Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.     

Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells

Adenovirus vectors have been used to transfer genes into both hematopoietic progenitor cells and tumor cells, including carcinoma cells that have metastasized to bone marrow (BM). However, the relative susceptibility of different subsets of hematopoietic cells is unknown. In permissive cells adenoviral-mediated gene transfer is mediated by the coxsackievirus and adenovirus receptor (CAR) protein and alpha(v) integrins expressed on the cell surface of the target cells. This prompted us to investigate the expression of CAR on subpopulations of hematopoietic cells, determine whether this protein played a role in adenovirus-mediated gene transfer of hematopoietic cells and whether we could modulate CAR to enhance gene transfer efficiency. In this report we show that CAR is expressed on approximately 40% of all human BM cells, including erythroid and myeloid cells, but not lymphoid cells. Of the CD34(+) cells, 10%-15% expressed CAR, but this did not include most colony-forming progenitor cells, nor the most primitive CD38(-) subpopulation. The presence of CAR correlated well with gene transfer efficiency, but we were unable to induce CAR expression on immature, noncommitted progenitor cells. In conclusion, our results show that primitive hematopoietic progenitor cells lack CAR expression, but that expression is acquired during erythroid and myeloid differentiation.     

Hematopoietic differentiation of embryoid bodies derived from the human embryonic stem cell line SNUhES3 in co-culture with human bone marrow stromal cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.     

Gene expression analysis of hematopoietic progenitor cells identifies Dlg7 as a potential stem cell gene

Inducible hematopoietic stem/progenitor cell lines represent a model for studying genes involved in self-renewal and differentiation. Here, gene expression was studied in the inducible human CD34+ acute myelogenous leukemia cell line KG1 using oligonucleotide arrays and suppression subtractive cloning. Using this approach, we identified Dlg7, the homolog of the Drosophila Dlg1 tumor suppressor gene, as downregulated at the early stages of KG1 differentiation. Similarly, Dlg7 was expressed in normal purified umbilical cord blood CD34+CD38- progenitors but not in the more committed CD34+CD38+ population. Dlg7 expression was not detected in differentiated cells obtained from hematopoietic colonies, nor was expression detected in purified T-cells, B-cells, and monocytes. When analyzed in different types of stem cells, Dlg7 expression was detected in purified human bone marrow-derived CD133+ progenitor cells, human mesenchymal stem cells, and mouse embryonic stem (ES) cells. Overexpression of Dlg7 in mouse ES cells increased their growth rate and reduced the number of EBs emerging upon differentiation. In addition, the EBs were significantly smaller, indicating an inhibition in differentiation. This inhibition was further supported by higher expression of Bmp4, Oct4, Rex1, and Nanog in EBs overexpressing Dlg7 and lower expression of Brachyury. Finally, the Dlg7 protein was detected in liver and colon carcinoma tumors but not in normal adjacent tissues, suggesting a role for the gene in carcinogenesis. In conclusion, our results suggest that Dlg7 has a role in stem cell survival, in maintaining stem cell properties, and in carcinogenesis. Disclosure of potential conflicts of interest is found at the end of this article.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

CDCP1 identifies a broad spectrum of normal and malignant stem/progenitor cell subsets of hematopoietic and nonhematopoietic origin

CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.     

脐血CD34+造血干/祖细胞基因表达图谱的研究

目的：通过脐血CD34^＋造血干／祖细胞的基因表达分析，理解造血干／祖细胞生物学特性。方法：利用MiniMACS免疫磁珠法从脐血细胞中分离CD34^＋造血干／祖细胞，提取总RNA，用SMART-PCR技术从微量RNA中扩增产生足够量的cDNA用于高密度点阵膜分析检测CD34^＋造血干／祖细胞表达的基因。结果：在所检测的588个基因中，发现63个基因具有显著的表达水平，其中18个基因强表达。这些基因主要涉及造血干细胞增殖、分化、应激响应、凋亡、转录调节以及细胞周期等。结论：对理解脐血干／祖细胞生物学性质以及指导造血干细胞体外培养提供了分子生物学基础。     

Identification and characterization of hemoangiogenic progenitors during cynomolgus monkey embryonic stem cell differentiation

We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2(high) CD34(-) and VEGFR-2(high) CD34(+) cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2(high) cells, particularly that of VEGFR-2(high) CD34(+) cells, in a dose-dependent manner. Although either population of VEGFR-2(high) cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2(high) CD34(+) cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2(high) CD34(-)or VEGFR-2(high) CD34(+) precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.     

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.     

