 x 1 min) to blue the nuclei and remove excess hematoxylin.
The slides went through one last round of 95% ethanol (1 min) before they were stained
with Eosin Y for 15 seconds. Excess Eosin Y (Harleco) stain was removed by 95%
Ethanol (2 x 1 mins) and slides were gradually dehydrated again in 95% Ethanol (2 x 30
secs), 100% Ethanol (5 x 1 min) and Xylene (3 x 2 mins). The slides were then cover
slipped using Cytoseal XYL mounting medium (Thermo Scientific, Waltham, MA) and
examined using Nikon Eclipse 50i upright microscope.
Chapter 3: Alternative Methods of Activation of the
Hematopoietic Stem Cell Niche Reveal a Spatial Localization
of Primitive Cell Subsets in the Adult Bone Marrow
3.1 Abstract
Osteoblasts are key constituents of the murine HSC endosteal niche, evidenced by the
fact that increasing their activity leads to an increase in the number of HSCs. However,
recent studies have also suggested a role for the bone resorbing osteoclast in the niche.
We pharmacologically activated osteoclasts and osteoblasts in the bones using receptor
activator of nuclear factor- B ligand (RANK-L) and parathyroid hormone (PTH),
respectively. We performed histological analyses to study the effect of these treatments
on bone composition and examined the effects on different HSC sub-populations. We
revealed that these treatments differentially affected the HSC sub-populations and that
these changes are specific to the region of bone analyzed. More specifically, the most
primitive HSCs were expanded presumably at the endosteal surface of cortical bone in
response to RANK-L treatment, whereas the more differentiated short-term HSCs or
progenitor cells are increased in the metaphysis region of the bones upon PTH treatment.
These data suggest a possible structural organization for the localization of primitive
hematopoietic cell subsets in distinct regions of the bone marrow, and therefore have
implications in stem cell based therapies aimed specifically at expanding distinct HSC
subsets.
3.2 Introduction
Within the BM, HSCs reside in close proximity to the endosteum in the endosteal HSC
niche (Scofield, 1978; Lord et al., 1975, Gong, 1978; Nilsson et al., 2001). The border
between the bone and the BM is also where bone remodeling is dynamically occurring by
constant interaction between the osteoblasts and the osteoclasts. During bone resorption,
the activity of these cells is intrinsically connected, as cells of the osteoblastic lineage
secrete RANK-L to promote differentiation of the osteoclasts (Teitelnaum et al., 2003).
Similarly, osteoclasts modulate the activity of osteoblasts through the induction of
retraction of the cells (Perez-Amodio et al., 2004).
There are multiple lines of evidence that implicate a role for the osteoclasts in the HSC
niche in addition to the osteoblasts. First, specific stimulation of osteoblasts through the
PTH receptor also increases osteoclast cell number (Calvi et al., 2001). Second, HSCs
potentially use extracellular calcium as a signal to localize to the HSC niche (Adams et
al., 2006), which would presumably be found close to sites of active bone resorption by
the osteoclasts. More direct evidence has come from recent studies where it has been
demonstrated that inhibition of osteoclast function reduces HSC numbers in vivo
(Lymperi et al., 2011). Mice treated with the bisphosphonate alendronate, which inhibits
bone resorption by the osteoclasts, displayed a reduced number and frequency of HSCs in
the BM. Also, treatment of mice with alendronate both reduced the ability of transplanted
HSCs to engraft in the BM and abolished the ability of PTH to enhance the number of
HSCs in vivo. These data highlight that osteoclasts, possibly through their interaction
with osteoblasts, are a key component of the HSC niche.
Taken together, these data demonstrate that the HSC niche may be closely linked with
sites of active bone remodeling, yet the actual location of the HSC niche in the BM
remains unknown. Whether the endosteal surface of the cortical bone or the trabecular
bone of the metaphysis regions of bones, or both, represent the actual location of the
HSCs is still subject to speculation. That there may be a spatial regulation of primitive
hematopoietic cells has been shown by Haylock and colleagues who demonstrated that
hematopoietic stem and progenitor cells isolated from the endosteal niche have superior
hematopoietic potential compared to their central counterparts (Haylock et al., 2007), a
finding that is supported by previous studies
4
. More recently, they analyzed stem cells
isolated from these different regions and showed that immunophenotypically purified
HSCs within the endosteal BM region have higher proliferative capacity and homing
efficiency compared to the same population isolated from the central BM suggesting that
HSCs show functional differences depending on their origin within the BM (Grassienger
et al., 2010).
To further understand the interaction between the stem cells and the niche, we
pharmacologically activated osteoblasts and osteoclasts and demonstrated that
manipulating the HSC niche in different manners affected primitive cell subsets
differently. We further provide evidence for the localization of primitive hematopoietic
cells according to the specific region of the bone, with the more primitive HSCs
preferentially localizing to the endosteal surface of the cortical bone, while the more
differentiated ST-HSCs specifically localized in the trabecular area of the metaphysis of
the long bones.
3.3 Materials and Methods
3.3.1 Animals
Six- to eight-week-old WT male C57Bl/6 and B6.SJL mice (Taconic, Oxnard, CA) were
obtained and used as detailed in section 2.1.
3.3.2 In vitro treatment of BM stromal cell cultures
Femurs and tibias were dissected from 6 to 8 week-old male C57Bl/6 mice as described
in section 2.2 and the BM-MNCs were flushed from the central cavity with M5300 long-
term culture medium containing penicillin/streptomycin. BM-MNCs were cultured at a
concentration of 5x10
6
cells/ml at 37
o
C/5% CO2 in a humidified atmosphere and treated
with 0.1µM rat PTH (1-34) (Bachem Bioscience, Torrance, CA), 50ng/ml recombinant
murine RANK-L (Peprotech, Rocky Hill, NJ) or equal volume PBS control. The cell
cultures were maintained for 2 weeks, changing the medium every 2 to 3 days.
3.3.3 TRACP and ALP staining
Adherent BM stromal cells were trypsinized and plated at a concentration of 1x10
5
cells/well in a 48-well plate. TRACP and ALP staining was performed using TRACP and
ALP double-stain kit (TAKARA BIO INC, Japan) according to the manufacturer’s
instructions as detailed in section 2.6.
3.3.4 Osteoclastogenesis from splenocytes
Spleens were isolated from 6 to 8 week-old male C57Bl/6 mice and homogenized in
MEM-Hank’s salts medium (Gibco, Carlsbad, CA). The homogenate was filtered through
a 70µm filter, red cells were lysed, and the MNCs were cultured (3 spleens to a 100-mm
dish) with -MEM medium (Cellgro, Manassas, VA) containing 10% FBS and 100ng/ml
macrophage colony stimulating factor (M-CSF; PeproTech). After 4 days, the
macrophage cultures were trypsinized, re-plated in 96-well plates (1.5x10
4
cells/well) in
-MEM medium containing 10% FBS, 30ng/ml M-CSF and 100ng/ml RANK-L and
maintained for a further 5 days.
3.3.5 CFU-C assay
BM-MNCs were obtained from the hind-limbs of C57Bl/6 mice and resuspended in
MethoCult
TM
GF M3434 (StemCell Technologies) with added RANK-L (50ng/ml), PTH
(0.1mM) or saline control. CFU-C assay was performed using these cells as detailed in
section 2.4 and the number of CFU-Cs were scored at day 10 according to standard
criteria.
3.3.6 CAFC assay
CAFC assay was performed, according to section 2.5, using the treated BM cell cultures
as stromal layers. Upon seeding the stromal layers with BM-MNCs obtained from
C57Bl/6 mice, cultures were maintained in a humidified atmosphere at 37°C/5% CO2 and
the presence of CAFCs were scored on week 5. The frequency of CAFCs was calculated
using L-Calc software (StemCell Technologies). When splenocyte-derived osteoclasts
were used in combination with control cell cultures, a one-to-one mixture of the two
different cell cultures were made and plated at a concentration of 2.5x10
4
cells/well in
96-well plates and then irradiated at 15 Gy.
3.3.7 In vivo treatment.
For PTH treatment, mice were injected with rat PTH (1-34) at a dose of 40µg/kg three
times per day for 10 days. Murine RANK-L (6µg) was injected twice a day for 5 days. In
all experiments control mice received sterile saline injections. All the injections were
performed intraperitoneally (Figure 3.1).
Figure 3.1 In vivo treatment plan. C57/Bl6 mice were treated with RANK-L (6 µg/BID for 5 days), PTH
(40 µg/Kg/TID for 10 days), or were control treated with saline.
3.3.8 Flow cytometry and cell sorting
BM-MNCs were stained and analysed with fluorescent labeled antibodies as described in
section 2.7. To sort different population of cells for transplantation, once stained
hematopoietic stem and progenitor cells were sorted using a FACSAria flow cytometer
(Becton Dickinson) based on established cell surface markers as described in section 2.7.
3.3.9 CRA
BM-MNCs were obtained from treated C57Bl/6 (CD45.2, donor) and non-treated B6.SJL
(CD45.1, competitor) mice. 200,000 cells from the donor mice and 500,000 cells from
the competitor were co-injected into the tail vein of B6.SJL mice that were lethally
irradiated with 9 Gy approximately 24 hours prior to transplantation. For the purified cell
transplantation, 100 LSKF
-
cells were injected into the tail vein of lethally irradiated
B6.SJL mice along with 500,000 competitor cells from a B6.SJL mouse. Engraftment
levels and multi-lineage reconstitution were assessed by monitoring the levels of donor
cell contribution to host hematopoiesis as described in section 2.8.
3.3.10 Histological analysis
Dissected tibias were fixed overnight in 10% formalin at 4
o
C. The bones were then
decalcified with EDTA over a two-week period and were processed and paraffin
embedded using standard histological procedures detailed in section 2.9. Longitudinal
bone sections were then cut at 5mm and used for further staining. H&E staining was
performed according to standard methods (section 2.9.1). TRACP staining and toluidine
blue staining were performed on the bone sections according to standard protocols
described below.
3.3.10.1 TRACP Staining
TRACP staining on the bone sections were done using TRACP and ALP double-stain kit
(TAKARA BIO INC, Japan). The slides were warmed up overnight and deparaffinized as
previously described (section 2.9.1). The TRACP substrate solution was then applied to
each section and the slides were incubated in 37
o
C for 60-90 minutes. The substrate was
removed and discarded and the slides were washed three times with sterilized distilled
water to terminate the reaction. The slides were then counter stained with Harris
Hematoxylin (30 sec) followed by washed with tap water and distilled water. They were
then dehydrated in 100% Ethanol (5x1 min) and Xylene (3x2 mins) and finally
coverslipped using Cytoseal XYL (Thermo Scientific) mounting medium and examined
using Nikon Eclipse 50i upright microscope. For osteoclast quantification, TRACP
+
cells
were identified as red stained cells (Figure 3.2.A) and counted in 3 consecutive
microscopic fields (at 4X) within the diaphysis area of the bone sections starting in the
region one section below the growth plate. In the metaphysis area, TRAP
+
cells were
counted across the whole bone section (at 20X) in the trabecular bone region right below
the growth plate.
3.3.10.2 Toluidine Blue Staining
Toluidine blue staining was performed on sections according to standard protocols.
Briefly, once deparaffinized, the bone sections were rehydrated in graded ethanols: 95%
ethanol (2x3 min), 70% ethanol (1x3 min), 40% ethanol (1x3 min) and then in tap water
(1x5 min). The sections were then stained in toluidine blue (1x5 min) and rinsed in water.
The sections were then blotted thoroughly (both sides) and passed through Butanol
(twice), Butanol/Toluene (once) and Toluene (twice) (all from EMD Millipore, Billerica,
MA) and finally cover slipped with toluene-based media Cytosol 60 (Thermo Scientific).
The toluidine blue stain is made according to the following protocol:
Buffer: Citric Acid 1.58 gr
Disodium Phosphate 0.75 gr
DH
2
O 1 L
pH to 3.7
Stain: Dissolve Toluidine Blue O 2gr
In Buffer (above) 100 ml
(Filter, adjust pH to 3.7 and store at room temperature)
The slides were examined using Nikon Eclipse 50i upright microscope and osteoblasts
were identified as plump cells lining the bone surface with elongated nuclei (Figure
3.2B). For quantification purposes, the osteoblasts were counted in 3 consecutive
microscopic fields (at 20X) within the diaphysis area of the bone sections starting in the
region one section below the growth plate. In the metaphysis area, osteoblasts were
counted in 3 consecutive microscopic fields (at 20X) in the trabecular bone region right
below the growth plate starting at the medial section of the bone.
3.3.11 Statistical analysis
Comparison of experimental groups was performed using the unpaired two-tailed
Student’s t-test as appropriate for the data set. A p-value of <0.05 was considered
significant.
