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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2556-2570
By
From the Department of Pediatrics, Section of Hematology/Oncology,
Herman B Wells Center for Pediatric Research, the Stem Cell Laboratory,
Cancer Research Building, and the Division of Biostatistics,
Regenstrief Institute for Health Care, Indiana University School of
Medicine, Indianapolis, IN; and Howard Hughes Medical Institute,
Indiana University School of Medicine, Indianapolis, IN.
Mobilized CD34+ cells from human peripheral blood (PB)
are increasingly used for hematopoietic stem-cell transplantation.
However, the mechanisms involved in the mobilization of human
hematopoietic stem and progenitor cells are largely unknown. To study
the mobilization of human progenitor cells in an experimental animal
model in response to different treatment regimens, we injected
intravenously a total of 92 immunodeficient nonobese diabetic/severe
combined immunodeficiency (NOD/SCID) mice with various numbers of
granulocyte colony-stimulating factor (G-CSF) -mobilized
CD34+ PB cells (ranging from 2 to 50 × 106
cells per animal). Engraftment of human cells was detectable for up to
6.5 months after transplantation and, depending on the number of cells
injected, reached as high as 96% in the bone marrow (BM), displaying
an organ-specific maturation pattern of T- and B-lymphoid and myeloid
cells. Among the different mobilization regimens tested, human
clonogenic cells could be mobilized from the BM into the PB (P
= .019) with a high or low dose of human G-CSF, alone or in
combination with human stem-cell factor (SCF), with an average increase
of 4.6-fold over control. Therefore, xenotransplantation of human cells
in NOD/SCID mice will provide a basis to further study the mechanisms
of mobilization and the biology of the mobilized primitive human
hematopoietic cell.
MOBILIZED HUMAN peripheral blood (PB)
CD34+ cells are increasingly used in clinical protocols as
a source of hematopoietic stem cells for allogeneic and autologous
transplantation.1, 2 More recently, this alternative source
of stem cells is being used as a target for genetic modification in
somatic cell gene therapy trials.3 Compared with bone
marrow (BM), the use of PB stem cells in patients undergoing autologous
transplantation has resulted in a significantly accelerated
engraftment.4-7 However, the mechanisms involved in the
mobilization of these cells, and the quality and biological
characteristics of the stem-cell subsets in the PB products are largely
unknown.8 Other clinically important issues include the
safety of the mobilizing regimens, the timing of mobilization and
collection, the amount of stem and progenitor cells that can be
harvested, and, ultimately, the adequacy of the product with respect to
the speed, level, and stability of engraftment. Moreover, with the
emergence of new candidate molecules for the mobilization of primitive
hematopoietic cells, such as interleukin-8, macrophage inhibitory
protein (MIP)-1 Mouse hematopoietic stem cells have functionally been defined as cells
that have the ability to engraft upon transplantation, proliferate, and
sustain multilineage hematopoiesis in vivo for an extended period. In
contrast, primitive human hematopoietic cells have only been
systematically studied using clonogenic assays or long-term BM
cultures.9,10 These assays have proven to be valuable in
estimating the quality and frequency of human hematopoietic cells with
extensive proliferative capacity. However, evidence from gene-marking
studies in human clinical trials suggests that primitive hematopoietic
cells cannot be adequately studied using in vitro assays, as no
correlation could be found between the expression of transgenes in
vitro and after transplantation in vivo.11-14 Similarly,
when comparing retroviral gene transfer in human long-term
culture-initiating cells (LTC-IC) and cells that are capable of
engrafting immunodeficient mice, LTC-IC but not in vivo repopulating
cells could be readily transduced.15 Although some
investigators have reported significant overlap between LTC-IC and
cells that give rise to engraftment in immunodeficient
mice,16 others have described these subsets to be
biologically distinct; differing in frequency,17
phenotype,18 and the ability to be maintained on human BM
stromal cells in vitro.19
To study the engraftment and differentiation of primitive human
hematopoietic cells in vivo, various models of immunodeficient mice
have been developed (see Wermann et al20 for a recent
review). These models include the classical C.B-17 scid/scid severe
combined immunodeficiency (SCID) mouse,21 the beige athymic
nude X-linked (bnx) immunodeficient mouse,22 the humanized
SCID (SCID-hu) mouse, in which human (fetal) hematopoietic tissues such
as liver, thymus, and bone fragments, are surgically
transplanted,23-25 and a transgenic SCID mouse that
expresses the genes for human IL-3, granulocyte-macrophage
colony-stimulating factor (GM-CSF), and stem-cell factor
(SCF).26 Recently, a new mouse strain
(NOD/LtSz-scid/scid, hereafter referred to as NOD/SCID) was
developed by crossing SCID mice with nonobese diabetic (NOD/Lt)
mice.27 It has been reported that the engraftment level of
human cells in the NOD/SCID model, after transplantation of
splenocytes,28 PB blood mononuclear cells,29
BM, or cord blood cells,15,26,27,30-32 was fivefold to
10-fold higher than could be achieved in the classical SCID mouse.
