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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1957-1966
HEMATOPOIESIS
A secreted and LIF-mediated stromal cell-derived activity that
promotes ex vivo expansion of human hematopoietic stem cells
Chu-Chih Shih,
Mickey C.-T. Hu,
Jun Hu,
Yehua Weng,
Paul J. Yazaki,
Jeffrey Medeiros, and
Stephen J. Forman
From the Department of Hematology/Bone Marrow Transplantation, City
of Hope National Medical Center, Duarte, CA; Department of Molecular
Biology, Beckman Research Institute at City of Hope, Duarte, CA;
Department of Cell Biology, Amgen Inc, Thousand Oaks, CA; and
Department of Pathology, University of Texas M. D. Anderson Cancer
Center, Houston, TX.
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Abstract |
The development of culture systems that facilitate ex vivo
maintenance and expansion of transplantable hematopoietic stem cells
(HSCs) is vital to stem cell research. Establishment of such culture
systems will have significant impact on ex vivo manipulation and
expansion of transplantable stem cells in clinical applications such as
gene therapy, tumor cell purging, and stem cell transplantation. We
have recently developed a stromal-based culture system that facilitates
ex vivo expansion of transplantable human HSCs. In this stromal-based
culture system, 2 major contributors to the ex vivo stem cell expansion
are the addition of leukemia inhibitory factor (LIF) and the AC6.21
stromal cells. Because the action of LIF is indirect and mediated by
stromal cells, we hypothesized that LIF binds to the LIF receptor on
AC6.21 stromal cells, leading to up-regulated production of stem cell
expansion promoting factor (SCEPF) and/or down-regulated production of
stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a
secreted SCEPF activity in the conditioned media of LIF-treated AC6.21
stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell
expansion depends on the concentration of the secreted SCEPF activity
in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is
mediated by a single factor or by multiple growth factors, our results
demonstrate that stromal cells are not required for this secreted
SCEPF activity to facilitate ex vivo stem cell expansion.
(Blood. 2000;95:1957-1966)
© 2000 by The American Society of Hematology.
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Introduction |
During the last 2 decades, hematopoietic stem cell
transplantation (HSCT) has been shown to provide definitive benefit for a variety of malignant and nonmalignant hematologic diseases and myelopoietic support for patients undergoing high-dose
chemotherapy.1,2 However, several inherent limitations
associated with HSCT have restricted its general use.3,4
These limitations include: (1) lack of sufficient donors for all
recipients; (2) requirement for either operative bone marrow (BM)
harvests or pheresis procedures to obtain sufficient stem cells to
achieve benefit after transplant; (3) a period of BM aplasia leading to
severe, prolonged neutropenia and thrombocytopenia; and (4) the
potential for tumor contamination in autologous HSCT. An increasing
interest exists in strategies to manipulate HSCs and hematopoietic
progenitor cells in vitro for clinical purposes. The ability to
generate and expand transplantable HSCs ex vivo from a small number of
HSCs could have enormous potential in a variety of clinical
settings.5 Ex vivo generated and expanded HSCs could
support multiple cycles of chemotherapy, provide transplantation options for patients without matched donors, facilitate transduction of
vectors into HSCs for gene therapy, and provide a tumor-free product
for transplantation. Finally, transplantation with ex vivo expanded
stem cells might abrogate the extended neutropenia and
thrombocytopenia.6-8
We have previously developed a stromal-based culture system that
facilitates ex vivo expansion of CD34+ thy-1+
cells using long-term hematopoietic reconstitution in severe combined
immunodeficient (SCID)-hu mice as an in vivo assay for transplantable human HSCs.9 The addition of leukemia
inhibitory factor (LIF)10 to purified CD34+
thy-1+ cells isolated from human fetal BM on AC6.21
stroma,11 a murine BM-derived stromal cell line, caused
expansion of cells with the CD34+ thy-1+
phenotype.9 Addition of other cytokines, including
interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte macrophage
colony-stimulating factor (GM-CSF), and stem cell factor (SCF), to LIF
in the cultures caused a 150-fold expansion of cells retaining the
CD34+ thy-1+ phenotype.9 The ex
vivo expanded fetal BM CD34+ thy-1+ cells gave
rise to multilineage differentiation, including myeloid, T, and B
cells, when transplanted into SCID-hu mice.9 Another human
HSC candidate, CD34+ CD38
cells,12,13 displayed a similar magnitude of
phenotypic and functional expansion,9 suggesting that ex
vivo expansion of transplantable HSCs under this culture system is a
general phenomenon. We have previously demonstrated that both murine
LIF, which cannot bind to human LIF receptor, and human LIF, which can
bind to murine LIF receptor, are equally capable of facilitating the
expansion of transplantable human HSCs in this culture system, which
suggests that LIF mediates its action through the murine
stroma.9 On the basis of these previous results, we have
hypothesized that binding of LIF to the receptor on AC6.21 stromal
cells leads to up-regulated production of stem cell expansion promoting
factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). The present studies were undertaken to test
this hypothesis. Here we report the detection of a secreted, LIF-mediated, stromal derived SCEPF activity in the conditioned media
(SCM-LIF) of AC6.21 stromal cell cultures. This secreted SCEPF activity
is able to expand transplantable human CD34+
thy-1+ cells without the stromal cells. The magnitude of
CD34+ thy-1+ cell expansion depends on the
concentration of the SCEPF in the SCM-LIF. The absolute number of
transplantable CD34+ thy-1+ cells increases
more than 200-fold after 3 weeks of culture. Furthermore, our data
demonstrate that 6 known prominent stem cell cytokines, including IL-3,
IL-6, GM-CSF, SCF, flt3 ligand (FL),14,15 and
thrombopoietin (TPO),16-18 either alone or in combinations,
cannot account solely for this secreted SCEPF activity in the SCM-LIF.
