IFN- Negatively Modulates Self-Renewal of Repopulating Human Hemopoietic Stem Cells
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免疫学杂志 2005年第2期
Abstract
Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN- might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN- on HSC maintenance and function. Although purified cord blood CD34+CD38– cells underwent cell divisions in the presence of IFN-, cycling HSCs exposed to IFN- in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN- accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN- can negatively affect human HSC self-renewal.
Introduction
Life-long hemopoiesis is dependent on hemopoietic stem cells (HSCs)3 that self-renew to maintain the HSC pool and, by multilineage differentiation, give rise to all the myeloid and lymphoid cell lineages of the blood (1). Although tightly regulated, little is known about the specific regulatory pathways controlling HSC fate decisions. Loss of function studies have demonstrated important roles of multiple stimulatory cytokines, such as c-kit ligand (or stem cell factor, SCF) and thrombopoietin (TPO), in maintenance of the HSC pool (2, 3). However, because HSCs are thought not to have unlimited self-renewal potential, it appears likely that also negative regulators might be involved in controlling the HSC pool. TNF (4, 5, 6), TGF- (7, 8, 9), Fas (10, 11, 12, 13), and IFNs (14, 15, 16, 17) have all been demonstrated to have suppressive effects on in vitro and in vivo hemopoiesis, and TNF- (18, 19), Fas (10, 11), and IFN- (11, 20, 21) have been implicated as important mediators of acute and chronic bone marrow (BM) failure syndromes, such as graft-vs-host disease and aplastic anemia.
Whereas TNF- recently has been demonstrated to negatively affect human (22, 23) and murine (24) HSCs, the ability of IFN- to modulate HSC fate and function remains to be investigated. Previous studies have demonstrated that depending on the conditions and the specific progenitors investigated, IFN- can either promote (25, 26) or inhibit (15, 16, 27) the growth of hemopoietic progenitors, including primitive human CD34+CD38– cells. However, to what degree such effects of IFN- on growth of progenitor/stem cells are associated with altered HSC numbers or function has not been explored.
Studies on HSCs in vitro have previously been hampered by the fact that most established culture conditions result in dramatic losses of HSCs (28, 29, 30). However, identification of combinations of early acting cytokines and improved overall HSC culture conditions promoting mouse HSC self-renewal have facilitated in vitro studies of the regulation of HSC fate decisions (31, 32, 33, 34). Importantly, such studies have confirmed the ability of growth-stimulating cytokines, such as SCF, TPO, flt3 ligand (FL), IL-11, and IL-3, to support mouse HSC self-renewal cell divisions (32, 33, 34). Development of similar conditions promoting self-renewal of candidate human HSCs (32, 35, 36, 37, 38, 39) as well as improvement of human HSC assays (40, 41, 42, 43) is now facilitating studies of human HSC regulation.
Using in vitro conditions recently demonstrated to efficiently promote proliferation of candidate murine and human HSCs with sustained HSC function (32, 33), we in this study explored, for the first time, the potential ability of IFN- to directly affect self-renewal of in vivo repopulating human HSCs.
Materials and Methods
Hemopoietic growth factors and IFN-
Human rSCF (rhSCF), rhG-CSF, rhFL, rhIL-3, IL-6, and rhGM-CSF were generous gifts from Amgen; rhTPO from Genentech; and recombinant human erythropoietin (rhEPO) from Boehringer Mannheim. rhIFN- was purchased from R&D Systems.
Isolation and purification of human stem and progenitor cells
Umbilical cord blood (CB) samples were obtained from normal full-term deliveries following informed consent from the mothers and with approval of the ethics committees at the University Hospital of Lund and Helsingborg Hospital. CD34+ CB cells were enriched according to previously described protocols (23, 32). Briefly, CB mononuclear cells were isolated from samples using Ficoll-Hypaque density gradient centrifugation (lymphoprep, 1.077 ± 0.001 g/ml; Nycomed Pharma). Positive selection of CD34+ cells was performed by a MACS-CD34 isolation kit (Miltenyi Biotec), according to the manufacturer’s instructions. CB CD34+ cells were run through a second column to obtain higher purity (91–94%) of CD34+ cells. CD34-enriched cells were incubated with anti-CD38 PE and anti-CD34 FITC mAbs or with isotype-matched irrelevant control Abs (all from BD Biosciences), and subsequently sorted on a FACSVantage cell sorter (BD Biosciences). A conservative approach was taken to exclusively sort the 3% lowest CD38-expressing CD34+ cells (CD34+CD38–), in an effort to obtain a highly purified population of primitive progenitor/stem cells. The purity of CD34+CD38– cells in the experiments was 96–98%.
In vitro expansion cultures
CD34+ and CD34+CD38– CB cells were cultured at 1000 cells/ml for 7 or 12 days in serum-free (SF) medium (IMDM; BioWhittaker) supplemented with 10 mg/ml BSA, 10 μg/ml human insulin, 200 μg/ml human transferrin (BIT; StemCell Technologies), and a mixture of cytokines (100 ng/ml rhSCF, 100 ng/ml rhFL, 100 ng/ml rhTPO, and 20 ng/ml rhIL-3, defined as SFT3) in the absence or presence of 1000 U/ml rhIFN-. Following culture, cells were enumerated and evaluated functionally in in vitro long-term cultures or by transplantation into NOD-SCID mice. In some experiments, cultured cells were further examined with regard to apoptosis, cell cycle status, and differentiation.
Single-cell clonogenic assays
As described previously (23), CD34+CD38– CB cells were seeded in Terasaki plates (Nunc) at a density of 1 cell/well in 20 μl of SF medium (X-vivo 15; BioWhittaker) and 1% BSA (StemCell Technologies), supplemented with a mixture of cytokines (50 ng/ml rhSCF, 50 ng/ml rhFL, 50 ng/ml rhTPO, and 20 ng/ml rhIL-3) and different concentrations of rhIFN-. Wells (120/group) were scored for cell growth following 11–12 days of incubation at 37°C in a humidified atmosphere with 5% CO2 in air.