Figure 3.2 Histostaining
of osteoclasts and osteoblasts. Osteoclasts and osteoblasts were quantified using
TRACP and Tolouidine Blue staining of the bone sections. The bone sections were stained for TRACP and
Toloudine Blue. Representative photomicrographs show (A) TRACP
+
cells (arrowheads) and (B)
osteoblasts (arrowheads) along the endosteal surface of the bone.
3.4 Results
3.4.1 Increased osteoclast cell number in BM stromal cell cultures augments the
support of primitive hematopoietic cells.
Although previous reports demonstrated that inhibiting osteoclast function leads to a
decrease in HSC frequency (Lymperi et al., 2011), it has not been established whether
activating osteoclast function leads to an increase in HSCs. Therefore, we first wished to
compare the effects of an osteoclastic specific activator (RANK-L) to an osteoblastic
specific activator (PTH) in the ability of BM stromal cells to support primitive
hematopoietic cells in vitro. To verify the effects of the different stimulatory factors on
osteoclast and osteoblast activity, we examined TRACP and ALP activity in the treated
BM stromal cell cultures in vitro. When cells were treated by either of these factors,
osteoclastic activity of the cell cultures were elevated as shown by an increase in TRACP
staining, although RANK-L stimulated the cells to a higher degree (greater than 3-fold
compared to control) (Figure 3.3, 3.4A). Similarly, osteoblastic activity was significantly
elevated when cell cultures were treated by both RANK-L and PTH, as evidenced by an
increase in ALP staining when compared to control cultures (Figure 3.3, 3.4B).
Figure 3.3 TRACP and ALP activity of treated BM-MNC cultures. Stromal cell cultures were stained for
TRACP and ALP activity. Representative staining performed in triplicate of 3 independent experiments is
shown.
Figure 3.4 Quantification of TRACP
+
and ALP
+
cells in treated BM-MNC cultures. (A) TRACP
+
and (B)
ALP
+
cells were counted in 2 microscopic fields (at 40X) for each repeat in each experiment (n= 9; from 3
independent experiments). Data represent the mean ± s.e.m.
We next investigated the ability of these treated cultures to support primitive
hematopoietic cells in vitro. Using the CAFC assay, the highest frequency of CAFCs was
found when BM-MNCs were cultured on RANK-L treated stromal layers (Figure 3.5A).
To rule out the possibility that the results observed is due to a direct effect of the
treatments on hematopoietic cells, we performed the CFU-C assay. As shown in Figure
3.5B, RANK-L and PTH treatment had no effect on in vitro growth potential of the
cultures.
We then examined whether osteoclasts alone are able to maintain HSCs. A population of
osteoclasts devoid of osteoblasts was derived from spleen mononuclear cell cultures
(Figure 3.6A). When these cell cultures were used as stromal layers in a CAFC assay,
they were unable to support primitive hematopoietic cells in vitro at any time point (data
Figure 3.5 Effects of in vitro treatments on growth and functional potential of BM-MNCs. (A) CAFC
assays were performed with treated BM-MNC cultures serving as the stromal layers (n=7-11; from 4
independent experiments). (B) CFU-C assay was performed on BM-MNCs treated with RANK-L or PTH
and was compared to control cultures (n = 7 from 3 independent experiments). Data represent the mean
± s.e.m.
not shown). However, when these cells were added to control cultures, these stromal
layers were able to better support hematopoietic cells (Figure 3.6B).
Together, this data suggests that increased osteoclastic cell activity leads to an enhanced
support of primitive hematopoietic cells, although the coordinated interaction of
osteoblastic and osteoclastic activity may be required to maintain HSCs in vitro.
3.4.2 In vivo RANK-L and PTH treatments have differing effects on BM stromal cell
composition.
To examine the effects of altering the stromal composition of the HSC niche in vivo, we
used a modified shortened treatment regimen, as compared to previous publications
(Calvi et al., 2003; Adams et al., 2007), outlined in Figure 3.2. To validate the effects of
this treatment regimen on the stromal cell composition, we performed histochemical
Figure 3.6 Splenocyte-derived osteoclasts enhance hematopoietic support of BM-MNC cultures.
Osteoclasts were derived from spleen cultures and then stained for ALP and TRAP activity. (A)
Representative picture of the TRACP and ALP staining performed on these splenocyte-derived osteoclasts
(representative of 3 independent experiments). (B) CAFC assays were performed with splenocyte-derived
osteoclasts added to control BM-MNC cultures serving as the stromal layers (n=4; from 2 independent
experiment). Data represent the mean ± s.e.m.
*
p<0.05 compared to the control group.
analysis of tibia sections. RANK-L treated mice showed a slight decrease in the amount
of bone in the trabecular area compared to controls due to osteoclastic resorption.
However, when mice were treated with PTH, an increase in the amount of bone
formation in the trabecular area was observed as evidenced by H&E staining (Figure 3.7).
Next, we examined whether these treatments affected the osteoclast and osteoblast
populations in the BM of the treated mice. Using expression of TRACP as a marker of
osteoclasts, we found that RANK-L treated mice demonstrated an unchanged number of
Figure 3.7 Effects of In vivo RANK-L and PTH treatments on bone structure. H&E (top), TRACP
(middle) or Toluidine Blue staining (bottom) of sections of paraffin-embedded decalcified tibias (original
magnification 4X).
TRACP
+
cells while PTH treated mice had a higher number in the trabecular area (Figure
3.8A). Toluidine blue staining was used to examine the osteoblastic population in the
bone section. In the RANK-L treated mice, we found no change in the number of
osteoblastic cells in the trabecular bone region, while PTH treated mice showed an
increase relative to control mice (Figure 3.8C). We then quantified the number of
TRACP
+
cells and osteoblasts along the endosteal surface of the cortical bone, where
HSCs have been shown to be located (Lord et al., 1975; Nilsson et al., 2001). Both
RANK-L and PTH treated mice displayed a significant increase in the number of TRAP
+
cells at the endosteal surface of the cortical region of the bone (Figure 3.8B).
Interestingly, the osteoblast numbers also followed the same trends; RANK-L and PTH
treatment increasing the number of osteoblasts at the endosteal surface of the cortical
bone (Figure 3.8D). These data suggests that the effects of our treatment regimens are
different according to region of the bone, with RANK-L treatment having no effect or
leading to a reduction in trabecular bone in the metaphysis, but an increase in bone
remodeling activity at the endosteal surface of the cortical bone in the diaphysis, while
PTH treatment increases bone formation and remodeling activity both in the diaphysis
and the trabecular bone of the metaphysis.
3.4.3 Primitive hematopoietic cell subset specific effects of RANK-L and PTH
treatment in vivo.
To address how the changes in the bone structure and stromal cell composition affected
the primitive hematopoietic stem and progenitor cells in the BM in vivo, we
immunophenotypically analyzed BM-MNCs using flow cytometry. To quantitate the
frequency of LT-HSCs, ST-HSCs, and MPPs, we analyzed the Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
+
CD48
-
(LSKCD150
+
CD48
-
), Lin
-
Sca-1
+
c-Kit
+
Flk-2
-
(LSKF
-
), LSKCD150
-
CD48
-
Figure 3.8 Effects of In vivo RANK-L and PTH treatments on BM stromal cell composition In vivo.
Quantification of TRACP
+
cells in (A) the trabecular bone region of metaphysis and (B) at the endosteal
surface of cortical bone. Quantification of osteoblasts in (C) the trabecular bone region of metaphysis and
(D) at the endosteal surface of cortical bone (n=19-21; from 2 independent experiments). Data represent
the mean ± s.e.m.
**
p<0.001 compared to the control group.
or LSKF
+
populations of cells, respectively (Christensen et al., 2001; Kiel et al., 2005b).
Following RANK-L treatment, we observed a significant increase in the frequency of the
most primitive LSKCD150
+
CD48
-
and LSKF
-
populations of hematopoietic cells in the
BM (Figure 3.9A,B). In contrast, following PTH treatment no effects were seen in the
frequencies of the primitive HSCs, yet we observed significant increases in the
frequencies of the more differentiated LSKCD150
-
CD48
-
and LSKF
+
ST-HSCs or MPPs
(Figure 3.9C,D).
Analysis of the more differentiated Lin
-
c-Kit
+
or LSK
-
F
+
cells, which represent the
committed progenitor populations, did not demonstrate any specific alteration in terms of
cell frequencies following any treatment (Figure 3.10).
Figure 3.9 Effects of In vivo RANK-L and PTH treatments on hematopoietic stem and progenitor cell
populations. BM-MNCs were harvested from the tibias of the treated mice and labeled with hematopoietic
cell surface markers (Lin, c-Kit, Sca-1, Flk2, CD41, CD48, and CD150). The cells were then analyzed by
flow cytometry. Measurement of (A) Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
+
CD48
-
(LSK CD150
+
CD48
-
), (B) Lin
-
Sca-1
+
c-Kit
+
Flk2
-
(LSKF
-
), (C) Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
-
CD48
-
(LSKCD150
-
CD48
-
), and (D) Lin
-
Sca-1
+
c-Kit
+
Flk2
+
(LSKF
+
). Cell content of BM-MNCs represented as percentage of BM-MNCs (n=20;
from 4 independent experiments). Data represent the mean ± s.e.m.
We then examined the changes in primitive hematopoietic cells function by CRA.
Following transplantation of an excess of competitor cells over the BM-MNCs from
control, RANK-L or PTH treated mice, we observed an increased level of short-term
engraftment (weeks 4 to 8) from BM-MNCs of PTH treated mice. No significant
differences were observed at these time points for cells from the RANK-L treated mice
compared to controls (Figure 3.11).
Figure 3.10 Effects of In vivo RANK-L and PTH treatments on committed progenitor cell populations.
BM-MNCs were harvested from the tibias of the treated mice and labeled with primitive hematopoietic cell
surface markers. (A) Lin
-
Sca-1
-
c-Kit
+
Flk2
+
(LS
-
KF
+
) and (B) Lin
-
c-Kit
+
hematopoietic progenitor cells
(n=20 from 4 independent experiments). Data represent the mean ± s.e.m.
However, examination of long-term engraftment (weeks 12 to 24) demonstrated
significant increases from BM-MNCs from both the RANK-L and PTH animals (Figure
3.11), which were evident in both lymphoid and myeloid lineages (Figure 3.12).
Figure 3.11 Effects of In vivo RANK-L and PTH treatments on primitive hematopoietic cell function.
BM-MNCs from treated C57Bl/6 mice were transplanted in a competitive repopulation assay into B6.SJL
recipients. Flow cytometric analysis of peripheral blood was performed every 4 weeks up to 24 weeks post-
transplantation. Measurement of whole BM-MNC engraftment levels based on percentage of CD45.2 cells
present in the peripheral blood (n=10-12; from 3 independent experiments). Data represent the mean ±
s.e.m.
**
p<0.001 compared to the control group. †p<0.001 compared to the control group when all
treatment groups had a p< 0.001.
While these data closely mirrored that observed for the immunophenotypic analysis of
RANK-L treated mice, there was a discrepancy between the number and function of the
most primitive LT-HSCs in the PTH treated mice. To address whether the in vivo
treatments resulted in alterations in the function of the primitive hematopoietic cells, we
repeated the competitive repopulation analysis using equal numbers of purified LSKF
-
cells. Analysis of the engraftment of these purified cells demonstrated that LSKF
-
cells
Figure 3.12 Multi-lineage engraftment of BM-MNCs isolated from RANK-L and PTH treated mice.
Whole BM-MNCs were harvested from tibias of the treated C57Bl/6 mice and co-transplanted with BM-
MNCs from a B6.SJL mouse into B6.SJL recipients in a competitive manner. Flow cytometry analysis of
peripheral blood was performed every 4 weeks up to 24 weeks post-transplantation. Measurement of (A)
myeloid , (B) B-lymphoid, and (C) T-lymphoid engraftment levels based of percentage of CD45.2 cells
present in each lineage in the peripheral blood (n=10-12; from 3 independent experiments). Data
represent the mean ± s.e.m. **p<0.001 compared to the control group. †p<0.001 compared to the control
group when all treatment groups had a p<0.001.
from various groups had no engraftment advantage compared to the control LSKF
-
cells
suggesting that the changes observed in the whole BM-MNC engraftment levels are due
to changes in primitive HSC cell numbers as oppose to their function (Figure 3.13).
The reasons for the discrepancy in the frequency of LT-HSCs identified by
immunophenotypic versus functional analysis following PTH treatment are not clear.
However, since the competitive repopulation assay is the most rigorous test of HSC
activity, we concluded that PTH treatment increased both LT-HSC and ST-HSC
frequencies.
Figure 3.13 Effects of in vivo RANK-L and PTH treatments on primitive hematopoietic cell function.
