Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors

We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M-CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL-3)/granulomonocyte-CSF (GM- CSF)/erythropoietin (Epo), FL potentiates colony-forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, whereas M-CSF alone stimulates prevalent monocytic differentiation but little cell proliferation: combined M- CSF+FL treatment causes both proliferation and almost exclusive monocytic differentiation (97% monocytes in fetal calf serum-rich (FCS+) culture conditions, mean value). At primitive HPC level, FL potentiates the clonogenetic capacity of colony-forming units-blast (CFU-B) and high proliferative potential colony-forming cells (HPP-CFC) in primary and secondary culture; KL exerts a similar action, and additive effects are induced by FL combined with KL. More important, addition of FL alone causes a significant amplification of the number of long-term culture-initiating cells (LTC-ICs), ie, putative repopulating HSCs, whereas this effect is not induced by KL. The FL effects correlate with flt3 mRNA expression in HPCs differentiating throught the erythroid or GM pathway in liquid suspension culture: (1) flt3 mRNA is expressed in freshly purified, resting HPCs; after growth factor stimulus the message (2) is abruptly down-modulated in HPC erythroid differentiation, but (3) is sustainedly expressed through HPC GM differentiation and abolished in GM precursor maturation. This pattern contrasts with the gradual downmodulation of c-kit through both erythroid and GM HPC differentiation. The results indicate that FL exerts a stimulatory action on primitive HPCs, including a unique expanding effect on putative stem cells, whereas its distal proliferative/differentiative action is largely restricted to CFU-GM and monocytic precursors. The latter effect is potentiated by KL and M- CSF, thus suggesting that the structural similarities of FL, KL, M-CSF, and their tyrosine kinase receptors may mediate positive interactions of these growth factors son monocytic differentiation.     

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs.     

Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.     

类风湿关节炎患者外周血CD34+造血干/祖细胞的检测及其意义

目的 探讨类风湿关节炎(RA)外周血造血干/祖细胞(HSC/HPC)的数量及细胞膜CD34平均荧光强度(MFI)变化及其与临床指标的关系,以期阐明其在RA中的意义.方法 收集34例RA患者和16名健康对照者外周血,利用单克隆抗体标记CD34+细胞,流式方法测定CD34+HSC/HPC所占外周血淋巴细胞的比例及膜CD34的MFI,分析其与外周血细胞计数、疾病活动病程和药物应用的关系.数据分析采用t检验和方差分析,相关性分析采用Pearson相关分析.结果 ①RA患者CD34+细胞在外周血中淋巴细胞中所占的比例比健康人偏低[分别为(0.13±0.09)%和(0.38±0.21)%,P＜0.05],但两者所占外周血淋巴细胞的比例均低于0.5%;但MFI偏高(分别为57±33和31±11,P＜0.05).②RA患者CD34+细胞在外周血淋巴细胞中所占的比例与外周血红细胞计数、血红蛋白浓度呈正相关性,和C反应蛋白呈负相关;其MFI与健康评价问卷表(HAQ)评分、X线分期呈正相关,与血小板计数呈负相关.③RA患者CD34+细胞在外周血淋巴细胞中所占比例的降低程度以及MFI与疾病的活动性、病程和用药情况无明显相关性(P＞0.05).结论 造血干细胞可能在RA的发病机制中起作用.     

Stimulating spectrum of human recombinant multi-CSF (IL-3) on human marrow precursors: importance of accessory cells

Recently, human multi-CSF was obtained by molecular cloning. In the present study, the effects of multi-CSF in vitro were investigated by comparative culture of whole bone marrow or progenitor cells obtained by sorting the cell fraction that binds the monoclonal antibody (MoAb) B13C5 (CD 34). Multi-CSF stimulated erythroid (BFU-E), multipotential (CFU-GEMM) and eosinophil (CFU-Eo) colonies in cultures of the progenitor cell enriched fraction, whereas (besides BFU-E, CFU-GEMM, and CFU-Eo) granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), and macrophage (CFU-M) colony-forming cells also were stimulated by multi- CSF when unfractionated bone marrow was cultured. Reconstitution of the progenitor cell fraction (B13C5 positive) with the B13C5-negative population restored the broad spectrum of progenitor cell stimulation. This suggested that accessory cells are required for expression of the full spectrum of progenitor cell stimulation by multi-CSF. Subsequently, specific marrow cell populations, including T lymphocytes, granulocytic cells, and monocytes, were prepared by using selected MoAbs in complement-mediated lysis or cell sorting, added to cultures of hematopoietic progenitors and tested for accessory cell function. The results demonstrate that small numbers of monocytes permit the stimulation of CFU-G, CFU-GM, and CFU-M by multi-CSF. These monocyte-dependent stimulating effects on CFU-G, CFU-GM, and CFU-M could also be achieved by adding recombinant GM-CSF as a substitute for monocytes to the cultures. Therefore, multi-CSF most likely has direct stimulative effects on BFU-E, CFU-GEMM, and CFU-Eo and indirect effects on CFU-G, CFU-GM, and CFU-M in the presence of monocytes.     