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Materials and methods |
Preparation of stroma-conditioned media from untreated (SCM) and
LIF-treated stromal cell cultures (SCM-LIF)
Stroma-conditioned media were harvested from a confluent layer of
mouse stromal cell line AC6.21. Briefly, 5 × 105 to
1 × 106 stromal cells were distributed into a T75
flask in long-term culture medium (LTCM) consisting of RPMI 1640 (GIBCO/BRL, Gaithersberg, MD), 5 × 105 mol/L
2-mercaptoethanol, 10 mmol/L HEPES, penicillin (50 U/mL) and
streptomycin (50 mg/mL), 2 mmol/L sodium pyruvate, 2 mmol/L glutamine,
and 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere
with 5% CO2. Confluent stromal layers formed after 1 week.
A complete medium change was made with fresh LTCM containing 10 ng/mL
of LIF when the layers were confluent, after which half the medium
conditioned by the stromal layers was harvested every 3 days and
replaced with fresh LTCM containing 10 ng/mL of LIF for a total of 4 weeks. The SCM-LIF was centrifuged at 1300 rpm for 10 minutes to remove
nonadherent cells and filtered through a 0.45-µm pore filter with low
protein binding (Sterivex-HV; Millipore, Bedford, MA). SCM-LIF was
stored at  20°C until use. In some cases, concentrated
SCM-LIF was prepared. Pooled crude supernatants were first concentrated
with a DC10 concentrator using a 100 000-d nominal molecular weight
cutoff (NMWC)19 hollow-fiber cartridge (Amicon Inc,
Danvers, MA), and then the concentrate was clarified by refiltering
with a 5000 NMWC cartridge.19,20 These concentration steps
reduced the volume 40-fold. Stroma-conditioned media (SCM) from AC6.21
cultures without LIF were also harvested and processed with the same
protocols and used in experiments as described in the text.
Preparation of human HSCs
Human fetal bones were dissected from 21- to 24-week-old fetuses
obtained by elective abortion with approved consent (Anatomic Gift
Foundation, White Oak, GA). To purify human HSCs, BM cell suspensions
were prepared by flushing split long bones with RPMI 1640 containing
2% heat-inactivated FCS (Gemini Bio-Products, Inc, Calabasas, CA).
Low-density (< 1.077 g/mL) mononuclear cells were isolated
(Lymphoprep; Nycomed Pharma, Oslo, Norway) and washed twice in staining
buffer (SB) consisting of Hanks' balanced salt solution (HBSS) with
2% heat-inactivated FCS and 10 mmol/L HEPES. Samples were then
incubated for 10 minutes with 1 mg/mL heat-inactivated human
gammaglobulin (Gamimune; Miles Inc, Elkhart, IN) to block Fc receptor
binding of mouse antibodies. Fluorescein isothiocyanate (FITC)-labeled
CD34 monoclonal antibodies (MoAbs) and phycoerythrin (PE)-labeled thy-1
MoAbs were then added at 0.5 to 1 µg/106 cells in 0.1 to
0.3 mL SB for 20 minutes on ice. Control samples were incubated in a
cocktail of FITC-labeled and PE-labeled isotype-matched MoAbs. Cells
were washed twice in SB, resuspended in SB containing 1 µg/mL
propidium iodide (Molecular Probes Inc, Eugene, OR), and sorted using
the tri-laser fluorescence-activated cell sorter (FACS) MoFlo
(Cytomation, Inc, Fort Collins, CO). Live cells (ie, those excluding
propidium iodide) were always greater than 95%. Sort gates were set
based on the mean fluorescence intensity of the isotype control sample.
Cells were collected in 12- or 24-well plates in RPMI 1640 containing
10% FCS and 10 mmol/L HEPES, counted, and reanalyzed for purity in
every experiment. Typically, 450 000 to 500 000 CD34+
thy-1+ cells were obtained from a single donor. MoAbs for
CD34 were purchased from Becton Dickinson (Mountain View, CA). MoAbs
for thy-1 and isotype controls were purchased from Pharmingen (San Diego, CA).
Stromal-based HSC expansion culture system
Sorted cells were cultured on a preestablished monolayer of a mouse
stromal cell line, AC6.21, as described previously.9 Briefly, 3 × 104 to 4 × 104
stromal cells were plated in 24-well plates 1 week before the experiment in 1 mL of LTCM. Twenty or 300 CD34+ thy-1+ cells were
distributed in 1 mL of LTCM into each well in 24-well plates with a
preestablished AC6.21 monolayer. A cytokine cocktail including IL-3,
IL-6, GM-CSF, and SCF was added immediately after seeding the sorted
cells into the 24-well plates at a final concentration of 10 ng/mL of
each growth factor. LIF was then added to the positive control wells at
a final concentration of 10 ng/mL. The LTCM in the negative control
wells contained only the cytokine cocktail without LIF. Human
recombinant IL-3, IL-6, GM-CSF, SCF, and LIF were purchased from R&D
Systems (Minneapolis, MN). Half of the culture medium was replaced
weekly with fresh LTCM containing the same cytokine cocktail with or
without LIF for positive and negative control wells, respectively.
SCM-based HSC expansion culture system
Twenty or 300 freshly purified CD34+ thy-1+
cells were distributed into each well in a 24-well plate with 2 mL of
LTCM containing 10 ng/mL of IL-3, IL-6, GM-CSF, SCF, and different
concentrations of SCM-LIF. Culture media containing 5%, 10%, and 25%
of SCM-LIF were prepared by mixing fresh LTCM with appropriate amounts
of unconcentrated SCM-LIF. Culture media containing 50%, 100%, 200%, and 400% of SCM-LIF were prepared by mixing fresh LTCM with respective amounts of concentrated SCM-LIF. A complete medium change was made
every 3 days and replaced with fresh LTCM containing the cytokine
cocktail and respective amounts of SCM-LIF. The proliferative and
phenotypic characteristics of these cultures were analyzed 3 weeks later.
For antibody-blocking experiments, neutralizing antibody against mouse
IL-3, IL-6, GM-CSF, SCF, FL, and TPO (R&D Systems) or control goat
immunoglobulin (R&D Systems) was used at 0.1 to 10 µg/mL in 200%
SCM-LIF. Cultures were fed daily by replacement of half of the medium.
The proliferative and phenotypic characteristics of the cultures were
analyzed 3 weeks later. The neutralizing activity of the antibody for
each cytokine has been determined by R&D Systems under a specific set
of conditions. The neutralizing activities for each antibody as
determined by R&D Systems and standardized to a 1-µg/mL dose are the
following: 7.5, 5, 25, 25, 25, and 3 ng/mL for GM-CSF, IL-3, IL-6, SCF,
FL, and TPO, respectively.
