GENERATION OF RED BLOOD CELLS FROM HUMAN EMBRYONIC STEM CELLS

从人胚胎干细胞产生红细胞

• 批准号: 7716462
• 负责人: Igor I. Slukvin
• 金额: $ 4.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-23 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7716462
• 关键词: Adult; Blood Transfusion; Bone Marrow; CD34 gene; Cell Line; Cells; Clinical; Coculture Techniques; Computer Retrieval of Information on Scientific Projects Database; Condition; Development; Dexamethasone; Embryo; Endothelial Cells; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Evaluation; Flow Cytometry; Funding; Gene Expression; Generations; Genes; Globin; Grant; Hematopoietic; Institution; Insulin; Interleukin-3; Interleukin-6; Leukocytes; Magnetism; Mesenchymal; Molecular; PTPRC gene; Polymerase Chain Reaction; Population; Production; Proliferating; Publications; Purpose; Research; Research Personnel; Resources; Sorting - Cell Movement; Source; Staging; Stem cells; Stromal Cells; System; Transferrin; United States National Institutes of Health; day; fetal; human embryonic stem cell; progenitor

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. 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, start up funding and federally approved hES cell lines. No publications yet.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 建立有效分化为RBC的系统，并有可能用于输血。 作为第一步，我们将HES细胞与OP9骨髓间隙细胞系共培养6天，以诱导其对CD34+细胞的分化，其中包括CD34+ CD43+ CD43+造血祖细胞的种群间充质细胞（小于15％）。随后，使用磁性排序分离CD34+细胞，并在补充了SCF，TPO，EPO，IL-3，IL-3，IL-6，Ex-Cyte，Ex-Cyte，Insulin，Dexamethersone和Transerrin的SFEM中培养的SFEM中进行培养五天。从第6天开始，将细胞在没有TPO，IL-3和IL-6的同一介质中扩展。培养10天后，大多数细胞是未成熟的CD71+ CD235a+红细胞祖细胞，表达高水平的胚胎�-和胎儿�-珠蛋白，以及由PCR确定的低水平的成人oom -Globin。红细胞祖细胞在培养物中持续增殖长达60天，导致4000倍以上的膨胀。膨胀后，» - 珠蛋白的表达增加，并且 - 斑块蛋白的表达发展并最终在培养的第50天消失。扩张细胞的形态学评估显示，在不同的成熟阶段（包括红细胞和正常细胞）处的红细胞祖细胞的同质群体。通过流式细胞仪，基本上所有细胞均为CD71+ CD235A+和CD34-和CD45-，证实它们代表了无白细胞的纯颗粒状祖细胞的纯种群。所描述的实验系统将有助于调节胚胎细胞扩展的基因研究，以及调节造血性造血蛋白基因表达和转换的分子机制，以及造血性早期发育过程中的转化，以及从hESC中大规模生产红细胞的大规模生产hESC的临床目的。这项研究使用了WNPRC干细胞资源，启动资金并获得联邦批准的HES细胞系。尚无出版物。