In the present paper we used the NOD/SCID mouse to provide a detailed
study of the engraftment and maturation of human mobilized PB
CD34+ cells in the mouse and to investigate whether
engrafted animals can be used as a model to study the mobilization of
human hematopoietic progenitor cells in vivo. Our results show that
NOD/SCID mice can be highly engrafted by PB CD34+ cells
with differentiation along multiple lineages in a
microenvironment-specific fashion, without supplemental human
cytokines. In addition, using various regimens that have been
previously shown to induce the mobilization of clonogenic cells in
humans or primates, our data show for the first time that human
progenitor cells can be mobilized from the BM of the NOD/SCID
transplanted with PB stem cells by treatment of the animals for 4 to 6 days with G-CSF, or G-CSF and SCF. This model will enable future
studies directed at investigating the biological mechanisms that
modulate the mobilization of human progenitor cells in vivo.
Animals.
A breeding colony of NOD/LtSz-scid/scid (NOD/SCID)
mice27 was established at the Laboratory Animal Research
Center at the Indiana University School of Medicine (Indianapolis, IN)
from breeding pairs kindly provided by Dr Leonard D. Shultz (The Jackson Laboratory, Bar Harbor, ME). Animals were housed in
a positive airflow ventilated rack (Lab Products, Maywood, NJ)
and bred and maintained in microisolators under specific pathogen-free
conditions. Animals were tested for the absence of mouse (CD4 + CD3+) T cells in the PB. Typically, T cells were
undetectable (<1%, in lymphocyte light-scatter gate) in 50% to 60%
of the animals (12-16 weeks of age). About 30% to 40% of the mice had
a low level of circulating T cells (1%-5%), whereas 10% of the mice
contained 14% ± 6% T cells. Before transplantation, 10- to
15-week-old male or female NOD/SCID mice received a sublethal dose of
300 cGy total-body irradiation (TBI) at 86 cGy/minute using a GammaCell
40 (Nordion International Inc, Ontario, Canada) equipped with two
opposing 137Cesium sources. Radiation-associated mortality
was not observed at this dose. In one specific experiment, animals were
splenectomized 4 weeks before transplantation. All animal experiments
were performed in accordance with institutional guidelines approved by
the Animal Care Committee of the Indiana University School of Medicine.
Transplantation of human cells.
Using protocols approved by the Institutional Review Board of the
Indiana University School of Medicine, healthy adult human volunteers
were treated for 5 days with human granulocyte colony-stimulating factor (hG-CSF) (Filgrastim, Neupogen; Amgen, Thousand Oaks, CA; 10 µg/kg/d, subcutaneously). White blood cells were collected by
apheresis and CD34+ cells were isolated by immunomagnetic
methods using the Isolex 300i cell selection device (Baxter
Immunotherapy, Irvine, CA). For these experiments, cell selection kits
and disposables were kindly provided by Baxter Immunotherapy. The
average purity of the CD34+ cells was 89% ± 7% with an overall recovery of 53% ± 19% from a total of 18 apheresis products containing 3.9 ± 1.7 × 1010
nucleated cells (mean ± standard deviation [SD]). After
separation, CD34+ cells were washed twice and injected into
the lateral tail vein of preirradiated NOD/SCID mice at 2 to 50 × 106 cells/animal in 0.5 mL Hanks' Balanced Salt Solution
(HBSS), 25 mmol/L HEPES, 20 U/mL heparin. In one specific experiment
CD34 Flow cytometric analysis.
