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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1957-1966
HEMATOPOIESIS
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.
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)
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 Preparation of stroma-conditioned media from untreated (SCM) and
LIF-treated stromal cell cultures (SCM-LIF)
Preparation of human HSCs
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.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.
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.
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.
In vivo transplantation potential of CD34+
thy-1+ cells before and after cultures
Effect of LIF on the production of 6 known prominent stem cell
cytokines by AC6.21 stromal cells
Neutralizing antibody to each of the 6 known prominent stem cell
cytokines cannot block ex vivo stem cell expansion
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).
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
We thank Drs David DiGiusto, John Rossi, John Zaia, and Ravi Bhatia 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.
Submitted May 12, 1999; accepted November 13, 1999.
Supported by grants from the National Cancer Institute: NCI PPG CA 30206, NCI CA 33572, and NCI CA 71.866.
Reprints: Chu-Chih Shih, Department of Hematology/Bone Marrow Transplantation, City of Hope National Medical Center, and Department of Molecular Biology, Beckman Research Institute at City of Hope, 1500 E Duarte Rd, Duarte, CA 91010; e-mail: cshih{at}coh.org.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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  J. J. Schuringa, K. Y. Chung, G. Morrone, and M. A.S. Moore Constitutive Activation of STAT5A Promotes Human Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation J. Exp. Med., September 7, 2004; 200(5): 623 - 635. [Abstract] [Full Text] [PDF]
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P. Kurre, S. Burdach;, C.-C. Shih, and S. J. Forman A potential role for leukemia inhibitory factor in the increased clonogenicity of human fetal progenitor cells Blood, August 1, 2000; 96(3): 1199 - 1200. [Full Text] [PDF]
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Top
Abstract
Introduction
cells,
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)