To determine the effect of exogenous cytokines on ex vivo stem cell
expansion, we added each of the 6 cytokines, either alone or in various
combinations, to the culture medium (either 200% SCM plus 10 ng/mL of
LIF or 200% SCM-LIF) at concentrations of 10, 50, and 100 ng/mL. A
complete medium change was made every 3 days, and cultures were
analyzed 3 weeks later for their proliferative and phenotypic
characteristics. Recombinant human FL and TPO were purchased from R&D Systems.
Proliferative analysis, phenotypic analysis, and sorting of ex
vivo expanded human fetal HSCs
The extent to which different concentrations of SCM-LIF supported in
vitro expansion of purified human fetal BM stem cells was determined by
counting the total number of hematopoietic cells present in 10 individual wells in each culture. At the end of the 3-week culture
period, hematopoietic cells were harvested individually from these
wells, cell number was counted, and then cells were analyzed for
lineage content by flow cytometry. Half of the cells from each well
were stained with FITC- or PE-labeled MoAbs against CD19 and CD33, and
the other half were stained with antibodies against CD34 and thy-1.
Analysis was gated on the hematopoietic cells, excluding the stromal
cells for the positive and negative control samples, and the quadrants
were set based on the mean fluorescence intensity of the isotype
control samples. FITC- and PE-labeled MoAbs against CD19 and CD33 were
purchased from Pharmingen. Cells were analyzed on a FACScan fluorescent
cell analyzer. To purify the ex vivo expanded HSCs from those cultures,
cells from each culture condition including positive control, negative
control, and SCM-based cultures were pooled, cell number was counted,
and then cells were sorted for CD34+ thy-1+
phenotype as described earlier.
In vivo reconstitution assay in SCID-hu mice
Human fetal bone, thymus, and liver tissues were dissected from 18- to 24-week-old fetuses obtained by elective abortion with approved
consent (Anatomic Gift Foundation). A sample of each received fetal
tissue was stained with a panel of MoAbs to HLA to establish the donor
allotype. These fetal tissues were used for construction of the SCID-hu
mice. C.B-17 scid/scid mice were bled in our facility under sterile
conditions. Mice used for human tissue transplantation were 6 to 8 weeks of age, and SCID-hu thymus/liver (thy/liv) and bone-model mice
were constructed as previously described9,21,22 and in
accordance with the guidelines set forth by the City of Hope Research
Animal Care Committee. At the time of surgery, the animals were weighed
and anesthetized with a mixture of ketamine (50 mg/kg) and xylazine HCl
(25 mg/kg) administered intraperitoneally. For thy/liv mice, individual
pieces (1 to 2 mm) of human fetal thymus and autologous liver were
placed under the kidney capsule of C.B-17 scid/scid mice and allowed to
engraft for 3 months before stem cell reconstitution. For bone-model
mice, pieces of fetal bone were placed subcutaneously and allowed to
vascularize for 2 to 3 months. Animals were preconditioned by
total-body irradiation with 350 rads 4 to 6 hours before they were
subjected to stem cell reconstitution. The ability of the purified
human fetal BM HSCs, CD34+ thy-1+ population
(stem cell donor is always selected to be HLA-MA2.1-positive), either
freshly purified or ex vivo expanded, to reconstitute thymus and BM was
tested by direct inoculation into irradiated grafts (thy/liv and bone;
the graft is always selected to be HLA-MA2.1-negative). For
reconstitution experiments, 10 000 cells were used because we have
previously established that 10 000 CD34+
thy-1+ cells, either purified from fresh fetal BM or after
ex vivo expansion in the stromal-based culture system, can reproducibly
establish long-term hematopoietic reconstitution in more than 90% of
SCID-hu mice and give rise to about 50% donor-derived hematopoietic
cells in reconstituted animals. Control animals were injected with HBSS only. Engraftment was analyzed at 3 to 4 months after injection. Human
bones were removed and split open to flush the marrow cavity with SB.
Collected cells were spun down, and the pellet was resuspended for 5 minutes in a red blood cell lysing solution. Cells were washed twice in
SB and counted before being stained for 2-color immunofluorescence with
directly labeled MoAbs against HLA allotypes in combination with CD19
and CD33. Human thymus grafts were recovered, reduced to a cellular
suspension, and subjected to 2-color immunofluorescence analysis using
directly labeled MoAbs against HLA allotypes in combination with CD3,
CD4, and CD8. FITC- and PE-conjugated irrelevant mouse immunoglobulins
were used as negative controls. Cells were analyzed on a FACScan
fluorescent cell analyzer. FITC- or PE-labeled CD19, CD33, CD3, CD4,
and CD8 were purchased from Pharmingen.
Quantitative measurement of cytokines by enzyme-linked immunosorbent
assay (ELISA)
The amount of various cytokines in the SCM and SCM-LIF was
determined by the sandwich ELISA technique, using combinations of
unlabeled and biotinylated MoAbs to different epitopes of each cytokine. Colorimetric ELISA kits for murine IL-3, IL-6, GM-CSF, SCF,
and TPO were purchased from R&D Systems, and assays were performed
according to the manufacturer's instructions. For murine FL, an
affinity-purified goat polyclonal antibody raised against a peptide
mapping at the amino terminus of murine FL (Santa Cruz Biotechnology,
Inc, Santa Cruz, CA) was used as capture antibody. Immulon 4 plates
(Dynatech Laboratories, Inc, Chantilly, VA) were coated overnight at
room temperature with 0.4 to 2 µg/mL of the above capture antibody. A
biotinylated anti-mouse FL polyclonal antibody from R&D Systems was
used as the detection antibody at 50 ng/mL, and assays were performed
according to the manufacturer's instructions. Recombinant murine FL
was purchased from R&D Systems and used to establish the standard titration.
