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University of Southern California Dissertations and Theses
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Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells
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Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells
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Content
EFFECTS OF A GSK-3 INHIBITOR ON RETROVIRAL-MEDIATED GENE
TRANSFER TO HUMAN CD34+ HEMATOPOIETIC PROGENITOR CELLS
by
Yeong “Christopher” Choi
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2009
Copyright 2009 Yeong “Christopher” Choi
ii
Table of Contents
List of Figures iv
Abstract vi
Chapter One – Introduction
1(i) Introduction to Hematopoietic Stem Cells 1
1(ii) Models of HSC Self-Renewal/Differentiation 3
1(iii) Hematopoietic Stem Cell Sources 5
1(iv) Cytokines/Growth Factors 7
1 (v) Genes Regulating HSC self-renewal 8
1 (vi) Ex vivo HSC Gene Therapy 10
Chapter Two – Wnt/ β-catenin and stem cells
2(i) Introduction to Wnt and Hematopoietic Stem Cells 13
2(ii) Wnts 15
2(iii) Wnt/ β-catenin Signaling in Different Tissues 17
2(iv) Wnt/ β-catenin Signaling in Hematopoietic Stem Cells 18
2(v) Small Molecules and HSC 19
2(vi) Effects of GSK3 Inhibitor BIO in Stem Cells 21
Chapter Three – Methods of β-catenin Upregulation
3(i) Introduction 23
3(ii) Materials and Methods 27
3(iii) Results 32
3(iv) Discussion 41
Chapter Four – Effects of BIO addition on UCB CD34+ progenitor
cells
4(i) Introduction 46
4(ii) Materials and Methods 48
4(iii) Results 57
4(iv) Discussion 72
iii
Chapter Five – Concluding Remarks and Future Directions
5(i) Hematopoietic Stem Cell Gene Therapy 76
5(ii) Perspectives of Small Molecules 76
5(iii) Application of Small Molecules for Gene Transfer to HSC 77
Bibiliography 79
iv
List of Figures
Figure 1-1. Schematic representation of Hematopoiesis: Lineage 2
differentiation of HSC.
Figure 1-2. Model of stem cell division. 4
Figure 3-1. siRNAs (Axin 1 through 4) to downregulate Axin 1 in HeLa 34
cells
Figure 3-2. The chemical structure of AR-A014418 also known as 35
GSK3β Inhibitor VIII.
Figure 3-3. AR-A014418 effect on K562 cells β-catenin concentration. 36
Figure 3-4. Effects of increasing AR-A014418 dose on K562 and 37
CD34+ cells β-catenin concentrations.
Figure 3-5. AR-A014418 exposure to CD34+ cells effect on cell cycle. 38
Figure 3-6. Effect of AR-A014418 addition on Stem/Progenitor Cell 40
Renewal.
Figure 4-1. Structure of BIO and dose escalation of BIO on CD34+ cells. 58
Figure 4-2. Analysis of β-catenin activity in BIO treated CD34+ cells. 59
Figure 4-3. Cell cycle analysis of CD34+ cells following BIO treatment. 61
Figure 4-4. Day 3 cell cycle analysis of CD34+ cells following BIO 62
treatment.
Figure 4-5. Tritiated thymidine analysis following BIO treatment in 63
CD34+ cells.
Figure 4-6. CFSE Analysis of CD34+ cells treated with BIO. 64
Figure 4-7. Apoptosis analysis of CD34+ cells treated with BIO. 66
Figure 4-8. In vitro transduction of CD34+ cells. 67
Figure 4-9. Percentage of Human CD45 Positive cells following 68
24hours post tail vein injection.
v
Figure 4-10. Percentage of Human CD45 Positive cells following 9-10 69
weeks post tail vein injection.
Figure 4-11. Lineage differentiation ability of BIO treated CD34+ cells 70
analyzed in a NSG xenotransplantation model.
Figure 4-12. Gene marking in a primary CD34+ xenotransplantation 71
mouse model.
vi
Abstract
Hematopoietic stem cells (HSC) are rare cells of the hematopoietic system with
the ability to self-renew and differentiate into all mature blood lineages. Although
HSC are an extremely attractive target for HSC gene therapy and despite
successful use of HSCs in the clinic and in clinical gene therapy trials, limitations
exist. One limitation is the transduction protocol used requiring a combination of
cytokines to promote HSC into cell cycle, since γ-retroviral vectors can only
transduce dividing cells. The Wnt/ β-catenin pathway has been shown to be
involved in stem cell fate determination and self-renewal. In these studies we
activate the Wnt/ β-catenin pathway using a GSK3 inhibitor, BIO, and test its
effects on γ-retroviral transduction, homing, engraftment and hematopoiesis
potential of human umbilical cord blood (UCB) CD34+ cells. We initially explored
different methods to activate the Wnt/ β-catenin pathway in CD34+ cells. We
designed and tested siRNA’s to Axin and used GSK-inhibitors in order to disrupt
the destruction complex thereby stabilizing β-catenin. GSK3 inhibitor BIO showed
the most promise. We found BIO treatment led to increased accumulation of β-
catenin in UCB CD34+ cells. We observed increased β-catenin activity following
BIO treatment, analyzed by TOPflash reporter assay and upregulation of Ccnd1
expression compared to the untreated control. We determined that BIO initially
increased UCB CD34+ cells progression through cell cycle but then imposed an
arrest in cell division. BIO decreased cell apoptosis and did not impair UCB
CD34+ cells homing, engraftment and hematopoiesis potential. BIO treatment of
vii
CD34+ cells allowed an increased extent of gene transfer using a γ-retroviral
vector. Future studies on the effects of the BIO induced cell arrest will allow for a
better understanding of the effects that GSK inhibition has on HSC function. BIO
mediated manipulation of the Wnt pathway shows promise as a useful method to
support γ-retroviral vector mediated gene transfer to HSC for gene therapy.
1
Chapter 1 – Introduction
1(i) Introduction to Hematopoietic Stem Cells
Stem Cells are rare cells with the crucial ability to self-renew and to
generate mature cells of any tissue through differentiation. Adult and embryonic
stem cells hold great promise for regenerative medicine, tissue repair, and gene
therapy (Cavazzana-Calvo, Hacein-Bey et al. 2000; Laughlin, Barker et al. 2001;
Filipovich 2008). In the hematopoietic system, these multipotent progenitors
comprise less than 0.05% of mouse bone marrow cells and can be divided into
three different populations, the long-term self-renewing HSCs, short-term self-
renewing HSCs, and multipotent progenitors without detectable self-renewal
potential (Reya, Morrison et al. 2001; Templin, Kotlarz et al. 2008). The develop-
mental stages of the hematopoietic hierarchy can be identified and arranged in a
hierarchical tree, beginning with the long-term self-renewing HSCs. The
progression leads to stem cells that gradually lose one or more developmental
options becoming lineage committed cells (Figure 1-1). Studies suggest that the
decision of these HSCs to differentiate or self-renew is regulated by both intrinsic
and extrinsic signals (O'Reilly, Harris et al. 1997; Van Den Berg, Sharma et al.
1998; Bhardwaj, Murdoch et al. 2001; Antonchuk, Sauvageau et al. 2002; Reya
and Clevers 2005). Identifying the mechanisms of regulation and the factors
involved in stem cell self-renewal will significantly promote the therapeutic
potential of stem cells.
2
A common approach used to identify HSC is through a combination of cell
surface antigens, the most common being the sialomucin CD34 glycoprotein
(Baum, Weissman et al. 1992). Antibodies against the CD34 surface protein are
used to purify this population of cells by either FACS sorting or using magnetic
beads and columns (Nakano 2003; Madkaikar, Ghosh et al. 2007; Kurtzberg
2009). The groups of cells isolated using these anti-CD34 antibodies contain the
long-term self-renewing HSCs, short-term self-renewing HSCs, and multipotent
progenitors and some non-stem cells. Cells that express CD34 and have low or
absent levels of CD38 appear to be a more enriched population of HSC
(Berenson, Andrews et al. 1988; Berenson, Bensinger et al. 1991; Lansdorp,
Schmitt et al. 1992). Although these markers can be used to identify potential
Figure 1-1.Schematic representation of the production of all blood cells.
Figure 1-1. Schematic representation of Hematopoiesis: Lineage differentiation of
HSC. Lineage restricted multi-potent progenitors are depicted: BFU-E = burst-forming unit-
erythroid. Meg-CFC= Megakaryocyte. Eo-CFC= Eosinophil. GM-CFC=Granulocyte and
Macrophage (Figure from Reya et al. 2002).
3
HSC, the markers are not specific to unique stem cell function so cannot be said
to truly identify a stem cell. These markers are useful but improvements in
identifying stem cells will be beneficial for future HSC studies.
1(ii) Models of HSC self-renewal/differentiation
In the adult bone marrow, under normal conditions, the number of HSC
remains fairly constant since most bone marrow (BM) HSC are quiescent
(Spangrude, Smith et al. 1991; Weissman, Spangrude et al. 1991; Reya,
Morrison et al. 2001; Nakano 2003). Passegue et. al., 2005, have shown that
most HSCs are capable of long term engraftment of irradiated recipients and are
in the G
0
phase of the cell cycle (Passegue, Wagers et al. 2005). However, it has
been indicated that the majority of these cells divide regularly, but in a slow
manner (Cheshier, Morrison et al. 1999). So the question arises as to how these
HSC divide and self renew?
HSCs can promote the self renewal process by symmetric and/or
asymmetric division (Knoblich 2008; Wu, Egger et al. 2008). Symmetric cell
division gives rise to two identical daughter cells both retaining stem cell
properties. Asymmetric cell division is where an individual HSC gives rise to non-
identical daughter cells. One of these cells keeps the HSC identity while the other
undergoes differentiation into a progenitor cell. There are two hypothetical
mechanisms by which asymmetric cell division may occur. The first is divisional
asymmetry where stem cell determinants (ie. proteins or factors) are distributed
unequally during cell division. The daughter cell which does not receive the stem
4
cell determinants, leaves the stem cell niche and enters an environment favoring
differentiation. The daughter cell that receives the stem cell determinants, retain
the HSC fate. The second is environmental asymmetry, where initially one HSC
produces two identical daughter cells. One of the daughter cells enters a different
environment which favors differentiation and the other daughter cell remains in
the HSC niche and retains its stem cell fate (Figure 1-2) (Morrison and Kimble
2006; Knoblich 2008; Wu, Egger et al. 2008).
Figure 1-2. Model of stem cell division.
5
A stem cell niche is a term used to describe a specific site where stem
cells reside and the niche interacts with stem cells to determine their fate. The
niche is formed by supporting cells that produce exogenous signals and provide
a microenvironment for stem cells (Spradling, Drummond-Barbosa et al. 2001;
Lin 2002; Li and Xie 2005). There are several in vitro coculture experiments
which support the niche hypothesis (Dexter, Moore et al. 1977; Moore, Ema et al.
1997; Li, Johnson et al. 2004).
1(iii) Hematopoietic Stem Cell Sources
Autologous or allogeneic hematopoietic stem cell transplantation has been
used successfully in the treatment of hematological diseases, such as primary
immunodeficiency diseases, and metabolic disorders of children and adult
patients (Brown and Boussiotis 2008; Filipovich 2008; Kurtzberg 2009).
Historically, the bone marrow has represented the main source of HSCs in
pediatric and adult individuals (Daley and Scadden 2008). However, in many
cases, a suitable donor is unavailable. Therefore, many researchers have looked
for alternative sources of HSCs for use in human BMT.
One alternative source of human HSCs is the peripheral blood (PB). PB is
considered to be a practical source of HSCs, especially because methods exist
which can mobilize significant numbers of stem/progenitor cells into circulation
following administration of G-CSF. A difficulty with this source is that some
patients show poor mobilization and stem cells (ie. cells in graft) obtained from
6
the PB are more likely to cause graft-versus-host disease than stem cells
obtained from the bone marrow (Cutler and Antin 2001).
Another source of HSC is from umbilical cord blood (UCB), which has
been proven to be an alternative HSC source for patients without matched sibling
donors. This source has many advantages for transplantation, but because of its
small volume, the recipients are generally pediatric patients (Laughlin 2001; Tse
and Laughlin 2005). Rarely can doctors extract more than a few million HSCs
from a placenta and umbilical cord, which is too few to use in a transplant for an
adult. Therefore, as a solution to these problems, ex vivo expansion of HSCs
would be an obvious area for further study.
The identification, cloning, and production of recombinant cytokines and
molecular factors, together with the identification and purification of HSCs and
progenitor cells have greatly increased our understanding of the hematopoietic
system. For example, Fms-like tyrosine kinase 3 (Flt3) ligand, Stem Cell Factor
(SCF) and Thrombopoietin (TPO) are growth factors found to be necessary in the
survival and/or proliferation of HSC (Murray, Young et al. 1999). Additionally,
factors such as β-catenin, Hedgehog and Notch are candidates evidenced to be
involved in stem cell self renewal (Bhardwaj, Murdoch et al. 2001; Antonchuk,
Sauvageau et al. 2002; Jho, Zhang et al. 2002). As a result of these fundamental
discoveries, many investigators are studying the ex vivo manipulation of HSCs
for potential therapeutic purposes. In general, the objectives of these
manipulations are to expand HSCs responsible for long-term hematopoietic
repopulation.
7
1(iv) Cytokines/Growth Factors
Various combinations of cytokines have marked effects on inhibition of
apoptosis and stimulation of self-renewal of HSC in short term ex-vivo cultures.
These growth factors are necessary for the survival and proliferation of HSC
(Madkaikar, Ghosh et al. 2007). As mentioned above, some growth factors
involved in HSC expansion are Flt3 ligand, SCF and TPO. Each of these
cytokines has been shown to support the survival and/or proliferation of HSCs
(Murray, Young et al. 1999; Madkaikar, Ghosh et al. 2007).