Long-term culture-initiating cell (LTC-IC) assay
Establishment and maintenance of long-term cultures were performed according to previously described procedures (23). Briefly, stroma cell feeders were established by seeding a mixture (1:1) of two irradiated (8,000 cGy) murine fibroblast cell lines (M2-10B4 and sl/sl; kindly provided by D. Hogge, Vancouver, Canada), engineered to produce high levels of human G-CSF, IL-3, and SCF (42), into 96-well collagen-coated microtiter plates (Nunc) containing LTC medium (Myelocult; StemCell Technologies) supplemented with freshly dissolved 10–6 M hydrocortisone 21-hemisuccinate (Sigma-Aldrich). These cell lines have been demonstrated to detect candidate HSCs with enhanced efficiency when compared with stromal derived from primary BM cells (42). Each well contained 10,000 cells from the mixture of two cell lines. The freshly isolated or expansion equivalents of 50 CD34+CD38– CB cells cultured in SFT3 in the absence or presence of rhIFN- for 7 days were seeded per stroma well (four replicates per group) and incubated for 6 wk at 37°C in a humidified atmosphere with 5% CO2 in air. Cocultures were maintained by weekly 50% medium changes.
Following 6 wk of culture, adherent and nonadherent cells from each well were transferred to methylcellulose cultures containing rhSCF, rhFL, rhIL-3, rhGM-CSF, rhG-CSF (all at 10 ng/ml), and rhEPO (5 U/ml). To ensure formation of a reliable number of colonies from the long-term culture, the content of each well was transferred to methylcellulose cultures at both a low (20% of cells) and a high concentration (80% of cells). LTC colony-forming cells (LTC-CFCs) were scored after an additional 10–12 days of culture. Stroma cells without hemopoietic cells and cytokines were used as a negative control for the CFC assay.
NOD-SCID-repopulating assay
NOD-SCID mice (originally from The Jackson Laboratory, Bar Harbor, ME) were bred and housed under sterile conditions and maintained on autoclaved food and acidified water. All animal procedures were performed with consent from the local ethics committee at Lund University. At 8–12 wk of age, mice were irradiated with 350 cGy from a 137Cs source. The transplantation of 5 x 104 to 1 x 105 CD34+ or 5000 CD34+CD38– CB cells together with 1 x 106 irradiated (1500 cGy) accessory cells (CD34-depleted CB cells) in 0.5 ml of medium was performed by tail vein injection within 4 h of irradiation. Mice were sacrificed after 6 wk by asphyxiation with CO2, and femora and tibiae were collected; and engraftment was investigated by flow cytometry (FACSCalibur; BD Biosciences) using CellQuest analysis software (BD Biosciences), as described previously (23, 40, 41). Briefly, BM cells were counted and blocked with anti-mouse CD16/CD32 (BD Pharmingen,) and ChromPure mouse IgG, whole molecule (Jackson ImmunoResearch Laboratories). Subsequently, cells were stained with FITC-conjugated anti-human CD45 and CD71 Abs (BD Biosciences) as well as anti-mouse CD45.1-PE Ab (BD Pharmingen). BM cells from untransplanted mice (negative controls) and mixtures of 0.1% human cells in NOD-SCID BM (positive controls) were included. If engraftment was detected as human CD45/CD71 positive (detection level 0.05%), lineage analysis with anti-human CD34 FITC (progenitors), anti-human CD19 PE (B cells; BD Biosciences), anti-human CD15 PE, and anti-human CD66b FITC (myeloid; both BD Pharmingen) combined with anti-human CD45 allophycocyanin (BD Biosciences) was performed. For all samples, 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) was included to gate out dead cells. A minimum of 5 x 104 BM cells was examined for each sample. Only mice with both positive myeloid and lymphoid engraftment (defined as >10 positive events each per 5 x 104 viable BM cells with maximum 1 event in corresponding controls) were evaluated as positive. If no engraftment was detected by flow cytometry or if the myeloid engraftment was questionable, BM cells were plated in methylcellulose supplemented with human-specific cytokines (25 ng/ml rhSCF, 25 ng/ml rhIL-3, 50 ng/ml rhGM-CSF) and 5 U/ml rhEPO at a density of 105 cells/35-mm plate, four replicates per group. CFU granulocyte-macrophage and burst-forming unit erythroid were scored after 10–12 days. No colonies were observed in the absence of cytokines or from BM of untransplanted mouse cultured with the same cytokines (L. Yang and S. E. W. Jacobsen, unpublished observations).
Flow cytometric evaluation of differentiation, apoptosis, and cell cycle status of cultured CB progenitors
To evaluate differentiation, CD34+ CB cells were cultured for 9 or 12 days in SF medium (X-vivo 15; BioWhittaker) with 1% BSA (StemCell Technologies) or IMDM (BioWhittaker) with 20% FCS (BioWhittaker), supplemented with SFT3 and SFT3 + IL-6 + GM-CSF + G-CSF, respectively, in the absence or presence of 1000 U/ml rhIFN-. Following culture, cells were stained with anti-human CD34 FITC or allophycocyanin and a lineage mixture containing PE- or FITC-conjugated Abs (against CD11b, CD14, CD15, CD33, CD41, CD66b, and glycophorin A) and 7-AAD (to exclude nonviable cells). Control samples of cultured cells were stained with irrelevant isotype-matched control Abs. Samples were analyzed on a FACSCalibur
Apoptosis of CD34+ CB cells was assessed by measuring redistribution of phosphatidylserine using annexin V-PE (BD Pharmingen) and uptake of 7-AAD, as described before (23, 44), with minor modifications. Briefly, cultured CD34+ CB cells (5 days) were first stained for surface markers using anti-human CD34 allophycocyanin and anti-lineage Abs, and subsequently stained with annexin V-PE (5 μl) and 7-AAD (5 μg/ml) in 100 μl of annexin V-binding buffer (BD Pharmingen) for 15 min, resuspended in annexin V-binding buffer, and analyzed on a FACSCalibur.
Cell cycle status of freshly isolated or cultured (5 days) CD34+ CB cells was determined, as described (45), with minor modifications. Cells were stained with anti-human CD34 allophycocyanin, followed by fixation and permeabilization with Cytofix/Cytoperm kit (BD Pharmingen) for 30 min. After washing, cells were stained with FITC-conjugated anti-Ki67 (Beckman-Coulter) or an isotype-matched irrelevant control Ab for 30 min. After 3 h of incubation in PBS containing 5% FCS supplemented with 7-AAD (5 μg/ml) at 4°C under dark conditions, samples were analyzed on a FACSCalibur.