Purified LSKF
-
cells from treated C57Bl/6 mice were transplanted in a competitive repopulation assay into
B6.SJL recipients. Flow cytometric analysis of peripheral blood was performed every 4 weeks up to 24
weeks post-transplantation. Measurement of LSKF
-
cells engraftment levels based on percentage of
CD45.2 cells present in the peripheral blood (n=4-5). Data represent the mean ± s.e.m.
3.4.4 Primitive LT-HSCs and the more differentiated ST-HSCs or MPPs localize to
distinct regions of the BM.
From our data, we observed an enhanced osteoclast and osteoblast number at the
endosteal surface of the cortical bone in the diaphysis following both RANK-L and PTH
treatment (Figure 3.8B,D; Table 3.1), yet we only observed an increase in osteoclast and
osteoblast number in the trabecular bone region of metaphysis following PTH treatment
(Figure 3.8A,C). Correlating with this was our observations that LT-HSC frequency was
increased following RANK-L and PTH treatment, yet an increased ST-HSC/MPP
frequency was only observed following PTH treatment (Figure 3.9; Table 3.1).
These data suggested a possible regional localization of the primitive hematopoietic cells,
with the LT-HSC niche localized to the endosteal surface of cortical bone and the ST-
Treatment Bone Region Affected HSC Type Affected
PTH
Diaphysis
Metaphysis
LT- HSC
ST-HSC
Rank Ligand Diaphysis LT-HSC
Table 3.1 Summary of in vivo results. Osteoclasts and osteoblasts numbers were increased in the
diaphysis area in response to PTH and Rank Ligand, which was associated with an elevated
frequency of LT-HSCs in that region. Osteoclasts and osteoblasts numbers were increased in
the metapysis area in response to PTH which in turn was correlated with increased ST-
HSC/MPP numbers in that area.
HSC/MPP niche localized to the trabecular bone of the metaphysis. To examine this
directly, mice were treated in vivo as before, then the tibias were dissected out and cut 3
mm below the proximal head to separate the metaphysis and the diaphysis regions of the
bone (Figure 3.14).
Figure 3.14 Separation of metaphysis and diaphysis sections of the long bones. Tibias from mice that had
been treated with saline control, RANK-L, or PTH were cut 3 mm below the head of the tibia to separate
the metaphysis and the diaphysis regions of the bones. H & E staining of paraffin-embedded sections of
decalcified metaphysis and diaphysis regions is shown.
We then repeated the flow cytometry analysis for the frequency of the primitive
hematopoietic cell subsets. These studies indicated that RANK-L treatment preferentially
increased the primitive stem cell numbers (LSKCD150
+
CD48
-
or LSKF
-
) in the diaphysis
region of the BM (Figure 3.15A,B). On the other hand, PTH treatment specifically
increased the ST-HSC (LSKCD150
-
CD48
-
) population in the metaphysis (Figure 3.15C),
while the MPP (LSKF
+
) cells were expanded equally in both metaphysis and diaphysis of
the bone (Figure 3.15D). Therefore, these data confirm that primitive HSC subsets are
located in distinct regions of the BM and are therefore differentially affected by
alterations of specific regions of the bone.
Figure 3.15 Effects of In vivo RANK-L and PTH treatments on hematopoietic cell population in distinct
bone regions. The tibias from mice that had been treated by RANK-L and PTH were cut 3 mm below the
proximal head of the bone to separate the metaphysis and the diaphysis section of the bones. BM-MNCs
were then harvested from these sections and analyzed for primitive hematopoietic cell surface markers as
described previously. Measurement of (A) Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
+
CD48
-
(LSKCD150
+
CD48
-
), (B)
Lin
-
Sca-1
+
c-Kit
+
Flk2
-
(LSKF
-
), (C) Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
-
CD48
-
(LSKCD150
-
CD48
-
), and (D)
Lin
-
Sca-1
+
c-Kit
+
Flk2
+
(LSKF
+
). Cell content of BM-MNCs represented as percentage of BM-MNCs
(n=20; from 4 independent experiments). Data represent the mean ± s.e.m.
**
p<0.001 compared to the
control group.
3.5 Discussion
While the role of osteoblasts in maintaining the HSC niche in vivo has been extensively
examined (Calvi et al., 2003; Zhang et al., 2003; Visnjic et al., 2004), it has been shown
that global osteoblastic expansion itself is not sufficient to expand HSCs, and that there
may be an additional role for osteoclastic regulation of the HSC niche (Lymperi et al.,
2008). In this study osteoblastic function was enhanced using Sr, an anabolic agent that
also inhibits osteoclast function. While increases in bone formation were observed in Sr
treated mice, no effects were seen in the HSC population. Correlating with this was the
observation that Sr-treated osteoblastic cells did not display an increase in RANK-L
expression, whereas PTH treatment (which increases HSC count in the niche) did.
Previous studies have also attempted to link activation of osteoclasts with regulating
primitive hematopoietic cell number in the BM. Kollet and colleagues demonstrated that
in vivo administration of RANK-L led to mobilization of hematopoietic progenitors,
specifically LSK cells, to the peripheral circulation (Kollet et al., 2006). Interestingly,
following RANK-L treatment they also observed an increase in primitive LSK cells in
the BM, suggesting a role for osteoclasts in regulating primitive hematopoietic cell
number. Here we demonstrate that specific activation of osteoclasts with RANK-L leads
to an enhanced ability of BM stromal cell layers to support primitive hematopoietic cell
activity in vitro, and also led to a specific increase in the most primitive LT-HSCs in vivo.
Taking our data together with these and other studies (Lymperi et al., 2011) demonstrates
that the active presence and coordinated interaction of osteoclasts and osteoblasts are
required for maintaining the HSC niche. Therefore, this suggests that areas of active bone
remodeling are the location for the endosteal HSC niche. During the remodeling process,
mature osteoclasts at the bone surface resorb bone, which in turn activates cells of the
osteoblast lineage to form new bone (Novack & Teitelbaum, 2008). Linking bone
remodeling to HSC physiology in this way may lead to the expansion of the stem cells
during development or in injury settings.
However, if this is the case, there still remains the question of where exactly are the
primitive hematopoietic cells located? Are the HSCs located at the endosteal surface of
the cortical bone, or are they located beside the trabecular bone of the metaphysis region?
Recent data from Ellis and colleagues has suggested that primitive hematopoietic cells
demonstrate a preferential localization to the metaphysis of bones, however, the
conclusion were obtained from the localization of LSK cells following short-term homing
(Ellis et al., 2011). Our data examines the specific expansion of primitive cells under
homeostatic conditions and also suggests the presence of distinct niches in the BM for
primitive hematopoietic cells. However, we demonstrate that the more primitive HSCs
are distinctly localized at the endosteal surface of the cortical bone in the diaphysis region
of the long bones while the more differentiated ST-HSCs are specifically localized next
to the trabecular bone of the metaphysis area (Figure 3.16). A possible explanation for
this regional localization would be that the most primitive HSCs are actually localized in
the region of bone with the least remodeling activity, whereas the more differentiated ST-
HSCs are located in regions of active bone remodeling and thus may be readily activated
and expanded.
An implication of the observation that osteoclasts comprise the endosteal HSC niche is
that the HSCs are directly contributing to their own niche. Osteoblastic cells are derived
from the mesenchymal stem cells, whereas osteoclasts are differentiated from monocyte-
macrophage lineage and therefore ultimately the HSCs. Whether or not there is any
feedback mechanisms from the HSCs to the osteoclasts can only be speculated, however
Figure 3.16 Hypothesized model of localization of the adult HSC in the endosteal niche. We hypothesize
that the most primitive HSCs localize at the endosteal surface of cortical bone in the diaphysis area of the
long bone, while more differentiated stem cells are located at the trabecular bone in the metaphysis region
of the bones, distinguishing two distinct niches for different hematopoietic populations. (Cartoon courtesy
of Kaveh Shoorideh)
studies in the drosophila have similarly observed stem cell contributions to the niche
(Voog et al., 2008).
This study provides further insight to the cellular components regulating the HSC BM
niche and how they interact in order to maintain this microenvironment. We also
demonstrate the presence of two distinct niches that are home to the LT-HSCs and ST-
HSCs, suggesting a regional localization of primitive hematopoietic cell subsets in the
BM. These observations have important implications in understanding the regulation of
HSC physiology and identifying the exact location of the HSC niche.
Chapter 4: Spatially Distinct Niche Regulation of a
Stem Cell Population
4.1 Abstract
During adulthood, HSCs are found at the endosteal surface of the BM. While
hematopoiesis occurs in many bones, the process of bone formation can be split into
those bones that develop through endochondral ossification (long bones) and those that
form through membranous ossification (flat bones). We examined the role played by the
microenvironment in these two distinct bones and whether they have differing effects on
HSCs. We demonstrated that the frequency and the function of HSCs are comparable
between flat and long bones. However, analysis of gene expression revealed increased
expression of n-cadherin in flat bone-derived HSCs. In vitro, flat bone stromal cells also
showed 5-fold greater expression of n-cadherin than long bone, while other cadherins
such as ve-cadherin show no difference. This was confirmed by immunohistochemical
analysis. Using calvaria-derived stromal layers to support primitive hematopoietic cells in
vitro, we demonstrated that these cells were able to support cobblestone area-forming
cells 10-fold greater than stromal layers derived from femurs and tibia. Our data shows
that while HSCs isolated from different bones have identical potential, their maintenance
may be regulated by different mechanisms. It is anticipated that these studies will lead to
the definition of the molecular cues that govern HSC physiology in different locations
within the mammalian skeleton, thus providing an understanding not only into the
continual migration of HSCs between different niches, but also the regression of
hematopoiesis that occurs from the appendicular skeleton to the axial skeleton during the
adult human lifespan.
4.2 Introduction
During development, HSCs translocate from the FL to the BM, which remains the site of
hematopoiesis throughout adulthood (Mikkola & Orkin, 2006). In the BM the HSCs are
located at the endosteal surface (Lord et al., 1975; Gong, 1978) where cells of the
osteoblastic lineage comprise a key component of the stem cell niche (Calvi et al., 2003;
Zhang et al., 2003; Visnjic et al., 2004). It is because stems cells are dependent on their
niche that studies which explore the differences between spatially and anatomically
differing environments possess a significant importance.
Within the mammalian system there are two processes of bone formation, endochondral
ossification and intramembranous ossification. Bone formation starts with mesenchymal
cells gathering to form clusters called condensations (Hall & Miyake, 2000). In a few
areas, especially in the bones of the calvaria, the cells of these nodules directly
differentiate into bone-forming osteoblasts (intramembranous ossification). These cells
then initiate calcification and lay down an extracellular matrix specifically rich in
collagen type I (Silbermann & von der Mark 1990; Kronenberg, 2003).
The long bones, however, develop through endochondral ossification. During this
process, the condensations first become chondrocytes, the primary cell type of cartilage.
These chondrocytes then start proliferating forming a hyaline cartilage mould for bone
formation. Chondrocytes in the middle of this mould then stop proliferating and enlarge
(hypertrophy). These hypertrophic chondrocytes, then become the principle director of
bone formation, directing perichondrial cells to become osteoblasts that leads to the
mineralization of the surrounding matrix and attracting blood vessels through production
of vascular endothelial growth factor (VEGF). The hypertrophic chondrocytes in the
middle of the developing bone then undergo apoptosis leaving a distinct marrow cavity in
the center of the long bones (Silbermann & von der Mark, 1990; Kronenberg, 2003).
Studies have shown that there are molecular and enzymatic differences within these two
spatially and anatomically differing bones. Aside from the differential expression of
different collagen types, matrix metalloproteinases (MMPs) and cysteine proteinases
(CPs) are both necessary for the osteoclastic degrading of calvarial bone; however,
degradation of long bone is only dependent on CPs (Everts et al., 1999). Differences in
resorptive activities between these two sites were suggested to be due to phenotypic
differences between the osteoclast populations residing there. If, indeed, such differences
do exist, the important question arises as to whether different subsets of monocytes give
rise to various subsets of osteoclasts. These differences suggest that there may be
differences between different sites of BM hematopoiesis located in the spatially distinct
bones (Everts et al., 1999).
Kiel and colleagues examined the spatial differences in hematopoiesis in adult BM (Kiel
et al., 2005a). By examining the hematopoietic cells in various bones, they found that
there were no differences in the immunophenotype of the cells examined (including the
primitive HSCs). They also found no differences in gene expression or in vivo function of
HSCs isolated from different bones. However, in these studies only the sternum, pelvis
and femur were examined. Chan and colleagues, using an in vivo ectopic bone formation
assay demonstrated that cells isolated from bones that undergo endochondral ossification
are able to form BM, and thus presumably an HSC niche (Chan et al., 2009). Yet,
immunophenotypically identical cells isolated from the calvaria are able to form bone,
but do not form a marrow cavity and thus no hematopoiesis is observed.