Flt3 ligand stimulates primitive hematopoietic cells by activating the Flt3
receptor, thereby regulating the proliferation and differentiation of multiple
lineages of cells of the hematopoietic system. By itself, Flt3 ligand stimulates little
growth of HSC in vitro, but in combination with other cytokines it has a synergistic
effect (Gammaitoni, Bruno et al. 2003). Flt3L is widely expressed in human tissue
(Nielsen, Husemoen et al. 2000; Madkaikar, Ghosh et al. 2007). Nielsen et. al.,
2000, have shown that Flt3L preserves the uncommitted CD34+CD38- progenitor
cells during cytokine prestimulation for retroviral transduction (Nielsen,
Husemoen et al. 2000).
SCF is expressed by a number of cell types. It binds to a tyrosine kinase
receptor called c-Kit, which is expressed on all HSCs. SCF plays an important
role in the prevention of HSC apoptosis, although it does not play an essential
role in the generation of HSC (Hassan and Zander 1996; Madkaikar, Ghosh et al.
2007).
8
TPO is a critical cytokine that regulates megakaryocyte and platelet
development. All HSCs express TPO’s receptor Mpl. In vitro, TPO induces
proliferation of primitive progenitors and thus has become an important factor in
ex vivo HSC expansion (Murray, Young et al. 1999; Madkaikar, Ghosh et al.
2007).
The combination of Flt3 ligand, SCF and TPO have been shown to be
synergistic in the expansion of HSC (Gammaitoni, Bruno et al. 2003). Although
cytokines can be used to expand HSC, they have been found to promote lineage
differentiation in long term cultures (Gammaitoni, Bruno et al. 2003). A better
understanding of the molecular mechanisms of HSC self-renewal and
differentiation would be beneficial in the field of ex vivo HSC expansion. Direct
manipulation of the molecular factors and pathways involved in HSC self-renewal
would enable a more targeted approach to HSC ex vivo expansion.
1(v) Genes Regulating HSC self-renewal
Despite the progress that has been made in identifying and obtaining
enriched HSC populations, the molecular mechanisms involved in the
maintenance and development of HSC still remain unclear. However,
researchers have discovered promising factors such as β-Catenin, Hedgehog
and Notch which are evidenced to be involved in regulating self-renewal of
mouse and human stem cells (Antonchuk, Sauvageau et al. 2002; Reya, Duncan
et al. 2003; Zhang, Lo Muzio et al. 2005).
9
The canonical Wnt/ β-catenin pathway has been implicated in the
regulation of HSC self-renewal (Foltz, Santiago et al. 2002; Reya, Duncan et al.
2003; Reya and Clevers 2005). The receptor for Wnt is the Frizzled family of
seven-pass transmembrane proteins and LDL-receptor-related proteins. In the
absence of Wnt, β-catenin is associated with APC and Axin1. Together, they
form what is called the destruction complex. When Wnt is bound to its receptor,
the destruction complex is disrupted allowing β-catenin to translocate into the
nucleus joining and activating the LEF/TCF family of transcription factors (Jho,
Zhang et al. 2002; Reya and Clevers 2005).
The Hedgehog (Hh) pathway has been associated with cell cycle
regulation (Roy and Ingham 2002) and proliferation of HSCs in adulthood
(Bhardwaj, Murdoch et al. 2001). Hh is also associated with hematopoiesis
during embryogenesis (Dyer, Farrington et al. 2001; Gering and Patient 2005).
The pathway is initiated once Hh ligand binds to the Ptch receptor thereby
relieving Smo’s inhibition and leading to the activation of the Gli family of
transcription factors.
Notch pathway is highly expressed in osteoblasts and thought to be an
essential component of HSC niches. Its signaling is active in HSCs and is down
regulated as HSCs differentiate (Duncan, Rattis et al. 2005). Notch seems to play
two separate but distinct roles in the
hematopoietic system, it inhibits the
differentiation
of hematopoietic progenitors and participates in cell fate decisions
within progenitor populations (Stier, Cheng et al. 2002).
10
With the advancements of functional genomics and other developing
technologies, a deeper understanding of the process of stem cell self-renewal
and differentiation is at hand. With this expansion of knowledge into the
molecular and cellular mechanisms of HSC development, future protocols for the
generation, expansion and differentiation of HSC may be developed for clinical
use in bone marrow transplants or gene therapies.
1(vi) Ex vivo HSC Gene Therapy
The concept of gene therapy involves the introduction of a functional gene
into a target cell in order to restore deficient protein function in an otherwise
normal cell. Although the principle may seem straightforward, consistent
successful gene therapy relies on the gene transfer vehicle and cell transduction
conditions.
Since HSCs have the potential to reconstitute the entire hematopoietic
system, they make a promising target for ex vivo gene therapy. Stable gene
transfer into HSCs could provide a long term cure for a number of disorders
affecting components of the hematopoietic system, including inherited
immunodeficiencies, thalassemias, lysosomal storage diseases; any disease that
can be treated by a bone marrow transplantation can potentially be treated by
gene transfer into HSCs (Bordignon, Notarangelo et al. 1995; Kohn 2001; Aiuti,
Slavin et al. 2002; Aiuti, Cattaneo et al. 2009).
In most cases for HSC gene therapy, there are three important aspects
that must be met for an ideal gene transfer vehicle. The first is efficient gene
11
delivery and expression, termed transduction. The second is persistent
expression of the delivered gene. The third is that the addition of the therapeutic
gene should not interfere with the normal functions of the host cells. Thus, viral
vectors were chosen as a promising gene transfer vehicle since evolution has
made them an efficient system for gene transfer (Anderson 1984; Lotzova 1994).
Vectors derived from a number of viruses are used as gene transfer
vehicles for HSCs. These include adenoviruses, adeno-associated viruses,
murine γ-retroviruses, mainly Moloney murine leukemia virus (MuLV),
lentiviruses, such as human immunodeficiency virus 1 (HIV 1), spumaviruses,
such as foamy virus, and many others (Anderson 1984; Lotzova 1994). In all
cases, the recombinant viruses are formed by removing the unwanted viral
genes and replacing them with a therapeutic gene. When needed, safety
precautions were added to make these vectors replication incompetent (Logan,
Lutzko et al. 2002).
Currently, the only successful HSC gene therapy clinical trials have been
performed by using vectors based on the MuLV (Cavazzana-Calvo, Hacein-Bey
et al. 2000; Aiuti, Cattaneo et al. 2009). However, a weakness to using transfer
vectors based on the Murine Leukemia virus is that they are not able to
transduce nondividing cells. Since HSC are inherently in the quiescent state,
clinical protocols require the use of a pre-stimulation step using various cytokine
cocktails (eg. SCF, Flt3 ligand, TPO) to induce these HSCs into cell cycle,
thereby allowing increased gene transduction efficiency when using γ-retroviral
vectors. The drawback of this method is that it promotes HSC to lose their
12
potential long term repopulating capacity (Williams 1993; Tisdale, Hanazono et
al. 1998). An ideal γ-retroviral transduction culture condition would promote
expansion of long term repopulating HSC while promoting cell cycle progression
for increased transduction and increase survival.
13
Chapter 2 – Wnt/ β-catenin and stem cells
2(i) Introduction to Wnt and Hematopoietic Stem Cells
HSCs have been the most extensively studied and characterized type of
stem cells but the question still remains, how is stem cell self-renewal regulated?
It has been shown that a combination of cytokines can induce extensive
proliferation but is unable to prevent differentiation of HSCs in long-term cultures
(Gammaitoni, Bruno et al. 2003). Although progress has been made in identifying
conditions that maintain HSC activity during short term culture conditions
(Murray, Young et al. 1999; Gammaitoni, Bruno et al. 2003), determining the
ideal combinations of growth factors that promote significant expansion of
progenitors with transplantable HSC activity still remains elusive.
Studies investigating the molecular factors that control self-renewal have
greatly advanced our understanding of HSC development. Many potential
mediators of HSC homeostasis have been identified by changes in HSC growth
in transgenic and knockout mice. These discoveries have allowed further study of
these candidate molecules. For example, exogenous HoxB4 expression caused
an increase in the numbers of transplantable hematopoietic stem cells both in
vitro and in vivo (Sauvageau, Thorsteinsdottir et al. 1995; Antonchuk, Sauvageau
et al. 2001; Antonchuk, Sauvageau et al. 2002). Mice overexpressing bcl-2 have
shown increased numbers of HSCs (Domen, Cheshier et al. 2000), suggesting
stem cell numbers are affected by anti-apoptotic signals. Mice missing the cell
14
cycle regulator p21, show a higher rate of HSC proliferation and differentiation
and a lower self-renewal capacity. In the absence of p21, HSCs rapidly
proliferate and lineage differentiate suggesting that p21 is required for
maintaining HSC quiescence (Cheng, Rodrigues et al. 2000). While all these
factors may be important in the development of HSC, the precise mechanisms
and order of action have not been clearly defined.
Signaling pathways such as Notch, hedgehog and Wnt, in the context of
embryonic stem cells development have presented possible candidates for the
regulation of stem cell self-renewal. To study the effect of Notch expression in
vivo, Stier and colleagues used SCID mice which were defective in lymphocyte
development to reveal the effects of Notch on HSC expansion (Stier, Cheng et al.
2002). Stier et al., 2002, demonstrated that expressing Notch in HSCs leads to
expansion in vivo. However, in many cases Notch expression in HSC can also
lead to the rapid formation of T-cell leukemias in vivo.
Sonic hedgehog, another pathway discovered as a regulator of embryonic
development, has been shown as a potential mediator of HSC development
(Bhardwaj, Murdoch et al. 2001). HSC that were CD34
+
and CD38
-
(a more
enriched population of progenitor cells), displayed increased self-renewal in
response to hedgehog signaling in vitro, although a combination of six other
growth factors were added (Bhardwaj, Murdoch et al. 2001). Since both Notch
and Sonic hedgehog activation have been shown to maintain progenitors in an
undifferentiated state in both ESC and HSC (Karanu, Murdoch et al. 2000;
Antonchuk, Sauvageau et al. 2001; Antonchuk, Sauvageau et al. 2002; Stier,
15
Cheng et al. 2002), this supports the possibility that these signals may be
preserved and can promote self-renewal in other types of stem cells.
The Wnt family of proteins are involved in multiple developmental events
during embryogenesis (Reya, Duncan et al. 2003). The effects of Wnt signals are
pleiotropic, affecting cell fate specification, mitogenic stimulation and
differentiation. Researchers have shown that using inhibitors to the Wnt
signalling pathway in HSCs, reduces HSC growth in vitro and reduces
reconstitution in vivo (Reya, Duncan et al. 2003; Willert, Brown et al. 2003; Reya
and Clevers 2005). Activation of the Wnt pathway in HSCs increases expression
of genes (ie. HoxB4 and Notch1) implicated in HSC self-renewal of HSCs (Reya,
Duncan et al. 2003). This suggests that the Wnt/ β-catenin pathway is critical for
HSC homeostasis and self-renewal.
2(ii) Wnts
Wnt proteins are part of a large family of secreted signaling molecules
which are expressed in diverse tissues and play a role in multiple processes in
vertebrate and invertebrate development (Reya and Clevers 2005). The founding
member of the family, the mouse Wnt1 gene, was identified as a proto-oncogene
(Nusse and Varmus 1982; Nusse and Varmus 1992). Subsequently, the fly
homolog of Wnt1, wingless (wg), was described as regulating segment polarity in
Drosophila (Siegfried, Chou et al. 1992) and axis specification in Xenopus (Moon
and Kimelman 1998). In the mouse, Wnt proteins are ubiquitous. In fact,
mutations in the Wnt genes have been associated with defects in limb, somite
16
and axis formation and abnormal development of brain, kidney, and reproductive
tract in mice (Parr, Shea et al. 1993; Parr and McMahon 1994; Monkley, Delaney
et al. 1996; Yoshikawa, Fujimori et al. 1997). Deregulation of the Wnt pathway
have also been shown to have oncogenic effects in tissues such as colon,
breast, prostate, and skin (Korinek, Barker et al. 1998; Morin 1999; Morin and
Weeraratna 2003; Tsukamoto, Yamamoto et al. 2003; Cheng, She et al. 2008).
Wnts can bind two receptors. The first is the Frizzled family of seven-pass
transmembrane proteins, which contain an extracellular N-terminal cysteine-rich
domain that binds to Wnt proteins (Reya, Duncan et al. 2003; Reya and Clevers
2005). The second, LRP-5 and 6 is part of the low-density lipoprotein receptor-
related protein (or the LRP) family (Parr, Shea et al. 1993; Parr and McMahon
1994), a single-pass transmembrane protein. Researchers have shown that LRP-
5/6 and Frizzled are needed to activate the canonical Wnt/ β-catenin pathway
(Parr and McMahon 1994; Morin and Weeraratna 2003; Reya, Duncan et al.
2003).
In the canonical Wnt/ β-catenin pathway, in the absence of a Wnt signal, β-
catenin is associated with the scaffold protein Axin and the serine/threonine
kinase, glycogen synthase kinase-3 beta (GSK-3 β). Together, this group of
proteins is called the destruction complex. In this complex, β-catenin is
phosphorylated at its amine terminus by GSK-3 β and targeted for ubiquitination
and subsequent proteasomal degradation (Reya, Duncan et al. 2003; Reya and
Clevers 2005). A key component of this complex is Axin, which acts as a scaffold
enabling GSK-3 β to phosphorylate β-catenin. Thus, when Wnt is bound to its
17
receptors, the phosphorylation of β-catenin by GSK-3 β is inhibited. This prevents
the proteasomal degradation of β-catenin, resulting in the stabilization and
accumulation of β-catenin within the cell (Reya, Duncan et al. 2003; Reya and
Clevers 2005). β-catenin then translocates to the nucleus, in an unknown
mechanism, where it binds to the High Mobility Group (HMG) family of
transcription factors, the LEF (Lymphoid Enhancing Factor)/T-cell factor (TCF).