Results
IFN- inhibits clonal expansion of human candidate HSCs
We have recently demonstrated that in vitro culture under SF conditions in the presence of the early acting cytokines SFT3 efficiently promotes recruitment of murine and candidate human HSCs into proliferation with sustained stem cell function (32, 33). In this study, we used this system to investigate the effect of IFN- on self-renewal of human HSCs. CD34+CD38– CB cells proliferated extensively in the presence of SFT3, and this expansion was inhibited by 35% in response to IFN- (Fig. 1).
FIGURE 1. IFN- inhibits the in vitro cellular expansion of CD34+CD38– CB cells. A total of 1000 cells/ml was cultured in SF medium supplemented with SFT3 in absence or presence of 1000 U/ml IFN- for 7 days. Data represent the mean (SD) from three individual experiments. *, p < 0.05, comparing –IFN- and +IFN-.
To investigate whether IFN- might potentially block the first cell divisions of candidate HSCs, single cells were cultured in SFT3 in the presence or absence of different concentrations of IFN- (10–1000 U/ml; Fig. 2). Importantly, at all used concentrations, IFN- did not affect the number of proliferating clones. However, the size of the clones was reduced in a dose-dependent manner. These findings suggested that IFN- does not affect cytokine-induced recruitment of CD34+CD38– candidate HSCs into proliferation, but rather inhibits their clonal expansion.
FIGURE 2. IFN- does not affect cytokine-induced recruitment of CD34+CD38– candidate HSCs into proliferation, but inhibits their clonal expansion. CD34+CD38– CB cells were plated at a density of 1 cell/well in 20 μl of SF medium supplemented with SFT3 in the absence or presence of increasing concentrations of IFN-. Each group, consisting of 120 wells, was scored for clonal growth (clone number and size) after 11–12 days of culture. Large colonies were defined as clones covering >10% of the well. Results are presented as means (SEM) of three individual experiments. *, p < 0.05, comparing –IFN- and +IFN-.
IFN- negatively affects maintenance of CD34+CD38– CB LTC-IC under self-renewing conditions
To investigate the effect of IFN- on maintenance of candidate human HSCs, we cultured CD34+CD38– CB cells in SFT3 in the presence or absence of IFN-, and subsequently evaluated the LTC-IC colony-forming cells (LTC-CFCs) in such cultures. In agreement with previous studies (23, 32), SFT3 promoted maintenance of high levels of LTC-CFC activity after 7 days of culture, comparable to uncultured input cells with an average of 343 LTC-CFCs derived from the initiating 50 CD34+CD38– CB cells (Fig. 3). In the presence of IFN-, LTC-CFCs were reduced by as much as 89% (Fig. 3).
FIGURE 3. IFN- negatively modulates self-renewal of CD34+CD38– candidate stem cells with LTC-CFC activity. Fifty CD34+CD38– cells were cultured for 7 days in SF medium with SFT3 in the absence or presence of 1000 U/ml IFN- and subsequently evaluated for 6-wk LTC-CFC activity (see Materials and Methods). Also shown is the number of LTC-CFC generated from 50 freshly isolated CD34+CD38– cells. Data (CFC generated by 50 CD34+CD38– cells after 6-wk LTC) are presented as means (SEM) of two individual experiments, each with four replicates cultures. *, p < 0.05, comparing –IFN- and +IFN-.
IFN- negatively modulates maintenance of multipotent NOD-SCID-repopulating HSCs under self-renewing conditions
The NOD-SCID xenograft assay is thought to detect more primitive human hemopoietic cells than the LTC-IC assay (40, 41, 46). In addition, the NOD-SCID assay allows evaluation of in vivo multilineage-repopulating ability of candidate human HSCs. In a total of three experiments using CD34+ cells and one using highly purified CD34+CD38– cells, SFT3 cultures maintained high levels of multilineage NOD-SCID reconstitution activity that was almost completely abolished in the presence of IFN- (Table I and Fig. 4). This was reflected in the mean human reconstitution being reduced from 11 to 0.3%. Furthermore, whereas 100% of mice transplanted with SFT3-cultured cells were multilineage reconstituted, only 18% of mice transplanted with cells exposed to IFN- were positive for human myeloid reconstitution (Table I).
Table I. IFN- potently inhibits self-renewal of in vivo multilineage NOD-SCID reconstituting cells
FIGURE 4. IFN- negatively modulates self-renewal of multipotent NOD-SCID-repopulating CD34+CD38– CB cells. FACS profile shows multilineage human engraftment of representative mice transplanted with expansion equivalent of 5000 CD34+CD38– cells cultured for 7 days in SFT3 in the absence or presence of 1000 U/ml IFN- (see Materials and Methods). Of all mice transplanted with CD34+CD38– cells exposed to IFN-, the one shown here had the highest level of human engraftment.
IFN- promotes differentiation of human hemopoietic progenitor/stem cells under self-renewing conditions
To investigate potential mechanisms by which IFN- might negatively affect maintenance of HSCs, we examined whether IFN- might promote differentiation at the expense of self-renewal. Following 12 days of culture under self-renewing conditions (SFT3), 18% of cells continued to express the stem/progenitor cell Ag CD34, which was down-regulated upon myeloid differentiation. In contrast, only 2% of cells cultured under the same conditions, but in the presence of IFN-, remained CD34+ (Table II), compatible with IFN- promoting differentiation rather than self-renewal of CD34+ stem/progenitor cells. To further investigate this, CD34+ cells were also cultured under conditions more efficiently promoting lineage differentiation (SFT3 + IL-6 + G-CSF + GM-CSF). Under these conditions, the maintenance of cells with a stem/progenitor CD34+ as well as lineage-negative phenotype was reduced significantly in the presence of IFN- (Table II). Specifically, whereas 40% of CD34+ cells cultured in the absence of IFN- remained lineage negative, as much as 92% of cells cultured in the presence of IFN- became positive for myeloid lineage Ags. In further support of IFN- promoting myeloid differentiation and in line with the reduced CD34 expression, the number of CFU-C was reduced almost 3-fold in IFN--containing cultures (Table II).