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Results |
Effect of SCM-LIF on ex vivo proliferation and differentiation of
human fetal BM CD34+ thy-1+ cells
The initial studies were focused on the detection of any potential
SCEPF activity in SCM-LIF. As reported previously, only 10% of the
wells initiated with 20 CD34+ thy-1+ cells per
well in co-culture with AC6.21 stromal cells were CD34+
thy-1+-positive after 5 weeks of culture.9 To
minimize the number of wells in the assay, we used 300 CD34+ thy-1+ cells per well. On the basis of
the binomial distribution, we expected that every well in the culture
should be CD34+ thy-1+-positive. The addition
of IL-3, IL-6, GM-CSF, and SCF to the LTCM was used to shorten the time
required for the assay from 7 weeks to 3 weeks.9 Three
thousand freshly purified CD34+ thy-1+ cells
were distributed into 10 wells (300 cells per well in a 24-well plate
with 1 mL of LTCM) on a preestablished AC6.21 stromal layer in the
presence of LIF (10 ng/mL) and a cytokine cocktail including 10 ng/mL
of IL-3, IL-6, GM-CSF, and SCF. These 10 wells served as the positive
control for the quality of the sorted CD34+
thy-1+ cells and the activity of the stromal-based culture
system to establish ex vivo expansion of CD34+
thy-1+ cells.9 Another 3000 freshly purified
CD34+ thy-1+ cells were similarly distributed
but without LIF. These 10 wells served as the negative
control.9 To investigate the responsiveness of
CD34+ thy-1+ cells to SCM-LIF, we used 8 different concentrations, ranging from 0% to 400%, of SCM-LIF. For
each concentration of SCM-LIF, 3000 CD34+
thy-1+ cells were distributed into 10 wells (300 cells per
well) in a 24-well plate without stroma. The proliferative and
phenotypic characteristics of these cultures were analyzed 3 weeks
later. As shown in Figure 1, the
proliferative potential of purified CD34+
thy-1+ cells in the SCM-based culture system was
proportional to the concentration of SCM-LIF. The total number of
hematopoietic cells apparently increased from 0% to 200% SCM-LIF and
reached a plateau at 200% SCM-LIF, which was very similar to the
total number of cells in the positive control cultures.
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| Fig 1.
Effects of SCM-LIF on the proliferative potential of
human fetal BM CD34+ thy-1+ cells in vitro.
Stromal-based cultures in the presence of cytokine cocktail (10 ng/mL
of each cytokine), including IL-3, IL-6, GM-CSF, and SCF, were used as
controls. Positive control and negative control are cultures with or
without exogenous LIF (10 ng/mL), respectively. Data are presented as
the total number of hematopoietic cells per well (total of 10 wells) in
each culture condition for 3-week cultures. Results are expressed as
the mean ± SD.
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To assess the effects of SCM-LIF on the differentiation potential of
purified CD34+ thy-1+ cells in this SCM-based
culture system, we analyzed cells from individual wells by flow
cytometry for the presence of CD33-positive myeloid cells and
CD19-positive B lymphocytes. As expected from our previous
report,9 all wells (with 300 cells per well), regardless of
treatments, were lymphoid/myeloid wells (data not shown). The
percentage of CD33-positive myeloid cells within these mixed
lymphoid/myeloid wells ranged from 46% of those treated with 200%
SCM-LIF to 54% of those treated with 0% SCM-LIF; the percentage of
CD19-positive cells ranged from 15% of those treated with 200%
SCM-LIF to 10% of those treated with 0% SCM-LIF (data not shown). To
better evaluate the effect on differentiation, experiments were
repeated at a limiting-dilution density (20 cells per well), and the
results showed that CD33-positive and CD19-positive cells were
generated at similar levels (about 50% of the wells) in these cultures
regardless of different treatments (data not shown). These results
suggest that SCM-LIF is capable of providing a suitable environment for
multipotential CD34+ thy-1+ cells to
differentiate into both B cells and myeloid cells similar to the
stromal-based culture system.
Effect of SCM-LIF on ex vivo expansion of cells with
CD34+ thy-1+ phenotype
To determine whether SCM-LIF is capable of facilitating the
maintenance and expansion of cells with CD34+
thy-1+ phenotype, we performed flow cytometric analyses to
detect and quantify the number of CD34+ thy-1+
cells in each individual well in these cultures. As expected
from our previous report with the stromal-based culture
system,9 cells with CD34+ thy-1+
phenotype were detected only in all of the positive control wells and
not in the negative control cultures (Table
1). The frequency of CD34+
thy-1+-positive wells in the SCM-based cultures was
proportional to the concentration of SCM-LIF in the cultures. The
frequency increased from 0% of cultures treated with 0% SCM-LIF to
100% of cultures treated with more than 100% of SCM-LIF (Table 1).
The percentage of CD34+ thy-1+ cells within the
CD34+ thy-1+-positive wells among those
SCM-based cultures also depended upon the concentration of SCM-LIF. The
percentage of CD34+ thy-1+ cells increased
significantly from 3.6% in cultures treated with 10% SCM-LIF to 18%
in cultures treated with more than 200% of SCM-LIF (Table 1;
P < .00001). These results demonstrate that SCM-LIF alone
is sufficient to facilitate ex vivo expansion of cells with
CD34+ thy-1+ phenotype and that the magnitude
of expansion of CD34+ thy-1+ cells is
proportional to the concentration of SCM-LIF in these SCM-based
cultures. These results suggest that it is a secreted, LIF-mediated,
stromal cell-derived SCEPF in the SCM-LIF that facilitates ex vivo
expansion of CD34+ thy-1+ cells in both the
stromal-based and SCM-based culture systems.
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Table 1.