2(iii) Wnt/ β-catenin Signaling in different tissues
The Wnt/ β-catenin signaling pathway has been linked to a number of
tissues. For example, the Wnt/ β-catenin signaling pathway has been associated
with the regulation of stem cell self-renewal in the hematopoietic system. Reya et
al, 2003, showed that transduction by a retroviral vector expressing a
constitutively activated form of β-catenin, resulted in an increase of self-renewal
of mouse hematopoietic progenitors/stem cells in vivo (Reya, Duncan et al.
2003). It has also been shown that there are increased levels of β-catenin in
cultured human keratinocytes with higher proliferative potential when compared
to those with lower proliferative capacity. Evidence has emerged that the Wnt
signaling pathway may be necessary in the maintenance or self-renewal of gut
stem cells. This conclusion is based on the finding that TCF-4-deficient mice
exhaust the undifferentiated progenitors in the crypts of the gut epithelium during
fetal development (Korinek, Barker et al. 1998). Researchers have also shown
that overexpression of activated β-catenin in transgenic mice displayed increased
cell cycle entry of neural precursors (Chenn and Walsh 2002). In preadipocytes,
18
Wnt10B has been shown to maintain these cells in an undifferentiated state.
When Wnt signaling is inhibited, these cells differentiate into adipocytes (Ross,
Hemati et al. 2000). Wnt3A have been shown to induce proliferation of B-cell
(Reya, Duncan et al. 2003). These studies support the hypothesis that the Wnt
signaling pathway may regulate self-renewal of stem and progenitor cells in
various tissues.
Wnt/ β-catenin Signaling in Hematopoietic Stem Cells
As mentioned, the Wnt pathway has been shown to play a role in the
development of various tissues but relatively little is known about its function in
the hematopoietic system. There is strong evidence that supports Wnt signaling
playing an important regulatory role in hematopoietic progenitors/stem cells
during both fetal and adult development.
During fetal hematopoiesis, in the yolk sac and the fetal liver (both sites of
hematopoiesis in the embryo), Wnt5A and Wnt10B are expressed (Austin, Solar
et al. 1997). An 11-fold expansion of fetal liver progenitors was seen when
cultured in conditioned media containing Wnt1, Wnt5A, or Wnt10B in addition to
stem cell factor, SLF (Austin, Solar et al. 1997). Colony forming assays showed
an increase in CFUs. This result suggested that in vitro, Wnt proteins cause
these cells to retain their immature functional characteristics. In humans, Wnt5A
exposure to CD34+Lin- human hematopoietic progenitors, promoted the
expansion of undifferentiated progenitors in its presence. CFU assays revealed a
10- to 20-fold higher number of CFU-GEMM (which indicates the presence of an
19
immature population). Wnt protein stimulation clearly increase immature
progenitors in these in vitro assays.
Reya et al, 2003, retrovirally transduced downstream components of the
Wnt pathway into mouse cKit+Lin-Sca+ (KLS) cells and analyzed their effects by
in vitro and in vivo assays. They found that overexpression of constitutively
activated β-catenin in long-term cultures of these mouse HSCs expands the pool
of HSCs in vitro and in vivo(Domen and Weissman 2000; Reya, Duncan et al.
2003). The constitutively activated β-catenin induced cells to enter the cell cycle
and grow in long-term cultures for 1 to 4 weeks and the control HSC did not
survive for more than 48 hours. These transduced HSCs retained HSC
functional characteristics, determined in a mouse transplant model. This study
demonstrated that β-catenin, a downstream component of the Wnt pathway
could regulate HSC bone marrow function, but depends on use of Bcl2
transgenic mice.
Small Molecules and HSC
Researchers have shown that small molecules can be used as modulators
of stem cell fate (Emre, Coleman et al. 2007; Schugar, Robbins et al. 2008). The
use of pharmacological chemicals to modulate stem cell fate has some distinct
advantages over genetic approaches. First, through high throughput screening of
existing compound libraries, pharmacological chemicals/small molecules capable
of regulating stem cell fate can be quickly identified. Second, by varying the
concentration of the small molecule, stem cell fate can be carefully and precisely
20
explored. Third, a single small molecule can ideally affect multiple proteins in the
same signaling pathway to synergistically control stem cell behaviors. Therefore,
the use of small molecules to modulate stem cell fate will greatly enhance basic
and applied research in stem cell biology (Emre, Coleman et al. 2007).
With a better understanding of the self-renewal, differentiation and
reprogramming mechanisms of stem cells, it would be possible to select specific
agents from a small molecule repository to modulate known programs of stem
cell fate. Recent studies have given credence to this approach in stem cell
biology research. In mouse ES cells for example, gene expression profilings of
both mouse and human ESCs have shown that the canonical Wnt/ β-catenin
pathway and related genes are expressed in their undifferentiated state
(Ramalho-Santos, Yoon et al. 2002). This implies that the canonical Wnt/ β-
catenin pathway plays a role in the self-renewal of mouse and human ESCs
(Sato, Meijer et al. 2004) and (Ramalho-Santos, Yoon et al. 2002). Sato et al.
used BIO, a specific inhibitor of GSK-3, to activate the Wnt/ β-catenin pathway.
BIO successfully maintained human and mouse ESCs to grow in an
undifferentiated state without the use of a Mouse Embryonic Fibroblast layer
during culture (Sato, Meijer et al. 2004). However, BIO is not highly specific and
may cross react with cyclin dependent kinases (CDK). To determine if other GSK
inhibitors could truly stimulate the Wnt/ β-catenin pathway, Ying et al. used a
more selective inhibitor of GSK-3 (CHIR99021) to maintain the self-renewal of
mouse ESCs (Ying, Wray et al. 2008). In this example, the use of a chemical
library with small molecules that have different selectivity for the same target
21
shows the usefulness of this drug discovery process. With a growing knowledge
and library of small molecules as well as an increasing knowledge of the stem
cell self cell fate mechanisms, small molecule biology shows a promising future.
Already, multiple studies have indicated that drug discovery has greatly
influenced stem cell biology. The process of discovering the drug target and
determining the drug activity will aide in the organized selection process of stem
cell fate modulators.
2(vi) Effects of GSK3 Inhibitor BIO in Stem Cells
6-bromoindirubin-3'-oxime (BIO), is a synthetic, cell permeable ATP
competitive GSK3 inhibitor. This small molecule is the first pharmacological
agent shown to maintain self-renewal in embryonic stem cells (Sato, Meijer et al.
2004). Sato et al., 2004, showed that BIO treated ESCs accumulated β-catenin in
the nucleus. This suggested that BIO activated the Wnt pathway in hESCs.
Furthermore, BIO treated hESCs cultures maintained high Oct-3/4 expression
and an undifferentiated morphology. When BIO was removed from the hESC
culture, ES cells would go on to normal differentiation.
In HSC, Trowbridge et. al. 2006, showed that In vivo administration of
GSK-3 β inhibitor improved the regenerative potential of both human and murine
HSCs in an NOD/SCID mouse model. Holmes et al., 2008, suggest that GSK-3
inhibition activates β-catenin in umbilical cord blood (UCB) CD34
+
cells. They
also observed transcription upregulation of two stem cell renewal candidate
factors, c-myc and HoxB4. And in an ex vivo NOD/SCID model, the addition of a
22
GSK-3 inhibitor preserves SCID repopulating cells (SRCs). Combined, these
results suggest BIO as a candidate molecule which modulates stem cell activity.
23
Chapter 3: Methods of β-catenin Upregulation
3(i) Introduction
Gene therapy, involving gene transfer into hematopoietic stem cells (HSC)
holds the promise to treat many genetic diseases. Since HSC are inherently in
the quiescent state, clinical protocols require the use of a pre-stimulation step
using various cytokine cocktails (e.g ckit ligand, flt3 ligand) to induce these cells
into cell cycle, thereby allowing increased gene transduction efficiency when
using retroviral vectors. The drawback of this method is that it promotes HSC to
lose their potential long term repopulating capacity.
The literature suggests that factors such as β-Catenin, HoxB4, Notch,
BMP and BMI-1 are involved in regulating self-renewal of mouse stem cells
(Antonchuk, Sauvageau et al. 2002; Reya, Duncan et al. 2003; Zhang, Lo Muzio
et al. 2005). This observation opens the possibility that a modified cytokine
cocktail in conjunction with modulation of one of the above factors or combination
of factors, may lead to HSC cell cycle without losing their ability for long term
repopulating capacity compared to the currently used cytokine cocktail regimen
used in human gene therapy clinical trials. We could exploit this observation to
increase the transduction efficiency of HSC in the use of gene therapy. Here we
looked for methods to increase β-catenin in CD34+ cells for future studies in
HSC gene therapy.
24
Wnt/ β -catenin involvement in HSC self-renewal
β-catenin is a multifunctional protein. It is an integral component of
adherens junctions as well as a pivotal member of the highly conserved Wnt/ β-
catenin signal transduction pathway. The cytoplasmic pool of β-catenin is
controlled by a large number of binding partners. Deregulation of β-catenin
signaling is an important event in the genesis of a number of malignancies. Most
recently, β-catenin has been found to be involved in stem cell self-renewal.
The Wnt/ β-catenin pathway has shown great promise for the prospect of
stem cell self-renewal. An extrinsic signaling molecule called Wnt signals this
pathway. The Wnts are growth factors that bind a transmembrane receptor called
Frizzled. This process produces a signal that is transduced to the nucleus via β-
catenin. The activation of this signaling pathway inhibits the phosphorylation of
β-catenin by glycogen synthase kinase (GSK-3 β), preventing its ubiquitinylation
and subsequent proteasomal degradation. This event allows β-catenin to
stabilize and enables it to form a nuclear complex with the lymphoid enhancer
factor-1/T-cell specific transcription factor (LEF-1/TCF) family of transcription
factors, inducing a genetic program that leads to HSC self-renewal.
A study by Reya et al., 2003, demonstrated that β-catenin expression
leads to self-renewal of HSCs. They transduced HSC derived from bcl-2
transgenic mice with a retroviral vector containing a constitutively active β-
catenin gene and found that while 34% of the HSCs transduced with a control
eGFP expressing retroviral vector were in S/G2/M phases of the cell cycle, 58%
of the HSCs expressing activated β-catenin were in the same phases of the cell
25
cycle, S/G2/M. Expression of β-catenin allows these HSC to pass the G1 cell
cycle restriction point. During an 8 week period, the HSCs with the activated β-
catenin showed 8 to 9 doublings to generate 100 times the number of input cells.
In contrast, the control vector transduced cells showed minimal growth beyond a
two-week period. When Reya et al. looked at the cell’s lineage markers, 75-80%
of the control vector transduced HSCs were positive for eGFP. In comparison, 5-
10% of the β-catenin-expressing HSCs expressed high levels of lineage markers.
These observations suggest that the expression of β-catenin in mouse HSCs
promotes self-renewal(Reya, Duncan et al. 2003).
GSK3 Inhibitors
One of the most common post-translational mechanisms used by cells to
regulate enzymes and structural proteins is by protein phosphorylation. Because
many diseases are associated with protein phosphorylation abnormalities,
pharmacological inhibitors of kinases and phosphatases have become a major
interest in drug discovery. Glycogen synthase kinase 3 (GSK3) is emerging as a
prominent drug target. It was first discovered as a kinase involved in the
regulation of glucose metabolism. Later, it was found to be a participant in
wnt/wingless signaling. It is in the center of many cellular and physiological
events, including Wnt and Hedgehog signaling, transcription, insulin action, cell-
division cycle, response to DNA damage, cell death, cell survival, patterning and
axial orientation during development, differentiation, neuronal functions, circadian
rhythm and others (Reya, Duncan et al. 2003; Reya and Clevers 2005).
26
In 2004, Sato et al demonstrated maintenance of pluripotency in human
and mouse embryonic stem cells (HESC and MESC, respectively) through
activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor called
6-bromoindirubin-3’oxime (BIO). BIO interacts with both GSK-3 α and β. BIO was
added to HESC or MESC’s in feeder cell free conditions, and it activated the
canonical Wnt pathway which was sufficient to maintain HESC and MESC self-
renewal. This conclusion was made based on these cells’ morphology and Oct-
3/4 expression, which is found in ESCs(Sato, Meijer et al. 2004). A more specific
GSK-3 β Inhibitor called GSK-Inhibitor VIII, also known as AR-A014418, has been
made commercially available. It inhibits GSK3 β in an ATP-competitive manner.
My hypothesis is that by decreasing the inhibitory effects of Axin1, GSK-
3 β or APC, it is possible to promote β-catenin stabilization, which would lead to
HSC self-renewal. Through this process, a γ-retroviral vector can be used to
transduce these cycling HSCs with increased transduction efficiency, which
would yield higher integration of the vector carrying a therapeutic gene, into
plurlipotent HSCs rather than a lineage committed progenitor cell population. At
the same time, we would promote the expansion of HSCs. This strategy would
have important implications in hematopoietic cell transplantation as well as gene
therapy. In this study, we attempt to find a method to increase β-catenin within
CD34+ cells. We also perform preliminary experiments to determine if we can
increase retroviral transduction of treated CD34+ cells.
27
3(ii) Materials and Methods
Cell culture.
HEK 293 cells (ATCC, Manassas, VA) were grown in Dulbecco's modified
Eagle's medium (DMEM) containing
10% (vol/vol) fetal bovine serum (FBS),
penicillin (100 U/ml),
streptomycin (100 µg/ml), and 2 mM L-glutamine. K562
erythromyeloid
cells, were obtained from ATCC, and
were grown in RPMI 1640
medium containing 10% FBS, penicillin
(100 U/ml), streptomycin (100 µg/ml), and
2 mM L-glutamine. HeLa cells were grown in Dulbecco's modified Eagle's
medium (DMEM) containing
10% (vol/vol) fetal bovine serum (FBS), penicillin
(100 U/ml),
streptomycin (100 µg/ml), 0.1mM Non-essential Amino Acids, 0.1mM
Sodium Pyruvate and 2 mM L-glutamine.