We examined the role played by the microenvironment in two distinct bone types
(femur/tibia vs. calvaria) and whether these microenvironments have differing effects on
the HSCs. Immunopheotypic analysis and functional assays as well as gene expression
analysis were used to compare the properties of the HSCs derived from these two bones
and the effects that the different microenvironments exert on these cells. It is anticipated
that we will be able to begin to define the molecular cues that govern HSC physiology in
different locations within the mammalian skeleton and thus provide an understanding into
the continual migration of HSCs between different HSC niches.
4.3 Materials and Methods
4.3.1 Animals
Six- to eight-week-old WT male C57Bl/6 and B6.SJL mice (Taconic, Oxnard, CA) were
obtained and used as detailed in section 2.1.
4.3.2 Bone harvesting and BM stromal cell cultures
Femurs and tibias were dissected from 6 to 8 week-old male C57Bl/6 mice as described
in section 2.2 and the BM-MNCs were flushed from the central cavity with culture
medium.
To dissect the calvaria, the skin above the skull was cut open with a No.10 scalpel and
the calvaria was exposed. Using a curved iris scissors, an incision was made starting at
the posterior part of the calvaria at the neck (occipital bone) around the parietal bones
(next to the ears) all the way to the anterior section of the calvaria (frontal bones). The
calvaria bone was lifted with a pair of tweezers and separated from the body. The bone
was cleaned of the remaining brain tissue and placed in culture medium. To obtain BM-
MNCs the bone was cut in several pieces and then crushed thoroughly using a mortar and
pestle. The cell suspension along with the bone segments was transferred to a 50 ml
conical tube (BD) and vortexed well. Finally, to remove any remaining bone fragments or
hair, the BM solution was filtered through a 70 µm cell strainer (BD).
BM-MNCs from femur/tibia or the calvaria were cultured ( long bone and flat bone
cultures, respectively) at a concentration of 5x10
6
cells/ml at 37
o
C/5% CO2 in a
humidified atmosphere in Myelocult
®
M5300 culture medium (Stem Cell Technologies)
containing 10% penicillin/streptomycin (Cellgro
®
by Mediatech Inc.). The cell cultures
were maintained for 2 weeks, changing the medium every 2 to 3 days.
4.3.3 TRACP and ALP staining
Adherent BM stromal cells from long bone and flat bone cultures were trypsinized and
plated at a concentration of 1x10
5
cells/well in a 48-well plate. TRACP and ALP staining
was performed using TRACP and ALP double-stain kit (TAKARA BIO INC) as detailed
in section 2.6.
4.3.4 CFU-C assay
BM-MNCs were obtained from the hind-limbs or calvaria of C57Bl/6 mice and
resuspended in MethoCult
TM
GF M3434 (StemCell Technologies). CFU-C assay was
performed using these cells as detailed in section 2.4 and the number of CFU-Cs were
scored at day 10 according to standard criteria.
4.3.5 CAFC assay
CAFC assay was performed, according to section 2.5 using the long bone or flat bone
BM cell cultures as stromal layers. Once irradiated, the stromal layers were seeded with
BM-MNCs obtained from the hind-limbs or calvaria of C57Bl/6 mice and the cultures
were then maintained in a humidified atmosphere at 37°C/5% CO2 and the presence of
CAFCs were scored on week 5. The frequency of CAFCs was calculated using L-Calc
software (StemCell Technologies).
4.3.6 Flow cytometry and cell sorting
BM-MNCs were stained and analyzed with fluorescent labeled antibodies as described in
section 2.7. To sort different population of cells for transplantation, once stained
hematopoietic stem and progenitor cells were sorted using a FACSAria flow cytometer
(Becton Dickinson) based on established cell surface markers.
4.3.7 CRA
BM-MNCs were obtained from tibia or calvaria of C57Bl/6 (CD45.2, donor) mice. Also
BM-MNCs were harvested from B6.SJL (CD45.1, competitor) mice. 200,000 cells from
the donor mice and 500,000 cells from the competitor were co-injected into the tail vein
of B6.SJL mice that were lethally irradiated with 9 Gy approximately 24 hours prior to
transplantation. Engraftment levels and multi-lineage reconstitution were assessed by
monitoring the levels of donor cell contribution to host hematopoiesis as described in
section 2.8.
4.3.8 Histological analysis
Dissected tibias and whole calvaria were fixed overnight in 10% formalin at 4
o
C. The
bones were then decalcified with Immunocal (Decal Corporation Group) over a three-day
period and were processed and paraffin embedded using standard histological procedures
detailed in section 2.8. Longitudinal bone sections were then cut at 5mm from the tibia
and used for further staining. For the calvaria, histological sections were made
perpendicular to the sagittal suture, which passes between the two parietal bones of the
skull. H&E staining was performed according to standard methods (section 2.9). N-
cadherin staining was performed on the bone sections as described below.
4.3.8.1 N-Cadherin Staining
The slides were warmed up overnight and deparaffinized as previously described (section
2.9.1). The slides were then microwaved twice in 1X Citrate Buffer (Diagnostic
BioSystems, Pleasanton, CA) at power level 6 for 35 seconds and let cool down for 30
minutes. A normal goat serum (Invitrogen, Camarillo, CA) solution in 1% BSA
(CALIBIOCHEM, Darmstadt, Germany) in PBS (1:20 dilution) was used as the blocking
solution. The slides were washed with 1X PBS with 0.5% Tween 20 (PBS-Tween 20,
TEKnova, Hollister, CA) once before they were incubated in the blocking solution for 60
minutes at room temperature. The slides were then drained and stained with anti-human
N-cadherin (YS) Rabbit IgG (1:50 dilution) (Immuno Biological Laboratoris Co. Ltd,
Minneapolis, MN) at 4
o
C overnight. The next day, the slides were first rinsed with PBS-
Tween 20 and then blotted with goat-anti-rabbit secondary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) for 30 minutes at room temperature. Meanwhile,
Vectastain ABC-AP solution (Vector Labs, Burlingame, CA) was prepared by adding
one drop (~50 µl) of reagent A and one drop of reagent B to 5 ml of PBS and mixing
them thoroughly. The solution was incubated at room temperature for 30 min for
activation. The Vecstatin ABC-AP was applied to the rinsed slides and incubated for 30
minutes at room temperature. The slides were then rinsed with PBS-Tween 20. The
Vector Red Alkaline Phosphatase Substrate (Vector Labs) was prepared in the dark
immediately before use by adding two drops of reagent 1,2 and 3 to 5 ml of 200 mM Tris
HCl (pH 8.5) and mixing the solution thoroughly after adding each reagent. The substrate
was added to the blots and slides were incubated in the dark for 10-30 minutes. Upon
color formation, the slides were rinsed with distilled water and then counterstained with
Methyl Green. Once the staining was completed, the slides were dehydrated and cover
slipped as described in section 2.9.1.
4.3.9 RNA Extraction
Total RNA was extracted with the use of the RNA Microprep Kit (Stratagene, Santa
Clara, CA). Long bone and flat bone cell cultures were trypsinized, as described in
section 2.5, and used as in vitro samples. BM-MNCs obtained from femur/tibia and
calvaria or sorted HSCs were used as in vivo samples. To lyse the cells, 0.7µl of beta
mercaptethanol ( -ME) was added to 100µl of Lysis Buffer for each sample of 5×10
5
cells. 100µl of Lysis Buffer -ME mixture was added to each cell sample and vortexed
vigorously until homogenized. An equal volume of 70% ethanol was added to the cell
lysate and mixed thoroughly by vortexing for 5 seconds. The mixture was then
transferred to a seated RNA-Binding Spin Cup and span down in a microcentrifuge at
maximum speed for 60 seconds. The spin cup was retained and the filtrate was discarded.
To remove DNA, 600µl of 1X Low-Salt Wash Buffer was added to the cells and span
down for 60 seconds at maximum speed. The spin cup was retained and the filtrate was
discarded. The spin cup was replaced in a new collection tube and span down one more
time for 2 minutes at maximum speed to dry the fiber matrix. For each sample, 5µl of
reconstituted RNase-free DNase I was gently mixed with 25µl of DNase Digestion Buffer
and then directly added onto the fiber matrix of the spin cup. The samples were then
incubated at 37°C for 15 minutes. 500µl of 1X High-Salt Wash Buffer was then added to
the samples and span down for 60 seconds at maximum speed. Once again, the spin cup
was retained and the filtrate was discarded. The samples were washed again twice, using
600µl and 300µl of 1X Low-Salt Wash Buffer for 60 seconds and 2 minutes respectively.
The spin cup was then transferred to a 1.5 ml collection tube and 30µl Elution Buffer,
which had been warmed up to 60°C, was directly added onto the fiber matrix. The
samples were incubated for 2 minutes at room temperature and finally span down for 60
seconds at maximum speed. The purified RNA in the Elution Buffer in the
microcentrifuge collection tube is stored in 80°C for long-term or at 20°C for short-
term use.
4.3.10 First-strand complementary DNA (cDNA) Synthesis
Total RNA was reverse-transcribed into cDNA with the use of the SuperScript VILO
cDNA synthesis kit (Invitrogen). For each single reaction, the following components
were combined in a tube on ice:
5X VILO™ Reaction Mix 4 µl
10X SuperScript® Enzyme Mix 2 µl
RNA x µl (usually 10 µl)
DEPC-treated water to 20 µl
The reaction was performed using an Eppendorf AG 22331 Mastercycler machine
(Eppendorf, Hamburg, Germany). The thermal cycling conditions to generate cDNA was
as follow: 10 minutes at 25°C followed by 60 minutes 42°C. To terminate he reaction the
tubes were then incubated at 85°C for 5 minutes. The cDNA samples were stored at
20°C until use.
4.3.11 Quantitative reverse transcription–polymerase chain reaction (QPCR)
To quantify the expression level of n-cad, p21, tek, notch1, myc, cyclind1, patch1
and hprt1, Taqman Gene Expression Assay primers (Applied Biosystems , Carlsbad, CA)
and probe sets (Roche Diagnostics, Pleasanton CA) were used. For each gene, the
reaction mix was prepared and added to each well of 384-well reaction plate on ice by
pipetting 0.5µl of 20X TaqMan Gene Expression Assay and 5µl of 2X Taqman Gene
Expression Master Mix (for a single reaction). The cDNA templates were then added to
each well and the plate was sealed. The plate was centrifuged at 400g for five minutes
and then loaded into the instrument. Levels of gene expression were quantified with the
use of the 7900HT real-time polymerase chain reaction system (Applied Biosystems). To
prepare the standard curves, first a stock of reference cDNA with a concentration of 0.05
µg/µl was made from the Quantitative PCR Mouse Reference Total RNA (Stratagene).
The reference cDNA solutions were then repeatedly diluted to solutions with a final
cDNA quantity of 50, 10, 2, 0.08, 0.016 ng.
The thermal cycling conditions to was as follow: 95°C for 10 minutes to activate the
polymerase followed by 40 cycles of one minute in 95°C (denaturation) and 20 minutes
in 60°C (Anealing/Extention).
4.3.12 Cell-cycle analysis
To stain for DNA content, cells were incubated with 10 µg/mL Hoechst 33342 (Sigma-
Aldrich, St. Louis, MO) at 37°C for 45 minutes. To stain for RNA content, pyronin Y
(Polysciences Inc, Warrington, PA) was added to the cells at a final concentration of 0.75
µg/mL and incubated at 4°C for 30 minutes. Cell-cycle status was examined with the use
of an LSR II flow cytometer (BD). An example of the gating strategy is shown below
(Figure 4.1).
4.3.13 Statistical analysis
Comparison of experimental groups was performed using the unpaired two- tailed
Student’s t-test as appropriate for the data set. A p-value of <0.05 was considered
significant.
Figure 4.1 Cell cycle analysis of hematopoietic cells. Cell cycle analysis was performed using Pyronin Y
as a RNA marker and Hoechst 33342 as a DNA marker. The G
0
cells are identified as Pyronin Y
-
/Hoechst
-
cells. The G
1
cells are identified as Pyronin Y
-
/Hoechst
+
cells. The S/G
2
/M
cells are identified as Pyronin
Y
+
/Hoechst
+
cells.
