单细胞聚团拟胚体形成诱导人多能干细胞造血分化的技术优化

Techniques optimizations of Spin-embroid body approach for hematopoietic differentiation of human pluripotent stem cell

目的优化利用单细胞聚团拟胚体(Spin-EB)培养诱导人多能干细胞造血分化的方式。方法采用Aggre Well TM800及V-96孔板两种培养板形成Spin-EB,在骨形成蛋白-4(BMP-4)、血管内皮生长因子(VEGF)及碱性成纤维生长因子(b FGF)组合培养基中诱导造血分化,通过流式细胞术检测不同分化条件下CD34+细胞的比例;选取CD34+细胞比例高的拟胚体(EB)形成方式,并通过造血集落形成实验及红细胞分化培养实验,验证EB来源的CD34+细胞的造血集落形成和红系分化能力。结果流式分析结果显示,在Aggre Well TM800培养板中培养所得的EB,其CD34+造血干祖细胞比例为22.6%,V-96孔板按照形成EB的细胞数目,CD34+造血干祖细胞比例分别为:3000个细胞/孔为10.8%、6000个细胞/孔为1.28%、9000个细胞/孔为1.23%。胚胎干细胞(ESC)来源的CD34+细胞形成的集落与脐带血CD34+造血干祖细胞形成的集落形态相似,并且在红系分化培养体系中分化为CD71+CD235a+的红细胞。结论 V-96孔板组EB形成和CD34+分化效率与用于EB形成的细胞数有相关性,3000个细胞/孔条件相对最优。采用Aggre WellTM800培养板形成EB及EB的造血分化效率均高于各组V-96孔板分化效率。

Objective To optimize hematopoietic differentiation of human pluripotent stem cells via spin-embryoid body （Spin-EB）. Methods AggreWellTMS00 and V-96 well culture plate were used to form Spin-EB, which was succes- sively induced to hematopoietic differentiation in culture medium contained BMP-4, VEGF and bFGF. Flow cytometry was used to analyze the proportion of CD34＋ hematopoietic stem/progenitor cells under the two different culture conditions. The higher test was seleted to verify its colony forming ability and erythroid differentiation ability via colony forming unit assay and erythroid differentiation culture system. Results The flow cytometry results showed that the proportion of CD34＋ hematopoieic stem/progenitor cells from AggreWellTMSO0 test was 22.6%, while the V-96 well test divided into three tests according to the number of ESC, the corresponding proportion were： 3000 cells/well was 10.8%, 6000 cells/well was 1.28%, 9000 cells/well was 1.23%. The morphology of the colony forming unit from CD34＋ cells o riginated from embryonic stem cell （ESC） was as similar as the umbilical cord blood CD34＋ cells. Simultaneously, CD34＋ cells originated from ESC could differentiate into CD71＋CD235a＋ erythrocytes in erythroid differentiati-on cul-ture system. Conclusion The forming ability of EB and the hematopoietic differentiation efficiency of CD34 ＋ cells is related to the number of ESC seeded in V-96 well culture plate, among which the 3000 cells/well test is relatively prestigeous. Furthermore, compared with the three tests of V-96 well, EB from AggreWellTM800 test has a better EB forming ability and higher hematopoietic differentiation efficiency of CD34＋ cells.