CD34
+
cells were isolated from human umbilical cord blood obtained
from
normal infant deliveries using Miltenyi MiniMACS magnetic separation
columns
(Miltenyi Biotech, Sunnyvale, Calif.) after Ficoll-Hypaque
(Amersham Pharmacia
Biotech, Piscataway, N.J.) density gradient
centrifugation. Use of these cord
blood samples was approved
by the Committee on Clinical Investigations at
Childrens Hospital,
Los Angeles, California to isolate CD34
+
cells.
Vector supernatant production.
Retroviral vector. The vector was produced as an amphotropic vector by the
PG13 packaging cell line. Vector-containing supernatants were collected as D-
10, conditioned for 24 hours at 37°C by subconfluent monolayers of the producer
28
cells. Supernatants were filtered through 0.45-µm filters and immediately frozen
at -70°C until needed.
Vector supernatant titer determination.
Vector supernatant titers were determined by endpoint dilution.
293 cells (ATCC
CRL-1573) were seeded at 10
5
cells/well in six-well
cell culture plates (Corning
Inc., Miami, Fla.) in DMEM supplemented
with 10% FBS, 100 U of penicillin/ml,
100 µg of streptomycin/ml,
2 mM L-glutamine and placed in a 37°C incubator for
12 h.
Cells were then transduced with 1-ml serial dilutions (i.e.,
10
-1
, 10
-2
, 10
-3
) of
vector supernatant and analyzed by flow
cytometry for eGFP expression 48 h
later. Titers were calculated
by multiplying the number of cells at the time of
vector supernatant
addition by the percentage of eGFP-positive cells determined
by flow cytometry divided by 100, multiplied by the dilution
factor to yield the
number of infectious units (IU) per milliliter.
The titer of the retroviral vector used
(PG13-MND-eGFP-SN) was 1 x 10
6
IU/ml.
Vector transductions.
For studies of vector transductions, CD34+ cells were transduced as follows: 10
5
cells were
plated in 100 µl of Xvivo15 in F,S,T or Scf at 100ng/mL in 48-well
plates
to which 100 µl of diluted vector supernatant
was added. Cells were
incubated for 12 h post-transduction, followed by the addition
of 200 µl of media.
Cells were transferred to a BBMM medium containing cytokines, SCF, IL-6 at
29
50ng/mL and IL-3 at 20ng/mL. Transduced cells were cultured for a
minimum of 5
days prior to any subsequent analysis.
Flow cytometry.
Transduced CD34 + cells were collected at 5 days post transduction, then
washed twice with PBS, and then resuspended in 100 μl of PBS and stained
directly with PE-conjugated anti-CD34+ mAb (Becton
Dickinson
Immunocytometry Systems, San Jose, Calif.). eGFP was detected for cells
transduced with an eGFP expressing vector. Measurements were performed on
a FASCalibur flow cytometer (Becton Dickinson, USA) with the Cellquest
software (version 3.0, B.D, USA). Initial gating was performed using forward and
side scatter to identify cell populations. A total of 2×10
4
events were collected.
Cell cycle analysis.
Aliquots of 2×10
5
cells were fixed in 70% ethanol in -20
o
C overnight then
centrifuged at 300g for 5min. The pellet was treated with PBS, RNase (200
μg/ml), Propidium Iodide (10 μg/ml) and 0.1% TritonX at 37
o
C for 40 min. Cells
were then washed in 4mL QS with PBS, centrifuged and resuspended in 500 μL
of PBS. DNA content and cell cycle distribution were analyzed using a Becton
Dickinson FACScan Plus flow cytometer. Cell cycle analysis was performed
using Modfit, on a minimum of 10,000 cells per sample.
30
Design and construction of siRNA.
siRNAs were designed by Qiagen. The Axin1 gene accession number (Gen Bank
accession #NM_003502) was sent to Qiagen to design four possible siRNAs
against Axin1. Four sequences labeled, AS Axin1-1 (AS1) s:
r(GGUGGGCGUGGCGUGCAAA)d(TT) as:
r(UUUGCACGCCACGCCCACC)d(TT), AS Axin1-2 (AS2) s:
r(AGGGCAGCUACAGAUACUA)d(TT) as:
r(UAGUAUCUGUAGCUGCCCU)d(TT), AS Axin1-3 (AS3) s:
r(GAUGGGAUAAGCCUGUUCA)d(TT) as
r(UGAACAGGCUUAUCCCAUC)d(TT), and Axin1-4 (AS4) s:
r(GAGGCUGAAGCUGGCGAGA)d(TT) as:
r(UCUCGCCAGCUUCAGCCUC)d(TT) were constructed. A control siRNA
(negAS) was purchased from Qiagen which was FITC labeled with
s:UUCUCCGAACGUGUCACGUdTdT as: ACGUGACACGUUCGGAGAAdTdT.
The constructed siRNA molecules were HPLC purified. Their concentration was
estimated by Ultraviolet Spectrophotometer. The lyophilized siRNAs were
resuspended in a buffer provided by Qiagen and immediately stored in -20
o
C to
prevent degradation. After thawing and use, the siRNAs were refrozen in -20
o
C.
Cell culture and siRNA transfection using Lipofectamine 2000.
HeLa cells (100,000) were plated on 48 well plates for 24 hours, then washed 2
times in PBS. These cells were then cultured in serum-free RPMI 1640 for 24
hours prior to siRNA addition. Transfection of HeLa cells with siRNA used the
31
Lipofectamine 2000 reagent (Invitrogen, USA). Added SiRNA (1.8 μg SiRNA) to
serum-free RPMI, use Lipofectamine 2000 and serum-free RPMI. Incubated for
5min at R/T. Combined then mixed and incubated for 22min at R/T. Cells were
rinsed twice with PBS, then added onto the cells that are in serum-free RPMI.
Incubated for 5 hours at 37
o
C/ 5% CO
2
. Removed supernatant and rinsed with
PBS. Added 500 μL of RPMI with 10% FCS. The transfected cells were cultured
and assayed for the appropriate activity at 48hours or 72hours post transfection.
Cell culture and siRNA transfection using the Amaxa electroporator.
HeLa cells were incubated in serum-free DMEM for 24 hours. Trypsinized the
cells and washed 2 times in PBS. Added 100 μL of Nucleofector solution R
(Amaxa Inc., USA) reagent per 1 x 10
6
HeLa cells and added 2 μg of the siRNA
duplex. Placed in the Amaxa electroporator (Amaxa Inc., USA) and used
program I13. Removed the cells with 500 μL of DMEM containing
10% (vol/vol)
fetal bovine serum (FBS), penicillin (100 U/ml),
streptomycin (100 µg/ml), 0.1mM
Non-essential Amino Acids, 0.1mM Sodium Pyruvate and 2 mM L-glutamine.
Incubated in 37
o
C/ 5% CO
2
.
Western Blot analysis.
Cells (minimum of 200,000 maximum of 600,000) were incubated with and
without soluble AR-A014418 for the
indicated times at 37°C, in 5% CO
2
. Pelleted
cells were then lysed
on ice for 10 minutes in 1% NP-40 lysis buffer (50 mM Tris-
HCL,
pH 7.4, 250 mM NaCl, 2 mM EDTA, 2 µg/mL aprotinin,1 mM
32
phenylmethylsulfonyl
fluoride [PMSF], 1 mM NaF, 0.5 µg/mL leupeptin, 1% NP-
40). The
cleared lysates were boiled in LDS sample buffer (Invitrogen, Carlsbad,
CA) at 95°C and electrophoresed
on 4-20% Tri-Glycine gels, then transferred
onto Immobilon-P
(Millipore Corporation, Bedford, MA). Immunoblotting was done
as
described using antibodies to Axin1 (H-98), β-catenin (E-5), and Actin (I-19),
from Santa Cruz Biotechnology, Santa Cruz, CA. Following incubation with HRP-
conjugated
secondary antibodies, proteins were detected
by the ECL reagent
(Pierce, Rockford, IL).
3(iii) Results
The Wnt/ β-catenin pathway is currently known to be involved in the self-
renewal of HSC (Reya et al, 2003). However, its direct role has yet to be clearly
demonstrated. It is known that increasing the amount of β-catenin within HSC
leads to self-renewal. We, therefore, investigated two different techniques to
increase the amount of β-catenin within HSC.
siRNA to Axin1 decreases Axin1 protein levels within HeLa cells
Axin1 is a component of the canonical Wnt/ β-catenin pathway. When
Axin1 binds with two other components of this pathway, APC and β-catenin,
these three molecules form the destruction complex. When the destruction
complex is formed, β-catenin is ultimately degraded. By inhibiting the formation of
the destruction complex, β-catenin is stabilized, leading to increased intracellular
concentrations of this protein.
33
We purchased four siRNAs, against Axin1 and a control siRNA, which has
no known human sequence homolog and contains a FITC label on its 3’ end.
When these siRNAs were added to HeLa cells, there was a pronounced
decrease in Axin1 protein levels using siRNA2 (AS2), whereas siRNA1, 3 and 4
(AS1, AS3 and AS4 respectively) had only moderate to low levels of decrease
(Figure 3-1A and B). These results were seen using either a transfection agent,
Lipofectamine 2000, or when using electroporation (Amaxa Inc.).
Our hypothesis was that by interrupting the APC, β-catenin and Axin
destruction complex, we would see an increase in cellular levels of β-catenin
because its ubiquitination and subsequent degradation would be prevented. In
HeLa cells, the endogenous level of β-catenin was high (Figure 3-1C). Therefore,
it was difficult to measure an increase in β-catenin concentration following Axin1
down-regulation using the Amaxa electroporator (Figure 3-1B). Although, in
figure 3-1C, when siRNA2 with a FITC conjugate (AS2-FITC) was used, there
was a noticeable reduction in β-catenin protein levels. This initial observation
suggests that the FITC label on AS2-FITC interacts with the Wnt/ β-catenin
pathway or is an artifact of this particular western blot analysis. This result
warrants further investigation.
While performing the siRNA transfections using Lipofectamine 2000 or the
Amaxa electroporator, cell death was observed using trypan blue exclusion (data
not shown). Due to the toxicity of both Lipofectamine 2000 and the
electroporation technique, we sought an alternative method of increasing β-
34
catenin levels within HSC by targeting the destruction complex in the Wnt/ β-
catenin pathway.
Figure 3-1. siRNAs (Axin 1 through 4) to downregulate Axin 1 in HeLa cells. HeLa cells
were plated in reduced serum media for 24 hours to enable cell cycle synchronization.
siRNA’s to Axin 1 were introduced using Lipofectamine 2000 or the Amaxa electroporator
transfection techniques and analyzed after 48 or 72 hours post transfection by western blot for
Axin 1 expression (a, b). β-catenin expression was also analyzed after 48 hours post siRNA
transfection using the Amaxa electroporator. A, western blot analysis for Axin1 in HeLa cells
treated with four different siRNAs to Axin1 using Lipofectamine 2000 shows that AS2 showing
the strongest effects. B, western blot analysis for Axin1 in HeLa cells treated with four different
siRNA’s to Axin using an Amaxa electroporator shows that AS2 showing the strongest effects.
C, western blot analysis for β-catenin in HeLa cells treated with siRNA2 and 4 showing the
saturation of the signal in HeLa cells. NT = no treatment. NegAS = siRNA with no known
human homolog. siRNA1-4 4 different siRNA sequences design against Axin1. AS2-FITC is
AS2 with a FITC tag. Actin is used for a loading control.
C
B
A
GSK3 β Inhibitors to increase β-catenin levels
It has been shown in both human ESCs (hESCs) and mouse ESCs
(mESCs) that inhibiting GSK3 maintains their undifferentiated phenotype and
35
sustains expression of the pluripotent state-specific transcription factors Oct-3/4,
Rex-1 and Nanog (Sato, Meijer et al. 2004). Several GSK inhibitors are currently
commercially available (Meijer, Flajolet et al. 2004). We initially chose an
inhibitor that had a low IC50 concentration and was reported as being specific to
GSK3 β. GSK inhibitor VIII, also known as AR-A014418 (Figure 2) is an inactive
ATP analog that functions by competitively binding the ATP binding site on
GSK3 β and interfering with its kinase activity.
GSK3 β Inhibitor VIII
Figure 3-2. The chemical structure of AR-A014418 also known as GSK3 β Inhibitor VIII.
K562, a human suspension cell line with multipotent hematopoietic
potential, was treated with 0, 104 or 208 μM of AR-A014418 (Figure 3-3). As
shown on the western blot analysis in fig. 3-3, there was a noticeable increase in
intracellular β-catenin levels with the addition of the inhibitor AR-A014418 (Figure
3-3) at 24 hours and 48 hours post AR-A014418 addition. When we performed a
dose response experiment in both K562 and CD34+ cells, we found a dose
dependent escalation of β-catenin (Figure 3-4).
36
Next, we treated primary CD34+ HSCs derived from cord blood at
increasing doses and found, by immunoprecipitation using an antibody to β-
catenin followed by western blot analysis, that there was also a dose dependent
escalation of β-catenin (Figure3- 4B). This suggests that intracellular β-catenin
concentrations in K562 and CD34+ cells are increased upon addition of AR-
A014418.
Figure 3-3. AR-A014418 effect on K562 cells β-catenin concentration. 500,000 K562
cells were plated on 12 well plates. 104 or 208 μM of AR-A014418 was added and after 24 or
48 hours a western blot was performed for β-catenin. Actin was used as a standard. A,
24hours after 104 μM and 208 μM of AR-A014418 was added to K562 cells, an increase in β-
catenin concentration was observed by western blot analysis B, 48hours after 104 μM and
208 μM of AR-A014418 was added to K562 cells, an increase in β-catenin concentration was
observed by western blot analysis.