4.4 Results
4.4.1 HSC frequency and function is comparable between flat and long bone.
Previous reports demonstrated that the frequency of LT-HSCs, identified as LSK
CD150
+
CD48
-
cells, are comparable between the femur and calvaria (Lo Celso et al.,
2009), however a more thorough immunophenotypic and functional analysis is required
to be able to fully compare the properties of the HSCs from these two different spatial
origins. We first immunophenotypically analyzed BM-MNCs from femur and calvaria
using flow cytometry. To quantitate the frequency of LT-HSC, ST-HSC, we analyzed the
Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
+
CD48
-
(LSKCD150
+
CD48
-
) and LSKCD150
-
CD48
-
subpopulations, respectively (Kiel et al., 2005b). To ensure that we were only examining
the hematopoietic cells, the percentage of long-term and short-term HSCs in the CD45
+
population of cells were calculated. Analysis of LSKCD150
+
CD48
-
and LSKCD150
-
CD48
-
cell frequencies did not demonstrate any specific difference between the cells
harvested from tibia vs. calvaria (Figure 4.2A). However, analysis of the LSK cells, that
also include more differentiated committed progenitor populations, demonstrated a
significant decrease in the frequency of these cells in the BM-MNCs of flat bone origin
(Figure 4.2B). These findings correlated with the functional analysis of the progenitor
cells in the CFU-C assay. Once CFU-Cs were counted after 10 days of culture in
methylcellulose, we witnessed that the cells originating from calvaria had a significantly
lower growth potential compared to their counterparts coming from femur and tibia
(Figure 4.2C).
We next examined differences in primitive hematopoietic cell function by competitive
repopulation analysis. Following transplantation of an excess of competitor cells over the
BM-MNCs harvested from femurs and calvaria of the mice, no significant difference was
observed between engraftment levels of these cells except for the first month (Figure
4.3).
Figure 4.2 Comparison of hematopoietic stem and progenitor cell population frequencies between cells
of different BM origin. BM-MNCs were harvested from the femurs/tibias and calvariae of WT C57/Bl6
mice and labeled with hematopoietic cell surface markers (CD45, Lin, c-Kit, Sca-1, Flk2, CD41, CD48,
and CD150). The cells were then analyzed by flow cytometry. Measurement of (A) Lin
-
Sca-1
+
c-
Kit
+
CD41
-
CD150
+
CD48
-
(LSK CD150
+
CD48
-
) and Lin
-
Sca-1
+
c-Kit
+
CD41
-
CD150
-
CD48
-
(LSKCD150
-
CD48
-
) and (B) Lin
-
Sca-1
+
c-Kit
+
(LSK) cell content of CD45
+
cells represented as percentage of CD45
+
cells (n=12; from 4 independent experiments); (C) CFU-C assay was performed on BM-MNCs extracted
from femurs/tibias and calvaria of WT C57/Bl6 mice (n = 10 from 5 independent experiments). Data
represent the mean ± s.e.m.
Finally, to examine whether the differences observed in the growth potential result from
alterations in the cell cycle profile of the BM-MNCs of different bones, cell-cycle
analysis was performed. The results demonstrated that the percentage of long bone and
flat bone cells that were in a G
0
or G
1
phase was comparable, although we witnessed a
mild increase in the long bone cells in the S/G
2
/M phase (Figure 4.4).
Figure 4.4 Comparison of cell cycle profile of BM-MNCs of different BM origin. Cell cycle profile of
BM-MNCs from different bones was examined mice (n = 4 from 2 independent experiments). Data
represent the mean ± s.e.m.
Figure 4.3 Comparison of primitive hematopoietic cell function between cells of different BM origin.
BM-MNCs from the femurs and calvariae of C57Bl/6 mice were transplanted in a competitive
repopulation assay into B6.SJL recipients. Flow cytometric analysis of peripheral blood was performed
every 4 weeks up to 20 weeks post-transplantation. Measurement of BM-MNC engraftment levels based
on percentage of CD45.2 cells present in the peripheral blood (n=10-12; from 3 independent
experiments). Data represent the mean ± s.e.m.
Together these data suggest that the BM-MNC extracted from various bone sites have
similar HSC content and reconstitution potential although the progenitor cells frequency
and function are reduced in the cells derived from calvaria.
4.4.2. n-cadherin expression is upregulated in flat bone hematopoietic cells.
We next evaluated whether the gene expression signature was also similar between the
cells of the long bones and flat bones. We selectively chose 7 genes that are known to be
part of important signaling pathways in HSC physiology. Total RNA was extracted from
femur/tibia and calvaria BM-MNCs and the expression levels of n-cad, p21, tek, notch1,
myc, cyclind1 and patch was compared to that of the housekeeping gene hprt1. Our data
showed that the BM-MNCs from the calvaria have a higher expression level of n-
cadherin compared to the femur/tibia bones, while the other showed no difference
(Figure 4.5).
Figure 4.5 Comparison of gene expression profile of BM-MNCs of different BM origin. Total RNA
was extracted from BM-MNCs from femur/tibia and calvaria bones. The expression level of n-cad, p21,
tek, notch1, myc, cyclind1 and patch1 was measured using quantitative RT-PCR, relative to hprt1 levels
(n=8 from 5 independent experiments). Data represent expression levels as fold-change compared to the
expression level in femur/tibia. Data represent the mean ± s.e.m.
N-cadherin (neural cadherin) is a member of adhesion proteins of the cadherin
superfamily and there is a vast body of evidence supporting its role in the HSC niche
(Zhang et al., 2003; Xie et al., 2009; Wilson et al., 2004). N-cadherin has also been
shown to involve in the adhesive interaction of human hematopoietic progenitor cells to
supporting mesenchymal stromal cells (Wein et al., 2010). We therefore decided to look
into the expression level of n-cadherin and other members of cadherin family (epithelial
cadherin: e-cadherin; vascular endothelial cadherin: ve-cadherin) in the BM cell cultures
derived from cells of femur/tibia or calvaria origin. Our data showed that the expression
level of n-cadherin is elevated in the flat bone BM cultures compared to long bone BM
cultures although we also witnessed a mild elevation of ve-cadherin expression in these
BM cultures (Figure 4.6).
Figure 4.6 Comparison of gene expression profile of stromal cells of different BM origin. Total RNA
was extracted from femur/tibia and calvaria stromal cultures and the expression level of n-cad, e-cad and
ve-cad was measured using quantitative RT-PCR, relative to hprt1 level (n=8 from 5 independent
experiments). Data represent expression levels as fold-change compared to the expression level in
femur/tibia. Data represent the mean ± s.e.m.
We further confirmed the changes seen in n-cadherin expression by immunostaining of
bone sections from tibias and calvariae (Figure 4.7A) as well as BM stromal cultures
(Figure 4.7B).
4.4.3 Calvaria- derived stromal cells better support HSC function in vitro
The differences observed between n-cadherin expression in the BM stromal cultures
prompted us to look at the composition of the stromal cultures. We therefore examined
the osteoclast and osteoblast activity in the BM stromal cell cultures from femur/tibia vs.
calvaria, by ALP and TRACP staining. As shown in figure 4.8, there was no difference
between the two different stromal cultures and hence, no disparity between the osteoblast
and osteoclast activity of the stromal cultures.
Figure 4.7 N-cadherin immunohistostaining of bone sections and BM stromal cultures of different BM
origin. (A) N-cadherin staining of sections of paraffin-embedded decalcified tibias and calvariae
(original magnification 4X) and (B) femur/tibia or calvaria BM stromal cultures. Representative
photomicrographs in (A) show n-cadherin staining in metaphysis and diaphysis areas of tibia sections as
well as calvaria section.
To examine the effects of differential n-cadherin expression by various stromal layers,
we then used the CAFC assay to investigate the ability of these cultures to support
primitive hematopoietic cells in vitro. The stromal layers derived from long bone or flat
bone cultures were seeded with fresh BM-MNCs from either femur/tibia or calvaria. As
shown in figure 4.9, when calvaria-derived cultures were used as stromal layers, the
CAFC frequencies were higher than when femur/tibia cultures were used.
Figure 4.8 ALP and TRACP activity of BM stromal cultures of different BM origin. Stromal cell
cultures from femur/tibia and calvaria were stained for ALP and TRACP activity. Representative
staining performed in triplicate of 3 independent experiments is shown.
Together these data suggest that the stromal layers originating from the calvaria are better
able to support primitive hematopoietic cells in vitro. However, this functional
enhancement cannot be attributed to differences in the cellular composition of these
stromal layers as they both have comparable osteoblastic and osteoclastic activity.
Figure 4.9 Comparison of in vitro functional potential of BM stromal cultures of different BM origin.
CAFC assays were performed with calvaria-derived (FB Stroma: Green) and femur/tibia (LB stroma:
Purple) cultures serving as the stromal layers Fresh BM-MNCs from calvaria (FB cells: Solid) or
femur/tibia (LB cells: Striped) were seeded on these stromal layers (n=7-11; from 4 independent
experiments). Data represent the mean ± s.e.m.
*
p<0.05 compared to LB stroma. Both FB stroma groups
had significantly higher CAFC frequencies when compared to both LB stroma. †p<0.05 compared to the
LB stroma group seeded with FB cells.
4.5 Discussion
Adult hematopoiesis is segregated among many different BM sites, yet it does in fact
regress from the appendicular skeleton to the axial skeleton during the adult human
lifespan (Bianco, 2009). Since the niche dictates the maintenance of the hematopoietic
activity and the lifelong stem-ness of the HSCs, a close examination of HSCs from
distinct BM compartments is required to see whether these various niches exert any
differeing effects on these cells.
Our data, along with other studies (Lo Celso et al., 2009), demonstrated that competitive
transplant of equal numbers of whole BM cells from calvaria and femur resulted in
comparable levels of engraftment at 16 weeks post-transplant. We also showed that the
stem cell activity is similar at these two sites (Figure 4.2A and 4.3). However, we
observed a higher LSK cell frequency in the cells from the femur/tibia (Figure 4.2B),
which could be due an increased number of committed progenitor cells as confirmed by
CFU-C assay (Figure 4.4A).
Our data demonstrates a difference in expression of N-cadherin in femur/tibia BM versus
calvaria BM. This may explain the controversial role of N-cadherin in HSC biology (Li
& Zon, 2010). A key paper, which described the role of the osteoblast in the HSC niche,
described the niche cells as spindle shaped N-cadherin expressing osteoblastic cells
(Zhang et al., 2003). Similarly, genetic knockdown studies have demonstrated that
N-cadherin is required for functional engraftment of the BM following transplantation
(Hosokawa et al., 2010). However, other studies have demonstrated a lack of evidence
that HSCs actually express N-cadherin and therefore do not use this to interact with
osteoblasts (Kiel et al., 2007). Similarly, conditional knockout studies have demonstrated
that HSCs do not depend upon N-cadherin for their maintenance (Kiel et al., 2009). All
these studies have always used HSCs from femur and never examined N-cadherin
downregulation in the calvaria . Here, we provide evidence that there are differences in
N-cadherin expression between long and flat bone, which may play a role in the
regulation of the HSCs in different anatomic sites. However, to further clarify this role,
analysis of N-cadherin expression levels in purified populations of HSCs and also further
assessment of its role by using genetic knockdown models is required.
Together, our data suggest that while HSCs isolated from different bones appear to have
identical potential, maintenance of HSCs in the calvaria depends upon N-cadherin, while
long bone HSCs do not. This may explain some of the discrepancies found in the filed
about the role of N-cadherin in HSC niche and provide the basis for future studies
examining the role of other signaling pathways between anatomically different sites of
hematopoiesis.
Chapter 5: A Novel Method to Image Hematopoietic Stem
Cells in the Adult Bone Marrow Stem Cell Niche
5.1 Abstract
The stem cell niche has been hypothesized to be the region where HSCs are able to self-
renew. However, attempts to visualize HSC self-renewal in the adult BM niche have not
been successful. Here, we have developed a novel imaging technique using Second
Harmonic Generation (SHG) nanoprobes to enable the imaging of HSCs in all BM
microenvironments of the adult mouse. These SHG nanoprobes circumvent many of the
limitations of classical fluorescence probes, providing unique advantages for imaging of
primitive hematopoietic cells in vivo in different bones of the mouse. Our data
demonstrates that these nanoprobes are non-toxic to primitive hematopoietc cells as
evidenced by in vitro functional analyses. Since the SHG nanoprobes do not photo-bleach
and are resistant to temperature and pH fluctuations, this had enabled us to label and
detect HSCs in bone slides to visualize the interactions between these cells and other cell
types present in the niche through various immunohistochemical stainings. More
significantly, the long wavelength that these nanoprobes are imaged at (>800 nm) has
permitted cells deep inside of intact tissues to be visualized, including the femurs and
tibias of mice. In summary, we have been able to image HSCs using a non-invasive
technique in multiple different bone types that will ultimately permit the dynamic
imaging of the HSCs in their native niche microenvironment.
5.2 Introduction
To capture the dynamic nature of HSCs in their niche in the live animal, we require an
imaging technique that has enough depth resolution to penetrate through the bone and its
surrounding muscle tissue. It also needs to produce images with single cell resolution that
allow studying the niche at the cellular level. This technology should have low
phototoxicity in order to track rare HSCs in vitro and in vivo, and rapid image acquisition
to enable imaging of the dynamic nature of the cells. In addition, it should offer the
tracking of injected or transplanted stem cells for a long period of time, enabling long-
term follow-up of tissue function and host survival.
