基础医学与临床 ›› 2012, Vol. 32 ›› Issue (9): 1030-1035.

• 研究论文 • 上一篇    下一篇

高效制备人诱导多能干细胞并诱导分化为心肌细胞

方月琴1,郭娟宁2,孙晓明3,侯一平4,顾准1   

  1. 1. 江苏健雄职业技术学院
    2. 新乡医学院法医学教研室
    3. 新加坡国家心脏中心
    4. 四川大学华西基础医学与法医学院
  • 收稿日期:2011-06-13 修回日期:2011-12-29 出版日期:2012-09-05 发布日期:2012-08-28
  • 通讯作者: 顾准 E-mail:guzhuntx@126.com
  • 基金资助:
    新加坡科研基金;河南省教育厅课题

Induction of human fibroblast cells to pluripotent stem cells and differentiation into cardiomyocytes

  • Received:2011-06-13 Revised:2011-12-29 Online:2012-09-05 Published:2012-08-28
  • Supported by:
    Singapore National Research Foundation;Grant of Department of Education, Henan Province

摘要: 摘要:目的 提高人诱导性多能干细胞(human induced pluripotent stem cells, hiPSC)转化效率,缩短其产生周期,并避免引入外源基因。方法 利用GP2-293细胞包装Oct4,Sox2,Klf4和c-myc四种因子的逆转录病毒,对人成纤维上皮细胞(human fibroblast feeder cells, HFF)进行感染,在转化过程中加入SB431542,PD0325901和thiazovivin物质,待形态与胚胎干细胞(embryonic stem cells, ESC)相似的克隆出现并长至足够大,进行独立培养,检测其标记蛋白,定向诱导分化能力和核型分析。结果 HFF经四种因子转化之后,在20天时出现形态与ESC克隆相似的hiPSC,比Yamanaka方法时间缩短了10天,效率提高约12倍。它们与ESC同样表达标记蛋白质Oct4,SSEA3,Tra-1-60和Tra-1-81;体外成功定向分化为心肌细胞。核型分析表明所获得hiPSC的染色体核型正常。结论 本课题所建立的hiPSC生成体系转化效率高、所需周期短,并成功诱导分化成具有节律性搏动的心肌细胞,为心血管疾病模型的建立和研究提供了实验基础。

关键词: 关键词:人诱导性多能干细胞, 转化效率, 形成周期, 心肌细胞分化

Abstract: Abstract: Objective To increase the transduction efficiency of iPSC formation, decrease the time schedule and avoid the possibility of exogene introduction. Methods Human fibroblast cells were infected with retrovirus of Oct4, Sox2, Klf4 and c-myc which were packaged by GP2-293 cells. During the transduction, small molecules of SB431542,PD0325901 and thiazovivin were added into hESC medium, until iPSC colonies generated big enough to be cut and paste. The stable iPSC colonies were detected with the expression of ESC protein markers, cardiomyocytes differentiation and karotyping analysis. Results On day 13 small iPSC colonies were shown up, and on day 20 they were ready to be cut and transferred to cell culture dishes. The reprogramming period was shortened for 10 days, and the efficiency was increased by 12 times. The iPSC shared protein expression with hESC on Oct4,SSEA3,Tra-1-60 and Tra-1-81. As well, both iPSC and hESC were differentiated into cardiomyocytes successfully with similar MEA data. And both showed normal karotyping results. Conclusion The iPSC reprogramming system established are efficient and time-saving for study on disease-specific model and holding clinic poteintial.

Key words: Key words: human induced pluripotent stem cells, transduction efficiency, time schedule, cardiomyocytes differentiation

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