A B
When we looked at the viability of the HSCs at increasing doses of AR-
A014418, the viability of these cells was between 75% to 100% (data not shown),
at concentrations less than or equal to 10.4 μM. Inhibitor levels above 10.4 μM
had toxic effects on the HSCs as determined by trypan blue exclusion. Therefore,
in subsequent experiments, 10.4 μM of inhibitor was used to increase β-catenin
levels within HSCs.
37
We next wanted to see if the treatment of CD34+ cells with the inhibitor
alone would enable S/G2/M cell cycle progression (Figure 3-5), CD34+
expansion/proliferation (Figure 3-6A and C) and increased retroviral transduction
using a retroviral vector that expresses eGFP (Figure 3-6B).
Figure 3-4. Effects of increasing AR-A014418 dose on K562 and CD34+ cells β-catenin
concentrations. 500,000 K562 or CD34+ cells were plated on 12 well plates where
increasing concentrations of AR-A014418 was added to the cells and incubated for 48hours.
Western blot analysis was then performed for β-catenin. A, Western analysis of K562 cells
treated with increasing concentrations of AR-A014418. Dose dependent upregulation of β-
catenin was observed. Actin was used as a loading control. B, Immunoprecipitation using β-
catenin was performed following a western blot for β-catenin in CD34+ cells, 48hours post
addition of AR-A014418. A dose dependent upregulation of β-catenin was observed.
Samples were equalized by loading an equivalent of 100,000 CD34+ cells per well. NT = not
treatment. No ab. = an IP control where no antibody to β-catenin was added.
B
A
AR-A014418 treated CD34+ cells effect on cell cycle progression
After observing AR-A014418 effects on CD34+ cells’ intracellular β-
catenin concentrations, we wanted to determine the effect of this inhibitor on cell
cycle progression of CD34+ cells. CD34+ (200,000) cells were plated on
fibronectin, incubated in either a standard growth medium with Flt, SCF and TPO
at 50ng/mL or in minimal medium with SCF at 50ng/ml in Xvivo15 with or without
addition of AR-A014418 at 10.4 μM. After 72hrs, these cells were analyzed for
38
their cell cycle status (Figure 3-5) by staining with propidium iodide and
measuring by flow cytometry. Modfit was used to analyze the data. After 72hrs,
the SCF arm with and without AR-A014418 treatment (45% and 42% S/G2/M,
respectively) showed a 3% difference in the percentage of the cells in S/G2/M
phase of the cell cycle. Similarly in the Flt, Scf, Tpo arm of the experiment, there
was no difference in the percentage of the cells in S/G2/M phase with and
without treatment of the AR-A014418 inhibitor (Figure 3-5). This result indicates
that the addition of AR-A014418 to CD34+ cells does not induce their cell cycle
progression.
Figure 3-5. AR-A014418 exposure to CD34+ cells effect on cell cycle. 200,000 CD34+
cells were plated in either SCF or Flt, SCF, and TPO at 50ng/mL for 72 hours with the
addition of AR-A014418 at 10.4 μM. An increase in S/G2/M was not observed in either
cytokine arms with the addition of AR-A014418, compared to the no treatment control.
%
39
AR-A014418 treatment effect on CD34+ cells expansion
In 2003, Reya et al demonstrated that β-catenin expression using a
retroviral vector in HSC derived from bcl-2 transgenic mice increased the total
number of HSCs compared to the control vector arm. Although bcl-2 is a factor
known to affect cell cycle, we wanted to see if the addition of AR-A014418 alone
would have an effect on expansion of CD34+ cells in human HSC. We observed
that the addition of AR-A014418 had little effect on the percentage of CD34+
cells (Figure 3-6A) found in either of the Flt, SCF, TPO or the SCF cytokine arms.
When looking at the total number of CD34+ cells, there was no difference when
comparing the AR-A014418 treated cells vs. non-treated cells in either of the
cytokine arms (Figure 3-6C). In the F,S,T arm of this experiment, there was
approximately a 5 fold expansion of CD34+ cells with or without AR-A014418
treatment. In the SCF arm, there was approximately a 2 fold expansion of CD34+
cells with or without treatment (Figure 3-6C). This further supports the
observation that this inhibitor alone is not sufficient to promote CD34+ cells self-
renewal after 5 days post treatment with the inhibitor under these culture
conditions.
AR-A014418 treated CD34+ cells effect on retroviral transduction
We also wished to determine the effects AR-A014418 treatment on HSC
retroviral transduction of HSC using a vector that expresses eGFP. CD34+
(100,000) cells were incubated in either Flt, Scf ,Tpo or Scf alone for 16 hours on
fibronectin. The cells were transduced with a retroviral vector expressing eGFP
40
at a concentration of 0.5 x 10^6 IU/mL. After a 24-hour incubation period, the
cells in all arms were placed in a standard basal bone marrow medium (BBMM)
containing cytokines (50ng/mL of SCF, 20ng/mL of IL-3 and 50ng/mL of IL-6).
Figure 3-6. Effect of AR-A014418 addition on Stem/Progenitor Cell Renewal. 100,000
CD34+ cells were plated on 12 well plates and either treated or not with AR-A014418 for 16
hours. The supernatant was then removed and 1 x 10
6
IU/mL of a PG13-MND-eGFP-SN
vector was added to the corresponding wells. These cells were incubated for 24hours. Next,
all the samples were placed in a common growth medium, BBMM with cytokines for 5 days.
Cells were then either stained with CD34-PE, then FACs analyzed (A) or the total number of
CD34+ cells were calculated (B) and the transduction percentage was determined by FACS
analyzing the respective cells for eGFP positivity (C). A, The percentage of CD34+ cells was
determined and after AR-A014418 addition little to no difference in the percentage of CD34+
cells when comparing the AR-A014418 treated vs. the non-treated cells was observed. B,
There was lower transduction of AR-A014418 treated CD34+ cells in both cytokine
conditions. C, There was little to no difference observed in the total number of CD34+ cells
when comparing the AR-A014418 treated vs. the non-treated cells.
41
The BBMM culture condition was used to ensure that any differences seen in the
results of the experiment were due to the initial 24 hour treatment with and
without the inhibitor. After 5 days, the total number of CD34+ cells was
determined by counting and flow cytometry analysis. Retroviral transduction
efficiency was also assessed by flow cytometry for eGFP expression. In both the
F,S,T and the SCF arms, there was a decrease in retroviral transduction
measured by eGFP positivity when AR-A014418 was added (Figure 3-6B). This
suggests that AR-A014418 has an inhibitory effect on retroviral transduction.
These results show that the addition of AR-A014418 alone is not sufficient to
promote HSC cell cycle progression, expansion/proliferation or increased
retroviral transduction. Perhaps other extrinsic and intrinsic factors are needed in
conjunction with AR-A014418 addition on HSC in order to induce self-renewal.
3(iv) DISCUSSION
We show that siRNAs can downregulate a component of the destruction
complex of the Wnt/ β-catenin pathway, namely Axin1. We used two independent
methods to get the siRNA into the cells and prove that this could be
accomplished. Using both methods, we observed identical results where siRNA2
also referred to as AS2 showed the most noticeable reduction in cellular Axin1
protein concentration measured by western blot analysis. Unfortunately, both
methods of delivery, Oligofectamine2000 and the Amaxa electroporator, showed
toxicity towards the HeLa cells. In the case of the Amaxa, approximately half the
cells were lost due to the harsh electroporation conditions (data not shown).
42
We found that in HeLa cells, the endogenous levels of β-catenin were
present at high concentrations so it would be difficult to demonstrate an increase
in β-catenin concentrations due to the decrease of Axin1. To demonstrate that
decreasing Axin1 by siRNA treatment would lead to an increase of intracellular β-
catenin concentration, a different cell line where β-catenin is not present at high
concentrations would be necessary such as K562 cells. With a lower baseline of
intracellular β-catenin concentration in K562 cells, the difference in β-catenin
concentration with or without siRNA treatment would be less likely due to cell
cycle status and more of a result of treatment with the inhibitor. Due to the
toxicity of both siRNA delivery methods, an alternative method of increasing β-
catenin within primary cells may be necessary.
Our study shows that a component of the Wnt/ β-catenin signaling pathway
can be upregulated with the addition of an exogenous factor. We used a novel
method of increasing β-catenin within HSCs for molecular and clinical study.
Addition of AR-A014418 was shown to increase β-catenin in both K562 cells as
well as primary HSCs in a dose dependent manner. In CD34+ cells, 10.4 μM of
AR-A014418 upregulated β-catenin and had little to no severe toxic effects as
compared to the Amaxa electroporation technique.
We next wanted to determine the effects of AR-A014418 addition on
CD34+ cells cell cycle progression, proliferation/expansion and their ability to be
transduced with a retroviral vector expressing eGFP. We treated CD34+ cells
with AR-A014418, grown in either Flt, Scf, Tpo, which is the standard cytokine
cocktail used in our gene therapy clinical trials and Scf alone which was found to
43
be the minimum cytokine treatment needed for HSC maintenance (Baba, Garrett
et al. 2005).
In a study by Reya et al., they expressed β-catenin using a retroviral
vector and measured a 2-fold increase of the cell population in S/G2/M phase of
the cell cycle as well as increased HSC expansion compared to the control
vector transduced arm. With the addition of AR-A014418 to CD34+ cells, we did
not observe increased S/G2/M cell cycle progression or CD34+ expansion.
Although, in prior experiments, when AR-A014418 was added to CD34+ cells, an
increase in β-catenin levels was seen by western blot analysis, the possibility
arises that in this particular experiment the addition of the inhibitor had no effect.
Another possibility may be that if β-catenin was increased, this increase alone
may not have any effect on cell cycle progression or CD34+ proliferation.
Alternatively the proliferative effects of the cytokines may be masking the
proliferative effects of the AR-A014418 GSK3 β inhibitor. For the model which
Reya et al used, it is possible that the overexpression of an antiapoptotic factor,
bcl-2 had an effect on their results. In lymphocytes, Bcl-2 delays their transition
from the quiescent state into the cell cycle (O'Reilly, Harris et al. 1997). Reya et
al, observed that the Wnt/ β-catenin signaling pathway is involved in HSC self
renewal, but the overexpression of bcl-2 may also be needed in conjunction with
β-catenin expression. In the future, these experiments for cell cycle progression,
expansion and increased retroviral transduction in AR-A014418 treated CD34+
cells need to be repeated where a fraction of the cells will be used for western
blot analysis to determine that the addition of the inhibitor upregulates β-catenin.
44
AR-A014418 should also be used in addition with a method of overexpressing
bcl-2 to simulate the model used in the Reya et al. study but in human HSC.
Evidence also suggests that increased β-catenin expression may be pro-
apoptotic or toxic (Trowbridge, Moon et al. 2006). It may be possible that the
optimal concentration of AR-A014418 and/or cytokine cocktail has yet to be
identified.
AR-A014418 addition to increase β-catenin in CD34+ HSC did not
increase cell cycle progression, proliferation and retroviral transduction in this
study under these culture conditions. I believe this was due to the necessity
forother extrinsic and intrinsic factors such as bcl-2 or specific cytokine cocktails
that may be optimal for stem cell maintenance and self-renewal. These
observations does not preclude the involvement of β-catenin in HSC self-renewal
and maintenance, but rather shows the necessity for further study of this system.
There is an ever growing body of literature suggesting the involvement of β-
catenin in self-renewal of mouse and human ESCs as well as in mouse and
human HSCs. GSK3 inhibition as a method of increasing β-catenin in stem cells
has important implications for human hematopoietic cell transplantations as well
as gene therapy. These GSK3 inhibitors such as AR-A014418 and BIO may be
used to expand CD34+ cells from donors simply by adding them to purified
CD34+ cells in ex-vivo. Then the expanded stem cells may be re-infused back
into the patients to treat the disease. The GSK inhibitors may also be used to
replace some or all the cytokines needed for HSC gene therapies which cause
HSC differentiation during the transduction process. These experiments are a
45
first step showing that the exogenous addition of a small molecule drug to CD34+
cells can increase β-catenin within HSCs without the use of a retroviral or lenti-
viral vector expressing the β-catenin gene. This method of adding a GSK
inhibitor, may be used to transiently increase β-catenin concentrations within
CD34+ cells. This method of increasing β-catenin will facilitate future studies of
β-catenin’s role in stem cell self-renewal.
46
Chapter 4: Effects of BIO Addition on UCB CD34+
Progenitor Cells
4(i) Introduction
HSC are characterized by their unique ability to self-renew and by their
pluripotentiality. Because HSC can differentiate into all of the cells in the
hematopoietic system, they make a good choice for gene therapy in the
treatment of blood cell diseases. Successes in clinical trials of gene therapy
using γ-retroviral vectors to introduce genes into human HSC indicate that it is a
promising therapeutic approach in the treatment of inherited immunodeficiencies
(Cavazzana-Calvo, Hacein-Bey et al. 2000; Aiuti, Slavin et al. 2002), and
potentially for hemoglobin disorders and other blood cell diseases.
For successful gene transfer using γ-retroviral vectors, the HSC must
progress through the cell cycle so that the therapeutic gene may be delivered to
the chromosomal DNA in the nucleus of the HSC. To initiate cell cycle
progression of the normally quiescent HSC, a combination of cytokines is
typically employed in ex vivo transduction protocols. Although use of these
cytokines can increase γ-retroviral transduction of HSC, the cytokine cocktails
may also drive HSC into commitment to lineage differentiation, thereby reducing
the pluripotentiality of the transduced HSC. An improved HSC gene transduction
protocol would induce cycling of the normally quiescent HSC, but without causing
lineage differentiation. An ideal protocol would also support high levels of
47
survival of the HSC, promote symmetrical expansion, and preserve the
capabilities of the HSC to home and engraft after in vivo re-infusion.