Effects of SCM-LIF on the maintenance and expansion of
freshly purified human fetal BM CD34+
thy-1+ cells in
vitro
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To further confirm that the mechanism is the up-regulated production of
SCEPF and not the down-regulated production of SCEIF, we mixed 200% of
SCM-LIF with either 200% or 400% SCM and determined the activity of
these conditions to facilitate ex vivo stem cell expansion. Table
2 clearly demonstrates that it is the
presence of SCEPF and not the absence of SCEIF in SCM-LIF that is
responsible for facilitating ex vivo stem cell expansion. Cultures
treated with 200% SCM-LIF displayed the highest magnitude of
CD34+ thy-1+ cell expansion, and 200% SCM-LIF
was derived from a concentration step by collecting proteins with
molecular weight between 5000 and 100 000 d (see "Materials and
methods").19,20 These results show that the size of the
protein for this secreted SCEPF is between 5000 and 100 000 d. Because
there are approximately 350 000 cells per well within these
CD34+ thy-1+-positive wells in those cultures
treated with more than 200% of SCM-LIF (Figure 1), the absolute number
of CD34+ thy-1+ cells in each well of these
cultures averaged about 65 000 (Table 1 and Figure
2), representing a greater than 200-fold
expansion in this population during the 3 weeks in culture (each well
was initiated with 300 purified CD34+ thy-1+
cells). Because cultures treated with 200% SCM-LIF gave rise to the
highest magnitude of expansion, this was selected as the optimal
condition for subsequent experiments. To better evaluate the degree of
stem cell expansion in SCM-LIF, experiments were repeated at a
limiting-dilution density (20 initial cells per well) with 200%
SCM-LIF. As expected from our previous report,9 only about
10% (13/125) of the wells were CD34+
thy-1+-positive, with an average of 18%
(18% ± 4%) of CD34+ thy-1+ cells within
these wells (data not shown). Because there are approximately
250 000 cells per well in each CD34+
thy-1+-positive well (data not shown), the absolute number
of CD34+ thy-1+ cells in each well averaged
about 45 000, representing a 2250-fold expansion. The overall bulk
equivalent is in excess of a 225-fold expansion of CD34+
thy-1+ cells in the cultures with 200% SCM-LIF. These
results demonstrate that SCM-LIF is capable of providing a
suitable environment for ex vivo expansion of CD34+
thy-1+ cells similar to the stromal-based culture system.
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Table 2.
SCM-LIF maintains its activity to facilitate ex vivo
expansion of freshly purified human fetal BM CD34+
thy-1+ cells in the presence of
SCM
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| Fig 2.
The number of CD34+ thy-1+
cells in the cultures is proportional to the concentration of SCM-LIF.
Data for the number of CD34+ thy-1+ cells in
the CD34+ thy-1+-positive wells are presented
as the mean ± SD of the total number of CD34+
thy-1+-positive wells in each culture condition. See
Figure 1 legend for additional information.
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In vivo transplantation potential of CD34+
thy-1+ cells before and after cultures
To determine whether ex vivo expanded CD34+
thy-1+ cells in the SCM-based cultures maintained their in
vivo transplantation potential and to further quantify the number of
transplantable CD34+ thy-1+ cells from
SCM-based cultures, we compared the in vivo engrafting activity among
freshly purified CD34+ thy-1+ cells and ex vivo
expanded CD34+ thy-1+ cells from both
stromal-based and SCM-based cultures from the same donors. Data from 3 independent experiments using cells from different donors were
compiled. As expected from our previous report,9 there was
no donor effect on the ex vivo expansion of CD34+
thy-1+ cells in the stromal-based cultures (positive
control; Table 3). Similarly, cells from
different donors did not display a donor effect on the ex vivo
expansion of CD34+ thy-1+ cells in the
SCM-based culture system (Table 3). As expected from our previous
report,9 when 10 000 freshly purified human fetal BM
CD34+ thy-1+ cells were injected into SCID-hu
mice, it gave rise to about a 90% reconstitution rate in both thy/liv
and bone-model mice with 40% to 50% donor-derived cells in the
reconstituted animals for all 3 donors (Table
4). Ex vivo expanded CD34+
thy-1+ cells from both stromal-based cultures (positive
control) and SCM-based cultures (200% SCM-LIF) gave almost identical
results as the freshly purified CD34+ thy-1+
cells from the same donors in terms of both the frequency of reconstitution and the percentage of donor-derived cells in the reconstituted animals (Table 4). These results indicate that ex vivo
expanded CD34+ thy-1+ cells in either the
stromal-based or SCM-based culture system are very similar to the
freshly purified CD34+ thy-1+ cells from the
same donors, both qualitatively and quantitatively. These results
further demonstrate that the secreted SCEPF activity in the conditioned
medium of LIF-treated AC6.21 stromal cell cultures is clearly capable
of facilitating, qualitatively and quantitatively, ex vivo expansion of
transplantable HSCs as the stromal co-culture system in the presence of
LIF, and show that this secreted, LIF-mediated, stromal cell-derived
SCEPF is responsible for ex vivo expansion of transplantable HSCs in
both the stromal-based culture system and SCM-based culture system.
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Table 4.
