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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1623-1636
Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human
Hematopoietic Stem Cells
By
Chu-Chih Shih,
Mickey C.-T. Hu,
Jun Hu,
Jeffrey Medeiros, and
Stephen J. Forman
From the Department of Hematology/Bone Marrow Transplantation and the
Department of Anatomic Pathology, City of Hope National Medical Center,
Duarte, CA; the Department of Cell Biology, Amgen Inc, Thousand Oaks,
CA; and the Department of Molecular Biology, Beckman Research Institute
at City of Hope, Duarte, CA.
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ABSTRACT |
We have developed a stromal-based in vitro culture system that
facilitates ex vivo expansion of transplantable CD34+
thy-1+ cells using long-term hematopoietic reconstitution
in severe combined immunodeficient-human (SCID-hu) mice as
an in vivo assay for transplantable human hematopoietic stem cells
(HSCs). The addition of leukemia inhibitory factor (LIF) to purified
CD34+ thy-1+ cells on AC6.21 stroma, a
murine bone marrow-derived stromal cell line, caused expansion of
cells with CD34+ thy-1+ phenotype. Addition
of other cytokines, including interleukin-3 (IL-3), IL-6,
granulocyte-macrophage colony-stimulating factor, and stem cell factor,
to LIF in the cultures caused a 150-fold expansion of cells retaining
the CD34+ thy-1+ phenotype. The ex
vivo-expanded CD34+ thy-1+ cells gave rise
to multilineage differentiation, including myeloid, T, and B cells,
when transplanted into SCID-hu mice. Both murine LIF (cannot bind to
human LIF receptor) and human LIF caused expansion of human
CD34+ thy-1+ cells in vitro, suggesting
action through the murine stroma. Furthermore, another human HSC
candidate, CD34+ CD38 cells, shows a
similar pattern of proliferative response. This suggests that
ex vivo expansion of transplantable human stem cells under this
in vitro culture system is a general phenomenon and not just specific
for CD34+ thy-1+ cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DEVELOPMENT of ex vivo culture conditions
that facilitate in vitro maintenance and expansion of long-term
transplantable hematopoietic stem cells (HSCs) is a crucial component
and a major challenge in stem cell research. This is a necessary first
step toward a better understanding of the regulatory process governing the development of all hematopoietic lineages from HSCs. Furthermore, establishment of such culture conditions is a prerequisite for potential ex vivo manipulation and expansion of transplantable HSCs in
several clinical applications such as gene therapy, tumor cell purging,
and stem cell transplantation.
Several clinical transplantation studies have shown that human
hematopoietic cells can be cultured ex vivo with or without stroma and
still retain the capacity to engraft human recipients, although it is
not known whether the number of transplantable HSCs has
changed.1-3 Furthermore, all of these studies involve autologous transplantation, making it difficult to determine whether long-term repopulation was derived from cultured cells or from surviving endogenous stem cells. Whether primitive human hematopoietic cells are actually expanded during ex vivo culture is controversial because different assays and culture conditions have been used in
different studies.4-6 It has recently been shown that
highly purified subfractions of CD34+ cells possess the
greatest proliferative potential, resulting in a large expansion of
colony-forming cells (CFC), while long-term culture-initiating cells
(LTC-IC) show either a slight reduction7 or moderate
increase.6 Although these in vitro assays provide an
essential quantitative assessment of the functional properties of the
expanded cells, they do not evaluate repopulation capacity. Appropriate
assays are needed and must be used for the direct quantitation of the
most primitive and transplantable stem cells.
HSCs are defined as cells having both capacity for self-renewal and
ability to differentiate into at least 8 distinct hematopoietic cell
lineages. Hematopoietic progenitors in human bone marrow (BM) can be
identified by the expression of the CD34 antigen. However, the majority
of the CD34+ BM cells are committed to specific
hematopoietic cell lineages. Using different assays8 for
the properties expected from HSC, enrichment for pluripotent progenitor
cells in the CD34+ cell fraction has been successfully
performed by eliminating the CD34+ cells expressing
lineage-associated antigens,9,10 HLA-DR,10 CD38,11 CD45RA,12 or CD71,10,12 or
lacking thy-1.13,14 Because none of these assays can
provide an accurate determination of the HSC identity, and no single
assay has been used across the board to compare different HSC
phenotypes, the utility of these assays is still being debated.
In the mouse, no in vitro system has been developed that adequately
recapitulates stem cell behavior, and, currently, the only reliable
functional assay system for the most primitive stem cell compartment is
long-term in vivo transplantation. Experimental in vivo hematopoietic
assays are not ethical with human subjects. Studies of human stem cell
renewal, differentiation, and maintenance would be facilitated by the
availability of a relevant animal model in which assays similar to
those used for secondary transfer and long-term reconstitution of mice
can be employed. Over the past decade, several groups have transplanted
human hematopoietic cells into immunodeficient mouse strains in an
attempt to develop a relevant and reproducible in vivo transplantation
model. A number of in vivo assays for human HSC are now
available.15-18 However, in these systems, the only
hematolymphoid microenvironments in which the human cells can
differentiate are those of murine origin. Because of the complexity of
the regulatory mechanisms surrounding hematopoiesis, a murine-origin
microenvironment may not be physiologically sufficient to sustain human
stem cell self-renewal and differentiation. This might explain why
large doses of exogenous human cytokines,15,17 high numbers
of unfractionated input cells,15,17 and cotransplantation with stromal cells engineered to produce human cytokines16
are needed to sustain human hematopoiesis in these murine systems. Recent data suggest that the nonobese diabetic severe combined immunodeficiency (NOD/SCID) mouse is a better host than the SCID mouse
for this type of research.18-20 Physiologically, it would be preferable to sustain human HSCs in a hematopoietic microenvironment of human origin in a murine model. To address this problem, a "humanized" murine model, the SCID-hu mouse, was developed by implantation of intact human hematolymphoid tissues into SCID mice.21 Studies have shown that the SCID-hu mouse is a
useful and relevant in vivo system for assaying the developmental
potential of transplantable human HSCs and that assays similar to those used for secondary transfer and long-term reconstitution of mice can be
employed.13,22 It is notable that lymphoid development from
primitive cells is better demonstrated in the SCID-hu mouse model.13,22
Recently, a SCID repopulating cell (SRC) assay19 has been
used to perform a quantitative assessment of the repopulation capacity
of ex vivo-cultured cells initiated with CD34+
CD38 cells.23 This study showed a 4- and
10-fold increase in the number of CD34+
CD38 cells and CFC, respectively, as well as a 2- to
4-fold increase in SRC after 4 days of culture.23 However,
after 9 days of culture, all SRC were lost, despite increases in total
cells, CFC counts, and CD34+ cells.23 These
results support the notion that appropriate quantitative assays for
transplantable stem cells are essential for the development of culture
conditions that support primitive cells.23 In this study,
our strategy is the following: First, we would like to establish a
quantitative assay based on in vivo reconstitution in the SCID-hu mouse
model to quantify the number of transplantable human stem cells. This
assay will be used to measure the number of stem cells before and after
culture from the same donors. Second, we would like to develop a
culture system based on a murine BM-derived stromal cell line and
exogenous cytokines. Here we report the development of a novel culture
system for facilitating ex vivo maintenance and expansion of
transplantable human CD34+ thy-1+ cells. In
this ex vivo culture system, the absolute number of cells with
CD34+ thy-1+ phenotype increases 150-fold after
5 weeks of culture. Those ex vivo-expanded CD34+
thy-1+ cells not only maintain their CD34+
thy-1+ phenotype, but also preserve their engrafting
ability for long-term hematopoietic reconstitution in the SCID-hu mice.