Lymphokine(s) from isolated T lymphocyte subpopulations support multilineage hematopoietic colony and megakaryocytic colony formation

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.     

Superior effect of 9‐cis retinoic acid (RA) compared with all‐trans RA and 13‐cis RA on the inhibition of clonogenic cellgrowth and the induction of apoptosis in OCI/AML‐2 subclones: is the p53 pathway involved?

In the present study, the effects of 9-cis retinoic acid (RA) and 13-cis RA on acute myeloblastic leukaemia (AML) cell growth and the induction of apoptosis as well as its relationship with bcl-2 and p53 were compared with those of all-trans RA (ATRA). The study was performed with the subclones of the retinoid-sensitive OCI/AML-2 cell line. The most prominent inhibitory effect on clonogenic cell growth and morphological apoptosis was shown by 9-cis RA. In addition, Western blotting revealed the most obvious translocation of p53 from cytosol to nucleus in the case of 9-cis RA, which was the only retinoid able to change the conformation of p53 from mutational to wild type, as demonstrated by flow cytometry. There was no difference between the retinoids in the downregulation of bcl-2 as analysed by Western blotting and flow cytometry. The RA receptor (RAR)-alpha antagonist had no effect on apoptosis in any of the three retinoids studied using the annexin V method. In conclusion, this study shows that 9-cis RA was a more potent agent than ATRA or 13-cis RA in inducing growth arrest and apoptosis in the OCI/AML-2 subclones. The effect was associated with the downregulation of bcl-2 and was hardly mediated through the RAR-alpha receptor, but might be related to the activation of p53.     

Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony- stimulating factor by human bone marrow stromal cells

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).     

All-trans retinoic acid at low concentration directly stimulates normal adult megakaryocytopoiesis in the presence of thrombopoietin or combined cytokines.

In order to investigate the direct effects of retinoids on normal adult hematopoietic progenitors, purified CD34+ cells were seeded in serum-free cultures in the presence of pharmacological (10(-6)) M or physiological (10(-12)) M concentrations of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) plus combinations of specific cytokines. 10(-6) M ATRA and 9-cis RA significantly decreased the number of granulomacrophagic, erythroid and megakaryocytic (CFU-meg) progenitors. On the other hand, 10(-12) M ATRA significantly promoted the growth of CFU-meg, in the presence either of thrombopoietin or of IL-3+ GM-CSF, and induced a reproducible stimulation of the immature CD34+DR- subset. In conclusion, our findings suggest that retinoic acids probably play a direct role in normal adult hematopoietic development at both physiological and pharmacological concentrations. The stimulatory effect on megakaryocytopoiesis should be considered in the perspective of a potential use of low-dose ATRA, combined with thrombopoietin or other cytokines, in pathological conditions where the megakaryocytic compartment is impaired and the stimulation of megakaryocytopoiesis is requested.     

Additive Inhibitory Effect of Experimentally Induced Hepatic Cirrhosis by Agonists of Peroxisome Proliferator Activator Receptor γ and Retinoic Acid Receptor

Peroxisome proliferator activator receptor (PPAR) ligands prevent liver fibrosis, while the role of all-trans retinoic acid (ATRA) and its metabolite 9-cis retinoic acid (9-cis RA) is less clear. We have investigated the ability of the combination of PPARγ ligand rosiglitazone (RSG) and of ATRA to prevent liver fibrosis. In vivo treatment with RSG or ATRA reduced fibrotic nodules, spleen weight, and hydroxyproline levels in rat model of thioacetamide-induced liver fibrosis. The combination of ATRA + RSG caused the strongest inhibition, accompanied by decreased expression of collagen I, α-smooth muscle actin, TGFβ1, and TNFα. In vitro studies showed that PPARγ ligand 15-deoxy-Δ12,14-prostaglandinJ(2)[PJ(2)] and RXR ligand 9-cis RA or PJ(2) and ATRA inhibited proliferation of hepatic stellate cells HSC-T6. 9-cis RA inhibited c-jun levels and also inhibited expression of its receptor RXRα in HSC-T6 cells. The combination of PPAR-γ and RAR agonists demonstrated an additive effect in the inhibition of TAA-induced hepatic fibrosis, due to inhibition of HSC proliferation and reduction of profibrotic TGFβ1 and proinflammatory TNFα.     

Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.     