Comparative quantitation of the number of transplantable
CD34+ thy-1+ cells among freshly purified
CD34+ thy-1+ cells and ex vivo expanded
CD34+ thy-1+ cells derived from
stromal-based (positive control) and SCM-based culture systems from the
same
donors
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Effect of LIF on the production of 6 known prominent stem cell
cytokines by AC6.21 stromal cells
It has been reported previously that LIF treatment of stromal cells
up-regulates a large number of known cytokines and down-regulates many
others.23 Our first step to characterize this secreted SCEPF activity was to rule out or rule in the contribution from various
known early-acting factors. It was reported previously that the
addition of LIF to SyS-1 stromal cells enabled the in vitro maintenance
of competitive repopulating murine stem cells.23 Evidence
was presented to suggest that synergy between IL-6 and SCF, both of
which are up-regulated by LIF on SyS-1 stroma, most likely accounted
for the LIF-mediated activity in maintaining competitive repopulating
murine stem cells in vitro.23 Flt3 is a recently discovered
member of the class III tyrosine kinase receptor
family.24,25 The flt3 receptor appears to be selectively expressed on primitive murine HSC and progenitor cells26
and is largely restricted in human hematopoietic cells to the
CD34+ progenitor population.27 FL has been
shown to enhance the proliferation of HSC28,29 and
progenitor cells30,31 in vitro and to mobilize HSC and
progenitor cells in vivo.32-34 Recently, Shah et
al35 have demonstrated that FL is able to induce
proliferation of quiescent human CD34+
CD38cells that are not responsive to other
early-acting cytokines and to improve the maintenance of progenitor
cells in vitro. TPO has been shown to function not only as a
proliferative and differentiative factor for megakaryocytes, but also
as a survival factor for highly purified HSCs from both
mouse36-38 and human39-41 in vitro. Several lines of evidence demonstrate the effect of TPO in stem cell
proliferation in vivo. Administration of TPO has been shown to shorten
the time for hematopoietic recovery in myelosuppressed
animals.42 Targeted disruptions of TPO or of its receptor,
c-mpl, result not only in thrombocytopenia and megakaryocytopenia, but
also in a decreased number of hematopoietic stem or progenitor
cells.43,44 In this study, experiments were performed to
determine whether IL-6, SCF, FL, TPO, and 2 other cytokines including
GM-CSF and IL-3 (which were used in our previous study),9
either alone or in combinations, might account solely for the secreted
SCEPF activity in the SCM-LIF. Because this secreted SCEPF activity is
mediated by LIF on stromal cells, the logical criterion for a cytokine
to be essential for this SCEPF activity is that the expression of the
candidate cytokine must be up-regulated by the stromal cells upon LIF
stimulation. ELISA was used to determine whether the expression of
these 6 cytokines was up-regulated in the LIF-stimulated AC6.21 stromal cells and to measure the amount of these cytokines in the SCM and
SCM-LIF. Among these 6 cytokines, IL-6 and SCF were the only 2 that
could be detected by ELISA, and both were up-regulated by the stromal
cells upon LIF stimulation. Because the amount of IL-6 in SCM was below
the sensitivity of ELISA for IL-6 (3.1 pg/mL), the production of IL-6
by the stromal cells was up-regulated more than 40-fold upon LIF
stimulation (Table 5). There was a 3-fold
increase in SCF production by the LIF-stimulated stromal cells (Table
5). This suggests that both IL-6 and SCF may represent components in
this secreted SCEPF activity. Although the other 4 cytokines were not
detectable by ELISA in either SCM or SCM-LIF (Table 5), we cannot
completely rule out the possibility that these 4 cytokines were being
generated by the stromal cells and were up-regulated by LIF treatment,
albeit at extremely low concentrations that were below the sensitivity
of ELISA. Therefore, each of these 4 cytokines, even though at low
concentrations, might remain possible components in this secreted SCEPF
activity.
Neutralizing antibody to each of the 6 known prominent stem cell
cytokines cannot block ex vivo stem cell expansion
To directly examine the role of these 6 cytokines in facilitating ex
vivo stem cell expansion, CD34+ thy-1+ cells
purified from human fetal BM were cultured on 200% SCM-LIF for 3 weeks
in the absence or presence of 0.1 to 10 µg/mL of neutralizing antibody against each cytokine. All cultures, regardless of treatment, gave similar results. That is, 100% of wells (20/20) are
CD34+ thy-1+-positive, with an average of 9%
(9 ± 2%) of CD34+ thy-1+ cells in each well
(data not shown). This result shows that ex vivo stem cell expansion
was not affected by the addition of various concentrations (0.1, 1, and
10 µg/mL) of each neutralizing antibody against GM-CSF, IL-3, IL-6,
SCF, FL, and TPO to the cultures in both the frequency of
CD34+ thy-1+-positive wells and the percentage
of CD34+ thy-1+ cells in the wells. This result
demonstrates that neutralizing antibody to each of the 6 known
prominent stem cell cytokines cannot block the ex vivo stem cell
expansion facilitated by the SCEPF activity in the SCM-LIF and suggests
that none of these 6 cytokines is essential for the SCEPF activity in
the SCM-LIF.
GM-CSF, IL-3, IL-6, SCF, FL, and TPO, either alone or in
various combinations, cannot facilitate ex vivo stem cell expansion
To further demonstrate that these 6 known prominent stem cell
cytokines are not essential for the SCEPF activity in the SCM-LIF, we
performed experiments with the addition of these 6 cytokines to the
cultures. CD34+ thy-1+ cells purified from
human fetal BM were cultured in 200% SCM containing 10 ng/mL of LIF
for 3 weeks in the presence of 10 ng/mL of these 6 cytokines, either
alone or in various combinations. Cells were also cultured with 200%
SCM-LIF as a positive control. Results from this set of experiments
showed that cells with CD34+ thy-1+ phenotype
could be detected only in the positive control culture and not in any
other culture conditions treated with cytokines, including 6 with a
single cytokine, 15 with any combination of 2 cytokines, 20 with any
combination of 3 cytokines, 11 with any combination of 4 cytokines, 3 with any combination of 5 cytokines, and 1 with all 6 cytokines
together (data not shown). Consistent with the antibody-blocking
experiments above, these results demonstrate that these 6 cytokines,
either alone or in various combinations, are not sufficient to
facilitate ex vivo stem cell expansion (data not shown). Similar data
were obtained when higher concentrations of cytokines, at 50 and 100 ng/mL, were used. Taken together, these results further demonstrate
that these 6 known prominent stem cell cytokines, including IL-3,
IL-6, GM-CSF, SCF, FL, and TPO, are not the essential components for
the SCEPF activity in the SCM-LIF.
Several combinations of the 6 cytokines can enhance the
proportion of CD34+ thy-1+ cells in
cultures with SCM-LIF
In our previous study with the stromal-based culture system, we
found that the addition of GM-CSF, IL-3, IL-6, and SCF to the stromal
cells in the presence of LIF was capable of increasing the proportion
of CD34+ thy-1+ cells as compared with the
culture with LIF alone.9 Although the addition of the 6 known stem cell cytokines, either alone or in any possible
combinations, to SCM was not capable of maintaining cells with
CD34+ thy-1+ phenotype in the cultures,
experiments were performed to determine whether the addition of these 6 cytokines, either alone or in any possible combinations, to
SCM-LIF had any effect on the proportion of CD34+
thy-1+ cells in this SCM-based culture system. As
expected, 100% of the wells (20/20) in all cultures were
CD34+ thy-1+-positive (data not shown). The
percentage of CD34+ thy-1+ cells in each well
averaged about 9% (9% ± 2%) in most of the cultures,
including the control (cultures without any exogenous cytokine).