Table II. IFN- promotes differentiation of in vitro expanded CD34+ CB cells
IFN- did not seem to significantly affect apoptosis of cultured progenitors because a combined annexin V and 7-AAD staining demonstrated comparable numbers of apoptotic cells in control and IFN--supplemented cultures (Fig. 5). Cellular maintenance also appeared unaffected by IFN-, as CD34+CD38– cells cultured in the absence and presence of IFN- showed indistinguishable forward scatter profiles (L. Yang and S. E. W. Jacobsen, unpublished observations). Furthermore, IFN- did not affect the fraction of cells in S/G2/M of CD34+ progenitors cultured in SFT3 (Fig. 6). Thus, IFN- might, at least in part, negatively affect the maintenance of primitive hemopoietic progenitor/stem cells by promoting their differentiation rather than self-renewal.
FIGURE 5. IFN--induced inhibition of human CD34+ progenitor/stem cells is not accompanied by enhanced apoptosis. CD34+ CB cells cultured for 5 days in STF3 in the absence or presence of IFN- (1000 U/ml) were stained with anti-CD14/15/66b (myeloid differentiation Ags), annexin V, and 7-AAD (see Materials and Methods), and analyzed by FACS for potential apoptotic (annexin V+ and 7-AAD–) cells. Lineage-positive cells were excluded from analysis. Results are from one of three experiments with similar results.
FIGURE 6. IFN- does not affect the cell cycle status of in vitro expanded CD34+ CB cells. Cell cycle status of freshly isolated and cultured (5 days in SFT3 in the absence or presence of IFN-) CD34+ CB cells stained with anti-CD34, anti-Ki67, and 7-AAD (see Materials and Methods). Ki67 and 7-AAD staining was specifically investigated on CD34+ gated cells. Numbers in quadrants represent mean values from three experiments.
Discussion
Clinical and experimental studies have strongly implicated IFN- as a key mediator of BM failure in a number of diseases associated with inflammation, such as graft-vs-host disease, myelodysplasic syndromes, and aplastic anemia (10, 11, 21, 47), and recent studies suggest that the ability of drugs, such as cyclosporine, to inhibit IFN- production might explain their beneficial effects on hemopoiesis in patients with immune-mediated BM suppression (47). Furthermore, IFN- produced by human stromal microenvironment has been demonstrated to negatively affect hemopoiesis (16, 47). In agreement with this, IFN- inhibits the in vitro growth of hemopoietic progenitors, including primitive human CD34+CD38– BM cells (15). However, whether or not such growth inhibition is associated with sustained, enhanced, or reduced HSC function had not been investigated before these studies.
Using recently developed conditions promoting in vitro self-renewal of candidate human HSCs (32), we found that IFN- negatively affects in vitro maintenance of cycling human CB HSCs capable of multilineage reconstitution in vivo. Importantly, IFN- did not affect cytokine-induced recruitment of CD34+CD38– HSC into proliferation, but rather reduced the number of subsequent cell divisions, as reflected in reduced clonal expansion. However, the relatively limited reduction in cellular proliferation was accompanied by a striking loss in HSC function as well as enhanced differentiation, suggesting that IFN- might negatively affect HSC maintenance, by promoting HSC commitment and differentiation rather than self-renewal.
Whereas we in recent studies demonstrated a comparable negative effect of TNF on CB and adult BM candidate HSCs (23), we in this study only investigated the effect on CB stem and progenitor cells. Thus, although we would postulate that IFN- would have similar suppressive effects on BM HSCs, as in this study demonstrated for CB stem and progenitor cells, this remains to be established.
Importantly, the conclusion that IFN- potently suppresses HSC self-maintenance in vitro was supported by evaluation of two key properties of human HSCs, the ability to maintain long-term cultures in vitro (42) and their in vivo multilineage-repopulating activity (41, 46). Although our data support that the ability of IFN- to reduce the reconstituting potential of self-renewing HSCs is due to enhanced differentiation, it remains possible that IFN- might also affect HSC potential through effects on HSC adhesion/engraftment, although IFN- was only present in the culture supporting self-renewing divisions and not in the LTC-IC assays.
In these studies, we found no indication that the suppressive role of IFN- involves effects on cell cycle or apoptosis. However, these conclusions are significantly limited by the fact that functionally defined HSCs represent a minority of the investigated CD34+ as well as CD34+CD38– populations. Thus, we cannot exclude HSC-specific effects of IFN- on cell cycle and apoptosis, as such effects might have been obscured by the presence of large numbers of more committed progenitors.
Although the present results demonstrated that IFN- at high concentrations negatively affects HSC maintenance, it remains uncertain to what degree these concentrations of IFN- are relevant for acute and chronic inflammatory BM-suppressive syndromes, in particular because the local concentrations of IFN- in the BM have not been established.
In conclusion, the present studies provide the first evidence that the suppressive effects of IFN- in BM failure syndromes might involve direct targeting and suppression of repopulating HSCs.
Acknowledgments
We thank Zhi Ma and Anna Fossum for expert assistance with cell sorting, and Gunilla G?rdebring, Lilian Wittman, and Ingbritt ?strand-Grundstr?m for technical assistance and animal care. We thank Amgen, Genentech, and Boehringer Mannheim for generous provision of cytokines for these studies. We are grateful to the staff and donors at the Department of Gynecology, Lund University Hospital and Helsingborg Hospital, for help with providing CB.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the Georg Danielsson Foundation; the Gunnar, Arvid, and Elisabeth Nilsson Foundation; the John and Augusta Persson Foundation; the O. and E. and Edla Johansson Foundation; the Thelma Zoega’s Foundation; the Tobias Foundation; the Greta and Johan Kock’s Foundations; Government Public Health Grant; Sk?nes Landsting; the Swedish Foundation for Strategic Research; the Swedish Cancer Society; Swedish Society of Pediatric Cancer and the Medical Faculty, University of Lund. We declare that we have no competing financial interests.
2 Address correspondence and reprint requests to Dr. Sten Eirik W. Jacobsen, Lund Stem Cell Center, Biomedical Center B10, Lund University, 221 84 Lund, Sweden. E-mail address: Sten.Jacobsen@stemcell.lu.se
3 Abbreviations used in this paper: HSC, hemopoietic stem cell; 7-AAD, 7-aminoactinomycin D; BM, bone marrow; CB, umbilical cord blood; CFC, colony-forming cell; EPO, erythropoietin; FL, flt3 ligand; LTC-IC, long-term culture-initiating cell; rh, recombinant human; SCF, stem cell factor; SF, serum free; SFT3, SCF + FL + thrombopoietin + IL-3; TPO, thrombopoietin.