Currently, there are two major categories of imaging technology used to image HSCs:
The first is the use of confocal microscopy to image stem cells that are auto-fluorescent
(Challen & Goodell, 2008) or have been fluorescently labeled (Nilsson et al., 2001); the
second is the use of bioluminescent animals and magnetic resonance imaging (MRI) to
image into the BM of live animals (Daldrup-Link et al., 2005; Partlow et al., 2007;
Schroeder, 2008). Each of these methods has their own advantages and disadvantages.
While confocal microscopy will offer the resolution needed to image stem cells at a
single-cell level, it does not have the required depth-resolution needed to image through
cancellous bone where the HSCs are thought to reside (Figure 5.1). On the other hand,
MRI of BM offers enough depth to be able to delineate between the yellow and red
marrow, providing information about the basic anatomic definition of the bone (Vogler
III & Murphy, 1988) but studying cellular interactions between the stem cells and their
environment is not possible (Ntziachristos et al., 2005; Schroeder, 2008). Another
requirement is the temporal resolution of the imaging technique. Temporal resolutions of
less than a second, required for imaging of regeneration, can only be achieved through
confocal microscopy (Schroeder, 2008).
To overcome these challenges, here we propose the use of a novel imaging technology
based on second harmonic generating (SHG) nanoprobes to study HSCs in their niche.
Similar to two-photon microscopy, SHG is also a nonlinear optical process. However, it
employs pulsed lasers in the infrared wavelength range, which will give them relatively
deep optical penetration and reduced phototoxicity (Pantazis et al., 2010). Moreover, the
response time of SHG is at the femtosecond level; four to five orders of magnitude faster
than the nanosecond response time of fluorescence, allowing very fast and sensitive
detection (Figure 5.2). Reduced phototoxicity and fast response time of SHG microscopy
make this technique the superior choice for long time-lapse imaging. In comparison to the
Figure 5.1 Spatiotemporal resolution of imaging modalities used for HSC visualizing. The imaging
resolution is inversely correlated with how deep into the tissue we can visualize. While imaging
modalities such as MRI can image deep into the tissue they lack the single-cell resolution that 2-photon
confocal microscopy offers.
conventional fluorescent dyes that are currently used for labeling, SHG nanoprobes get
excited at longer wavelengths [820nm in case of barium titanate (BaTiO
3
) crystals used
in these experiments], which allow deep tissue imaging. They will then emit at half the
wavelength and twice the power (Figure 5.2).
SHG nanoprobes are fundamentally different from fluorescence-based probes and have
many photophysical advantages compared to them. First, in comparison to fluorescence-
based dyes such as quantum dots, they do not photobleach or saturate. This will allow
increasing the laser power to detect faint signals from the tissue. While amplifying the
power leads to an increased background noise in the fluorescent dyes the narrow
Figure 5.2 Two-photon excited fluorescence versus SHG. Compared to two-photon excited
fluorescence, all of the incident radiation energy at frequency i
is converted in the process of SHG to
radiation at the SHG frequency 2 i
. And whereas two-photon excited fluorescence involves real energy
transition of electrons, SHG involves only virtual energy transition. As a result, using ultrafast
(femtosecond) pulsed lasers, the response time of SHG is at the femtosecond level, about several orders
of magnitude faster than the nanosecond response time of fluorescence, allowing very fast and sensitive
detection.
emission spectrum in the SHG nanoprobes allows constriction of the detection spectrum,
which in turn yields an outstanding signal-to-noise ratio (Pantazis et al., 2010).
While SHG microscopy has previously been adopted to observe intrinsic SHG signals,
(Lo Celso et al., 2009), SHG nanoprobes have never been used to characterize biological
targets in whole organisms. Here, we are proposing the use of BaTiO
3
crystals as SHG
nanoprobes to label HSCs, visualize their localization in a variety of whole bones, and
look into the interaction of these cells with different components of the HSC niche in
these bones.
5.3 Materials and Methods
5.3.1 Animals
week-old male C57Bl/6 mice (Taconic, Oxnard, CA) were obtained and
used in accordance with the University of Southern California IACUC guidelines. Mice
were housed in sterilized microisolator cages and received autoclaved food and water ad
libitum.
5.3.2 SHG nanoprobe labeling
Femurs and tibias were dissected from 6 to 8 week-old male C57Bl/6 mice as described
in section 2.2 and the BM-MNCs were flushed from the central cavity with M5300 long-
term culture medium containing penicillin/streptomycin. To sort the cells BM-MNCs
were stained and analysed with fluorescent labeled antibodies as described in section 2.7.
A 0.01 mM stock of 30 nm BaTiO
3
nanocrystals (Nanostructured and Amorphous
Materials, Inc., Houston, TX) was prepared in PBS and ultrasonicated using a
Professional Grade Ultrasonic Jewelry Cleaner (Bogure Systems, Inc., Patterson, NJ).
As shown in figure 5.3, BM-MNCs or sorted cells were then incubated with the crystals
at a final concentration of 1 mM. The cells were incubated with the nanoprobes for 2
hours in 37°C. If needed, for endosome labeling, Alexa-546nm Dextran 10,000 MW
(Invitrogen) was added at this incubation step at a concentration of 1:1000.
To separate the mononuclear cells from excess SHG nanoprobes, these cells were then
separated by ficoll separation. Briefly, after incubation the cells and SHG nanoprobes
suspension were spun down and resuspended in 5 ml of medium. This cell suspension
was then slowly layered on top of 5 ml of lymphocyte separation medium (LSM;
Cellgro
®
by Mediatech Inc.) in a 15 ml conical tube (BD Bioscience) and span down at
800 G for 20 minutes.
Figure 5.4 Ficoll separation of BM-MNCs. Mononuclear cells are separated by performing a ficoll
separation. The labeled cells are in the mononuclear cell layer separated in between the medium on the
top and LSM on the bottom.
Figure 5.3 An overview of SHG nanoprobe labeling. Labeling BM-MNCs/sorted HSCs were labeled
with BaTiO
3
nanoprobes at a final concentration of 1 mM and incubated at 37°C for 2 hours.
Using a bulb pipette the thin layer in between the LSM and the medium on the top is
removed. This thin layer contains all the mononuclear cells including our labeled cells.
The cells were then washed in 10 ml of medium to remove excess LSM and resuspended
in 1 ml of PBS (Figure 5.4).
5.3.3 CFU-C assay
SHG- labeled and control non-labeled BM-MNCs were resuspended in MethoCult
TM
GF
M3434 (StemCell Technologies). CFU-C assay was performed using these cells as
detailed in section 2.4 and the number of CFU-Cs were scored at day 10 according to
standard criteria.
5.3.4 CAFC assay
CAFC assay was performed, according to section 2.5 using OP9 stromal cultures as
stromal layers. The stromal layers were plated at a concentration of 2.5x10
4
cells/well in
flat bottom 96-well plates and irradiate at 9 Gy. Once irradiated, the stromal layers were
seeded with SHG- labeled or control non-labeled BM-MNCs. The cultures were then
maintained in a humidified atmosphere at 37°C/5% CO2 and the presence of CAFCs were
scored on week 5. The frequency of CAFCs was calculated using L-Calc software
(StemCell Technologies).
5.3.5 Apoptosis assay
1× 10
6
SHG- labeled or control non-labeled BM-MNCs were suspended in 100 µl of 1X
binding buffer and stained with 7-amino-actinomycin D (7-AAD) and PE Annexin V (all
from BD Biosciences) at a 5:100 concentration. The cells were vortexed and incubated at
room temperature for 15 minutes. With the use of an LSRII flow cytometer (BD
Biosciences), the apoptotic cells were identified as 7-amino-actinomycin negative and PE
Annexin V positive cells.
5.3.6 Image acquisition in cell preparation
BM-MNCs that were incubated with BaTiO
3
nanocrystals and Alexa-546nm Dextran
10,000 MW for 2 hours were then used for imaging. To do so 100µl of the cell sample
was placed in the center of a 8-well Lab-Tek™ II Chamber Slide™ with a thickness No.1
coverslip bottom (nunc Inetrnational, Rochester, NY) to cover the bottom of the well.
The images of the cells were obtained with a LD C-APO 40 ×/1.1 objective on a Zeiss
LSM710 confocal setup. For three-channel confocal image acquisition, a femtosecond
pulsed laser line at 820 nm (for imaging BaTiO
3
nanocrystals) from a Ti:Sapphire laser
(Coherent, Inc., Santa Clara, CA) and a continuous Diode Pumped Solid State (DPSS)
561-10 He/Ne laser line (for imaging of Alexa-546nm Dextran 10,000 MW) were used
sequentially.
Upon image acquisition, the bone was outlined using the Magic Wand tool in Adobe
Photoshop and the SHG signals were detected using the semi automated spot
segmentation tool in Imaris software (BitPlane AG, Zurich, Switzerland).
5.3.7 Image acquisition in bone slices
Dissected bones were fixed overnight in 10% formalin at 4
o
C. The bones were then
decalcified with Immunocal over a three-day period and were processed and paraffin
embedded using standard histological procedures detailed in section 2.8. Longitudinal
bone sections were then cut at 5mm. These bone slices were then deparafinized as
described in section 2.8.1 and used for imaging. H&E staining [according to standard
methods (section 2.8.1)] was performed on some slices before image acquisition if
needed.
The images of the slices were obtained with a Plan-Apochromat 20 ×/0.8NA objective on
a Zeiss LSM710 confocal setup. For imaging BaTiO
3
nanocrystals, a femtosecond pulsed
laser line at 870 nm with a 422-441nm emission filter was used. SHG signal detection
was performed in epidirection.
5.3.8 Bone clearing and imaging the SHG nanoprobes in whole bone samples
To image whole bone samples, once dissected, the bones were fixed in 10% formalin
overnight and where then transferred to PBS. To clear the bones, they were gradually
dehydrated in 60, 80, 90 and 100% Ethanol (10 minutes each) and finally incubated in a
1:2 ratio of benzyl alcohol: benzyl benzoate (BABB) (both from EMD Millipore)
overnight at room temperature. The bones were then imaged in the same solution in a 2-
well Lab-Tek™ II Chamber Slide™ as described in section 5.2.7.
5.3.9 Statistical analysis
Comparison of experimental groups was performed using the unpaired two- tailed
Student’s t-test as appropriate for the data set. A p-value of <0.05 was considered
significant.
5.4 Results
5.4.1 SHG nanoprobes are uptaken by primitive hematopoietic cells.
We first wished to demonstrate that the cells take up SHG nanoprobes, therefore we
imaged BM-MNCs incubated with BaTiO3 crystals. These cells were also prepared with
the addition of Alexa-546nm Dextran (10,000 MW) in the primary SHG nanoprobe
incubation step to mark endosomes. As clearly seen in figure 5.5 the SHG nanoprobes did
in fact cluster exclusively within endosomes of the BM-MNCs that successfully took up
the crystals (Figure 5.5), possibly through fluid-phase endocytosis (Berlin and Oliver,
1980), as evidenced by co-localization of the SHG signal (Figure 5.5B, white) and the
Alexa-546nm Dextran signal (Figure 5.5C, red).
5.4.2 SHG nanoprobes are not toxic to primitive hematopoietic cells.
The use of any imaging modality requires that it is non-toxic to the cells of interest.
Therefore, we wished to specifically evaluate the toxicity of the SHG nanoprobes that are
to be used to label primitive hematopoietic cells. To validate that the 30nm BaTiO3
nanocrystals do not have deleterious effects on the HSCs, we assessed the growth and
differentiation potential of labeled BM-MNCs by primitive hematopoietic cell assays
(CAFC and CFU-C). As demonstrated in figures 5.6A-B, we observed that labeling of
these cells had no effect on their growth or differentiation potential.
We also assessed the percentage of apoptotic cells following incubation by AnnexinV
and 7-AAD labeling. Apoptotic cells were identified as AnnexinV
+
7-AAD
-
cells and as
Figure 5.5 SHG nanoprobe cell labeling. BM-MNCs were prepared as described in section 5.2.2
with the addition of Alexa-546nm Dextran (10,000 MW) in the primary SHG nanoprobe incubation
step. (A) Bright field image of the cell. (B) Alexa-546 Dextran signal (red). (C) SHG nanoprobe
signal (white). (D) Bright field merged with SHG and Alexa-546nm signal. Note that all SHG
nanoprobes accumulate inside endocytic compartments that are positively labeled with the fluid-phase
endocytic marker, Alexa-546nm Dextran. Scale bar, 5µm. (Picture courtesy of William P Dempsey)
Figure 5.6 Effects of BaTiO
3
labeling on growth and functional potential of hematopoietic cells. BM-
MNCs were labeled as described and assessed for growth in (A) CFU-C and (B) CAFC culture systems.
(n = 8 from 3 independent experiments). Data represent the mean ± s.e.m.
shown in figure 5.7, incubation of BM-MNCs with BaTiO
3
crystals did not change the
percentage of these apoptotic cells.