However, the addition of several combinations of cytokines to
SCM-LIF in this SCM-based culture system was clearly capable of enhancing the proportion of CD34+ thy-1+
cells, similar to the phenomenon observed in the stromal-based culture
system. Table 6 shows the combinations of
cytokines that significantly increased the proportion of
CD34+ thy-1+ cells in these cultures from 9%
to about 18%. Addition of IL-3 and IL-6 or TPO to SCF significantly
increased the proportion of CD34+ thy-1+ cells
from 9% to 14% (P = .0001). Although the addition of more cytokines to IL-3 + IL-6 + SCF or to TPO + SCF seemed to give a
greater proportion of CD34+ thy-1+ cells,
overall the differences were not significant (P = .32).
View this table:
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Table 6.
Conditions that significantly enhance the proportion of
cells with CD34+ thy-1+ phenotype in the
cultures
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Discussion |
The hematopoietic stem cell is characterized by its ability to
self-renew and to generate cells of all hematopoietic lineages. The
mechanisms that regulate stem cell self-renewal versus differentiation are poorly understood. In vivo, hematopoiesis occurs close to the BM
microenvironment, which presumably provides all the signals necessary
for proliferation and differentiation of stem cells. Long-term bone
marrow cultures (LTBMC) closely mimic many aspects of the BM
microenvironment45 and have been shown to be capable of
supporting HSC self-renewal, proliferation, and differentiation in
vitro.46,47 However, stromal layers derived from LTBMC
consist of a heterogeneous mixture of cells and present difficulties
for the identification of cytokines that may promote HSC self-renewal or differentiation in this setting.48 Studies using cloned
murine stromal cell lines have further confirmed that stromal cells are functionally heterogeneous in terms of their ability to support lymphoid and/or myeloid differentiation11,49,50 and
proliferation of HSCs.23,51,52 It has been hypothesized
that distinct stromal cells form niches within the microenvironment
that selectively regulate stem cell functions.48,53-55 We
have previously shown that the AC6.21 stromal cell line provides an
environment for a single multipotential CD34+
thy-1+ cell to differentiate into both B lymphocyte and
myeloid cells, a phenomenon similar to the in vivo BM
environment.9 This AC6.21 stromal cell line might represent
a specific and relatively rare subpopulation of stromal cells that
constitute the stem cell-supporting niches in the BM
microenvironment.48,53 Using AC6.21 stromal cells, we have
recently developed an in vitro culture system in which purified human
fetal BM CD34+ thy-1+ cells are expanded
150-fold in the presence of LIF.9 Furthermore, we have
demonstrated that LIF facilitates ex vivo CD34+
thy-1+ cell expansion indirectly via AC6.21 stromal
cells.9 LIF is a polyfunctional regulator of cell growth
and has been shown to have a broad spectrum of effects on a variety of
cell types.56,57 Prior studies have shown that LIF has
little or no effect on murine hematopoietic progenitor cell growth yet
enhances hematopoiesis in vivo, suggesting that LIF might have an
indirect role in hematopoiesis.58-61
On the basis of our own results and those of others, we have
hypothesized that LIF stimulates the AC6.21 stromal cells either to
produce SCEPF or to inhibit the production of SCEIF. In this study, we
focused on the detection and characterization of the putative SCEPF or
SCEIF. We demonstrated that SCM-LIF is sufficient to support ex vivo
proliferation (Figure 1) and multilineage differentiation of purified
CD34+ thy-1+ cells and to cause ex vivo
expansion of CD34+ thy-1+ cells independent of
AC6.21 stromal cells (Table 1). The magnitude of CD34+
thy-1+ cell proliferation (Figure 1) and expansion (Table
1) was proportional to the concentration of SCM-LIF. These results
suggest that there is an SCEPF activity in the SCM-LIF. Results from
mixing SCM-LIF and SCM (Table 2) clearly confirmed that it is the
presence of SCEPF and not the absence of SCEIF in SCM-LIF that is
responsible for facilitating ex vivo stem cell expansion. Furthermore,
we demonstrated that this secreted SCEPF activity can support ex vivo
proliferation and differentiation of purified CD34+
thy-1+ cells and promote the expansion of cells with
CD34+ thy-1+ phenotype (Figure 2). In addition,
these ex vivo expanded CD34+ thy-1+ cells not
only maintained the input CD34+ thy-1+
phenotype, but also preserved their in vivo transplantation potential as freshly purified CD34+ thy-1+ cells (Tables
3 and 4). Our data from hematopoietic reconstitution in SCID-hu mice
showed that ex vivo expanded cells from both stromal-based and
SCM-based culture systems and freshly purified CD34+
thy-1+ cells were qualitatively and quantitatively similar
(Table 4). In our previous studies in the stromal-based culture system,
there was a greater than 200-fold expansion of CD34+
thy-1+ cells in 5-week cultures. Similarly, there was a
217-fold expansion of CD34+ thy-1+ cells in the
3-week SCM-based cultures initiated with 300 CD34+
thy-1+ cells per well (Table 1). These results demonstrate
that the degree of stem cell expansion supported by the SCM-based
culture system is as effective as that in the stromal-based culture
system. The degree of stem cell expansion in the SCM-based culture
system was further examined at a limiting-dilution density (20 cells per well), and our results showed a 225-fold expansion under this culture condition (data not shown). Although the cultures initiated at
20 cells per well had a higher degree (225-fold) of stem cell expansion
than the cultures initiated with 300 cells per well (217-fold), the
difference in the degree of stem cell expansion between these
conditions was not statistically significant (P = .825).
These results indicate that there is no inhibitory or enhancing effect
on ex vivo stem cell expansion by the interactions among hematopoietic
cells in this SCM-based culture system and suggest that this SCM-based
culture system might be ready for scaled-up expansion in a clinical application.