At 6 to 8 weeks after transplantation, tissues were harvested for
analysis. Blood (0.5 mL-1.0 mL) was collected from the subclavian vessels after the animals were anaesthetized with tri-bromo-ethyl alcohol (Sigma, St Louis, MO) and injected intravenously
with 20 U of heparin sodium (Fujisawa USA, Deerfield, IL). Erythrocytes were depleted from the blood by a 2-minute incubation in 155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA at
4°C. In addition, single-cell suspensions were prepared from the
BM, spleen, and thymus. For flow cytometric analysis, cells were
preincubated for 30 minutes at 4°C in phosphate-buffered saline
(PBS) containing 0.1% (wt/vol) bovine serum albumin (BSA), 10% mouse
serum (Caltag, South San Francisco, CA), and 10% rat serum (Caltag).
Cells were then incubated with monoclonal antibodies (MoAbs) [specific
or isotype controls] conjugated to either fluorescein isothiocyanate
(FITC), phycoerythrin (PE), or PerCP (at 0.5 µg/0.5-1.0 × 106 cells) for 30 minutes at 4°C, washed and analyzed
for three-color fluorescence on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA). Routinely, 40,000 events per sample were
collected. The lack of cross-reactivity of human-specific antibodies
with mouse cells was confirmed in every experiment by staining BM cells from a nontransplanted irradiated control animal. In addition, every
analysis included isotype control antibodies to assess the level of
background fluorescence. Engraftment of human cells was defined by the
presence of at least 1% nucleated cells showing expression of CD45
over the background. FITC- and PE-conjugated mouse (IgG1 and IgG2b) and
rat (IgG2a) isotype control antibodies were purchased from Caltag, and
PerCP-conjugated mouse IgG1 was obtained from Becton Dickinson. The
IgG2a rat anti-mouse CD18 (mCD18)-FITC antibody was purchased from
Caltag. All other antibodies were specific for human cell-surface
antigens. CD10-FITC, CD19-PE, and CD38-PE (all IgG1 isotype) were
purchased from Caltag; anti-human IgM-FITC (IgG1) and HLA-DR-FITC
(IgG2b) were purchased from Pharmingen (San Diego, CA); goat anti-human
IgD-PE was purchased from Southern Biotechnology (Birmingham, AL);
Kappa-FITC and Lambda-FITC were bought from Kallestad (Redmond, WA);
CD11b-FITC (IgG2b), CD16-FITC, and Glycophorin A (Gly A)-PE (both IgG1)
were purchased from Immunotech (Westbrook, ME); and CD34-FITC,
CD8-FITC, CD4-PE, CD7-PE, CD33-PE, CD20-PerCP, CD3-PerCP, CD45-PerCP
(all IgG1 isotype), were bought from Becton Dickinson.
Mobilization.
In six individual experiments, groups of animals were treated with the
following mobilizing regimen: Cyclophosphamide (Cytoxan; Mead Johnson,
Princeton, NJ) at 200 mg/kg intraperitoneally followed 2 days later by
4 days of hG-CSF (Filgrastim Neupogen, Amgen) at 250 µg/kg/d
subcutaneously33; hG-CSF at 25 µg/kg/d subcutaneously or
250 µg/kg/d subcutaneously for 4 days34; or hG-CSF at 25 µg/kg/d subcutaneously or 250 µg/kg/d subbcutaneously together with
pegylated-human stem-cell factor (PEG-hSCF; Amgen) at 20 µg/kg/d subcutaneously for 4 to 6 days.34,35 Animals were
individually weighed before injection. Cyclophosphamide was diluted in
PBS and administered once intraperitoneally, PEG-hSCF was administered
once-a-day subcutaneously in HBSS containing 25 mmol/L HEPES and 0.1%
BSA, and hG-CSF was admistered twice-a-day subcutaneously in the
morning and evening diluted in 0.9% NaCl with 5% fetal calf serum
(FCS) at pH 4.5 (to ensure stability of the protein). For analysis,
tissues and PB were harvested the day after the last injection.
Cultures.
PB and BM from transplanted animals were tested for the presence of
human progenitor cells in semi-solid tissue culture medium consisting
of Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island,
NY), 25% FCS, 10% human plasma, 2% Pen/Strep, 5 × 10-5 mol/L Histology.