The canonical β-catenin pathway plays a critical role in the regulation of stem
cell maintenance and proliferation. β-catenin over-expression using retroviral-
mediated gene transfer led to maintenance of murine HSC and increased cell
cycle progression (Reya, Duncan et al. 2003). One of the primary factors that
inhibits the action of β-catenin is the protein glycogen synthase kinase-3 beta
(GSK-3 β), which phosphorylates β-catenin and leads to its retention in the
cytoplasm for proteosomal destruction, rather than allowing it to enter the
nucleus to stimulate gene expression. Recent publications suggest β-catenin as
an important regulator of HSC function (Reya, Duncan et al. 2003; Trowbridge,
Xenocostas et al. 2006).
In this paper, we assessed the potential to increase γ-retroviral vector-
mediated gene transfer to human umbilical cord blood-derived CD34+ cells by
increasing the intracellular levels of β-catenin through inhibition of GSK-3 β using
6-bromoindirubin-3’ -oxime (BIO) (Meijer, Skaltsounis et al. 2003). We also
determined the effects that this BIO treatment had on the CD34+ cells, in terms
of cell cycle status, intracellular signaling, apoptosis as well as their in vivo
homing and engraftment in an immune-deficient murine host. We observed that
BIO did increase gene transfer to the CD34+ cells, in parallel with an increase in
the fraction of cells that underwent an initial round of cell cycle progression. No
adverse effects were seen from the exposure of the cells to BIO, suggesting that
48
manipulation of the β-catenin pathway may provide a useful method to support γ-
retroviral vector mediated gene transfer to HSC for gene therapy.
4(ii) Materials and Methods
Cell culture.
CD34
+
cells were isolated from human umbilical cord blood (UCB) obtained
from
normal infant deliveries using Miltenyi MiniMACS magnetic separation
columns
(Miltenyi Biotech, Sunnyvale, Calif.) after Ficoll-Hypaque
(Amersham Pharmacia
Biotech, Piscataway, N.J.) density gradient
centrifugation. Use of these cord
blood samples was approved
by the Committee on Clinical Investigations at
Childrens Hospital
Los Angeles, California. All recombinant human cytokines (flt-
3 ligand, Stem Cell Factor (Gammaitoni, Bruno et al.), and thrombopoietin) were
purchased from R&D Systems [R&D], Minneapolis, MN, USA. CD34+ cells were
treated with 6-bromoindirubin-3-oxime (BIO, Sigma-Aldrich, St. Louis MO, USA),
the inactive control 1-methyl-6-bromoindirubin-3'-oxime (meBIO,Sigma-Aldrich,
St. Louis MO, USA) (Figure 1A) or equal volume of vehicle control (<0.1% DMSO
(Edwards Lifesciences, Irvine, CA, USA) by culturing them in X-vivo 15 medium
(Cambrex, East Rutherford, NJ, USA) containing either only “low dose” Flt3
ligand ([3ng/mL];, or with “high dose” SCF ([50 ng/mL], [50 ng/mL] Flt-3 ligand,
and [50 ng/mL] thrombopoietin on recombinant fibronectin fragment CH-296 (FN)
[20ug/ml] (Takara Bio Medical, Otsu, Japan) for up to 6 days depending on the
assay.
49
TOPflash and FOPflash assay
TOPflash (Upstate Cell Signaling Solutions, Billerica, MA) and FOPflash (Upstate
Cell Signaling Solutions, Billerica, MA) are commercially available constructs
used to assess
TCF/ β-catenin dependent signaling that drive the expression
of
firefly luciferase. TOPflash is a TCF reporter plasmid containing wild-type TCF
binding sites and FOPflash contains mutated TCF binding sites. Both constructs
contain the thymidine kinase minimal promoter upstream of a firefly luciferase
reporter gene. The Renilla luciferase (pRL-CMV vector, Promega, Madison, WI,
USA) transfection control vector
is driven by the cytomegalovirus (CMV)
immediate-early promoter
region expressing the Renilla luciferase enzyme.
Electroporation was performed using the Amaxa nucleoporator (AMAXA
Gmb H Cologne, Germany) according to the manufacturers' protocol using the
Human CD34 Cell Kit with program U-08 to electroporate 1 × 10
6
cells in 100 μL
nucleoporation solution and 1 μg of pRL-CMV and 3 μg of TOPflash or FOPflash.
Electroporated cells were immediately treated with BIO, the inactive control
meBIO,or equal volume of DMSO and cultured in 1mL of X-vivo 15 medium
containing “low dose” Flt3 ligand ([3ng/mL] on FN for 2 days in 37
o
C/5% CO
2
.
Treated cells (500,000 cells) were then washed with dPBS and processed
using the Promega dual luciferase kit (Promega, Madison, Wi, USA) as per
manufacturers’ protocol. Renilla and firefly luciferase expression was measured
using the TD 20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA).
50
Vector supernatant production.
The retroviral vector MND-eGFP-SN was produced as a GALV-pseudotyped
vector by the PG13 packaging cell line (Malik, Fisher et al. 1998). Vector-
containing supernatants were collected in DMEM, 10% FCS, 100 U of
penicillin/ml, 100 µg of streptomycin/ml,
2 mM L-glutamine (D-10), conditioned
for 24 hours at 37°C by subconfluent monolayers of the producer cells.
Supernatants were filtered through 0.45-µm filters and immediately frozen at -
70°C until needed.
Vector supernatant titer determination.
Vector supernatant titers were determined by endpoint dilution. HEK
293 cells
(ATCC CRL-1573, Manassas, VA) were seeded at 1 x 10
5
cells/well in six-well
cell culture plates (Corning Inc., Miami, Fla., USA) in D10 and placed in a 37°C
incubator for 12 h.
Cells were then transduced with 1 ml serial dilutions (i.e.,
10
-1
,
10
-2
, 10
-3
) of vector supernatant and analyzed by flow
cytometry for eGFP
expression 48 h later. Titers were calculated
by multiplying the number of cells at
the time of vector supernatant
addition by the percentage of eGFP-positive cells
determined
by flow cytometry divided by 100, multiplied by the dilution
factor to
yield the number of infectious units (IU) per milliliter.
The titer of the retroviral
vector used (PG13-MND-eGFP-SN) was 2 x 10
6
IU/ml.
51
Vector transductions.
For studies of vector transductions, CD34+ cells were transduced as follows: 0.7
to 1 x 10
5
cells were
plated in 100 µl of X-vivo 15 medium containing either only
“low dose” Flt3 ligand ([3ng/mL], or with SCF ([50 ng/mL], [50 ng/mL] Flt-3 ligand,
and [50 ng/mL] thrombopoietin in 48-well plates on FN,
to which 100 µl of diluted
vector supernatant
was added. Cells were incubated at 37
o
C for 12 h post-
transduction, followed by the addition
of 200 µl of the X-vivo 15 medium.
Transduced cells were cultured for 4 to 6 days prior to any subsequent analysis.
Immunoprecipitation and Immunoblot blot analysis.
CD34+ cells (500,000) were incubated with 0, 0.3, 1, 3 and 10 μM of BIO
dissolved in DMSO as per manufacturers’ protocol. Treated cells were then
cultured in X-vivo 15 medium containing Flt3 ligand ([50ng/mL], on FN for 48 hrs
at 37°C, in 5% CO
2
. Cultured cells (100,000) were then lysed
on ice for
10 minutes in 1% NP-40 lysis buffer (50 mM Tris-HCL,
pH 7.4, 250 mM NaCl,
2 mM EDTA, 2 µg/mL aprotinin,1 mM phenylmethylsulfonyl
fluoride [PMSF],
1 mM NaF, 0.5 µg/mL leupeptin, 1% NP-40). Whole-cell lysates were pre-cleared
with Protein G sepharose beads for 4 minutes at 4°C. The supernatant was
transferred to a new tube and then incubated with antibody to β-catenin E5
[1.5 μg] (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, and then
incubated with Protein G sepharose beads for 30 minutes, vortexing every 5
minutes at 4°C. The complex was washed 3 times with lysis buffer and then
resuspended in 2X LDS sample buffer (Invitrogen, Carlsbad, CA) at 95°C.
52
Immunoprecipitates were separated on 4-20% tris-glycine SDS-PAGE gel and
transferred to Immobilon-P membrane
(Millipore Corporation, Bedford, MA). Blots
were probed with a β-catenin E5 antibody (1:1,000), blocked in 5% milk and
0.1% Tween 20. Following incubation with HRP-conjugated
secondary antibodies
(type? Source?), proteins were detected
by the ECL plus reagent (Pierce,
Rockford, IL, USA) as per manufacturers’ protocol.
Real-Time PCR
Total RNA was extracted from 1 x 10
6
CD34+ cells using RNA STAT60 (Tel-test,
Friendswood, TX) per manufacturer's protocol. RNA quantity and quality was
assessed using a ND-1000 Spectrophotometer (NanoDrop Technologies Inc,
Wilmington, DE). Five nanograms purified mRNA per 20- μl reaction volume were
used in the synthesis of first-strand cDNA using Oligo (dT) reverse-transcriptase
kit at 37°C for 60 minutes (Invitrogen). All Reactions utilized the Universal PCR
Master Mix (Applied Biosystems, Inc. (ABI), Fullerton, CA) and were run under
default conditions in the 7900HT Sequence Detector System (ABI). Primers for
GAPDH were used as loading control, and the level of expression for
the genes
Ccnd1, Hes1and Gli3 was assessed
(ready-made real-time primer/probe sets;
ABI).
Amplification efficiency was determined by the delta delta Ct method of
the
real-time PCR amplification plots (reference to this). Fold increase or
decrease in
gene expression were calculated using the untreated control
as a baseline.
53
Flow cytometry analysis of transduced CD34+ cells.
Transduced CD34+ cells were cultured for 6 days in X-vivo15 with 3ng/mL Flt3L,
and then collected, washed twice with PBS, and resuspended in 100uL of PBS
and stained directly with PE-conjugated anti-CD34+ mAb (clone 8G12, Becton
Dickinson Immunocytometry Systems, San Jose, Calif. USA). eGFP was
detected for cells transduced with an eGFP expressing vector. Measurements
were performed on a FACS Calibur flow cytometer (Becton Dickinson, Franklin
Lakes, NJ, USA) with the Cellquest software (version 3.0, B.D., USA). Initial
gating was performed using forward and side scatter to identify viable cell
populations.
Cell cycle analysis.
CD34+ cells were treated with BIO, the inactive control meBIO, or equal volume
of DMSO vehicle control by culturing them in X-vivo 15 medium containing either
only “low dose” Flt3 ligand ([3ng/mL], or with “high dose” SCF ([50 ng/mL], [50
ng/mL] Flt-3 ligand, and [50 ng/mL] thrombopoietin on FN for 4 days. 50 μl
aliquots of CD34+ cells were removed daily, fixed in 70% ethanol in -20
o
C
overnight, then centrifuged at 300g for 5min. The cell pellet was resuspended in
PBS and treated with RNase (200 μg/ml), Propidium Iodide (10 μg/ml) and 0.1%
TritonX-100 at 37
o
C for 40 min. Cells were then washed in 4mL PBS, centrifuged
and resuspended in 500 μL of PBS. DNA content and cell cycle distribution were
analyzed using a Becton Dickinson FACScan flow cytometer. Cell cycle analysis
54
was performed using Modfit Lt (Version 3.1, Verity Software House,Topsham,
ME, USA), on a minimum of 10,000 cells per sample.
Tritiated Thymidine Proliferation assay.
DNA synthesis was measured by tritiated thymidine incorporation in serum-free
medium. CD34
+
cells (2.5-7.5 x 10
4
) were cultured in 200 ul of X-vivo 15 medium
containing 3ng/mL Flt3 ligand in addition to varying concentrations of Bio, MeBio
or DMSO (<0.1%) as indicated on FN. 50 uCi 3H-thymidine was added to the
cells which were incubated for 18-24 hours and then lysed and transferred to
filters for scintillation counting. Four independent experiments were performed in
triplicate and values were adjusted for a starting CD34
+
number of 2.5x10
4
cells/well.
CFSE assay of cell division.
CD34
+
selected cells were labeled with CFSE (Carboxyfluorescein succinimidyl
ester, Molecular Probes Inc., Eugene, OR) at a cell concentration of 3 × 10
6
/mL
and a dye concentration of 1.25 μM in X-vivo15 at room temperature for 10
minutes, with occasional mixing. Ten ml of X-Vivo 15 medium containing 1%
BSA was added to the cells, which were then centrifuged at 400g for 10 minutes.
Cells were counted, resuspended in X-Vivo 15/1% BSA at a concentration of 2 ×
10
5
cells/ml, placed into one ml cultures in 24-well tissue culture plates (Corning),
and cultured in the presence of various cytokine combinations with and without
BIO as described previously for 6 days. Compensation was adjusted to allow
55
CFSE fluorescence to be read only in the FITC channel. Analysis was performed
on a FACS-Calibur flow cytometer and Modfit Lt software
Apoptosis Assay.
Apoptotic cells were detected using an Annexin V-fluorescein-isothicyanate
(FITC) and propidium iodide (PI) kit (BD Biosciences) according to the
manufacturer’s protocol. Annexin V-FITC negative/PI negative cells are
considered alive. Annexin V-FITC positive/PI negative cells are identified as
being in early apoptosis, and Annexin V-FITC/PI positive cells are in late
apoptosis or are already dead.
CFU-GEMM assay.
To assess the clonogenic potential of hematopoietic progenitor cells following Bio
treatment and retroviral transduction, CD34
+
cells were cultured in X-Vivo15
medium containing either only “low dose” Flt3 ligand ([3ng/mL], or with SCF ([50
ng/mL] , [50 ng/mL] Flt-3 ligand, and [50 ng/mL] thrombopoietin in 48-well plates,
to which 100 µl of diluted vector supernatant
was added following 3 day of
culture. Cells were then incubated at 37
o
C overnight and washed with 10ml of
dPBS. Samples of 300 to 1,000 cells were plated in duplicate for colony forming
unit (CFU) analysis in methylcellulose (Stem Cell Technologies Vancouver BC
Canada). CFU-granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-
GEMM), burst-forming unit erythroid (BFU-E), CFU-colony-forming unit erythroid
56
(CFU-E) and CFU-granulocyte-macrophage (CFU-GM) colonies were
enumerated 14-15 days later using an inverted phase contrast microscope.