Furthermore, we have identified that leukemia inhibitory factor (LIF)
is the responsible cytokine for maintaining and expanding those
transplantable CD34+ thy-1+ cells in this ex
vivo culture system and that LIF exerts its role in expanding
transplantable CD34+ thy-1+ cells by indirectly
affecting the stromal AC6.21 cells. Our results also suggest that ex
vivo expansion of transplantable human stem cells under this in vitro
culture system is a general phenomenon and not just specific for
CD34+ thy-1+ cells.
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MATERIALS AND METHODS |
Preparation of human hematopoietic cells and fluorescence-activated
cell sorting.
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, White Oak, GA). A sample of each received
fetal tissue was stained with a panel of monoclonal antibodies (MoAbs)
to HLA to establish the donor allotype. The fetal tissues were used
either for construction of the SCID-hu mice or for preparation of human
HSCs. To purify human HSCs, BM cell suspensions were prepared by
flushing split long bones with RPMI 1640 (GIBCO/BRL, Gaithersberg, MD)
containing 2% heat-inactivated fetal calf serum (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 MoAbs and phycoerythrin (PE)-labeled
thy-1 MoAbs (or PE-labeled CD38 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, and
then resuspended in SB containing 1 µg/mL propidium iodide (Molecular
Probes Inc, Eugene, OR) and sorted using the tri-laser
fluorescence-activated cell sorter 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 and CD38 were purchased from Becton Dickinson (Mountain View, CA).
MoAbs for thy-1 and isotype controls were purchased from Pharmingen (San Diego, CA).
In vitro human hematopoietic progenitors/mouse stroma cocultures.
Sorted cells were cultured on a preestablished monolayer of mouse
stromal cell line AC6.21. Briefly, 5 × 103 to 1 × 104 stromal cells were plated in 96-well
flat-bottom plates 1 week before the experiment in 100 µL of
long-term culture medium (LTCM) consisting of RPMI 1640, 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% FCS. Twenty CD34+
thy-1+ cells were distributed in 100 µL of LTCM into each
well in 96-well flat-bottom plates with preestablished AC6.21
monolayer. Individual growth factor or combinations of growth factors
were added immediately after seeding the sorted cells into the
microtiter plates at a concentration of 10 ng/mL of each growth factor.
Human recombinant interleukin-3 (IL-3), IL-6, granulocyte-macrophage
colony-stimulating factor (GM-CSF), stem cell factor (SCF), and LIF
were purchased from R&D Systems (Minneapolis, MN). Half of the culture
medium was replaced weekly with fresh LTCM containing the respective growth factor(s). To minimize disturbance to the cultures during the
weekly medium change, 100 µL of old medium from each well was removed
slowly from the top of the well with a multiple channel pipetter, and
100 µL of fresh medium was then slowly added to each well.
Proliferative analysis, phenotypic analysis, and sorting of ex
vivo-cultured human fetal HSCs.
The extent of each growth factor or combinations of growth factors to
support in vitro expansion of purified human fetal BM stem cells was
determined weekly by counting the total number of hematopoietic cells
present in 15 individual wells in each culture. For counting the
content of hematopoietic cells in these wells, cells were harvested
individually from these wells by gently pipetting off the wells without
destruction of the stromal layer. At the end of the culture period,
either week 5 or week 7, individual wells were analyzed for lineage
content by flow cytometry. Cells from each well were harvested
individually by vigorously pipetting of the wells including stromal
cells. 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 or CD38. Analysis was gated on
the hematopoietic cells, excluding the stromal cells, and the quadrants
were set based on the mean fluorescence intensity of the isotype
control samples. FITC- or PE-labeled MoAbs against CD19 and CD33 were purchased from Pharmingen (San Diego, CA). Cells were analyzed on a
FACScan fluorescent cell analyzer (Cytomation, Inc, Fort Collins, CO).
To purify the ex vivo-expanded HSCs from the culture, cells from each
well were harvested individually by vigorously pipetting off the wells
including stromal cells at the end of the culture. One-twentieth of the
cells (200,000 cells per well, 1/20 fraction of each well contains
about 10,000 cells) from each well was divided into 2 fractions. One
fraction was stained with antibodies against CD34 and thy-1 or CD38,
and another fraction was stained with FITC- or PE-labeled MoAbs
against CD19 and CD33 and analyzed on the FACScan cell analyzer. The
cells from the wells containing all 3 populations, including
CD33+ cells, CD19+ cells, and CD34+
thy-1+ cells or CD34+ CD38
cells, were pooled together (usually about 10% of the
wells in the cultures initiated with CD34+
thy-1+ cells and 12% of the wells in the cultures
initiated with CD34+ CD38 cells) and
sorted for HSCs, either CD34+ thy-1+ or
CD34+ CD38 cells, as described above.
In vivo reconstitution assay in 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 the construction of SCID-hu thymus/liver (thy/liv)
and bone model mice were constructed as previously
described13,22 and in accordance with the guidelines set
forth by the City of Hope Research Animal Care Committee. At the time
of surgery, 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,
including CD34+ thy-1+ and CD34+
CD38 populations (stem cell donor is always selected
to be HLA-MA2.1-positive), either fresh uncultured 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). A limiting dilution
experiment was conducted to determine quantitatively the transplantable
cells in the freshly purified human fetal BM CD34+
thy-1+ population. Four different cell doses were evaluated
(10,000, 3,000, 1,000, and 300 cells in 10 µL of HBSS were injected
per human graft). For other reconstitution experiments, 10,000 cells were used because 10,000 CD34+ thy-1+ cells
purified from fresh fetal BM reproducibly establish long-term hematopoietic reconstitution in greater than 90% of SCID-hu mice in
our laboratory. Control animals were injected with HBSS only. Engraftment was analyzed at 3 to 4 months postinjection. 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 Igs 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 (San Diego, CA).
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RESULTS |
Limiting dilution experiments in SCID-hu mice.
Limiting dilution experiments were performed to determine if the in
vivo reconstitution assay in SCID-hu mice is capable of quantifying
transplantable stem cells in the heterogeneous CD34+
thy-1+ population. CD34+ thy-1+
cells (HLA-MA2.1-positive) were sorted from 22- to 24-week-old fetal
BM samples and 4 different cell doses (10,000, 3,000, 1,000, and 300 cells per graft) were evaluated. Thirty SCID-hu mice (15 thy/liv and 15 bone mice) were used for each cell dose, and a total of 120 mice were
used for the CD34+ thy-1+ cells purified from
each fetal BM sample. The ability of the injected cells to engraft the
SCID-hu mice was determined by flow cytometry 3 months after cell
injection. Four different fetal BM samples were used and compared in
these experiments (Table 1). For donor no.