In selected experiments, tissues from transplanted animals
and controls were fixed and kept in 8% formaldehyde, 15%
(wt/vol) sucrose in PBS. For histopathological examination, mouse
humeri were decalcified, embedded in paraffin, and sectioned. The
slides were stained with hematoxylin and eosin using standard
procedures.
Statistical analysis.
Statistical variation in the text is indicated by the SD, whereas
differences between groups and data in figures are expressed as the
mean ± standard error of the mean (SEM). Differences between percentages were calculated using the Wilcoxon test, whereas
differences between other groups were compared using either a
Student's t-test or analysis of variance (ANOVA). The
frequency of the NOD/SCID-repopulating cell was estimated using a
generalized additive statistical model36 to describe the
relationships between the number of CD34+ cells injected,
the purity of the CD34+ cells, and engraftment.
Specifically, the logit transformed percentage of human cells was
modeled as the sum of smoothing spline and loess functions of the
number of input CD34+ and non-CD34+ cells,
using the SAS (SAS Institute, Cary, NC) statistical package. Finally,
differences in mobilization between growth-factor treated and control
groups were calculated based on a generalized linear model37 using SAS.
Conditioning of NOD/SCID mice.
The SCID mutation has been described to affect radiation-induced DNA
repair.38 In initial experiments we monitored the survival of 10- to 15-week old NOD/SCID mice after various doses of TBI. After a
dose of 450 cGy or higher all animals died between 1 and 2 weeks after
irradiation. At 350 cGy to 400 cGy a late mortality (between 35 and 50 days after irradiation) was observed in about 20% of the animals,
whereas after 300 cGy all animals survived for at least 8 weeks. In
addition, in pilot studies we transplanted groups of 300 cGy, 200 cGy,
and 100 cGy irradiated animals and one group of nonirradiated animals
(3 animals per group) with 12 × 106 human
G-CSF-mobilized PB CD34+ cells per mouse. At 6 weeks after
transplantation no human cells could be detected in the BM of the
nonirradiated animals, but chimerism in the 300 cGy, 200 cGy, and 100 cGy groups was 63.9%, 56.0%, and 36.5% (± 11.3%, pooled SD),
respectively. Based on these results we established 300 cGy as a safe
and adequate radiation dose for our subsequent transplantation
experiments.
Engraftment of human hematopoietic cells in BM, spleen, and PB of
NOD/SCID.
To study the level of engraftment of human PB cells in various
hematopoietic organs of the NOD/SCID mouse, sublethally (300 cGy)
irradiated animals were intravenously injected with increasing numbers
of CD34+ cells. At 6 to 8 weeks after transplantation, PB,
BM, and spleen were harvested and analyzed by flow cytometry for the
presence of human cells (>1% of all nucleated cells) using
antibodies against human pan-leukocyte marker CD45 and mouse CD18
(Fig 1A, B, C). Open circles indicate untreated animals,
solid circles and triangles indicate animals that were treated for 4 to
6 days with G-CSF or G-CSF and SCF, respectively, which was part of a
mobilization study that will be described below. The level of
engraftment in BM and spleen (as determined by the percentage
CD45+ mCD18
Overall distribution and maturation of myeloid and B-lymphoid
lineages in the hematopoietic organs.