All-trans retinoic acid (ATRA) enhances maintenance of primitive human hematopoietic progenitors and skews them towards myeloid differentiation in a stroma-noncontact culture system

OBJECTIVE: We have previously shown that hematopoietic progenitor cells (HPCs) from umbilical cord blood (UCB) can be maintained in a cytokine-supplemented stroma-noncontact (SNC) system. Here, we tested if all-trans retinoic acid (ATRA), known to improve expansion of murine hematopoietic stem cells, would enhance human HPC maintenance in a SNC culture system. METHODS: CD34+CD38-Lin- cells from UCB were cultured in transwells above AFT024 in the presence of Flt-3 ligand (FLT) and thrombopoietin (TPO), with or without ATRA. Total nucleated cells (TNC), colony-forming units (CFUs), long-term culture-initiating cells (LTC-ICs), myeloid-lymphoid initiating cells (ML-ICs) and SCID repopulating cells (SRCs) were evaluated 1 to 5 weeks after culture. RESULTS: All-trans retinoic acid (1 mumol/L) reduced expansion of CD34+CD38-Lin- TNC and CFUs after 2 to 5 weeks of culture. However, it significantly increased LTC-IC expansion after 1 to 3 and, even more so, 5 weeks of culture. ATRA also increased recovery of more primitive ML-ICs and SRCs. Increased HPC recovery appeared dependent on the presence of stromal cells, as LTC-IC expansion was significantly reduced when ATRA was added to stroma-free cultures. CONCLUSION: All-trans retinoic acid increases expansion of early HPCs in a stromal cell-dependent fashion.     

PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)     

Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.     

Effect of mast cell growth factor (c-kit ligand) on clonogenic leukemic precursor cells.

Mast cell growth factor (MGF), the ligand for the c-kit receptor, has been shown to be a hematopoietic growth factor that preferentially stimulates the proliferation of immature hematopoietic progenitor cells (HPC). We studied the effect of MGF on the in vitro growth of clonogenic leukemic precursor cells in the presence or absence of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or erythropoietin (EPO). Leukemic blood and bone marrow cells from patients with various types of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) in chronic phase, as well as bone marrow samples from patients with myelodysplastic syndromes (MDS) were studied. MGF as a single factor did not induce significant colony formation by clonogenic leukemic precursor cells. In the presence of IL-3 and/or GM-CSF, MGF weakly stimulated the colony formation by clonogenic precursor cells from patients with AML. In contrast, in the presence of IL-3 and/or GM-CSF, MGF strongly induced both size and number of leukemic colonies from patients with CML in chronic phase. Furthermore, in the presence of EPO, MGF strongly stimulated erythroid colony formation by CML precursor cells. Cytogenetic analysis of the colonies showed that all metaphases after 1 week of culture were derived from the leukemic clone. In patients with MDS, MGF strongly stimulated myeloid colony formation in the presence of IL-3 and/or GM-CSF (up to fourfold), and erythroid colony formation in the presence of EPO (up to eightfold). Not only the number, but also the size of the colonies increased. In the presence of MGF, the percentage of normal metaphases increased in three patients tested after 1 week of culture compared with the initial suspension, suggesting that the normal HPC were preferentially stimulated compared with the preleukemic precursor cells. In the absence of exogenous EPO and in the presence of 10% human AB serum, MGF in the presence of IL-3 and/or GM-CSF induced erythroid colony formation from normal bone marrow and patients with MDS or CML, illustrating that MGF greatly diminished the EPO requirement for erythroid differentiation. These results indicate that MGF may be a candidate as a hematopoietic growth factor to stimulate normal hematopoiesis in patients with acute myeloid leukemia, or with myelodysplastic syndromes.     

Role of endothelial cells in human hematopoiesis: modulation of mixed colony growth in vitro

The identification of clonal human multipotent hematopoietic progenitors has permitted an analysis of the growth factor requirements for these cells. Human endothelial cell cultures were used to examine the effects of media conditioned by the endothelial cells on human multipotent (CFU-mix) and committed erythroid (BFU-E, CFU-E) and myeloid (CFU-GM) precursors. These studies demonstrate that endothelial cells produce proteins of approximately 30,000 daltons, with isoelectric focusing points of 4.5 and 7.2, which stimulate the growth of human BFU-E and CFU-mix. A heat-labile protein(s) of 30,000 and 15,000 daltons stimulated the proliferation and differentiation of granulocyte-macrophage (CFU-GM) colonies. No erythropoietin was detected in endothelial cell supernatants. This suggests that endothelial cells, a normal component of marrow stroma, play an active role in the modulation of human hematopoietic stem cell growth.     

Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU- E]), and granulocyte-macrophage (colony-forming unit-granulocyte- macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU- GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU- GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.