1, donor reconstitution derived from freshly purified CD34+
thy-1+ cells was evident in 87% (13 of 15), 20% (3 of
15), 7% (1 of 15), and 0% (0 of 15) of the bone grafts and in 93%
(14 of 15), 20% (3 of 15), 7% (1 of 15), and 0% (0 of 15) of the
thymic grafts from an injected cell dose of 10,000, 3,000, 1,000, and
300, respectively. The percentage of donor-derived cells in the bone
grafts of reconstituted animals was 41% ± 10% (ranging from 30%
to 54%), 9% ± 3% (ranging from 6% to 12%), 2.2% from an
injected cell dose of 10,000, 3,000, and 1,000, respectively. The
percentage of donor-derived cells in the thymic grafts of reconstituted
animals was 50% ± 8% (ranging from 40% to 58%), 12% ± 4%
(ranging from 8% to 16%), and 3.2% from a injected cell dose of
10,000, 3,000, and 1,000, respectively. The data from the other 3 donors were almost identical to donor no. 1 and showed no significant
donor effect in this assay.
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Table 1.
In Vivo Hematopoietic Reconstitution Rate in the SCID-hu
Mice With Limiting Numbers of Freshly Purified Human Fetal BM
CD34+ thy-1+ Cells
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Calibration between cell dose and reconstitution rate in SCID-hu
mice.
To determine the appropriate cell dose that can provide a quantitative
measurement for transplantable stem cells for subsequent in vivo
reconstitution assay in SCID-hu mice, calibration curves for cell doses
and reconstitution rates in both thy/liv and bone models were
established by logistic regression based on the data from limiting
dilution experiments (Table 1). The calibration curves for both thy/liv
and bone models are almost identical (Fig 1). These curves suggest that cell doses from 1,000 to 10,000 could be
used to quantify the number of transplantable stem cells with a 50%
reconstitution rate at approximately 5,000 cells. Because the
10,000-cell dose gives rise to a higher reconstitution rate of about
90% and the percentage of donor-derived cells in the reconstituted
animals only reaches 40% to 50%, this suggests that the 10,000-cell
dose is not at the saturation level yet in this in vivo
reconstitution assay. We have decided to use 10,000 cells as the
standard cell dose for subsequent in vivo transplantation experiments.
This assay was then used to quantify the number of transplantable
CD34+ thy-1+ cells, before and after culture,
from the same donors.
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| Fig 1.
Calibration curves of limiting numbers of freshly
purified human fetal BM CD34+ thy-1+ cells
with hematopoietic reconstitution rates in the SCID-hu mice. The
calibration curves are established by a logistic regression method
based on the engraftment data shown in Table 1. Both calibration curves
fit well, with no evidence of overdispersion.
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Characteristics of AC6.21 stromal cell line.
The present study was designed to develop a simple culture system to
facilitate in vitro maintenance and expansion of transplantable stem
cells. Our strategy was to use cloned stromal cell lines because the
stromal cell line offers several advantages over the heterogeneous
adherent cell monolayer derived from BM.24 Several stromal
cell lines have been shown to support in vitro
myelopoiesis,25-28 B lymphopoiesis,28 or in
some cases both.29-31 We have performed single-cell
(CD34+ thy-1+) sorting experiments in the
AC6.21 stromal cell line, derived from the Whitlock-Witte culture
system,32 coculture assay. Individual CD34+
thy-1+ cells were deposited onto a preestablished AC6.21
monolayer without the addition of exogenous cytokines. Our unpublished
data suggest that 1 in 20 CD34+ thy-1+ cells
was able to initiate long-term cultures and become growth-positive (>100 progeny), and 50% of these growth-positive wells gave rise to
both B lymphocytes and myeloid cells. Our unpublished results are
similar to a previously published report using a different stromal cell
line, SyS-1.13 Because the AC6.21 stromal cell line can
provide 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, we
hypothesized that it might also provide a natural environment for a
primitive stem cell to self-renew, which in turn will facilitate the
development of an ex vivo culture system for HSC expansion. We have
decided to use AC6.21 as a stromal support in developing an ex vivo
culture system. Based on our own results from single-cell coculture
experiments, we have decided to initiate the cultures with 20 purified
human fetal BM CD34+ thy-1+ cells in each well
and anticipated that 100% of the wells would be growth-positive in 5 weeks. From the single-cell coculture experiments, our results showed
that coculture with AC6.21 was not able to maintain cells with a
CD34+ thy-1+ phenotype in the culture. To
evaluate whether the addition of cytokines to the culture will help to
maintain the cells with stem cell phenotype in this coculture system,
IL-3,33,34 IL-6,33 GM-CSF,35
SCF,34,36,37 and LIF38,39 were selected as exogenous cytokines based on their potential for expanding
hematopoietic progenitor cells from in vitro studies.
Effect of cytokine combinations on ex vivo proliferation of
CD34+ thy-1+ cells.
We evaluated the effects of the 5 individual cytokines on the
proliferative capacity of CD34+ thy-1+ cells in
AC6.21 stroma cocultures. Kinetic studies
(Fig 2) showed no obvious proliferation for
the first 2 weeks in culture, regardless of the treatments. This was
followed by a continuous increase in total cell numbers, which reached
about 5,000 cells per well by week 7. By week 7, almost all wells
(>95%) for each treatment scored as growth-positive (>5,000
cells/well) as expected for CD34+ thy-1+ cells
from our own unpublished data and a previously published report.13 However, the culture treated with LIF showed a
late-phase exponential growth from the fourth week and reached about
100,000 cells per well by week 7. This result suggests that among the 5 cytokines evaluated, LIF is the only cytokine by itself that can
facilitate the proliferation of purified CD34+
thy-1+ cells. Even though IL-3, IL-6, GM-CSF, and SCF,
individually, cannot promote cell growth in this coculture system
compared with the control (without exogenous cytokine), they were able
to establish synergistic effects on HSC expansion with LIF. Kinetic
studies (Fig 3) show the synergistic
effects of LIF with other cytokines on the proliferative capacity
of purified human CD34+ thy-1+ cells.
The addition of other cytokines to LIF accelerates the proliferative
kinetics of purified human fetal BM CD34+
thy-1+cells, and these cultures enter an exponential phase
of growth from the second week in culture (v the fourth week
with LIF alone). At the end of the fifth week in culture, more than
95% of the wells for all treatments except LIF alone are confluent
(about 200,000 cells per well). However, it is noteworthy that those cultures treated with combinations of LIF and other cytokines still
remain quiescent during the first week in culture (v 2 weeks with LIF alone).
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| Fig 2.