To investigate the capacity of human PB stem cells in NOD/SCID mice to
differentiate into multiple lineages, and to compare the
differentiation of human cells in different hematopoietic organs, cells
in BM, spleen, and PB of transplanted animals were tested for the
expression of the human myeloid-specific marker CD33 and B
lymphoid-specific marker CD19 by flow cytometry.41 The
distribution of CD33+ and CD19+ cells showed
that the majority of human cells in the BM (n = 50) and spleen (n = 10)
had differentiated into the B-lymphoid lineage
(Fig 4A). On the other hand, in the PB (n = 50) myeloid and lymphoid cells were equally represented. When comparing
BM and spleen, the percentage of myeloid cells in the BM (28.2 ± 16.4, mean ± SD, n = 50) was significantly higher (P = 0.3)
than in the spleen (16.0 ± 10.3, n = 10), indicating that
myelopoiesis preferentially took place in the BM. In animals that were
treated with either G-CSF or G-CSF and SCF in our mobilization study
(described below), the percentage of human myeloid cells in the BM had
increased to 39.1% ± 17.0% (n = 18). This is significantly higher
than the percentage in untreated animals (P = .02),
which indicated that the treatment had triggered human myelopoiesis. In
addition, in highly engrafted animals (with more than 70% human cells)
the percentage of myeloid cells in the BM (56.1% ± 9.2%
CD33+, n = 9) was significantly higher than in animals
engrafted at lower levels (32.8% ± 18.8% CD33+, n = 41; P< .001), suggesting that the graft itself contributed to
the microenvironment in supporting myelopoiesis. A more detailed flow
cytometric analysis of the cells engrafted in the BM, which was based
on the expression of CD33 and HLA-DR for myeloid cells and CD10 and
CD20 for B-lymphoid cells (Fig 4B), showed that the majority of both
myeloid and B-lymphoid cells had an immature phenotype. Although in the
myeloid lineage mature monocytes and granulocytes could be identified
based on the level of expression of CD33 and HLA-DR, maturation in the
B-lymphoid lineage in the BM appeared to be incomplete as judged by the
absence of the more mature CD20+ CD10
Flow cytometric analysis of various lineages in bone marrow and
spleen.
A detailed flow cytometric analysis of the human hematopoietic cells in
BM and spleen of a representative animal is shown in
Fig 5A through E. The
differentiation of human B lymphocytes can be characterized by an
increase in expression of CD20 on CD10+ cells, followed by
a subsequent loss of CD10 from the cells expressing high levels of
CD20.41 We show that both BM and spleen contained immature
CD10+20
Flow cytometric analysis of human cells in the thymus.
Human T lymphocytes, as defined by the expression of CD4 and CD3, could
never be detected in BM, spleen, or PB of engrafted animals. However,
human thymocytes could be detected in the thymus of highly engrafted
animals (Fig 6A and B). Statistical
analysis showed a positive correlation between the level of engraftment in the BM and the presence of human cells in the thymus (Fig 6A, left
panel; P < .05). Of the 13 animals analyzed, thymocytes could be detected in 5 out of 6 animals that had been engrafted in the BM
with at least 40% human cells. Interestingly, human thymocytes could
also be detected in the thymus of animals that had only 25% human
cells in the BM, but which had previously been treated for 6 days with
human G-CSF and PEG-SCF (as part of a mobilization experiment), whereas
the thymus of untreated animals contained virtually no human cells (Fig
6A, right panel). Phenotypic analysis of these thymocytes, as shown for
a representative animal in Fig 6B, showed a large percentage of
CD4+ CD8+ double-positive cells and cells that
coexpress CD8 and CD7. In addition, the majority of
CD4+8
Mobilization of mouse progenitors in nontransplanted NOD/SCID.
To study mobilization, we first established that mouse progenitor cells
could be mobilized in a normal nontransplanted NOD/SCID. To test
whether preirradiation interfered with this ability we also included a
group of animals that had been sublethally irradiated 6-weeks before
treatment. Both nonirradiated and sublethally irradiated animals were
administered a single injection of cyclophosphamide (at 200 mg/kg
interperoneally) followed 2-days later by 4 days of G-CSF (at 250 µg/kg/d). This mobilization protocol has previously been used by
Neben et al33 to mobilize cells in C57BL/6 mice. Animals
were sacrificed on day 6 after cyclophosphamide treatment and
progenitor cells in both PB and BM were enumerated in methylcellulose cultures (Fig 7A). The results show that
mouse progenitor cells could be efficiently mobilized in unirradiated
as well as irradiated animals (1,120- and 300-fold increase in PB
progenitors, respectively). The difference between unirradiated and
irradiated animals was not statistically significant (P = .07).
As expected, the increase in progenitor cells was accompanied by an
increase in spleen weight (Fig 7B) and number of white cells in the
blood (Fig 7C). These results indicate that the NOD/SCID mouse allowed
murine progenitor cells to be mobilized into the periphery after an
appropriate stimulus, and that circulating progenitor cells were
present in sufficient numbers to be detected by standard colony assays
even though the animals had not been splenectomized.
Mobilization of human progenitors in transplanted NOD/SCID.
To detect mobilization of human progenitor cells we used a tissue
culture medium containing the human-specific growth factors SCF and
IL-3 plus Epo, that selectively supported the growth of human but
not mouse progenitor cells (data not shown). For mobilization, NOD/SCID
mice were treated with various regimens that have previously been shown
to mobilize primitive hematopoietic cells (see Materials and Methods).