To characterize the nature of this secreted SCEPF activity, we
investigated the possible contribution from 6 known prominent stem cell
cytokines, including GM-CSF, IL-3, IL-6, SCF, FL, and TPO, either alone
or in combinations, to this secreted SCEPF activity. It has been
reported that addition of LIF to SyS-1 stromal cells enabled the in
vitro maintenance of competitive repopulating murine stem
cells.23 Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to demonstrate that macrophage colony-stimulating factor (M-CSF), interleukin-7 (IL-7), SCF, flt3/flk2 receptor, interleukin-2 (IL-2), IL-6, granulocyte colony-stimulating factor (G-CSF), and LIF were up-regulated on SyS-1 stromal cells upon LIF
stimulation.23 Evidence was presented to suggest that
synergy between IL-6 and SCF, both of which are up-regulated by LIF on SyS-1 stroma, most likely accounts for the LIF-mediated activity in
maintaining competitive repopulating murine stem cells in
vitro.23 Several studies have shown that FL is capable of
facilitating proliferation of hematopoietic stem and progenitor cells
in vitro28-31 and mobilization of hematopoietic stem and
progenitor cells in vivo.32-35 TPO, although a potent
factor for megakaryocytopoiesis,18 also stimulates division
of primitive human hematopoietic stem and progenitor
cells.39-41 Because this secreted SCEPF activity is
mediated by LIF on stromal cells, the logical criterion for a cytokine
to be essential for this secreted SCEPF activity is that its expression
must be up-regulated by the stromal cells upon LIF stimulation. ELISA
was used to determine not only whether the expression of the cytokine
was up-regulated upon LIF treatment, but also the actual amount of the
cytokine in the conditioned media from LIF-treated stromal cell
cultures, which is a prerequisite for knowing the amount of antibody
needed in antibody-blocking experiments. As shown in Table 5, only IL-6
and SCF gave measurable amounts of cytokines by ELISA, and both were
up-regulated by the stromal cells upon LIF stimulation. Although the
amount of proteins for the other 4 cytokines could not be determined by
ELISA, it remains possible that these 4 cytokines might be up-regulated by the stromal cells upon LIF stimulation, but at extremely low concentrations that are below the sensitivity of ELISA. To directly examine the possible contribution from these 6 cytokines to this secreted SCEPF activity, we added neutralizing antibodies against each
cytokine to the 200% SCM-LIF and determined their activity to
facilitate ex vivo expansion of CD34+ thy-1+
cells. Results from this set of experiments showed that neutralizing antibody to each of the 6 known prominent stem cell cytokines cannot
inhibit the ex vivo stem cell expansion facilitated by the SCEPF
activity in the SCM-LIF. Based on the amount of the cytokines present
in the SCM-LIF determined by ELISA and the neutralizing activity of the
antibody against each cytokine, 1 µg/mL of each neutralizing antibody
was already above saturation level for each cytokine. These results
demonstrate that none of these 6 cytokines is essential for the SCEPF
activity in the SCM-LIF. To further rule out the contribution from
these 6 known cytokines to this SCEPF activity, all possible
combinations of these 6 cytokines at 10, 50, and 100 ng/mL were added
to 200% SCM plus 10 ng/mL of LIF, and their activity to maintain and
expand cells with CD34+ thy-1+ phenotype was
determined. Our results demonstrate that these 6 cytokines, either
alone or in various combinations, are not capable of maintaining
CD34+ thy-1+ cells in the cultures (data not
shown). We have recently used the more sensitive RT-PCR method to
further investigate the expression of IL-3, FL, TPO, and GM-CSF in the
stromal cells before and after LIF treatment (data not shown). SCF was
used as the positive control to define the conditions for RT-PCR. We
have established an optimal condition in which there is a 3-fold
increase in SCF expression upon LIF stimulation, consistent with the
result from ELISA. Under this RT-PCR condition, there is no detectable
signal for IL-3, GM-CSF, and TPO in the stromal cells with or without
LIF treatment. The expression of FL is detectable by RT-PCR and is not
altered upon LIF stimulation. We conclude that these 6 known prominent stem cell cytokines, including IL-3, IL-6, GM-CSF, SCF, FL, and TPO,
are not required for the SCEPF activity in the SCM-LIF.
Recently, a number of cytokines and different cytokine combinations
have been shown to inhibit apoptosis of hematopoietic stem and
progenitor cells. TPO promotes clonal growth of murine marrow Sca+
Lin cells in vitro by suppression of
apoptosis.62 SCF can directly promote survival of
hematopoietic progenitor cells in the absence of cell
division.63,64 Combinations of cytokines including TPO,
SCF, and other early-acting cytokines have been demonstrated to
facilitate the survival of human CD34+ cells following cell
division ex vivo.39-41 In this study, several combinations
of cytokines including TPO, SCF, and other early-acting cytokines have
been identified that significantly increased the proportion of
CD34+ thy-1+ cells in these cultures from 9%
to about 18% (Table 6). It is very likely that the addition of these
cytokines to the cultures can promote the survival of cells with
CD34+ thy-1+ phenotype and subsequently
increase the proportion of CD34+ thy-1+ cells
in these cultures as compared with the cultures without these cytokines.
In this study, we have demonstrated that 6 known early-acting
cytokines, including GM-CSF, IL-3, IL-6, SCF, FL, and TPO, are not
required for the secreted SCEPF activity in the SCM-LIF, and suggest
the presence of other secreted, LIF-mediated, stromal cell-derived
factor(s) in the SCM-LIF that promote ex vivo expansion of
transplantable human HSCs. At the present time, we have no data to
demonstrate that a single novel protein is the candidate for this
putative SCEPF activity in facilitating ex vivo expansion of
transplantable human fetal BM HSCs, and it remains possible that this
putative SCEPF activity might involve a synergism among multiple
factors, including known and novel cytokines. Based on the method we
have used to concentrate the SCM-LIF, our results suggest that the
molecular weight for the factor(s) of this secreted SCEPF activity is
between 5000 and 100 000 d. Although the nature of the putative SCEPF
activity is not yet defined, the results presented in this study
support the hypothesis that binding of LIF to the receptor on AC6.21
stromal cells leads to the production of a secreted SCEPF activity that
facilitates ex vivo expansion of transplantable HSCs, and that this
secreted SCEPF activity is not mediated by cell-cell or
cell-extracellular matrix interactions. The identification of the
component(s) for this secreted SCEPF activity will undoubtedly lead to
a better understanding of the intricate regulatory process governing
the development of all hematopoietic lineages from HSCs. Furthermore,
the ability to reconstitute this SCEPF activity in vitro with defined
components will facilitate ex vivo manipulation and expansion of
transplantable HSCs in several critical clinical applications,
including gene therapy, tumor cell purging, and stem cell transplantation.
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Top
Abstract
Introduction