Mouse Xenograft Transplantation.
Ten-week-old NOD/SCID/ γc
null
mice (NSG) mice (Ito, Hiramatsu et al. 2002) were
irradiated sublethally with 300 cGy from a Cesium
137
source and 100,000 CD34
+
cells were transplanted intravenously. Nine to ten weeks after transplantation,
mice were euthanized and the bone marrow (BM) was collected for analyses of
engraftment and the presence of the retroviral vector, using real-time PCR. All
BM cells collected from the mice were analyzed using FACS Calibur or FACS
Scan. For each analysis, viable cells defined by forward- and side-scatter were
further gated for human CD45
+
cells. Mouse anti-human monoclonal antibodies
used were: phycoerythrin (PE)-conjugated anti-CD13, CD14, CD15, CD19,
CD33, CD56; PerCP-conjugated anti-CD3 and CD34; APC-conjugated anti-
CD45 from Becton Dickinson PharMingen (San Jose, CA, USA). Analyses were
performed with the Cellquest software (version 3.0, B.D., NJ, USA) or FloJo
software (version 7.0, Tree Star, OR, USA.).
Proviral copy number
Proviral copy numbers were determined by real time quantitative PCR (qPCR).
DNA was extracted tissue samples (Qiagen DNeasy, ) and was quantitated using
fluorimetry and a DNA specific dye (Hoescht dye; Sigma). Quantitative PCR was
performed with primers and probe designed to amplify integrated the eGFP
57
sequence of PG13-MND-eGFP-SN. The sense primer is 5'-ctg ctg ccc gac aac
ca-3', the antisense primer is 5'- gaa ctc cag cag gac cat gtg-3 and the TAMRA
probe sequence is 5'-FAM - ccc tga gca aag acc cca acg aga - Tamra-3'. The
primer concentrations (sense and antisense) were 400 nM and the probe
concentration was 50 nM in all reactions. All reactions utilized Universal Master
Mix (Applied Biosystems, Inc., Fullerton, CA) and were run under default
conditions in the 7900HT Sequence Detector System (ABI). Each of the wells
contained 350 ng of template DNA and they were compared to 350 ng of DNA
from a set of copy number standards. The standards were produced using a
clonal population of 293 human embryonic kidney (HEK) cells containing one
copy of the pHR-CMV-eGFP provirus (as determined by Southern blot analysis),
diluted into DNA of untransduced 293 HEK cells, yielding a detection sensitivity
of 1:100,000 vector-containing cells or 0.00001 copies/cell.
4(iii) Results
BIO treatment activates β-Catenin in human umbilical cord blood CD34+
cells
BIO has been shown to reduce GSK-3 β kinase activity thus rescuing β-
catenin from proteasomal degradation in the canonical WNT pathway (Meijer,
Skaltsounis et al. 2003; Sato, Meijer et al. 2004; Holmes, O'Brien et al. 2008). To
determine if BIO treatment of umbilical cord blood (UCB) CD34+ cells leads to
accumulation of β-catenin, we cultured cells with an escalating dose of BIO and
measured immunoreactive β-catenin protein by immunoprecipitation followed by
58
western blot analysis. BIO treatment increased accumulation of β-Catenin in a
dose-dependent manner (Figure 1B). Although the highest dose of Bio tested, 10
uM, led to the highest levels of β-catenin protein, we observed cytotoxicity at this
dosage and therefore used 3.0 uM BIO for subsequent studies.
No table of contents entries found.
Figure 4-1. Structure of BIO and dose escalation of BIO on CD34+ cells. A. The
chemical structure of 6’ Bromo Indirubin 3’Oxime (BIO). B. Immunoprecipitation followed by
western analysis of CD34+ cells with an increasing dose of BIO.
To evaluate whether UCB CD34+ cells are capable of responding to BIO
treatment, we used the TOPflash reporter assay to determine the level of β-
catenin/TCF activity (Roose, Molenaar et al. 1998). We directly monitored TCF
transcriptional activity in the UCB CD34+ cells by transfecting a TCF reporter
59
(pTOPflash) into CD34+ cells following immediate exposure to BIO. As a control,
we used a reporter containing mutated TCF-binding sites (pFOPflash). We also
co-transfected a plasmid expressing the Renilla luciferase gene to normalize for
transfection efficiency.
Figure 4-2. Analysis of β-catenin activity in BIO treated CD34+ cells. A.
TOP/FOP Flash assay to analyze β-catenin activity in CD34+ cells. Luciferase
activity was measured 2 days following electroporation of the TOP/FOP Flash
plasmids. B. Quantitative real time PCR to determine Ccnd1, Hes1 and Gli3
transcription following BIO treatment.
B
A
60
Following 48 hours of culture after transfection of the reporter plasmids, a
2-fold increase of β-Catenin/TCF activity was observed in the cells treated with
BIO compared to the untreated control (Figure4-2A). BIO led to a 2-fold increase
in TOPflash/FOPflash activity, compared to untreated cells. Cells treated with
the negative control MeBIO or the vehicle alone, showed only baseline
TOPflash/FOPflash activity. Thus, BIO increased the activity of β-catenin in the
human UCB CD34+ cells.
To characterize the effects of BIO on the Wnt pathway in UCB CD34+
cells, expression of the Wnt regulated target gene Ccnd1 was examined by
reverse-transcriptase-linked Real-time PCR using RNA isolated from the treated
cells described above. Following BIO treatment, Ccnd1 was upregulated by 4-
fold compared to the untreated control CD34+ cells (Figure 4-2B).
To investigate the potential effects of BIO on the Notch and Hedgehog
pathways, which have been shown to be modulated by activation of the WNT
pathway, we quantitatively examined expression of the Notch-regulated gene
target Hes1 and the Hedgehog pathway regulated gene target Gli3, again
performing RT Real time PCR. Following BIO treatment, expression of Hes1 was
down-regulated by 2.3 fold and the expression of Gli3 was up-regulated by 3 fold
compared to the untreated controls (Figure 4-2B). These results suggest that BIO
treatment of UCB CD34+ cells modulates the Notch and the Hedgehog
pathways, although it is not evident whether this is a direct or secondary effect.
61
Stimulation with BIO increases initial cell cycle progression of human UCB
CD34+ cells, but arrests further cell division
To better understand the effects of BIO on the cell cycle characteristics of
UCB CD34+ cells, we measured S/G2/M progression using propidium iodide (PI)
Figure 4-3. Cell cycle analysis of CD34+ cells following BIO treatment. A. CD34+ cells
were cultured in 3ng/mL of Flt3L and cell cycle analysis was measured at the indicated days.
B. CD34+ cells were cultured in 50ng/mL of F,S,T and % S/G2/M progression was measured
at the indicated days.
B
A
62
staining and flow cytometric analysis. Cells were cultured with BIO, MeBio or
vehicle alone in the presence of Flt3L [3ng/mL –low cytokines] or in the presence
Figure 4-4. Day 3 cell cycle analysis of CD34+ cells following BIO treatment. A. CD34+
cells were cultured in 3ng/mL of Flt3La and cell cycle analysis was measured at the indicated
days. B. CD34+ cells were cultured in 50ng/mL of F,S,T and % S/G2/M progression was
measured at day 3.
B
A
63
of Flt3L, SCF and TPO (FST) [50ng/mL – high cytokines] on FN for 4 days,
mimicking a typical retroviral transduction protocol. Cell samples were obtained
daily and assayed for cell cycle status by flow cytometry.
In the presence of only a low cytokine concentration (flt-3 ligand at 3
ng/ml), untreated or vehicle-treated cells showed minimal cell cycle progression
(<10% S/G2/M by day 2) (Figure 4-3A). The addition of BIO to cells in low
cytokine conditions increased the percentage of cells in S/G2/M more than two-
fold to 23% after two days of culture. Addition of high concentrations of three
cytokines caused a greater increase in cell cycle progression, with more than
40% of the cells in S/G2/M by 48 hours. In the presence of these high cytokine
conditions, there were no discernible additional effects from the addition of BIO
Figure 4-5. Tritiated Thymidine analysis following BIO treatment in CD34+ cells.
CD34+ cells were cultured in 3ng/mL of Flt3La and Tritiated Thymidine analysis was
performed 3 days following BIO treatment.
64
(Figure 4-3B). Taking a closer look following 3 days of culture, the difference in
BIO addition from the control was statistically significant with the control at 12%
and BIO treated arm at 19% S/G2/M progression, p<0.05 (Figure 4-4A). Under
conditions of high cytokine culture, no significant difference in S/G2/M
percentage was observed (Figure4-4B).
To confirm that BIO treatment of UCB CD34+ cells can promote cell cycle
progression, we performed a tritiated thymidine incorporation assay to measure
DNA synthesis. In the low cytokine conditions, three days of BIO treatment
resulted in a 2.85 fold increase of DNA synthesis in BIO treated cells compared
to the untreated cells or MeBIO or vehicle-treated cells (Figure 4-5).
Figure 4-6. CFSE Analysis of CD34+ cells treated with BIO. CD34+ cells were cultured
in 3ng/mL of Flt3L and CFSE analysis was performed 6 days after BIO treatment. B.CD34+
cells were cultured in 50ng/mL FST and Tritiated Thymidine analysis was performed 6 days
following BIO treatment.
65
To study the effect of BIO on UCB CD34+ cell division and expansion, we
performed a CFSE assay of cell division. CD34+ cells were labeled with CFSE
and then cultured with or without BIO or MeBio for 6 days and then analyzed by
flow cytometry to examine cell division. CD34+ cells cultured in the low cytokine
conditions underwent 1-4 divisions during this time; cells cultured with high
cytokines had undergone 4-9 divisions. Unexpectedly, after 6 days of culture,
the BIO treated cells in low cytokine conditions had undergone just one cell
division, and were arrested compared to cells without BIO (Figure 4-6A). The cell
division arrest was observed as early as 4 days post BIO treatment (data not
shown). High concentrations of FST [50ng/mL] enabled the BIO treated cells to
undergo further cell divisions but there were less divisions than the cells exposed
to high cytokines without BIO (Figure 4-6B). MeBIO-treated cells performed
identically to the untreated controls under the respective cytokine conditions.
Thus, BIO induced an initial round of cell cycle progression, but then led to cell
cycle arrest.
BIO treatment decreases apoptosis in UCB CD34+ cells
The pathways that arrest cell cycle progression and/or induce cell death
after stress are known as cell cycle checkpoints. These checkpoints maintain the
fidelity of DNA replication, repair, and division. To determine whether the cell
division arrest observed of the BIO treated UCB CD34+ cells induced an
apoptotic response, we measured the externalization
of the membrane
phospholipid phosphatidylserine (Annexin V-FITC
assay) by flow cytometry. To
66
detect the early apoptotic
response following BIO addition, UCB CD34+ cells
were treated
with BIO for 6 days and then subjected to Annexin
V-FITC assay. As
shown in Figure 4-7, early apoptosis (Annexin V-FITC positive/PI negative) was
reduced by half,
compared to the DMSO treated and untreated cells. Therefore,
an anti-apoptotic effect is associated with BIO treatment of CD34+ cells
Figure 4-7. Apoptosis of CD34+ cells. CD34+ cells were cultured in 3ng/mL of Flt3L in
addition to BIO. Following 6 days of culture, early apoptosis was measured.
*
p < 0.05
BIO treatment improves in vitro retroviral vector-mediated transduction of
human UCB CD34+ cells
To explore the effects of BIO treatment on the transduction of human UCB
CD34+ cells by a γ-retroviral vector, we cultured cells in BIO, MeBIO, DMSO, or
untreated medium for 3 days and then added an eGFP expressing γ-retroviral
67
vector, incubated the cells for 3 days and analyzed them by flow cytometry. We
observed only a modest increase in the percentage of eGFP positive cells in the
presence of BIO compared to the controls (12% vs. 8%) (Figure 4-8A). However,
focusing the analysis on the more primitive population of progenitor cells that
Figure 4-8. In vitro transduction of CD34+ cells. CD34+ cells cultured in 3ng/mL of Flt3L
and on day 3, transduced with and PG13-MND-eGFP-SN vector. A. FACS analysis at day
6 following BIO treatment measuring the percent of eGFP positive cells. B. The total
number of CD34positive and eGFP positive dual positive cells.
A
B
*
p < 0.05
68
retained expression of CD34 revealed a significantly higher number of eGFP
positive cells following BIO treatment versus the untreated control. (Figure 4-8B,
P<0.05).
BIO treatment maintains homing and engraftment in UCB CD34+ cells.
To assess the effects of BIO treatment on the homing ability of the UCB
CD34+ cells, they were injected into the tail vein of NSG mice, and twenty-four
hours later bone marrow from these recipients was harvested. The human cells
that had homed and migrated to the bone marrow were quantified using flow
•
Figure 4-9. Percentage of Human CD45 Positive cells following 24hours post tail
vein injection. Homing was determined by culturing CD34+ cells with or without Bio for 4
days ex-vivo. Cells (5 x 10
4
) were then injected into NSG mice. 24hours later bone
marrow was harvested to determine the percentage of CD45+ cells.
69
cytometry to detect CD45+ cells. BIO treated UCB CD34+ cells showed no
significant difference in homing function compared to the controls, as determined
by the percentage of human CD45 cells present in the harvested bone marrow
(Figure 4-9).
To determine the effect of BIO on the engraftment capabilities of UCB
CD34+ cells following 4 days of total culture in the conditions mentioned above,
recipient mice were injected and 9 weeks later, BM from these recipients were
harvested and flow cytometry for hCD45 expression was evaluated. No
Figure 4-10. Percentage of Human CD45 Positive cells following 9-10 weeks post tail
vein injection. Engraftment was determined by culturing CD34+ cells with or without Bio
for 4 days ex-vivo. Cells (1 x 10
4
) were then injected into NSG mice. 9-10 weeks later, bone
marrow was harvested to determine the percentage of CD45+ cells.
