GENERATION OF RED BLOOD CELLS FROM HUMAN EMBRYONIC STEM CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目的：建立人胚胎干细胞（hESC）向红细胞（RBC）高效分化的诱导体系。 为了从hESC产生红系细胞，作为第一步，我们将hES细胞与OP 9骨髓基质细胞系共培养6天以诱导其向CD 34+细胞分化，所述CD 34+细胞包括CD 34 + CD 43+造血祖细胞群体（10-20%）、CD 34 + CD 43-KDR+内皮细胞群体（10- 2（高达60%）和⑶ 34 + ⑶ 43-KDR-间充质细胞（小于15%）。 随后，使用磁性分选分离CD 34+细胞，并在非粘附条件下在补充有SCF、TPO、EPO、IL-3、IL-6、EX-CYTE、胰岛素、地塞米松和转铁蛋白的SFEM中培养5天。从第6天起，在不含TPO、IL-3和IL-6的相同培养基中扩增细胞。培养10天后，大多数细胞是未成熟的CD 71 + CD 235 a+红系祖细胞，表达高水平的胚胎和胎儿<$-珠蛋白，和低水平的成人<$-珠蛋白，如通过PCR测定的。 红系祖细胞在培养物中持续增殖长达60天，导致超过4000倍的扩增。扩增后，<$-珠蛋白表达增加，<$-珠蛋白表达减少，并最终在培养第50天消失。扩增细胞的形态学评价揭示了在不同成熟阶段的红系祖细胞的同质群体，包括成红细胞和正常成红细胞。 通过流式细胞术，基本上所有细胞都是CD 71 + CD 235 a+和CD 34-和CD 45-，证实它们代表不含白细胞的纯红系祖细胞群。所描述的实验系统将可用于研究调控红系细胞扩增的基因，以及在早期造血发育期间调控珠蛋白基因表达和开关的分子机制，以及用于从hESC大规模生产红细胞用于临床目的。 这项研究使用了WNPRC干细胞资源。 这项研究使用了启动资金;目前还没有出版物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To establish system for efficient differentiation of hESC into RBCs with potential to use for blood transfusions. To generate erythroid cells from hESCs, as a first step, we cocultured hES cells with OP9 bone marrow stromal cell line for 6 days to induce their differentiation towards CD34+ cells, which included population of CD34+CD43+ hematopoietic progenitors (10-20%), CD34+CD43-KDR+ endothelial cells (up to 60%), and CD34+CD43-KDR- mesenchymal cells (less than 15%). Subsequently CD34+ cells were isolated using magnetic sorting and cultured in non-adherent conditions in SFEM supplemented with SCF, TPO, EPO, IL-3, IL-6, EX-CYTE, insulin, dexamethasone, and transferrin for five days. From the day 6, cells were expanded in the same media without TPO, IL-3 and IL-6. After 10 days of culture, most of the cells were immature CD71+CD235a+ erythroid progenitors expressing high level of embryonic ¿- and fetal ¿-globin, and low level of adult ¿-globin as determined by PCR. Erythroid progenitors continued proliferate in culture for up to 60 days resulting in more than 4000-fold expansion. Following expansion, ¿-globin expression increased and ¿-globin expression decreased and eventually disappeared by the day 50 of culture. Morphologic evaluation of expanded cells revealed homogenous population of erythroid progenitors at different stages of maturation, including erythroblasts and normoblasts. By flow cytometry, essentially all cells were CD71+CD235a+ and CD34- and CD45-, confirming that they represent pure population of erythroid progenitors free of leukocytes. The described experimental system will be useful for the studies of genes regulating erythroid cell expansion, as well as molecular mechanisms regulating globin gene expression and switches during early hematopoietic development as well for large scale production of red blood cells from hESCs for clinical purposes. This research used WNPRC Stem Cell Resources. This research used start-up funds; there are no publications yet.