Received for publication May 14, 2004. Accepted for publication November 2, 2004.
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Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN- might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN- on HSC maintenance and function. Although purified cord blood CD34+CD38– cells underwent cell divisions in the presence of IFN-, cycling HSCs exposed to IFN- in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN- accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN- can negatively affect human HSC self-renewal.
Introduction
Life-long hemopoiesis is dependent on hemopoietic stem cells (HSCs)3 that self-renew to maintain the HSC pool and, by multilineage differentiation, give rise to all the myeloid and lymphoid cell lineages of the blood (1). Although tightly regulated, little is known about the specific regulatory pathways controlling HSC fate decisions. Loss of function studies have demonstrated important roles of multiple stimulatory cytokines, such as c-kit ligand (or stem cell factor, SCF) and thrombopoietin (TPO), in maintenance of the HSC pool (2, 3). However, because HSCs are thought not to have unlimited self-renewal potential, it appears likely that also negative regulators might be involved in controlling the HSC pool. TNF (4, 5, 6), TGF- (7, 8, 9), Fas (10, 11, 12, 13), and IFNs (14, 15, 16, 17) have all been demonstrated to have suppressive effects on in vitro and in vivo hemopoiesis, and TNF- (18, 19), Fas (10, 11), and IFN- (11, 20, 21) have been implicated as important mediators of acute and chronic bone marrow (BM) failure syndromes, such as graft-vs-host disease and aplastic anemia.
Whereas TNF- recently has been demonstrated to negatively affect human (22, 23) and murine (24) HSCs, the ability of IFN- to modulate HSC fate and function remains to be investigated. Previous studies have demonstrated that depending on the conditions and the specific progenitors investigated, IFN- can either promote (25, 26) or inhibit (15, 16, 27) the growth of hemopoietic progenitors, including primitive human CD34+CD38– cells. However, to what degree such effects of IFN- on growth of progenitor/stem cells are associated with altered HSC numbers or function has not been explored.
Studies on HSCs in vitro have previously been hampered by the fact that most established culture conditions result in dramatic losses of HSCs (28, 29, 30). However, identification of combinations of early acting cytokines and improved overall HSC culture conditions promoting mouse HSC self-renewal have facilitated in vitro studies of the regulation of HSC fate decisions (31, 32, 33, 34). Importantly, such studies have confirmed the ability of growth-stimulating cytokines, such as SCF, TPO, flt3 ligand (FL), IL-11, and IL-3, to support mouse HSC self-renewal cell divisions (32, 33, 34). Development of similar conditions promoting self-renewal of candidate human HSCs (32, 35, 36, 37, 38, 39) as well as improvement of human HSC assays (40, 41, 42, 43) is now facilitating studies of human HSC regulation.
Using in vitro conditions recently demonstrated to efficiently promote proliferation of candidate murine and human HSCs with sustained HSC function (32, 33), we in this study explored, for the first time, the potential ability of IFN- to directly affect self-renewal of in vivo repopulating human HSCs.
Materials and Methods
Hemopoietic growth factors and IFN-
Human rSCF (rhSCF), rhG-CSF, rhFL, rhIL-3, IL-6, and rhGM-CSF were generous gifts from Amgen; rhTPO from Genentech; and recombinant human erythropoietin (rhEPO) from Boehringer Mannheim. rhIFN- was purchased from R&D Systems.
Isolation and purification of human stem and progenitor cells
Umbilical cord blood (CB) samples were obtained from normal full-term deliveries following informed consent from the mothers and with approval of the ethics committees at the University Hospital of Lund and Helsingborg Hospital. CD34+ CB cells were enriched according to previously described protocols (23, 32). Briefly, CB mononuclear cells were isolated from samples using Ficoll-Hypaque density gradient centrifugation (lymphoprep, 1.077 ± 0.001 g/ml; Nycomed Pharma). Positive selection of CD34+ cells was performed by a MACS-CD34 isolation kit (Miltenyi Biotec), according to the manufacturer’s instructions. CB CD34+ cells were run through a second column to obtain higher purity (91–94%) of CD34+ cells. CD34-enriched cells were incubated with anti-CD38 PE and anti-CD34 FITC mAbs or with isotype-matched irrelevant control Abs (all from BD Biosciences), and subsequently sorted on a FACSVantage cell sorter (BD Biosciences). A conservative approach was taken to exclusively sort the 3% lowest CD38-expressing CD34+ cells (CD34+CD38–), in an effort to obtain a highly purified population of primitive progenitor/stem cells. The purity of CD34+CD38– cells in the experiments was 96–98%.
In vitro expansion cultures
CD34+ and CD34+CD38– CB cells were cultured at 1000 cells/ml for 7 or 12 days in serum-free (SF) medium (IMDM; BioWhittaker) supplemented with 10 mg/ml BSA, 10 μg/ml human insulin, 200 μg/ml human transferrin (BIT; StemCell Technologies), and a mixture of cytokines (100 ng/ml rhSCF, 100 ng/ml rhFL, 100 ng/ml rhTPO, and 20 ng/ml rhIL-3, defined as SFT3) in the absence or presence of 1000 U/ml rhIFN-. Following culture, cells were enumerated and evaluated functionally in in vitro long-term cultures or by transplantation into NOD-SCID mice. In some experiments, cultured cells were further examined with regard to apoptosis, cell cycle status, and differentiation.
Single-cell clonogenic assays
As described previously (23), CD34+CD38– CB cells were seeded in Terasaki plates (Nunc) at a density of 1 cell/well in 20 μl of SF medium (X-vivo 15; BioWhittaker) and 1% BSA (StemCell Technologies), supplemented with a mixture of cytokines (50 ng/ml rhSCF, 50 ng/ml rhFL, 50 ng/ml rhTPO, and 20 ng/ml rhIL-3) and different concentrations of rhIFN-. Wells (120/group) were scored for cell growth following 11–12 days of incubation at 37°C in a humidified atmosphere with 5% CO2 in air.