70
significant difference in hCD45 expression was observed between the BIO
treated versus untreated control cells harvested from the recipient’s bone marrow
(Figure 4-10).
Figure 4-11. Lineage differentiation ability of BIO treated CD34+ cells analyzed in a
NSG xeno-transplantation model. Lineage differentiation capability was determined by
culturing CD34+ cells with or without Bio for 4 days ex-vivo. Cells (1 x 10
4
) were then
injected into NSG mice. 9-10 weeks later, bone marrow was harvested to determine the
percentage of hematopoietic cells: hCD45+ cells, Myeloid cells: hCD56+ and
hCD13,14,15,33+ and Lymphoid cells: CD19+ and CD3+. Control= cells cultured in
3ng/mL Flt3L. Bio [3µM]= cells cultured in 3ng/mL Flt3L in addition to BIO. F,S,T
[50ng/mL]= cells cultured in Flt3L, SCF and TPO [50ng/mL].
71
BIO treatment does not impair UCB CD34+ cells progenitor potential
To further study the effects of GSK3 β inhibition on the developmental
potential of the UCB CD34+ cells, we cultured cells for 4 days in BIO and injected
them into NSG mice. After 9-10weeks, bone marrow was harvested from the
mice and the lineages of engrafted human cells were determined. We
determined that all reconstituted recipients contained multi-lineage human donor
derived cells (Figure 4-11).
Figure 4-12. Gene marking in a primary CD34+ xenotransplantation mouse model.
CD34+ cells were cultured with or without Bio for 4 days ex-vivo. At day 3, cells were
transduced with a PG13-MND-eGFP-SN vector. Cells (1 x 10
4
) were then injected into
NSG mice. 9-10 weeks later, bone marrow was harvested to determine the proviral copy
number by real time PCR. Control= cells cultured in 3ng/mL Flt3L. Bio [3µM]= cells
cultured in 3ng/mL Flt3L in addition to BIO. F,S,T [50ng/mL]= cells cultured in Flt3L, SCF
and TPO [50ng/mL].
72
After 9 weeks, the mice were euthanized and the bone marrow cells were
analyzed to quantify the extent of transduction using real-time PCR to measure
vector proviral copy number. We observed a mean 50-fold increase of gene
marking in the marrow of mice receiving BIO-treated human cells compared to
the untreated arm but this difference was not statistically significant (Figure 8B).
4(iv) Discussion
In this study, we have found that the GSK3 inhibitor BIO promotes γ-
retroviral transduction of UCB CD34+ cells (Figure 4-8B) in vitro. We show that
BIO treatment of UCB CD34+ cells stimulate cell cycle progression which is
necessary for γ-retroviral transduction. In addition, we observed upregulation of
Ccnd1, a downstream target of the Wnt pathway and Gli3, a downstream target
of the Hedgehog pathway. These findings correlate with the Wnt and the
Hedgehog pathways being involved in stem cell self renewal and BIO activation
of these pathways. We and others observed (Holmes, O'Brien et al. 2008) a
reduction in apoptosis following the addition of BIO on UCB CD34+ cells.
While the study by Holmes et al. (Holmes, O'Brien et al. 2008) sought to
investigate BIO’s effect on UCB CD34+ cells expansion while cultured in high
concentrations of cytokines (FST), our goal with this study was to determine
whether the addition of BIO would be sufficient to increase γ-retroviral
transduction of UCB CD34+ cells under minimal cytokine conditions.
Following a typical transduction protocol, transduction of UCB CD34+ cells
treated with BIO showed a 2.5 fold increase of the number of transduced CD34+
73
cells compared to the non-treated cells. However, there was no statistical
difference in only the percentage of transduced CD34+ between the BIO treated
and non-treated cells.
In this study, we found that the GSK inhibitor BIO stimulated proliferation
and subsequent cell division arrest does not impair UCB CD34+ cells γ-retroviral
transduction accessibility, homing, engraftment and hematopoiesis potential.
Our study indicates that BIO treatment of UCB CD34+ cells results in the dose
dependent accumulation of β-catenin (Figure 4-1B). In addition we observed that
BIO initiated activation of β-catenin by TOPFLASH assay (Figure 4-2A) and
increased Ccnd1 expression, a target of the Wnt pathway (Figure 4-2B). The
increase in TOPFLASH luciferase activity and Ccnd1 expression lends further
credence to our suggestion that BIO stimulates the Wnt pathway (Sato, Meijer et
al. 2004; Holmes, O'Brien et al. 2008). GSK3 β has also been suggested to
modulate the Notch (Foltz, Santiago et al. 2002; Trowbridge, Xenocostas et al.
2006) and Hedgehog (Jia, Amanai et al. 2002; Trowbridge, Xenocostas et al.
2006) pathways. We discovered that BIO treatment of UCB CD34+ cells
decreases Hes 1, a downstream target of the Notch pathway (Karanu, Murdoch
et al. 2000) and increases Gli3, a target of the Hedgehog pathway (Figure 4-2B).
It has been previously reported these pathways, Wnt, Notch and Hedgehog, are
all involved in HSC function (Karanu, Murdoch et al. 2000; Varnum-Finney, Xu et
al. 2000; Bhardwaj, Murdoch et al. 2001). We demonstrate that BIO modulates
the Wnt, Notch and Hedgehog pathways. This suggests that multiple signaling
pathways are employed following BIO treatment.
74
Increased cell cycle was observed in BIO treated UCB CD34+ cells
(Figure 4-3A and B). Surprisingly, following this initial increase in cell cycle
progression, treated cells undergo a cell division arrest as early as 4 days post
treatment (unpublished data) and by day 6 the majority of cells arrests following
one cell division (Figure 4-6A). When cells were treated with high concentrations
of cytokines, FST [50ng/mL] in the presence of BIO, the cell division arrest was
transient, leading to a delay in proliferation (Figure 4-6B) as observed by Holmes
et al, 2008. We suggest that BIO arrests cell division, and high cytokine
concentrations allow the bypass of this arrest in UCB CD34+ cells. This
observation, incorporates and confirms BIO treatment of CD34+ cells leads to
increase cell cycle progression (Trowbridge, Xenocostas et al. 2006; Holmes,
O'Brien et al. 2008), and includes a new observation that BIO treatment leads to
an arrest in cell division following an initial increase in cell cycle progression,
providing a more complete understanding of BIO’s effect on UCB CD34+
proliferation and cell division. Cell division is composed of cell cycle checkpoints
and cell cycle delay/arrest is generally associated with DNA repair (Shackelford,
Kaufmann et al. 1999). We proceeded to study the effects of BIO on other UCB
CD34+ cells functions.
We found that BIO treated UCB CD34+ cells cultured for 6 days showed a
decrease in early apoptosis (Figure 4-7). Here we also show that BIO does not
impair UCB CD34+ cell ability to be transduced with a γ-retroviral vector ex vivo
(Figure4- 8). Although the percent of eGFP transduced CD34+ cells were
comparable, the number of CD34+/eGFP positive cells was approximately 2 folds
75
greater (Figure 4-8B). This result could perhaps be attributed to the reduction of
apoptosis following BIO treatment. When these transduced cells were injected
into mice, although an average of a 50 fold increase in marked cells was
observed compared to the untreated cells (Figure 4-12), there was no statistical
difference in marking compared to the controls following 9-10 weeks of
incubation.
In our study BIO treatment does not impair UCB CD34+ cells
hematopoiesis potential. Following 4 days of culture, cells were either plated for
CFU analysis or injected into NSG mice. There was no difference in CFU
formation between the BIO treated cells and the untreated cells (data not shown).
In mice, there was no difference in the percentage of hCD45 isolated, CD3 (T
cell), CD19 (B cell), CD56 (NK marker) or CD13, 14, 15, 33 (myeloid) after 9-10
weeks of incubation (Figure 4-11).
Our results show that BIO treatment of UCB CD34+ cells does not impair
their ability to home (Figure 4-9), engraft (Figure 4-10) or lineage differentiate
(Figure 4-11). Although we observed an increase in initial cell cycle of treated
UCB CD34+ cells, the following cell division arrest remains a barrier that must be
studied for future improvement of gene transfer potential. Our protocol allows us
to study this GSK inhibitor induced arrest and may be used for further inquiries.
We conclude that a pharmacological agent such as BIO may be a useful tool in
future HSC γ-retroviral gene therapy studies.
76
Chapter 5 – Concluding Remarks and Future Directions
5(i) Hematopoietic Stem Cell Gene Therapy
Hematopoietic stem cells gene therapy can potentially cure a variety of
human hematopoietic diseases. Even with the development of leukemia in a few
of the patients in the X-linked SCID retroviral gene therapy trial in France, stem
cell gene therapy still shows great potential for use in the clinic to treat diseases.
The main goals of HSC gene therapy should be to increase therapeutic efficacy
as well as safety. Understanding the mechanism of stem cell fate decisions can
potentially aide both these goals.
With advancing technology and drug discoveries, understanding the
mechanism of stem cell fate decision will be critical. Being able to identify then
study the factors involved in stem cell self-renewal will greatly benefit HSC gene
therapy. The focus of this work has therefore been to study the Wnt/ β-catenin
pathway using a small molecule inhibitor to GSK3 and observing its effect on
CD34+ cells expansion and potential use in gene therapy.
5(ii) Perspectives of small molecules
There is much evidence in the literature suggesting a role for Wnt/ β-
catenin signaling in the hematopoietic stem and progenitor cell growth and cell
fate decision process (Reya, Duncan et al. 2003; Reya and Clevers 2005; Zhang,
Lo Muzio et al. 2005; Trowbridge, Xenocostas et al. 2006; Tseng, Engel et al.
77
2006). These studies have opened the door to many important questions
regarding the mechanisms of HSC regulation. For example, to understand and
elucidate the mechanisms by which Wnt and β-catenin promote self-renewal,
identification and analysis of the targets that are regulated in HSCs is needed.
With the possibility that multiple signaling processes (e.g., Notch, Hedgehog) and
transcription factors (e.g., HoxB4) are regulators of HSC self-renewal, it is critical
to determine if, when and how these factors work together to regulate HSC
development. Having the tools to answer these questions will further the
understanding of the signals that regulate HSCs cell fate. With time, researchers
will be able to create optimal methods to expand HSC while maintaining their
undifferentiated state during culture. Small molecule drugs will have numerous
implications for future transplantation and regenerative therapies.
5(iii) Application of Small Molecules for Gene Transfer to HSC
As an alternative to cytokines for HSC transduction culture conditions,
small molecules which control stem cell self-renewal can help expand transduced
HSC. There is a high correlation between engraftment and cell dosage
(Nakamura, Bahceci et al. 2001). In the case of umbilical cord blood HSC gene
therapy, a consistent method of HSC expansion will be critical for use in not only
infant transplant but for adult transplants as well. With an ever growing library of
small molecules, a growing knowledge of HSC fate determinants will be
necessary. For BIO to be an effective mediator of gene therapy, the cell cycle
arrest following culture must be overcome. Perhaps, BIO in addition to cytokines
78
or BIO and another pharmacological drug that regulates HSC fate may be the
key. We do show that BIO in a low cytokine condition can increase the
transduction of CD34+ cells compared to the non-treated control but an optimal
culture condition is far from being identified. We show that a small molecule
inhibitor known to be involved in stem cell fate determination, BIO, can be used
in a gene therapy context. In the future, new small molecules which may control
stem cell fate may be studied using a similar model used in this study.
79
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Abstract (if available)
Abstract
Hematopoietic stem cells (HSC) are rare cells of the hematopoietic system with the ability to self-renew and differentiate into all mature blood lineages. Although HSC are an extremely attractive target for HSC gene therapy and despite successful use of HSCs in the clinic and in clinical gene therapy trials, limitations exist. One limitation is the transduction protocol used requiring a combination of cytokines to promote HSC into cell cycle, since γ-retroviral vectors can only transduce dividing cells. The Wnt/β-catenin pathway has been shown to be involved in stem cell fate determination and self-renewal. In these studies we activate the Wnt/β-catenin pathway using a GSK3 inhibitor, BIO, and test its effects on γ-retroviral transduction, homing, engraftment and hematopoiesis potential of human umbilical cord blood (UCB) CD34+ cells. We initially explored different methods to activate the Wnt/β-catenin pathway in CD34+ cells. We designed and tested siRNA’s to Axin and used GSK-inhibitors in order to disrupt the destruction complex thereby stabilizing β-catenin. GSK3 inhibitor BIO showed the most promise. We found BIO treatment led to increased accumulation of β-catenin in UCB CD34+ cells. We observed increased β-catenin activity following BIO treatment, analyzed by TOPflash reporter assay and upregulation of Ccnd1 expression compared to the untreated control. We determined that BIO initially increased UCB CD34+ cells progression through cell cycle but then imposed an arrest in cell division. BIO decreased cell apoptosis and did not impair UCB CD34+ cells homing, engraftment and hematopoiesis potential. BIO treatment of CD34+ cells allowed an increased extent of gene transfer using a γ-retroviral vector. Future studies on the effects of the BIO induced cell arrest will allow for a better understanding of the effects that GSK inhibition has on HSC function.
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Choi, Yeong "Christopher"
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Core Title
Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Molecular Microbiology
Publication Date
07/31/2009
Defense Date
06/16/2009
Publisher
University of Southern California
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beta-catenin,BIO,CD34,cell cycle,gene therapy,GSK3 inhibitor,hematopoietic stem cells,OAI-PMH Harvest
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Ou, Jing-Hsiung James (
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), Cannon, Paula M. (
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), Kohn, Donald B. (
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)
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cchoi@chla.usc.edu,yeongchoi@mednet.ucla.edu
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Choi, Yeong "Christopher"
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Tags
beta-catenin
BIO
CD34
cell cycle
gene therapy
GSK3 inhibitor
hematopoietic stem cells