Effects of 5 individual cytokines on the proliferative
potential of human fetal BM CD34+ thy-1+
cells in vitro. Data are presented as the total number of hematopoietic
cells per well (average of 15 wells) in each culture condition at each
weekly time point. The standard deviation for the 15 wells in the
LIF-treated cultures at each weekly time point is less than 8% of the
mean value. ( ), Control; ( ), IL-3; ( ), IL-6; ( ), GM-CSF;
( ), SCF; ( ), LIF.
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| Fig 3.
Synergistic effects of LIF with other cytokines on the
proliferative capacity of freshly purified human fetal BM
CD34+ thy-1+ cells. See Fig 2 legend for
additional information. (), LIF; (), LIF + IL-3 + IL-6;
(), LIF + IL-3 + IL-6 + GM-CSF; (), LIF + IL-3 + IL-6 + GM-CSF + SCF.
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Effect of cytokine combinations on differentiation.
To assess the effects of these cytokine combinations on the
differentiation potential of purified CD34+
thy-1+ cells, cells from individual wells were analyzed by
flow cytometry for the presence of CD33+ myeloid cells and
CD19+ B lymphocytes. Approximately 50% of the wells in
each treatment were mixed lymphoid/myeloid wells, ranging from 41% of
the GM-CSF-treated culture alone to 62% of those treated with LIF + IL-3 + IL-6 + GM-CSF + SCF (Table 2). The
percentage of CD33+ myeloid cells within these mixed
lymphoid/myeloid wells ranges from 40% of those treated with LIF + IL-3 + IL-6 to 55% of those treated with LIF + IL-3 + IL-6 + GM-CSF;
and the percentage of CD19+ cells ranges from 15% of those
treated with combinations of LIF with other cytokines to 8% of the
control group (Table 2). The frequency of mixed lymphoid/myeloid wells
and the percentages of the myeloid and B-cell populations within these
mixed lymphoid/myeloid wells among these different treatments are very
similar to the control. These results suggest that addition of cytokine
combinations in this coculture system does not dramatically alter the
differentiation potential of purified human fetal BM CD34+
thy-1+ cells.
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Table 2.
Effects of Combinations of Five Cytokines on the
Differentiative Potential of Freshly Purified Human Fetal BM
CD34+ thy-1+ Cells In Vitro
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Effect of cytokine combinations on expansion of cells with
CD34+ thy-1+ phenotype.
To determine if addition of exogenous cytokines to the coculture system
is capable of facilitating the maintenance and expansion of cells with
CD34+ thy-1+ phenotype, flow cytometric
analyses were performed to detect and quantify the number of
CD34+ thy-1+ cells in each individual well in
these cultures. As expected from single-cell coculture experiments,
cells with CD34+ thy-1+ phenotype were not
detectable in the control group (Table 3). Among the 5 cytokines evaluated, LIF is the only factor by itself that
can give rise to CD34+ thy-1+/positive wells.
The percentage of CD34+ thy-1+ cells in these
positive wells is about 7%. Because each well was initiated with 20 cells and only about 10% of the wells are CD34+
thy-1+/positive, this suggests that the frequency of cells
capable of regenerating CD34+ thy-1+ phenotype
is about 1 in 200 within the CD34+ thy-1+
population. The frequency of CD34+
thy-1+/positive wells in those cultures treated with
combinations of LIF with other cytokines ranges from 10% of those
treated with LIF + IL-3 + IL-6 + GM-CSF to 12% of those treated with
LIF + IL-3 + IL-6 + GM-CSF + SCF (Table 3). All CD34+
thy-1+/positive wells, regardless of treatments, contain
both CD33+ and CD19+ cells. The percentage of
CD34+ thy-1+ cells within the CD34+
thy-1+/positive wells increases significantly from 7% in
LIF-treated cultures to 15% in cultures treated with combinations of
LIF and other cytokines (Table 3; P < .00001). As there are
about 200,000 cells per well within these CD34+
thy-1+/positive wells in these cultures, the absolute
number of CD34+ thy-1+ cells in each well
averages about 30,000, representing a 1,500-fold expansion in this
population during the 5 weeks in culture (each well was initiated with
20 purified CD34+ thy-1+ cells). The overall
bulk equivalent (ie, not counting only the 10% to 12% of wells
containing CD34+ thy-1+ cells) is in excess of
a 150-fold expansion of CD34+ thy-1+ cells
under these culture conditions. Although the difference in the
frequency of CD34+ thy-1+/positive wells in
these cultures is not statistically significant (P = .85),
cultures treated with LIF + IL-3 + IL-6 + GM-CSF + SCF had a
higher frequency (12%) of CD34+
thy-1+/positive wells (Table 3) and were selected as the
optimal condition for subsequent experiments.
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Table 3.
Effects of Combinations of Five Cytokines on the
Maintenance and Expansion of Freshly Purified Human Fetal BM
CD34+ thy-1+ Cells In Vitro
|
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Ex vivo-expanded CD34+ thy-1+ cells give
rise to multilineage differentiation in SCID-hu mice.
To determine whether ex vivo-expanded CD34+
thy-1+ cells possess the same in vivo proliferative and
differentiation capacity as freshly purified human fetal BM
CD34+ thy-1+ cells, the ability of these cells
to engraft and initiate long-term hematopoietic reconstitution in
SCID-hu mice was analyzed by multiparameter flow cytometry. A
representative analysis of hematopoietic reconstitution in the SCID-hu
mouse transplanted with 10,000 ex vivo-expanded CD34+
thy-1+ cells is shown in Fig 4.
The thymic engrafting potential of ex vivo-expanded CD34+
thy-1+ cells is shown in Fig 4A. The engrafted human thymus
of this SCID-hu mouse contained 50% ex vivo-expanded
CD34+ thy-1+-derived thymocytes as detected by
expression of the donor HLA (MA2.1-positive). These cells were further
analyzed for their expression of T-cell markers CD3, 4, and 8, and they
displayed a normal T-cell maturation pattern. The BM engrafting
potential of ex vivo-expanded CD34+ thy-1+
cells is shown in Fig 4B and C. The engrafted human bone fragment of
this SCID-hu mouse contained 39% donor-derived CD19+ B
cells (Fig 4B) and 16% donor-derived CD33+ myeloid cells
(Fig 4C) as detected by donor HLA (MA2.1-positive). The control mice
did not receive any donor cells and did not show any detectable
donor-derived cells. These results show that ex vivo-cultured and
expanded CD34+ thy-1+ cells not only maintain
their CD34+ thy-1+ phenotype, but also preserve
their in vivo engrafting potential.
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| Fig 4.
Hematopoietic reconstitution in the SCID-hu mice with
10,000 ex vivo-expanded CD34+ thy-1+ cells
from 5-week cultures. (A) Intrathymic T-cell development of ex
vivo-expanded CD34+ thy-1+ cells. Graft
cells were analyzed by flow cytometry for T-cell markers, CD3, CD4, and
CD8, and donor marker (HLA-MA2.1-positive). The percentage of T cells
expressing detectable levels of donor-specific HLA class I antigen was
recorded. (B) B-cell differentiation and (C) myeloid differentiation of
ex vivo-expanded CD34+ thy-1+ cells in
implanted human fetal bone fragment. Graft cells were analyzed for
B-cell marker CD19 and myeloid marker CD33, and donor marker
HLA-MA2.1.
|
|
In vivo engrafting potential of CD34+ thy-1+
cells before and after culture.