Long-term culture-initiating cell (LTC-IC) assay
Establishment and maintenance of long-term cultures were performed according to previously described procedures (23). Briefly, stroma cell feeders were established by seeding a mixture (1:1) of two irradiated (8,000 cGy) murine fibroblast cell lines (M2-10B4 and sl/sl; kindly provided by D. Hogge, Vancouver, Canada), engineered to produce high levels of human G-CSF, IL-3, and SCF (42), into 96-well collagen-coated microtiter plates (Nunc) containing LTC medium (Myelocult; StemCell Technologies) supplemented with freshly dissolved 10–6 M hydrocortisone 21-hemisuccinate (Sigma-Aldrich). These cell lines have been demonstrated to detect candidate HSCs with enhanced efficiency when compared with stromal derived from primary BM cells (42). Each well contained 10,000 cells from the mixture of two cell lines. The freshly isolated or expansion equivalents of 50 CD34+CD38– CB cells cultured in SFT3 in the absence or presence of rhIFN- for 7 days were seeded per stroma well (four replicates per group) and incubated for 6 wk at 37°C in a humidified atmosphere with 5% CO2 in air. Cocultures were maintained by weekly 50% medium changes.
Following 6 wk of culture, adherent and nonadherent cells from each well were transferred to methylcellulose cultures containing rhSCF, rhFL, rhIL-3, rhGM-CSF, rhG-CSF (all at 10 ng/ml), and rhEPO (5 U/ml). To ensure formation of a reliable number of colonies from the long-term culture, the content of each well was transferred to methylcellulose cultures at both a low (20% of cells) and a high concentration (80% of cells). LTC colony-forming cells (LTC-CFCs) were scored after an additional 10–12 days of culture. Stroma cells without hemopoietic cells and cytokines were used as a negative control for the CFC assay.
NOD-SCID-repopulating assay
NOD-SCID mice (originally from The Jackson Laboratory, Bar Harbor, ME) were bred and housed under sterile conditions and maintained on autoclaved food and acidified water. All animal procedures were performed with consent from the local ethics committee at Lund University. At 8–12 wk of age, mice were irradiated with 350 cGy from a 137Cs source. The transplantation of 5 x 104 to 1 x 105 CD34+ or 5000 CD34+CD38– CB cells together with 1 x 106 irradiated (1500 cGy) accessory cells (CD34-depleted CB cells) in 0.5 ml of medium was performed by tail vein injection within 4 h of irradiation. Mice were sacrificed after 6 wk by asphyxiation with CO2, and femora and tibiae were collected; and engraftment was investigated by flow cytometry (FACSCalibur; BD Biosciences) using CellQuest analysis software (BD Biosciences), as described previously (23, 40, 41). Briefly, BM cells were counted and blocked with anti-mouse CD16/CD32 (BD Pharmingen,) and ChromPure mouse IgG, whole molecule (Jackson ImmunoResearch Laboratories). Subsequently, cells were stained with FITC-conjugated anti-human CD45 and CD71 Abs (BD Biosciences) as well as anti-mouse CD45.1-PE Ab (BD Pharmingen). BM cells from untransplanted mice (negative controls) and mixtures of 0.1% human cells in NOD-SCID BM (positive controls) were included. If engraftment was detected as human CD45/CD71 positive (detection level 0.05%), lineage analysis with anti-human CD34 FITC (progenitors), anti-human CD19 PE (B cells; BD Biosciences), anti-human CD15 PE, and anti-human CD66b FITC (myeloid; both BD Pharmingen) combined with anti-human CD45 allophycocyanin (BD Biosciences) was performed. For all samples, 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) was included to gate out dead cells. A minimum of 5 x 104 BM cells was examined for each sample. Only mice with both positive myeloid and lymphoid engraftment (defined as >10 positive events each per 5 x 104 viable BM cells with maximum 1 event in corresponding controls) were evaluated as positive. If no engraftment was detected by flow cytometry or if the myeloid engraftment was questionable, BM cells were plated in methylcellulose supplemented with human-specific cytokines (25 ng/ml rhSCF, 25 ng/ml rhIL-3, 50 ng/ml rhGM-CSF) and 5 U/ml rhEPO at a density of 105 cells/35-mm plate, four replicates per group. CFU granulocyte-macrophage and burst-forming unit erythroid were scored after 10–12 days. No colonies were observed in the absence of cytokines or from BM of untransplanted mouse cultured with the same cytokines (L. Yang and S. E. W. Jacobsen, unpublished observations).
Flow cytometric evaluation of differentiation, apoptosis, and cell cycle status of cultured CB progenitors
To evaluate differentiation, CD34+ CB cells were cultured for 9 or 12 days in SF medium (X-vivo 15; BioWhittaker) with 1% BSA (StemCell Technologies) or IMDM (BioWhittaker) with 20% FCS (BioWhittaker), supplemented with SFT3 and SFT3 + IL-6 + GM-CSF + G-CSF, respectively, in the absence or presence of 1000 U/ml rhIFN-. Following culture, cells were stained with anti-human CD34 FITC or allophycocyanin and a lineage mixture containing PE- or FITC-conjugated Abs (against CD11b, CD14, CD15, CD33, CD41, CD66b, and glycophorin A) and 7-AAD (to exclude nonviable cells). Control samples of cultured cells were stained with irrelevant isotype-matched control Abs. Samples were analyzed on a FACSCalibur
Apoptosis of CD34+ CB cells was assessed by measuring redistribution of phosphatidylserine using annexin V-PE (BD Pharmingen) and uptake of 7-AAD, as described before (23, 44), with minor modifications. Briefly, cultured CD34+ CB cells (5 days) were first stained for surface markers using anti-human CD34 allophycocyanin and anti-lineage Abs, and subsequently stained with annexin V-PE (5 μl) and 7-AAD (5 μg/ml) in 100 μl of annexin V-binding buffer (BD Pharmingen) for 15 min, resuspended in annexin V-binding buffer, and analyzed on a FACSCalibur.
Cell cycle status of freshly isolated or cultured (5 days) CD34+ CB cells was determined, as described (45), with minor modifications. Cells were stained with anti-human CD34 allophycocyanin, followed by fixation and permeabilization with Cytofix/Cytoperm kit (BD Pharmingen) for 30 min. After washing, cells were stained with FITC-conjugated anti-Ki67 (Beckman-Coulter) or an isotype-matched irrelevant control Ab for 30 min. After 3 h of incubation in PBS containing 5% FCS supplemented with 7-AAD (5 μg/ml) at 4°C under dark conditions, samples were analyzed on a FACSCalibur.