AGM区来源的基质细胞促进造血干细胞扩增的体外实验研究

目的： 探讨主动脉-性腺-中肾（aorta-gonad-mesonephros，AGM）来源的基质细胞对造血干细胞（HSC）增殖的促进作用，为探寻HSC的体外扩增方法奠定实验基础。 方法： 分别从孕11 d BALB/c小鼠胚胎AGM区及6周龄小鼠骨髓分离、培养基质细胞，流式细胞仪等对基质细胞进行鉴定；利用小鼠胚胎干细胞（ESC）向造血细胞定向分化的模型，结合高增殖潜能集落（HPP-CFC）、原始细胞集落（BL-CFC）形成实验及流式细胞仪分析CD34+、CD34+Sca-1+细胞比例，对比研究AGM及骨髓基质细胞对ESC来源的HSC的扩增作用。 结果： 小鼠AGM和骨髓基质细胞在形态及表型上基本相似，均符合基质细胞的特征。AGM和骨髓基质细胞均可促进ESC来源的HPP-CFC的形成，但AGM基质细胞还可促进ESC来源的 BL-CFC的形成；流式细胞仪检测发现：在骨髓基质细胞支持下，CD34+细胞增加了3-4倍，但CD34+/Sca-1+却无明显增加；而在AGM基质细胞支持下CD34+、CD34+Sca-1+细胞均明显增加了4-5倍。 结论： AGM基质细胞在有效扩增小鼠HSC同时，能很好地维持HSC自我更新及多向分化的潜能。     

Mesenchymal stromal cells can be derived from bone marrow CD133+ cells: implications for therapy

It is known that the bone marrow (BM) CD133(+) cells play an important role in the hematopoietic compartment, but this is not their only role. The cells indeed can take part in vascular reconstitution when they become endothelial cells (EC), in skeletal muscle fiber regeneration when there is a switch in muscle precursors, and to cardiomyocyte phenotypic conversion when differentiating in cardiomyocytes-like cells. While the role in hematopoiesis and vasculogenesis of the selected cells is well established, their ability to differentiate along multiple non-EC lineages has not yet been fully elucidated. The goal of this study is to assert whether human CD133(+)BM-derived cells are able to differentiate in vitro, besides to blood cells, cell lineages pertinent to the mesoderm germ layers. To this end, we isolated CD133(+) cells using a clinically approved methodology and compared their differentiation potential to that of hematopoietic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) obtained from the same BM samples. In our culture conditions, CD133 expression was consistently decreased after passage 2, as well as the expression of the stemness markers c-kit and OCT4, whereas expression of Stage Specific Embryonic Antigen 4 (SSEA4) remained consistent in all different conditions. Expanded CD133 were also positive for HLA-ABC, but negative for HLA-DR, in accordance with what has been previously reported for MSCs. Moreover, CD133(+) cells from human BM demonstrated a wide range of differentiation potential, encompassing not only mesodermal but also ectodermal (neurogenic) cell lineages. CD133 antigen could be potentially used to select a cell population with similar characteristics as MSCs for therapeutic applications.     

Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.     

Higher expression of transcription targets and components of the nuclear factor-kappaB pathway is a distinctive feature of umbilical cord blood CD34+ precursors

Delayed engraftment, better reconstitution of progenitors, higher thymic function, and a lower incidence of the graft-versus-host disease are characteristics associated with umbilical cord blood (UCB) transplants, compared with bone marrow (BM). To understand the molecular mechanisms causing these intrinsic differences, we analyzed the differentially expressed genes between BM and UCB hematopoietic stem and progenitor cells (HSPCs). The expressions of approximately 10,000 genes were compared by serial analysis of gene expression of magnetically sorted CD34(+) cells from BM and UCB. Differential expression of selected genes was evaluated by real-time polymerase chain reaction on additional CD34(+) samples from BM (n = 22), UCB (n = 9), and granulocyte colony stimulating factor-mobilized peripheral blood (n = 6). The overrepresentation of nuclear factor-kappaB (NF-kappaB) pathway components and targets was found to be a major characteristic of UCB HSPCs. Additional promoter analysis of 41 UCB-overrepresented genes revealed a significantly higher number of NF-kappaB cis-regulatory elements (present in 22 genes) than would be expected by chance. Our results point to an important role of the NF-kappaB pathway on the molecular and functional differences observed between BM and UCB HSPCs. Our study forms the basis for future studies and potentially for new strategies to stem cell graft manipulation, by specific NF-kappaB pathway modulation on stem cells, prior to transplant.     

Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells

Hematopoietic and endothelial cells may be derived from a common precursor cell (hemangioblast) during embryogenesis; however, some evidence suggests that hematopoietic cells may emerge from endothelial cells. The onset of definitive hematopoiesis at E8.25 in the murine embryo is marked by high-level CD41 expression. We questioned whether these hematopoietic cells were derived directly from mesoderm cells or emerged from endothelium. At 8.25 days post coitus (dpc), CD41 was coexpressed with CD31, CD34, and Flk1 in some intraluminal round cells that appeared to arise from flattened endothelial cells lining yolk sac capillary vessels. Cell-sorting studies revealed that all subpopulations of cells expressing CD41 possessed hematopoietic activity. Surprisingly, Tie2(+)Flk1(+) cells, a phenotype enriched in adult endothelial progenitors, also displayed some hematopoietic progenitor activity in vitro, but this activity was restricted to the CD41(+) fraction; only endothelial cells were derived from freshly isolated Tie2 (+)Flk1(bright) CD41() cells. Tie2(+)Flk1(dim)CD41() 8.25-dpc yolk sac cells devoid of hematopoietic progenitor activity gave rise to endothelial-like capillary networks in vitro and differentiated upon co-culture with OP9 stromal cells into definitive hematopoietic progenitors. These results demonstrate that CD41-expressing definitive hematopoietic cells appear to arise from endothelial cells lining nascent capillaries in vivo.