To further compare the number of transplantable CD34+
thy-1+ cells before and after culture, we have compared the
in vivo engrafting activity between freshly purified and ex
vivo-expanded CD34+ thy-1+ cells from the same
donors. Data from three independent experiments using cells from
different donors were compiled (Table 4).
As expected from previous limiting dilution experiments with 10,000 freshly purified CD34+ thy-1+ cells (Table 1),
all 3 donors gave rise to about a 90% reconstitution rate in both
thy/liv and bone mice with 40% to 50% donor-derived cells in the
reconstituted animals (Table 4). Ex vivo-expanded CD34+
thy-1+ cells from the same donors gave almost identical
results as the freshly purified CD34+ thy-1+
cells in both the frequency of reconstitution and the percentage of
donor-derived cells in the reconstituted animals (Table 4). These
results suggest that CD34+ thy-1+ cells, before
and after culture, are similar, both qualitatively and quantitatively.
In this set of experiments, we have also evaluated whether the ex
vivo-expanded hematopoietic cells from wells without detectable
CD34+ thy-1+ cells (about 90% of the wells in
this culture are CD34+ thy-1+/negative wells)
are capable of engrafting in SCID-hu mice. Our results show that donor
reconstitution was not detectable (0 of 60) in the SCID-hu mice when
transplanted with 10,000 ex vivo-expanded hematopoietic cells from
CD34+ thy-1+/negative wells for all 3 donors,
suggesting that transplantable cells, before and after culture, are
only present in the CD34+ thy-1+ population.
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Table 4.
Comparative Quantitation of the Number of Transplantable
Freshly Purified CD34+ CD38 Cells and
Transplantable Ex Vivo-Expanded Human Fetal BM CD34+
CD38 Cells From the Same Donors
|
|
Quantitative comparison of transplantable CD34+
thy-1+ cells before and after culture.
To directly quantify the number of transplantable CD34+
thy-1+ cells before and after culture, the in vivo
engraftment data (Table 4) were subjected to statistical analysis. The
calibration curves established from limiting dilution experiments (Fig
1) were used to estimate an equivalent dose of fresh cells based upon
the engraftment data derived from CD34+ thy-1+
cells before and after culture from the same donors. Separate logistic
regression curves were estimated for thy/liv and bone models, as shown
in Fig 5A and B, respectively. In both
cases, the estimation of equivalent dose of fresh CD34+
thy-1+ cells for 10,000 cultured CD34+
thy-1+ cells was larger than 10,000 cells (13,600 for the
thy/liv model, and 16,350 for the bone model). The lower (95%)
confidence bounds on equivalent fresh CD34+
thy-1+ cells for 10,000 cultured cells were 7,295 for the
thy/liv model and 7,905 for the bone model.40 These results
suggest that the ex vivo-expanded CD34+ thy-1+
cells have higher in vivo engrafting potential than the freshly purified CD34+ thy-1+ cells from the same
donors, and imply that there is a preferential expansion of
transplantable CD34+ thy-1+ cells rather than
just the cells with the CD34+ thy-1+ phenotype
under this culture system.
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| Fig 5.
Statistical measurements for the transplantable human
fetal BM CD34+ thy-1+ cells before and
after culture by a standard calibration method. (A) The measurements in
the SCID-hu thy/liv model. (B) The measurements in the SCID-hu bone
model. The statistical analyses are based on the data shown in Tables 1
and 6. Ninety-five percent lower confidence bounds were found by
applying a standard calibration method40 to
the exact binomial lower confidence bound for the cultured cell
engraftment probability, using a 97.5% confidence level for both
SCID-hu mouse models.
|
|
LIF exerts its role by an indirect route.
Our data have shown that LIF plays key roles in facilitating
maintenance and expansion of transplantable human CD34+
thy-1+ cells under this culture system. To understand how
LIF mediates its effects on maintenance and expansion of transplantable
human CD34+ thy-1+ cells under this ex vivo
culture system, our experimental approach was to determine whether LIF
acts directly on human CD34+ thy-1+ cells or
indirectly on the murine AC6.21 stromal cells. It has been previously
reported that human LIF (huLIF) can bind to the murine receptor and is
biologically active in mouse M1 cells.41 However, there is
no binding of murine LIF (muLIF) to the human receptor.42
This published information provided us with the rationale for using
muLIF in our culture system. If huLIF acts directly on human
CD34+ thy-1+ cells to promote their ex vivo
expansion, then muLIF will not be able to serve the same function
because muLIF cannot bind to human CD34+ thy-1+
cells.41,42 However, if huLIF exerts its roles indirectly by acting on AC6.21 stromal cells, then muLIF will be able to substitute for huLIF in facilitating ex vivo expansion of human CD34+ thy-1+ cells.41,42 Results
from muLIF experiments demonstrate that muLIF performs its function as
efficiently as huLIF in facilitating maintenance and expansion of
CD34+ thy-1+ cells in vitro
(Table 5). These results suggest that LIF
facilitates maintenance and expansion of CD34+
thy-1+ cells in this ex vivo culture system not by directly
acting on human CD34+ thy-1+ cells but by
indirectly affecting AC6.21 stromal cells. This conclusion is further
supported by the previous results from kinetic studies (Figs 2 and 3).
The lack of proliferative response of CD34+
thy-1+ cells for the first 1 to 2 weeks in culture suggests
that these cells are waiting for the AC6.21 stroma to establish a
suitable microenvironment, which in turn is induced by the presence of LIF.
In vivo reconstitution potential of CD34+
CD38 cells.
To determine if the activity to facilitate ex vivo expansion of
transplantable stem cells by this in vitro culture system is a general
phenomenon and not only specific to CD34+
thy-1+ cells, we decided to investigate the proliferative
responses of another HSC candidate (CD34+
CD38 population)11 under this ex vivo
culture system. Since we have established a quantitative assay for
transplantable stem cells based on the SCID-hu mouse model, our first
set of experiments was to demonstrate that CD34+
CD38 cells are capable of establishing long-term
hematopoietic reconstitution in SCID-hu mice. A representative analysis
of hematopoietic reconstitution in SCID-hu mice transplanted with
10,000 freshly purified CD34+ CD38 cells
is shown in Fig 6. The thymic engrafting
potential of CD34+ CD38 cells is shown
in Fig 6A. The engrafted human thymus of this SCID-hu mouse contained
30% donor-derived thymocytes as detected by expression of the donor
HLA (MA2.1-positive). These cells also displayed a normal T-cell
maturation pattern. The BM engrafting potential of fresh
CD34+ CD38 cells is shown in Fig 6B and
C. The engrafted human bone fragment of this SCID-hu mouse contained
18% donor-derived CD19+ B cells and 15% donor-derived
CD33+ myeloid cells as detected by donor HLA
(MA2.1-positive). In contrast, donor-derived cells were not detected in
the control mice transplanted with 10,000 CD34+
CD38+ cells. These results suggest that there is an
enrichment for the in vivo engrafting activity in the CD34+
CD38 population compared with the CD34+
CD38+ population. Our results showed that about 90% of the
SCID-hu mice, both bone and thy/liv mice, are reconstituted by
injecting 10,000 freshly purified human fetal BM CD34+
CD38 cells into each human graft (data not shown).