Results
IFN- inhibits clonal expansion of human candidate HSCs
We have recently demonstrated that in vitro culture under SF conditions in the presence of the early acting cytokines SFT3 efficiently promotes recruitment of murine and candidate human HSCs into proliferation with sustained stem cell function (32, 33). In this study, we used this system to investigate the effect of IFN- on self-renewal of human HSCs. CD34+CD38– CB cells proliferated extensively in the presence of SFT3, and this expansion was inhibited by 35% in response to IFN- (Fig. 1).
FIGURE 1. IFN- inhibits the in vitro cellular expansion of CD34+CD38– CB cells. A total of 1000 cells/ml was cultured in SF medium supplemented with SFT3 in absence or presence of 1000 U/ml IFN- for 7 days. Data represent the mean (SD) from three individual experiments. *, p < 0.05, comparing –IFN- and +IFN-.
To investigate whether IFN- might potentially block the first cell divisions of candidate HSCs, single cells were cultured in SFT3 in the presence or absence of different concentrations of IFN- (10–1000 U/ml; Fig. 2). Importantly, at all used concentrations, IFN- did not affect the number of proliferating clones. However, the size of the clones was reduced in a dose-dependent manner. These findings suggested that IFN- does not affect cytokine-induced recruitment of CD34+CD38– candidate HSCs into proliferation, but rather inhibits their clonal expansion.
FIGURE 2. IFN- does not affect cytokine-induced recruitment of CD34+CD38– candidate HSCs into proliferation, but inhibits their clonal expansion. CD34+CD38– CB cells were plated at a density of 1 cell/well in 20 μl of SF medium supplemented with SFT3 in the absence or presence of increasing concentrations of IFN-. Each group, consisting of 120 wells, was scored for clonal growth (clone number and size) after 11–12 days of culture. Large colonies were defined as clones covering >10% of the well. Results are presented as means (SEM) of three individual experiments. *, p < 0.05, comparing –IFN- and +IFN-.
IFN- negatively affects maintenance of CD34+CD38– CB LTC-IC under self-renewing conditions
To investigate the effect of IFN- on maintenance of candidate human HSCs, we cultured CD34+CD38– CB cells in SFT3 in the presence or absence of IFN-, and subsequently evaluated the LTC-IC colony-forming cells (LTC-CFCs) in such cultures. In agreement with previous studies (23, 32), SFT3 promoted maintenance of high levels of LTC-CFC activity after 7 days of culture, comparable to uncultured input cells with an average of 343 LTC-CFCs derived from the initiating 50 CD34+CD38– CB cells (Fig. 3). In the presence of IFN-, LTC-CFCs were reduced by as much as 89% (Fig. 3).
FIGURE 3. IFN- negatively modulates self-renewal of CD34+CD38– candidate stem cells with LTC-CFC activity. Fifty CD34+CD38– cells were cultured for 7 days in SF medium with SFT3 in the absence or presence of 1000 U/ml IFN- and subsequently evaluated for 6-wk LTC-CFC activity (see Materials and Methods). Also shown is the number of LTC-CFC generated from 50 freshly isolated CD34+CD38– cells. Data (CFC generated by 50 CD34+CD38– cells after 6-wk LTC) are presented as means (SEM) of two individual experiments, each with four replicates cultures. *, p < 0.05, comparing –IFN- and +IFN-.
IFN- negatively modulates maintenance of multipotent NOD-SCID-repopulating HSCs under self-renewing conditions
The NOD-SCID xenograft assay is thought to detect more primitive human hemopoietic cells than the LTC-IC assay (40, 41, 46). In addition, the NOD-SCID assay allows evaluation of in vivo multilineage-repopulating ability of candidate human HSCs. In a total of three experiments using CD34+ cells and one using highly purified CD34+CD38– cells, SFT3 cultures maintained high levels of multilineage NOD-SCID reconstitution activity that was almost completely abolished in the presence of IFN- (Table I and Fig. 4). This was reflected in the mean human reconstitution being reduced from 11 to 0.3%. Furthermore, whereas 100% of mice transplanted with SFT3-cultured cells were multilineage reconstituted, only 18% of mice transplanted with cells exposed to IFN- were positive for human myeloid reconstitution (Table I).
Table I. IFN- potently inhibits self-renewal of in vivo multilineage NOD-SCID reconstituting cells
FIGURE 4. IFN- negatively modulates self-renewal of multipotent NOD-SCID-repopulating CD34+CD38– CB cells. FACS profile shows multilineage human engraftment of representative mice transplanted with expansion equivalent of 5000 CD34+CD38– cells cultured for 7 days in SFT3 in the absence or presence of 1000 U/ml IFN- (see Materials and Methods). Of all mice transplanted with CD34+CD38– cells exposed to IFN-, the one shown here had the highest level of human engraftment.
IFN- promotes differentiation of human hemopoietic progenitor/stem cells under self-renewing conditions
To investigate potential mechanisms by which IFN- might negatively affect maintenance of HSCs, we examined whether IFN- might promote differentiation at the expense of self-renewal. Following 12 days of culture under self-renewing conditions (SFT3), 18% of cells continued to express the stem/progenitor cell Ag CD34, which was down-regulated upon myeloid differentiation. In contrast, only 2% of cells cultured under the same conditions, but in the presence of IFN-, remained CD34+ (Table II), compatible with IFN- promoting differentiation rather than self-renewal of CD34+ stem/progenitor cells. To further investigate this, CD34+ cells were also cultured under conditions more efficiently promoting lineage differentiation (SFT3 + IL-6 + G-CSF + GM-CSF). Under these conditions, the maintenance of cells with a stem/progenitor CD34+ as well as lineage-negative phenotype was reduced significantly in the presence of IFN- (Table II). Specifically, whereas 40% of CD34+ cells cultured in the absence of IFN- remained lineage negative, as much as 92% of cells cultured in the presence of IFN- became positive for myeloid lineage Ags. In further support of IFN- promoting myeloid differentiation and in line with the reduced CD34 expression, the number of CFU-C was reduced almost 3-fold in IFN--containing cultures (Table II).