5.4.3 BaTiO
3
SHG nanoprobes can be used to label cells for in vivo studies.
One of the challenges to study the localization of HSCs in adult bone using fluorescence-
based dyes is that the signal fades by the tissue being exposed to extreme changes in pH
Figure 5.7 Effects of BaTiO
3
labeling on cellular death. Percentages of apoptotic cells were also
quantified by flow cytometry in the BM-MNC population. (A) Representative flow cytometry plot of
BaTiO
3
–treated and nontreated cells. (B) Quantification of the AnnexinV
+
7-AAD
-
cells (n = 3 from 3
independent experiments). Data represent the mean ± s.e.m.
and heat during decalcification and processing. To examine whether we would
experience the same problem with SHG nanoprobes, BM-MNCs labeled with BaTiO
3
crystals were delivered to the marrow by tail vein injection and the recipient mouse was
sacrificed after 16 hours giving the cells enough time to home to the bone. The tibia of
the mouse was then dissected, fixed and decalcified before sectioning. The bone sections
were then imaged on a standard two-photon (2P) microscope. Because these SHG signal
is based off of inorganic crystals, unlike fluorescence-based dyes they are resistant to
changes to pH and heat, which means the signal was still maintained upon processing of
the bone tissue (Figure 5.8). It is noteworthy that because the SHG do not photo-bleach
and SHG nanoprobes have a superior signal-to-noise ratio, SHG imaging with tiling of
bone sections (Figure 5.8A-B) and semi-automated quantitative segmentation of the SHG
nanoprobe-labeled cells is possible. This would potentially permit the analysis of the
distribution of the cells in their niche.
Histochemical and immunohistochemistry (IHC) stainings are widely used to label
various components of the niche and look into the distribution of the HSCs relative to
these components. Yet, once again because of the extreme sensitivity of fluorescence-
based dyes to pH, pressure and heat changes, sustaining the fluorescent signal is a
constant challenge. However, here we demonstrated that the histochemical staining
process did not affect the SHG signal from the BaTiO
3
crystals. As shown in figure 5.9,
SHG imaging was performed on the same bone slice that underwent H&E staining, and
the SHG data were overlayed with the wide-field image (Figure 5.9B-D) to allow
identification of HSC localization with respect to marrow landmarks. Together, these
results demonstrated that BaTiO
3
SHG nanoprobes could be used to label cells to be
injected into recipient mice for in vivo studies. They also highlighted one of the main
advantages of SHG nanoprobes imaging over all other imaging probes: the SHG signal is
robust and remains visible following processing of samples for standard histochemical, or
immunohistochemical analysis.
Figure 5.8 Bone section imaging and image processing. (A) Overview of bone preparation, imaging,
and image processing. Paraffin-embedded, fixed bone samples are cut longitudinally resulting in 5µm
sections (left). Sections are imaged with a 2P microscope (870nm excitation wavelength) to obtain
bright field (bottom center) and SHG (top center) signal and individual frames are tiled to cover the
entire bone slice. SHG nanoprobe signal is segmented, and distances from the nearest bone surface are
measured in a semi- automated fashion (top right and zoom of yellow box bottom right). (B) Tiled
images from an exemplary tibia bone slice with bright field. (C) SHG signal from bone and SHG
nanoprobe-labeled BM-MNCs (white). (D) Segmented data showing 2D cellular localization (SHG
nanoprobes in red, bone in gray). Scale bar, 1500µm. (Picture courtesy of William P Dempsey)
Figure 5.9 Overlaying wide-field histological data with SHG tiled scan for topological
characterization of niche-dependent HSC localization. (A) Overview of the strategy to align wide-field
histological sections with SHG data to identify SHG nanoprobe-positive BM-MNCs in the niche. Raw
data of scanned (top left, cyan) and wide-field stained images (bottom left) are aligned using
histological landmarks as a guide. The right half of the figure shows a zoomed portion (yellow box
within whole slice cartoons) of each imaging set for clarity. The presence of SHG nanoprobe signal
(red) allows identification of a subpopulation of BM-MNCs that would be otherwise indistinguishable
from surrounding marrow cells (identified BM-MNCs shown in white for clarity). (B) Tiled images from
an exemplary tibia bone slice with SHG signal (870nm excitation wavelength) from bone in cyan and
SHG nanoprobe-labeled hematopoietic cells in red. (C) Wide-field H&E stained bone slice. (D)
Overlayed data showing 2D cellular localization within the BM cell population (SHG nanoprobes in
red, bone in cyan, marrow cells in purple). Note that in the unsegmented image, SHG nanoprobe signal
(red) is always adjacent to the nuclei of the labeled hematopoietic cells (inset in D, yellow arrowheads,
zoomed in region from yellow box in D), presumably within endosomes. Scale Bar, 1000µm (B, C, and
D) and 50µm (inset in D). (Picture courtesy of William P Dempsey)
5.4.4 BaTiO
3
SHG nanoprobes can be used to characterize the 3D environment of
the niche in whole bone samples.
Stem cells reside in a complex 3D microenvironment, therefore imaging stem cell-niche
interactions should be analyzed in intact 3D structures. To do so, the laser power that is
used to image the cells in whole bone should be amplified in order to penetrate through
the physical barriers of the bone. However, increasing the power is accompanied by
increasing the background noise and will result in saturation of the signal emitted from
fluorescence-based dyes.
To examine whether we encounter the same problems when using SHG nanoprobes,
labeled BM-MNCs were injected into the tail vein of recipient mice, the hind limb bones
were isolated after 16 hours and imaged using SHG microscopy. To reduce signal
scattering and autofluorescence the bones were first cleared using a BABB solution (1:2
ratio of benzyl alcohol: benzyl benzoate) (Figure 5.10). BABB solution is commonly
used for whole mount embryo clearing (Liang & Herr, 1994; Weisblat & Kuo, 2009) and
allows more SHG signal to be emitted through the bones. The cleared bones were then
imaged using a conventional 2P microscope. It has previously been shown that unlike
fluorescent approaches (e.g. cells tagged with GFP), SHG nanoprobe labeling results in a
signal that would saturate and exihibits virtually no background noise (Pantazis et al.,
2010).
Our data demonstrated that after sophisticated imaging and tiling of a cleared long bone
specimen outlined in figure 5.11A, we were indeed able to see labeled cells as deep as
150µm into the BM (Figure 5.11B, left), and that we could segment SHG nanoprobe
signal from the bone SHG signal in a semi-automated fashion (Figure 5.11B, right).
Additionally, we were able to reconstruct tiled bone optical slice images that were nearly
as sharp as those seen in our histological slices in figure 5.8 and 5.9 (Figure 5.11D-F).
Together our data demonstrated that SHG nanoprobes could be used to label and
visualize BM-MNCs deep inside tissue that were previously refractory to other imaging
techniques.
Figure 5.10 BABB bone clearing. The SHG signal is maintained once the bones were cleared using a
BABB solution (1:2 ratio of benzyl alcohol:benzyl benzoate) to minimize autofluorescence and signal
scattering. (Picture courtesy of William P Dempsey)
Figure 5.11 Characterization of a long bone HSC niche in optical cross section and optical
transverse section. (A) Overview of the preliminary strategy for fixed long bone deep tissue imaging.
The bone is harvested and fixed in 10% formalin without being decalcified (I). Next, the bone is
cleared in 1:2 BABB solution for ~2 days (II). Conventional 2P SHG microscopy (820nm excitation
wavelength) is used to image a sample stack of optical slices (zStack) from the metaphysis portion of the
humerus (yellow box in II). Signal from bone (cyan) and SHG nanoprobe-labeled hematopoietic cells
(red) can be visualized deep in the tissue by rotating an XYZ stack (III) 90 degrees (IV). (B) SHG signal
from SHG nanoprobes was observed throughout an entire exemplary cleared humerus bone zStack
(unprocessed SHG image on left, processed SHG image on right). After scanning and orienting the
zStack as in III-IV, a maximum intensity projection of 50µm (in y) was imposed to illustrate that
traditional 2P microscopy can see crystals >150µm beneath the surface of the cleared long bone tissue.
(C) Overview of strategy for long bone tiled imaging. After fixation and clearing, the bone can be
imaged using 2D tiling as with the fixed bone slices seen in Figures 5.7 (left). SHG data (bottom right)
can be segmented into bone (cyan) and SHG nanoprobe (red) signals for analysis. (D, E, and F) Tiled
optical section of the same exemplary whole humerus as in B, taken approximately 100µm from the
surface of the tissue. Unprocessed SHG data (D, white) can be segmented to separate the SHG
nanoprobe-labeled HSCs from the bone (F, red and cyan, respectively). Note that the bone signal is
intense in whole bone optical sections, making the segmentation difficult; however, SHG nanoprobe
signal can still be segmented correctly, as illustrated by the fact that the signal from segmented SHG
nanoprobes (red) only resides within bone marrow regions (dark areas in the bright field image in E).
Scale bar, 100µm (B) and 500µm (D, E and F). (Picture courtesy of William P Dempsey)
5.5 Discussion
To identify the precise location of the adult HSC niche and reveal the signaling pathways
that maintain HSCs in the niche, one requires an imaging technology that permits HSC
tracking with single-cell resolution, deep tissue penetration, and low phototoxicity in
vivo. However, the location of the HSC niche in the bone – that is surrounded by muscle
and skin – imposes physical limitations that need to be addressed if visualization of the
stem cells is to be achieved. Many studies have been proposed to try to overcome these
hurdles, including explant tissue studies (Yoshimoto et al., 2003; Xie et al., 2009),
intravital studies where portions of the bone are physically removed for easier imaging
(Askenasy et al., 2002; Köhler et al., 2009), and in vivo studies of the particularly thin-
boned calvaria (Lo Celso et al., 2009). However, none of these techniques are applicable
for imaging marrow from most bones in the mouse without extensive trauma to the tissue
that is imaged. Therefore an effective imaging technology that would be able to image
HSCs in all bones in vivo still needs to be developed.
Here, we have used a novel imaging probe to image HSCs in vitro and in vivo: very
bright SHG nanoprobes whose photophysical properties are fundamentally different from
that of the fluorescent agents currently used (Pantazis et al., 2010). We were able to
image HSCs using this non-invasive technique in multiple different bone types that
permitted the dynamic imaging of the HSCs in their native niche microenvironment.
Our data showed that these SHG nanoprobes get engulfed by BM-MNCs and are non-
toxic (Figures 5.4-5.6). Also, by labeling the BM-MNCs and injecting them back in the
mice, we were able to track the cells in their BM niche by imaging them in both bone
slices as well as in whole bone setting. In fact, using SHG nanoprobes as a cellular label,
we had for the first time visualized these cells in their naural 3D niche environment in
long bones.
However, there still exist caveats in the imaging modality in its current mode of use that
need to be resolved before any further biological questions could be asked and
conclusions be derived. First, all the toxicity studies performed as well as the cells that
have been injected and imaged are BM-MNC population that is a heterogeneous
population of cells. These cells have different uptake efficiencies as similar studies have
demonstrated with non-functionalized quantum dots (Nabiev et al., 2007). Therefore,
before we can use the SHG imaging technology, we should specifically target purified
stem cell populations and increase their labeling efficiency. One approach is to complex
the BaTiO
3
crystals to polycationic transfection agents through electrostatic interactions
such as poly-L-lysine (PLL) or protamine sulfate (Anderson et al., 2004; Arbab et al.,
2003; Arbab et al., 2004). This method is currently used and is proven to be an efficient
and effective technique for incorporating the superparamagnetic iron oxide (SPIO)
nanoparticles within endosomes, thereby labeling cells that can be detected by MRI
(Frank et al., 2002; Frank et al., 2003; Arbab et al., 2003). Alternatively, the BaTiO
3
crystals could be functionalized by being attached to antibodies specific for their target
HSCs.
Hematopoieitc stem and progenitor cells have been previously shown to localize to
various parts of the BM niche (Nilsson et al., 2001). In order to visualize these cells
simultaneously and examine the interaction between them, it is required that one can
visualize and distinguish these two cell populations apart. To do so a Multi-SHG
Detection Imaging (MSDI) modality, which has previously been described in Pantazis et
al. could be used (Pantazis et al., 2010). Briefly, two types of nanoprobes (Silicon
carbide, SiC and BaTiO
3
) with different wavelength-dependent SHG signal intensity
profiles can be discriminated by recording the SHG signal intensities at two different
excitation wavelengths (e.g. 800 nm versus 880 nm respectively). Using MSDI, we can
distinguish the relative location of different cell populations (e.g. HSC distances to
progenitor cells) to improve upon our understanding from the adult BM niche.