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| Fig 6.
Hematopoietic reconstitution in the SCID-hu mice with
10,000 freshly purified human fetal BM CD34+
CD38 cells. (A) Intrathymic T-cell development, (B)
B-cell differentiation, and (C) myeloid differentiation of purified
CD34+ CD38 cells in implanted human fetal
bone fragment. See Fig 4 legend for additional
information.
|
|
Effect of cytokine combination on ex vivo proliferation of
CD34+ CD38 cells.
To facilitate a relevant comparison between CD34+
CD38 population and CD34+
thy-1+ population, the same experimental procedures were
followed. The kinetic studies of cultures initiated with
CD34+ CD38 cells show an early wave of
proliferation in the first 2 weeks that was absent in the cultures
initiated with the CD34+ thy-1+ population
(Fig 7). This was followed by a second wave
of exponential growth from the end of the second week to the fourth
week, and by the fourth week most of the wells were confluent (Fig 7).
These results show that the growth response of CD34+
CD38 cells under this ex vivo culture system is 1 week faster than and distinguishable from the CD34+
thy-1+ population.
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| Fig 7.
Kinetics of the proliferative potential of purified human
fetal BM CD34+ CD38 cells in vitro. The
growth factor cocktail included IL-3, IL-6, GM-CSF, SCF, and LIF. Data
are presented as the total number of hematopoietic cells per well (mean
of 15 wells) at each weekly time point. The standard deviation for the
15 wells at each weekly time point is less than 12% of the mean value.
For comparison, the kinetic data of CD34+
thy-1+ cells as shown in Fig 4 have been superimposed
with the data obtained from CD34+ CD38
cells. (), CD34+ CD38; (),
CD34+ thy-1+.
|
|
Effect of cytokine combination on ex vivo differentiation of
CD34+ CD38 cells.
To determine the differentiation potential of purified
CD34+ CD38 cells under this ex vivo
culture system, cells from individual wells were analyzed by flow
cytometry for the presence of CD33+ myeloid cells and
CD19+ B lymphocytes. The phenotypic analyses of 5-week
cultures derived from 3 different donors are summarized in
Table 6. The frequency of mixed
lymphoid/myeloid wells in 5-week cultures is 12% (36 of 300), 10% (31 of 300), and 11% (34 of 300) for donors no. 21, 22, and 23, respectively. This frequency of mixed lymphoid/myeloid wells derived
from CD34+ CD38 cells (Table 6) is
significantly lower (P < .00001) than the respective 50%
from the CD34+ thy-1+ population (Table 2). We
have estimated that about 1 in 182 CD34+
CD38 cells is multipotential for both B-cell and
myeloid lineages (v 1 in 40 CD34+
thy-1+ cells) under this ex vivo culture condition. It is
also notable that the percentage of CD19+ B lymphocytes
(Table 6) is significantly lower (P < .00001) in these
cultures, averaging 8%, compared to 15% for the cultures initiated
with CD34+ thy-1+ cells (Table 2). However, the
percentage of CD33+ myeloid cells (Table 6) is
significantly higher (P < .0001) in these cultures, averaging
64%, compared to 45% for the cultures initiated with
CD34+ thy-1+ cells (Table 2).
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Table 6.
Effect of Growth Factor Cocktail on the Differentiation
Potential of Purified Human Fetal BM CD34+
CD38 Cells In Vitro
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|
Effect of cytokine combination on ex vivo expansion of
CD34+ CD38 cells.
To determine the maintenance and expansion potential of purified
CD34+ CD38 cells under this ex vivo
culture system, flow cytometric analyses were performed on the 5-week
cultures to quantify the number of CD34+
CD38 cells in the wells of these cultures. The
frequency of CD34+CD38-positive wells in
these cultures is 69% (207 of 300), 71% (214 of 300), and 72% (216 of 300) for donors no. 21, 22, and 23, respectively (Table 7). This result suggests that 1 in
29 CD34+ CD38 cells (v 1 in 200 CD34+ thy-1+ cells) is capable of expansion
under this ex vivo culture condition. The percentage of
CD34+ CD38 cells in myeloid wells
(CD34+CD38/positive wells without
detectable CD19+ cells), averaging 9%, is significantly
lower (P < .00001) than the respective percentage, averaging
15%, in the mixed lymphoid/myeloid wells. All of the mixed
lymphoid/myeloid wells (36 wells from donor no. 21, 31 wells from donor
no. 22, and 34 wells from donor no. 23) contain CD34+
CD38 cells. These data show that 1 in 29 CD34+ CD38 cells is capable of
proliferation, but only 1 in 182 CD34+
CD38 cells is capable of both proliferation and
multipotential differentiation. The higher frequency and percentage of
myeloid wells in these cultures are evidence suggesting that the
majority of CD34+ CD38 cells which are
capable of proliferation in the cultures have already committed to the
myeloid lineage. However, the CD34+ CD38
cells in those mixed lymphoid/myeloid wells present both expected characteristics of HSC, proliferation, and multipotential
differentiation. In those mixed lymphoid/myeloid wells, an average of
15% (30,000 cells) are CD34+ CD38
cells, representing a 1,500-fold expansion of cells with the CD34+ CD38 phenotype. The overall bulk
equivalent (ie, not counting only the 10% to 12% of mixed
lymphoid/myeloid wells) is in excess of a 150-fold expansion of
CD34+ CD38 cells under this culture
system.
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Table 7.
Effect of Growth Factor Cocktail on the Maintenance and
Expansion of Purified Human Fetal BM CD34+
CD38 Cells In Vitro
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|
In vivo engrafting potential of CD34+
CD38 cells before and after culture.
Our next experiment was to determine whether ex vivo-expanded
CD34+ CD38- cells still maintain their ability
to establish long-term hematopoietic reconstitution in SCID-hu mice,
and to determine whether ex vivo-expanded CD34+
CD38 cells from myeloid wells and mixed
lymphoid/myeloid wells differ in their in vivo engrafting activity. The
ex vivo-expanded CD34+ CD38 cells were
sorted from pools of myeloid wells and mixed lymphoid/myeloid wells,
respectively, derived from 5-week cultures. The sorted cells in HBSS
were then injected (10,000 cells/graft) into human thymus and bone
fragments in SCID-hu mice. Control mice were injected with HBSS only.
Thymic and BM engraftment by CD34+ CD38
cells in the SCID-hu mice were analyzed 3 to 4 months after stem cell
injection. Data from 3 independent experiments using cells from
different donors were compiled (Table 8).
Donor reconstitution derived from 10,000 freshly purified
CD34+ CD38 cells was evident in about
90% of the bone grafts and 90% of the thymic grafts for all 3 donors.