Table II. IFN- promotes differentiation of in vitro expanded CD34+ CB cells
IFN- did not seem to significantly affect apoptosis of cultured progenitors because a combined annexin V and 7-AAD staining demonstrated comparable numbers of apoptotic cells in control and IFN--supplemented cultures (Fig. 5). Cellular maintenance also appeared unaffected by IFN-, as CD34+CD38– cells cultured in the absence and presence of IFN- showed indistinguishable forward scatter profiles (L. Yang and S. E. W. Jacobsen, unpublished observations). Furthermore, IFN- did not affect the fraction of cells in S/G2/M of CD34+ progenitors cultured in SFT3 (Fig. 6). Thus, IFN- might, at least in part, negatively affect the maintenance of primitive hemopoietic progenitor/stem cells by promoting their differentiation rather than self-renewal.
FIGURE 5. IFN--induced inhibition of human CD34+ progenitor/stem cells is not accompanied by enhanced apoptosis. CD34+ CB cells cultured for 5 days in STF3 in the absence or presence of IFN- (1000 U/ml) were stained with anti-CD14/15/66b (myeloid differentiation Ags), annexin V, and 7-AAD (see Materials and Methods), and analyzed by FACS for potential apoptotic (annexin V+ and 7-AAD–) cells. Lineage-positive cells were excluded from analysis. Results are from one of three experiments with similar results.
FIGURE 6. IFN- does not affect the cell cycle status of in vitro expanded CD34+ CB cells. Cell cycle status of freshly isolated and cultured (5 days in SFT3 in the absence or presence of IFN-) CD34+ CB cells stained with anti-CD34, anti-Ki67, and 7-AAD (see Materials and Methods). Ki67 and 7-AAD staining was specifically investigated on CD34+ gated cells. Numbers in quadrants represent mean values from three experiments.
Discussion
Clinical and experimental studies have strongly implicated IFN- as a key mediator of BM failure in a number of diseases associated with inflammation, such as graft-vs-host disease, myelodysplasic syndromes, and aplastic anemia (10, 11, 21, 47), and recent studies suggest that the ability of drugs, such as cyclosporine, to inhibit IFN- production might explain their beneficial effects on hemopoiesis in patients with immune-mediated BM suppression (47). Furthermore, IFN- produced by human stromal microenvironment has been demonstrated to negatively affect hemopoiesis (16, 47). In agreement with this, IFN- inhibits the in vitro growth of hemopoietic progenitors, including primitive human CD34+CD38– BM cells (15). However, whether or not such growth inhibition is associated with sustained, enhanced, or reduced HSC function had not been investigated before these studies.
Using recently developed conditions promoting in vitro self-renewal of candidate human HSCs (32), we found that IFN- negatively affects in vitro maintenance of cycling human CB HSCs capable of multilineage reconstitution in vivo. Importantly, IFN- did not affect cytokine-induced recruitment of CD34+CD38– HSC into proliferation, but rather reduced the number of subsequent cell divisions, as reflected in reduced clonal expansion. However, the relatively limited reduction in cellular proliferation was accompanied by a striking loss in HSC function as well as enhanced differentiation, suggesting that IFN- might negatively affect HSC maintenance, by promoting HSC commitment and differentiation rather than self-renewal.
Whereas we in recent studies demonstrated a comparable negative effect of TNF on CB and adult BM candidate HSCs (23), we in this study only investigated the effect on CB stem and progenitor cells. Thus, although we would postulate that IFN- would have similar suppressive effects on BM HSCs, as in this study demonstrated for CB stem and progenitor cells, this remains to be established.
Importantly, the conclusion that IFN- potently suppresses HSC self-maintenance in vitro was supported by evaluation of two key properties of human HSCs, the ability to maintain long-term cultures in vitro (42) and their in vivo multilineage-repopulating activity (41, 46). Although our data support that the ability of IFN- to reduce the reconstituting potential of self-renewing HSCs is due to enhanced differentiation, it remains possible that IFN- might also affect HSC potential through effects on HSC adhesion/engraftment, although IFN- was only present in the culture supporting self-renewing divisions and not in the LTC-IC assays.
In these studies, we found no indication that the suppressive role of IFN- involves effects on cell cycle or apoptosis. However, these conclusions are significantly limited by the fact that functionally defined HSCs represent a minority of the investigated CD34+ as well as CD34+CD38– populations. Thus, we cannot exclude HSC-specific effects of IFN- on cell cycle and apoptosis, as such effects might have been obscured by the presence of large numbers of more committed progenitors.
Although the present results demonstrated that IFN- at high concentrations negatively affects HSC maintenance, it remains uncertain to what degree these concentrations of IFN- are relevant for acute and chronic inflammatory BM-suppressive syndromes, in particular because the local concentrations of IFN- in the BM have not been established.
In conclusion, the present studies provide the first evidence that the suppressive effects of IFN- in BM failure syndromes might involve direct targeting and suppression of repopulating HSCs.
Acknowledgments
We thank Zhi Ma and Anna Fossum for expert assistance with cell sorting, and Gunilla G?rdebring, Lilian Wittman, and Ingbritt ?strand-Grundstr?m for technical assistance and animal care. We thank Amgen, Genentech, and Boehringer Mannheim for generous provision of cytokines for these studies. We are grateful to the staff and donors at the Department of Gynecology, Lund University Hospital and Helsingborg Hospital, for help with providing CB.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the Georg Danielsson Foundation; the Gunnar, Arvid, and Elisabeth Nilsson Foundation; the John and Augusta Persson Foundation; the O. and E. and Edla Johansson Foundation; the Thelma Zoega’s Foundation; the Tobias Foundation; the Greta and Johan Kock’s Foundations; Government Public Health Grant; Sk?nes Landsting; the Swedish Foundation for Strategic Research; the Swedish Cancer Society; Swedish Society of Pediatric Cancer and the Medical Faculty, University of Lund. We declare that we have no competing financial interests.
2 Address correspondence and reprint requests to Dr. Sten Eirik W. Jacobsen, Lund Stem Cell Center, Biomedical Center B10, Lund University, 221 84 Lund, Sweden. E-mail address: Sten.Jacobsen@stemcell.lu.se
3 Abbreviations used in this paper: HSC, hemopoietic stem cell; 7-AAD, 7-aminoactinomycin D; BM, bone marrow; CB, umbilical cord blood; CFC, colony-forming cell; EPO, erythropoietin; FL, flt3 ligand; LTC-IC, long-term culture-initiating cell; rh, recombinant human; SCF, stem cell factor; SF, serum free; SFT3, SCF + FL + thrombopoietin + IL-3; TPO, thrombopoietin.
Received for publication May 14, 2004. Accepted for publication November 2, 2004.
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