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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
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摘要 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.
作者 孟强 王世春 王甜甜 陈艺心 张强 黄梅莓 赵树铭
机构地区 第三军医大学第一附属医院输血科中国人民解放军重庆血站
出处 《中国输血杂志》 CAS 北大核心 2015年第5期493-497,共5页 Chinese Journal of Blood Transfusion
基金 国家自然科学基金(81270649)
关键词 人诱导多能干细胞 红系集落 红系祖细胞株 滋养层细胞共培养 体外扩增 诱导因子 human induced pluripotent stem cells erythroid colony erythroid progenitor cell line trophoblast co-culture in vitro amplification inducing factor
分类号 R457.7 [医药卫生—治疗学] R331.1 [医药卫生—人体生理学]
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参考文献27

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中国输血杂志
2015年 第5期

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