These results are consistent with our unpublished data and are also
similar to the reconstitution rate when 10,000 CD34+
thy-1+ cells were injected. The percentage of donor-derived
hematopoietic cells was about 35% in both the bone and thymic grafts
for all 3 donors (Table 8). Compared with the percentage of
donor-derived cells in the animals transplanted with 10,000 CD34+ thy-1+ cells (40% in the bone mice and
50% in the thy/liv mice), the percentage of donor-derived cells from
CD34+ CD38 cells is lower. However, ex
vivo-expanded CD34+ CD38 cells purified
from mixed lymphoid/myeloid wells give the same results in both
reconstitution rate and percentage of donor-derived cells as freshly
purified CD34+ CD38 cells from the same
donors (Table 8). These results suggest that ex vivo-expanded
CD34+ CD38 cells from mixed
lymphoid/myeloid wells are similar, both qualitatively and
quantitatively, to freshly purified CD34+
CD38 cells from the same donors. In contrast,
reconstitution was only detected in 10% (3 of 30) of the bone grafts
and 0% (0 of 30) of the thymic grafts with ex vivo-expanded
CD34+ CD38 from myeloid wells. These in
vivo data, which are consistent with in vitro results obtained from
CD34+ CD38 cultures, suggest that
although CD34+ CD38 cells in the myeloid
wells are capable of regenerating CD34+
CD38 cells, the majority of these cells have already
committed to the myeloid lineage and are not able to differentiate into
lymphoid lineage, including T and B lymphocytes. Because ex
vivo-expanded CD34+ CD38 cells give
almost the same reconstitution rate and percentage of donor-derived
cells as freshly purified CD34+ CD38
cells from the same donors, it is reasonable to conclude that transplantable CD34+ CD38 cells have
been similarly expanded 1,500-fold as the cells with CD34+
CD38 phenotype in these mixed lymphoid/myeloid
wells. The bulk equivalent (ie, the overall culture, not just the 10%
to 12% of mixed lymphoid/myeloid wells) is in excess of a 150-fold
expansion.
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Table 8.
Comparative Quantitation of the Number of Transplantable
Freshly Purified CD34+ CD38 Cells and
Transplantable Ex Vivo-Expanded Human Fetal BM CD34+
CD38 Cells From the Same Donors
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|
| |
DISCUSSION |
Efforts to develop culture systems for the maintenance of
transplantable stem cells have met with limited success for the last 2 decades. One major problem in developing an ex vivo culture system for
HSC expansion has been finding an assay to quantitatively measure
transplantable stem cells before and after culture. It has been
repeatedly shown that combinations of cytokines can exert potent
stimulatory effects on stem/progenitor populations. Although in vitro
studies are often used to study stem cell biology, it is difficult to
relate these results to what occurs in the in vivo setting. Many
studies have shown that progenitor cells with myeloid-erythroid and
lymphoid potential can be expanded in vitro; however, the in vivo
transplantation potential of these ex vivo-expanded progenitor cells
remains obscure. In this study, we have first established a
quantitative assay for transplantable human stem cells using in vivo
reconstitution in SCID-hu mice as the model system. Limiting dilution
experiments in SCID-hu mice using 4 different fetal BM samples (Table
1) have established calibration curves for both thy/liv and bone models
by logistic regression (Fig 1). There was no significant donor effect
and no significant lack of fit in either case, and the calibration
curves are suitable baselines for quantitative measurement of
transplantable cells in a heterogeneous stem cell population. These
calibration curves also suggest that cell doses ranging from 1,000 to
10,000 used in establishing the calibration curves can be employed in
the assay to relatively quantify the number of transplantable
CD34+ thy-1+ cells in the heterogeneous
CD34+ thy-1+ population. This quantitative
assay system was used to quantify the number of transplantable human
stem cells before and after culture.
In this study, we used CD34+ thy-1+ cells from
human fetal BM as input HSC phenotype. The CD34+
thy-1+ population is very heterogeneous. Studies from many
groups have found that the CD34+ thy-1+
phenotype does not exclusively select primitive stem cells only. The
CD34+ thy-1+ population contains a proportion
of cells able to repopulate SCID-hu mice,13,22 a proportion
of extended LTC-IC (ELTC-IC),4 a large number of
LTC-IC,6 a large number of CFC,7 and even some
more differentiated cells.13 In this study, the 1,500-fold expansion in the CD34+ thy-1+/positive wells is
based on the phenotype of output cells subsequent to culture. Because
the CD34+ thy-1+ population is heterogeneous,
it remains possible that the 1,500-fold expansion might represent the
expansion of those more differentiated cells. To address whether the
proportion of cells capable of engrafting SCID-hu mice is similarly
expanded 1,500-fold as CD34+ thy-1+ cells after
culture, the engraftment potential of ex vivo-expanded CD34+ thy-1+ cells was determined in SCID-hu
mice. If the 1,500-fold expansion of CD34+
thy-1+ cells in the CD34+
thy-1+/positive wells is largely contributed by the other
cells and not by the proportion of cells capable of engrafting SCID-hu
mice, then we would expect to see a decrease in the frequency of
reconstituted SCID-hu mice injected with 10,000 ex vivo-expanded
CD34+ thy-1+ cells compared to the mice
injected with 10,000 freshly purified CD34+
thy-1+ cells. Our data (Table 4) from a comparative
analysis of 296 SCID-hu mice transplanted with CD34+
thy-1+ cells before and after culture show that the
proportion of CD34+ thy-1+ cells capable of
engrafting SCID-hu mice and the percentage of donor-derived
hematopoietic cells in the reconstituted animals remain the same before
and after culture (Table 4). Because the whole CD34+
thy-1+ population in the CD34+
thy-1+/positive wells has expanded 1,500-fold over the
5-week culture period and the proportion of cells capable of engrafting
SCID-hu mice remains the same within the CD34+
thy-1+ phenotype before and after culture, these results
suggest that the proportion of cells capable of engrafting SCID-hu mice
in the CD34+ thy-1+/positive wells has been
similarly expanded 1,500-fold. Statistical analysis based on the
calibration curves established by logistic regression from limiting
dilution experiments (Table 1) further supports that the equivalent
dose of fresh CD34+ thy-1+ cells for 10,000 cultured CD34+ thy-1+ cells was larger than
10,000 cells (13,600 for the thy/liv model, Fig 5A; and 16,350 for the
bone model, Fig 5B). These results suggest that there is a preferential
expansion of transplantable CD34+ thy-1+ cells
under this ex vivo culture system. Because we are not able to identify
a single cell type responsible for long-term engraftment in SCID-hu
mice, the experimental results presented here are not yet able to
unequivocally prove that the proportion of cells capable of engrafting
SCID-hu mice in the CD34+ thy-1+/positive wells
is expanded 1,500-fold as the CD34+ thy-1+
phenotype over the 5-week culture period.
