人诱导多能干细胞体外扩增生成红系集落并构建红系祖细胞株的研究

In vitro proliferation of human induced pluripotent stem cells into erythroid colony and establishment of a stable erythroid progenitor cell line

目的将人诱导多能干细胞(hi PSCs)分化成为红系祖细胞并构建稳定的红系祖细胞株。方法采用与滋养层细胞共培养的方式,添加10 ng/m L的TPO、5 U/m L的EPO、25 ng/m L的SCF、5 ng/m L的Flt-3、20 ng/m L的IL-3的诱导因子,诱导hi PSCs分化成为红系集落,构建稳定的红系祖细胞株;倒置显微镜观察获得的细胞株形态,瑞氏染色并计数,MTT法检测细胞活性,流式细胞仪检测细胞表面标志物。结果 hi PSCs诱导构建的红系祖细胞株遗传性状稳定并可长期传代,细胞呈集落样生长,外观上与正常红系祖细胞无差异。分类计数构建的红系祖细胞株比例(%):P0与P5代细胞为84.90±4.93 vs 72.20±6.24(P<0.05);流式检测,hi PSCs诱导构建的红系祖细胞株细胞表面标志物CD235a阳性率(%)表达:P0与P5代细胞为3.02±0.75 vs 5.87±0.37(P<0.05)。结论诱导hi PSCs生成的红系祖细胞株在表面标志继承性和遗传性状方面都是稳定的。

Objective To induce differentiation of human induced pluripotent stem cells（ hi PSCs） into erythroid progenitor cells and to establish a stable erythroid progenitor cell line. Methods The hi PSCs were co-cultured with trophoblast,and supplemented with 10 ng / m L of TPO,5U / m L of EPO,25 ng / m L of SCF,5ng / m L of Flt-3,and 20 ng / m L of IL-3 to induce differentiation of the hi PSCs into erythroid colony and to construct a stable erythroid progenitor cell line. Cell morphology was observed by inverted microscopy. The number of cells were counted after Wright staining. Cell activity was determined by MTT assay. Last,cell surface markers were determined by flow cytometry. Results The hi PSC-derived erythroid progenitor cells were stable and can be passed on for a long period. The cells grew in colonies,and were morphologically identical to normal erythroid progenitor cells. The rate of erythroid progenitor-like cells were 84. 90 ± 4. 93% and 72. 20± 6. 24%,respectively,in the P0 and P5 generations（ P〈 0. 05）. Flow cytometry indicated that the positive rates of cell surface CD235 a were 5. 87 ± 0. 37 and 3. 02 ± 0. 75,respectively,in the P0 and P5 generations of induced erythroid progenitor cells（ P 〈0. 05）. Conclusion Both the genetic traits and cell surface markers of the hi PSC-induced erythroid progenitor cell line are stable.