Treatment was initiated 6 to 8 weeks after transplantation and PB and
BM cells were harvested the morning after the last treatment day. The
distribution of human cells among PB, spleen, and BM of engrafted
animals, in treated and untreated groups of five individual
experiments, is shown in Table 2.
Figure 8 (A through E) shows
the average percentage of human cells in the BM (left panel), and the
number of human progenitor cells detected in BM and PB (right panel,
log scale, and linear scale, respectively) of the same experiments. In
animals treated with G-CSF, the average number of human cells in the PB
was significantly increased as compared with control (P < .01 and P< .05, respectively; Table 2, B and C). In addition,
human progenitor cells were found to be mobilized in animals treated
with a low or high dose of G-CSF (Fig 8B and 8C, respectively), alone
or in combination with SCF (Fig 8D and 8E, respectively). More
specifically, the number of progenitor cells in the PB increased from
1.3-fold in an animal treated with a high-dose G-CSF and SCF (Fig 8E)
to 7.9-fold in two animals treated with a high dose of G-CSF (Fig 8C).
For statistical analysis a generalized linear model37 was
used to account for the volume of blood collected, the number of cells
injected per animal, the number of colonies counted, and whether or not
animals had been treated with hematopoietic growth factors. When
combining the results of individual experiments, an estimated 4.6-fold
increase in progenitors could be found in the PB of animals treated
with either G-CSF or G-CSF and SCF as compared with control
(Table 3; P = .019), with a 95%
confidence interval of (1.24, 17.0). The total number of nucleated
cells and number of mouse cells in the BM or PB of animals treated with
G-CSF or G-CSF and SCF had not changed (P > .05, not shown).
The mobilized PB progenitor cells, as well as the progenitor cells in
the BM of untreated and growth-factor treated animals, were
predominantly CFU-GM (92.0% ± 4.2% (SEM; n = 9); 84.1% ± 4.3% (n = 17); and 85.6% ± 7.1% (n = 15), respectively), with
only a minority being BFU-E (10%-20%) and less than 1% being
CFU-Mix. In contrast to the mobilization described above, no progenitor
cells could be detected in the PB of animals treated with
cyclophosphamide and G-CSF (Fig 8A; Table 3, group I). In addition to
the lack of mobilization, the average number of human cells in PB and
BM had decreased significantly (Table 2; experiment A). In summary, our
experiments show that human hematopoietic progenitor cells can be
induced to egress from the BM space into the PB after stimulation with
human G-CSF or a combination of human G-CSF and pegylated-SCF, albeit
with a low frequency.
The mechanisms of mobilization of progenitor cells from the BM into the
blood are still poorly understood. A systematic study of the
physiologic or therapeutic stimuli that induce mobilization, as well as
the components that modulate the extent and timing of mobilization,
requires the development of an in vivo transplantation model. In the
present study we developed such a model by transplanting a total of 92 sublethally irradiated NOD/SCID mice with increasing numbers of
CD34+ cells harvested from adult G-CSF-mobilized healthy
donors. At 6 to 8 weeks after transplantation, the hematopoietic organs
were analyzed for the presence of differentiated human hematopoietic cells using multiparameter flow cytometry, and groups of engrafted animals were treated with various regimens to study the level of
mobilization of human hematopoietic progenitors into the circulation.
|
Abstract
Introduction
Abstract
, and Flt3 ligand (as recently reviewed by To et
al
cells were collected, irradiated with 20 Gy,
depleted of adherent cells by plastic-adherence for 30 minutes at
37°C, and injected intravenously (25 × 106
cells/animal) 4 hours before injection with CD34+ cells.
-mercapto-ethanol, 11 ng/mL human IL-3
(Peprotech, Rocky Hill, NJ), 100 ng/mL hSCF (Amgen), 4 U/mL human
erythropoietin (Amgen), and 0.8% methylcellulose (Stem Cell
Technologies, Vancouver, BC, Canada). Triplicate plates with 3 × 104 to 3 × 105 cells/dish were cultured
for 14 days at 37°C in a humidified environment with 5%
CO2. The number of colonies was determined by microscope.