To rule out the possibility that this in vitro culture system might be
specific for CD34+ thy-1+ cells and can only
facilitate the expansion of CD34+ thy-1+ cells,
we have investigated the proliferative responses of another human HSC
candidate, CD34+ CD38 cells, under this
in vitro culture condition. Because CD34+
CD38 cells have a different phenotype than
CD34+ thy-1+ cells, these cells show similar
but distinguishable growth kinetics compared with CD34+
thy-1+ cells. The kinetic studies demonstrate that cultures
initiated with the CD34+ CD38 cells show
an early wave of proliferation in the first 2 weeks followed by a
second wave of exponential growth from the end of second week to the
fourth week, and that the overall cell growth in the cultures initiated
with CD34+ CD38 cells is 1 week faster
than the cultures derived from CD34+ thy-1+
population (Fig 7). Under this ex vivo culture system, the frequency of
cells in the CD34+ CD38 population
capable of multipotential differentiation for B-cell and myeloid
lineages is estimated to be 1 in 182, which is significantly lower
(P < .00001) than the 1 in 40 for CD34+
thy-1+ cells (Tables 2 and 6). The frequency of cells in
the CD34+ CD38 population capable of
proliferation is estimated to be 1 in 29, which is significantly higher
(P < .00001) than the 1 in 200 for CD34+
thy-1+ cells (Tables 3 and 7). These data suggest that the
majority of CD34+ CD38 cells that are
capable of proliferation in the cultures have already committed to the
myeloid lineage, which might account for the higher frequency and
percentage of myeloid wells in these cultures. However, the frequency
of cells capable of proliferation and multipotential differentiation in
CD34+ CD38 and CD34+
thy-1+ population is very similar (1 in 182 in the
CD34+ CD38 population and 1 in 200 in
the CD34+ thy-1+ population). These data also
show that this ex vivo culture system is capable of measuring the 2 key
stem cell-specific features, proliferation and multipotential
differentiation, and suggest that this ex vivo culture system might
represent a relevant system for the identification of primitive stem
cells and for the relative position of different human HSC candidates
in the stem cell hierarchy. Most importantly, this study shows that
cells with the CD34+ CD38 phenotype are
expanded 1,500-fold in the mixed lymphoid/myeloid wells (Table 7). The
frequency of reconstituted animals and the percentage of donor
hematopoietic cells in the reconstituted animals derived from ex
vivo-expanded CD34+ CD38 cells purified
from mixed lymphoid/myeloid wells are apparently very similar to the
same parameters in the animals reconstituted with freshly purified
CD34+ CD38 cells from the same donors
(Table 8). Based on the similar frequency of their activity to
reconstitute the SCID-hu mice among freshly purified CD34+
thy-1+ cells and CD34+ CD38
cells, and ex vivo-expanded CD34+ CD38
cells from mixed lymphoid/myeloid wells, we conclude that
transplantable CD34+ CD38 cells have
been similarly expanded 1,500-fold as the cells with CD34+
CD38 phenotype in these mixed lymphoid/myeloid wells
(Table 8). The bulk equivalent (ie, the overall culture, not just the
10% to 12% of mixed lymphoid/myeloid wells) is in excess of a
150-fold expansion. These results suggest that ex vivo expansion of
transplantable human stem cells facilitated by this in vitro culture
system is a general phenomenon and not just specific for
CD34+ thy-1+ cells.
In this study, we have identified LIF as the determining cytokine in
maintaining and expanding transplantable human CD34+
thy-1+ cells in this ex vivo culture system. In addition,
our studies demonstrate that LIF exerts its role in expanding
transplantable human HSCs by indirectly affecting the stromal AC6.21
cells. Recently, it has been reported that addition of LIF to SyS-1
stromal cells enables the maintenance and 9-fold expansion of highly
enriched competitive repopulating murine stem cells.31 The
reverse transcription-polymerase chain reaction (RT-PCR) was used to
show that M-CSF, IL-7, SCF, flt3/flk2 receptor, IL-2, IL-6, G-CSF, and
LIF were upregulated on SyS-1 stromal cells upon LIF
stimulation.31 Evidence was presented to suggest that
synergy between IL-6 and SCF, both of which are upregulated by LIF on
SyS-1 stroma, most likely accounts for the LIF-regulated murine stem
cell maintenance and expansion in vitro.31 However, our
data show that AC6.21 stroma treated with a combination of IL-3, IL-6,
GM-CSF, and SCF is not capable of promoting ex vivo maintenance and
expansion of CD34+ thy-1+ cells (Table 5).
These results suggest that the synergy between IL-6 and SCF cannot
account for the 1,500-fold expansion of human CD34+
thy-1+ cells in our ex vivo expansion system. We
hypothesize that LIF binds to the receptor on AC6.21 cells and
initiates signal transduction pathways leading to upregulated
production of factor(s) that facilitate transplantable stem cell
expansion and/or downregulated production of factor(s) that inhibit
stem cell expansion. Although the molecular mechanisms of LIF-mediated
ex vivo maintenance and expansion of transplantable human fetal BM
CD34+ thy-1+ and CD34+
CD38 cells are not currently defined, it is likely
that this ex vivo culture system will provide a relevant tool for the
identification of novel factors involved in regulating the process of
self-renewal, proliferation, and differentiation in early hematopoietic
development, and will have important implications for ex vivo stem cell
expansion, gene therapy, and therapeutic transplantation. In this
study, human fetal BM has been used as the HSC source. However, fetal BM is not a relevant HSC source in clinical application. The
traditional sources of HSCs in clinical transplantation include
autologous and allogeneic adult BM and mobilized peripheral blood (PB).
Recently, human umbilical cord blood (UCB) has been shown to be an
alternative source of stem cells for clinical
transplantation.43 To evaluate the feasibility of using
this ex vivo culture system to expand transplantable CD34+
thy-1+ or CD34+ CD38 cells
for clinical transplantation, the ex vivo expansion potential of
purified CD34+ thy-1+ or CD34+
CD38 cells from adult BM, adult PB, and UCB under
this culture system should be determined.
| |
ACKNOWLEDGMENT |
We thank Drs Don Diamond, John Zaia, John Rossi, David DiGiusto, and
Catherine M. Verfaillie for review of the manuscript; Drs Jeffrey
Longmate and Joycelynne Palmer for their assistance in statistical
analysis; Lucy Brown and Jim Bolen for assistance in FACS analysis; Drs
Steve Novak and Tom LeBon for their assistance in the preparation of
the manuscript; supportive team members in the Department of Molecular
Biology at Beckman Research Institute at City of Hope for their
administrative assistance; and members in the Animal Research Center at
City of Hope for their assistance in animal care.
| |
FOOTNOTES |
Submitted May 26, 1998; accepted May 4, 1999.
Supported by Grants No. NCI PPG CA 30206, NCI CA 33572, and NCI CA
71866 from the National Cancer Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Chu-Chih Shih, PhD, Department of
Hematology/BMT, City of Hope National Medical Center, and Department of
Molecular Biology, Beckman Research Institute at City of Hope, 1500 E
Duarte Rd, Duarte, CA; e-mail: cshih{at}coh.org.
| |
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ABSTRACT
INTRODUCTION