In summary, we have demonstrated how the photophysical advantages of SHG
nanoprobes make them a perfect tool for labeling stem cells and visualizing them in their
3D niche environment. This new tool could potentially enable us to address questions that
are presently unanswerable with current imaging technologies: Identifying the location of
all HSC niches; the cellular participants surrounding them and teasing apart the systems
directing stem cell behavior.
Chapter 6: Concluding Remarks and Future Directions
The definition of “niche” was proposed more than thirty years ago in the hematopoietic
system as the structured environment that maintains the “stem-ness” of the stem cells and
controls their homeostasis (Schofield, 1978). However, the first experimental evidences
to support this concept came years later from studies of the Drosophila melanogaster
ovary (Xie & Spradling, 2000). Here, the anterior ovariolar somatic cells comprised of
three different cell types act as a germ line stem cell (GSC) niche with the cap cells
acting as the key component of the niche (Xie & Spradling, 2000). Soon after, the hub,
located at the tip of the Drosophila testis and the distal tip cell located at the tip of the
germ line organizing region in Caenorhabditis elegans was found to function as the niche
in supporting GSCs (Kiger et al.,2001; Tulina & Matunis, 2001; Crittenden et al., 2002).
In these contexts, the supporting stromal cells maintain stem cells and regulate their
proliferation and differentiation through providing them with (1) structural support
through anchoring the stem cells, (2) trophic support by secreting conductive molecules,
and (3) topographic information by sending proper polarity cues through physical
interaction with the stem cells (Lymperi et al., 2010).
Identifying such organized niche environments in mammalian systems has been more
challenging because of their complicated anatomical structure as well as the rarity of the
stem cells and lack of definitive identifying markers. However, extensive progress has
been made and many of the cellular components and the underlying molecular
interactions between the stem cells and their residing niche in the mammalian system
have been identified (Lie & Xie, 2005; Jones & Wagers, 2008).
The BM has long been known to be the home for adult HSCs. Studies have shown that
within the BM, HSCs are located near the bone surface (Lord et al., 1975, Gong, 1978;
Nilsson et al., 2001) or are associated with the sinusoidal endothelium (Kiel et al., 2005b;
Sipkins et al., 2005). At the endosteal surface, cells of osteoblastic lineage have been
proven to have a crucial role in maintaining the HSCs in their niche (Calvi et al., 2003;
Zhang et al., 2003; Visnjic et al., 2004). However, osteoblasts are not the only cells
present at the endosteal surface. The border between the bone and the BM is also where
bone remodeling is dynamically occurring by constant interaction between the osteoblasts
and the osteoclasts, and these cells are constantly modulating each other’s activity
through various molecular cross talk signals (Teitelnaum et al., 2003; Perez-Amodio et
al., 2004; Ryu et al., 2006).
To examine whether the active bone remodeling plays a role in maintaining the HSC
niche, we have pharmacologically activated osteoclasts and osteoblasts. Our data
(Chapter 3), together with other studies (Lymperi et al., 2011), indicate that dynamic
crosstalk and precise coordination of osteoclastic and osteoblastic activity is required for
maintaining the HSC niche. In fact, global expansion of HSCs without activating the
osteoclasts will not lead to an increase in the size of the stem cell pool (Chapter 3,
Lymperi et al., 2008). Therefore, we suggest that the sites of active bone remodeling are
the location for the HSC niche at the endosteal surface of the bone.
The question that also needs to be addressed would be the exact location of these niches
in the bone. Are the HSCs located at the endosteal surface of the cortical bone, or are
they located beside the trabecular bone of the metaphysis region? Our data suggests the
presence of distinct niches in the BM for primitive hematopoietic cells. We have
demonstrated that the more primitive HSCs are localized at the endosteal surface of the
cortical bone in the diaphysis region of the long bones, while the more differentiated ST-
HSCs are localized next to the trabecular bone of the metaphysis area (Figure 3.16). As
suggested earlier, a possible explanation for this regional localization would be that the
most primitive HSCs would preferentially localize to the region of bone with the least
extrinsic disturbance (e.g. remodeling activity) to maintain their quiescent state, whereas
the more differentiated ST-HSCs are located in regions of active bone remodeling and
thus would be readily recruited for hematopoietic expansion.
Although this study provides further insight to the cellular components regulating the
HSC BM niche, this model is solely based on immunophenotypic and functional analysis
of the HSCs. The gold standard to prove the existence of such regional localization of
primitive hematopoietic cell subsets in the BM would be tracking these cell populations
in their natural BM habitat.
Visualizing HSCs in the adult BM niche has always been challenging because of the
anatomical barriers imposed by bone itself and the tissues surrounding it. One of the
common ways to circumvent this issue is to image HSCs in the calvaria (Mazo et al.,
1998; Mazo et al., 2002; Lo Celso et al., 2009). However, flat bones such as calvaria are
formed through intramembranous ossification, which is different from endochondral
ossification of long bones (Hall & Miyake, 2000; Silbermann & von der Mark 1990;
Kronenberg, 2003). These two differing processes have been thought to be the source of
some hematopoietic differences (Everts et al., 1999; Chan et al., 2009). Therefore, one
should first confirm whether HSC behavior and its interaction with the cellular
components of the niche in the calvaria are comparable to that of long bones before
applying the data collected from imaging the calvaria to HSC physiology in the long
bones.
To do so, we have looked into the regulation of HSC physiology in the calvaria and
compared that to HSCs derived from the femur/tibia. Our data (Chapter 4) corroborate
other studies (Lo Celso et al., 2009) showing that primitive HSCs frequencies and their
cell-cycle profiles are comparable between the HSCs derived from the two sites.
However, gene expression profile and immunostaining studies revealed that n-cadherin
expression levels are much higher in the cells derived from the calvaria compared to the
femur/tibia. The elevated n-cadherin expression levels were accompanied with a superior
potential of calvaria-derived cultures to support hematopoietic cell growth in vitro. We
have hypothesized that although the cells derived from the two different sources seem to
have similar hematopoietic potential, HSC maintenance in the calvaria might be N-
cadherin dependent, while this does not seem to be the case in the long bone.
The role of N-cadherin in HSC physiology is controversial. Studies define the niche cells
as spindle shaped N-cadherin-expressing osteoblastic cells (Zhang et al., 2003) that are
required for functional engraftment of the BM following transplantation (Hosokawa et
al., 2010). On the other hand, other studies demonstrate a lack of evidence for N-cadherin
expression in highly purified HSCs (Kiel et al., 2007), and suggest that HSCs do not
depend upon N-cadherin for their maintenance (Kiel et al., 2009). All these studies,
however, have always examined HSCs from the femur and never looked into how N-
cadherin down-regulation might affect calvaria HSCs. We, on the other hand, have
provided evidence that there are differences in N-cadherin expression in the long and flat
bones, which may play a role in the regulation of the HSCs in different anatomic sites.
Although only speculated, if the regulatory mechanism in the calvaria is indeed different
from that of the femur/tibia, then the original question still remains as to whether one can
use the imaging data obtained from the calvaria to address questions about HSC
physiology in the long bones. Of course, an alternative that avoids such complications
would be to try to image HSCs in the long bones.
In this regard, the second harmonic generating (SHG) nanoprobe imaging technique
(Pantazis et al., 2010) has opened the door to capturing the dynamic nature of HSCs at
the single-cell level in all BM environments including long bones. We have demonstrated
that these nanoprobes are not toxic to BM-MNCs and can be visualized in bone slices as
well as whole bones (Chapter 5). Combining traditional histological and
immunohistochemical approaches with SHG nanoprobe imaging and sophisticated
analyses (e.g. quantitative segmentation of the SHG nanoprobe-labeled stem cells), we
would be able to localize HSCs in their natural niche and look into their distribution with
respect to other cellular components.
The ultimate goal of developing such an imaging technique, however, would be to
determine the location and the dynamic nature of the niche components in vivo. The
photophysical characteristics of SHG nanoprobes give them excellent long-term
photostability in vivo with unmatched signal-to-noise ratio. This will allow signal
detection in deep organs, real-time biodistribution monitoring and long-term tracking of
HSCs in vivo. This idea is not unrealistic, and in fact, our preliminary experiments have
confirmed the feasibility of using a microendoscope to detect the SHG signal through the
calvaria bone (Figure 6.1).
In this study, we placed a 350-µm-diameter doublet microendoscope (Grintech, Jena,
Germany) directly onto the surface of a fixed excised calvaria bone from a WT mouse
that had been injected with SHG nanoprobe-labeled BM-MNCs. As shown in figure 6.1,
we can indeed see SHG signals representing individual SHG nanoprobe-labeled cells
within the marrow using the microendoscope. A similar minimally invasive optical
microendoscopy setup has been successfully used before to observe second-harmonic
frequencies of light generated in the muscle fibers of live mice and humans (Llewellyn et
al., 2008).
Figure 6.1 SHG microendoscopy within a fixed, whole calvarium. (A) Cartoon of a microendoscope
apparatus. Incoming infrared laser light (red lines, 850nm excitation wavelength) is directed into the
back aperture of a microscope objective lens. This light is focused into the back of a microendoscope
(radius ~350µm), which then refocuses the light onto the sample, with a maximum working distance of
700µm. The signal from SHG nanoprobe-labeled HSCs (blue lines) is then detected in the epi-direction
via detectors behind the objective. The endoscope is held in place and is moved in tandem with the
objective using a motorized stage and clamp arm setup. (B) Photograph of the microendoscopy stage.
The microendoscope is held in place with an arm connected to a motorized stage that can move the
endoscope up and down. The 20x objective is suspended above the endoscope in an upright modality. A
2P laser at 850 nm is scanned and focused using the objective and the endoscope to visualize a 2D
window (up to 250µm x 250µm in x and y) into the tissue. (C) Photograph of the microendoscope
(nearly transparent and at the bottom of the tube). Note that the microendoscope has a length of only
5mm. (D) Cartoon depicting a calvaria and the general vicinity of where the imaging took place (yellow
arrow). (E) Maximum intensity z-projection of a 20 µm optical slice stack (5 µm between each optical
slice), clearly showing 10 SHG nanoprobe-labeled HSCs within the tissue. Scale bar, 20 µm. (Picture
courtesy of Dr. Mark J Schnitzer and William P Dempsey).
In summary, the work described in this thesis has evaluated various aspects of HSC
localization in the BM niche. It has provided evidence that there is a regional localization
of primitive hematopoietic cell subsets in the adult BM, suggesting specific niches for the
long-term and short-term HSCs in the cortical bone of the diaphysis area and the
trabecular bone of the metaphysis region, respectively. We have also proposed a spatially
distinct niche regulation of stem cell populations in the calvaria and the femur/tibia.
Using SHG nanoprobe imaging, we can now identify individual cell populations deep
within the marrow of various bones, which opens the possibility of continuous tracking of
HSCs in their niche in vivo. This new imaging modality will provide a major
advancement in understanding how HSCs interact with a variety of elements within the
dynamic environment of the BM.
Using the knowledge gained through these studies combined with deep tissue in vivo
imaging of HSC dynamics within the BM, we would be able to identify the precise
location of different hematopoietic cell populations in a variety of bone types and
examine the efficiency of novel treatments of blood related disorders and design new
therapeutic strategies that could enhance HSC-based therapies.
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Abstract (if available)
Abstract
Adult HSCs reside in the BM in a microenvironment known as the stem cell niche. Many studies have identified key components of the niche required to maintain the HSCs in their primitive state. However, other cellular components and the exact location of the HSC niche are yet to be clearly identified. My studies have demonstrated that the coordinated interaction between the osteoblasts and the osteoclasts is required for maintaining HSCs in their BM niche. This also highlighted a structural organization of the localization of primitive hematopoietic cell subsets in distinct regions of the BM. We further examined this spatial regulation of HSCs in two anatomically distinct sites, flat bone and long bones. Our data shows that while HSCs isolated from these two different bones have identical potential, their maintenance may be regulated by different mechanisms. These data further demonstrate that although flat bones are desirable sites for visualizing HSC in their niche, the data derived from imaging HSCs in these bones may not necessarily correlate with functional analysis data that is mainly derived from cells of the long bones. Therefore, we developed an imaging modality based on SHG nanoporbes that can potentially capture the exact localization of HSCs in their niche and their dynamic nature at single cell resolution within all bones. Understanding all of the interactions between the stem cells and their niche may yield both practical methods for manipulating stem cells to achieve therapeutic outcomes and also define a model for the impact of the microenvironment on stem cell biology.
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Rashidi, Narges
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Regional localization and regulation of hematopoietic stem cells in the bone marrow stem cell niche
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Keck School of Medicine
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Doctor of Philosophy
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Systems Biology and Disease
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2011-12
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11/14/2011
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hematopoietic stem cell,hematopoietic stem cell niche,OAI-PMH Harvest,osteoclast,second harmonic generating nanoprobes
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hematopoietic stem cell
hematopoietic stem cell niche
osteoclast
second harmonic generating nanoprobes