Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.     

Dendritic cell differentiation from hematopoietic CD34+ progenitor cells

Dendritic cells (DC) are the most powerful antigen presenting cells (APC) and play a pivotal role in initiating the immune response. In light of their unique properties, DC have been proposed as a tool to enhance immunity against infectious agents and in anticancer vaccine strategies. In the last few years, the development of DC has been extensively investigated. The present paper summarizes the most recent findings on the differentiation of myeloid DC from hematopoietic CD34+ progenitors and methods for DC generation in vitro. A better understanding of DC function has important implications for their use in clinical settings.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Detection of cytokines in supernatant from hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and endothelial progenitor cells

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.     

低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

Notch signaling specifies megakaryocyte development from hematopoietic stem cells

In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with gamma-secretase inhibitors or by expression of the dominant-negative Mastermind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.     

A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34(+) cells isolated from umbilical cord blood

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ～15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.     

Normal and leukemic CD34-negative human hematopoietic stem cells

Considerable progress has been made in recent years in purifying human and murine hematopoietic stem cells. The essential marker identified is the sialomucin CD34, which is expressed on primitive cells and downregulated as they differentiate into more abundant mature cells. CD34 is not unique to stem cells, however, as it is also expressed on clonogenic progenitors and some endothelial cells. Nevertheless, all clinical and experimental protocols are targeted to CD34+ cells enriched by a variety of selection methods. Recent studies in both the murine and human systems have indicated that some stem cells capable of multilineage repopulation do not express detectable levels of cell surface CD34. These studies challenge the dogma that all human repopulating cells are found in the CD34+ subset. However, the precise relationship between CD34- and CD34+ stem cells is still not well understood. In this review, the results on the discovery of the CD34- repopulating cell are summarized and the impacts this discovery may have, both clinically and in our understanding of the organization of the human hematopoietic system, are examined.     

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.     

骨髓间充质干细胞对脐血CD34+细胞分化为巨核细胞的影响

目的：探讨骨髓间充质干细胞对脐血CD34＋细胞诱导分化为巨核细胞的影响。方法：骨髓间充质干细胞培养采用低糖型DMEM培养基,待细胞满度达到约80%后加入脐血CD34＋细胞在一定的培养体系中进行实验,同时以无骨髓间充质干细胞的相应培养体系作为对照,培养14 d后观察结果。实验中共观察了两种不同的培养体系：基础培养液、基础培养液＋白细胞介素-11（IL-11）。其中基础培养液为含血小板生成素（TPO）、白细胞介素-3（IL-3）、干细胞因子（SCF）的低糖型DMEM。培养后单个核细胞数采用细胞计数仪分析,CD41＋细胞和血小板检测采用流式细胞仪,血小板功能评价采用凝血酶诱导的血小板凝集实验。结果：与相应的对照组比较,骨髓间充质干细胞实验组单个核细胞数增加不明显（P〉0.05）,而CD41＋细胞和血小板数量有明显的增加（P〈0.05）。显微镜下和流式细胞仪上均可观察到凝血酶诱导的血小板凝集现象。结论：骨髓间充质干细胞在实验培养体系中可以促进脐血中CD34＋细胞诱导分化为巨核细胞。     

Human mesenchymal stem cells license adult CD34+ hemopoietic progenitor cells to differentiate into regulatory dendritic cells through activation of the Notch pathway

The mechanisms underlying the immunomodulatory functions of mesenchymal stem cells (MSC) on dendritic cells (DC) have been shown to involve soluble factors, such as IL-6 or TGF-beta, or cell-cell contact, or both depending on the report referenced. In this study, we intend to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34(+) hemopoietic progenitor cells (HPC). MSC have been shown to inhibit interstitial DC differentiation from monocytes and umbilical CD34(+) HPC. In this study, we confirm that MSC not only halt interstitial DC but also Langerhans cell differentiation from adult CD34(+) HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80, and CD83 Ags on their cell surface. Accordingly, the functional capacity of CD34(+) HPC-derived DC (CD34-DC) to stimulate alloreactive T cells was impaired. Furthermore, we showed that 1) MSC inhibited commitment of CD34(+) HPC into immature DC, but not maturation of CD34-DC, 2) this inhibitory effect was reversible, and 3) DC generated in coculture with MSC (MSC-DC) induced the generation of alloantigen-specific regulatory T cells following secondary allostimulation. Conditioned medium from MSC cultures showed some inhibitory effect independent of IL-6, M-CSF, and TGF-beta. In comparison, direct coculture of MSC with CD34(+) HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC-DC. Finally, DAPT, a gamma-secretase inhibitor that inhibits Notch signaling, was able to overcome MSC-DC defects. In conclusion, our data suggest that MSC license adult CD34(+) HPC to differentiate into regulatory DC through activation of the Notch pathway.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.     

人造血干／祖细胞多态性的研究：Ⅷ.人正常骨髓CD34^＋造血细胞体视学的特征

人ＣＤ３４＋造血细胞是具有高度自我更新,多向分化及重建长期造血与免疫学功能的独特体细胞。为系统探索ＣＤ３４＋造血细胞的形态,细胞化学及超微结构特征,新近我们设计组合并建立了ＣＩＭＳ－１００ＦＡＣＳ４４０无菌二次分选术,可使所获ＣＤ３４＋５造血的纯度达１００％。