两种非整合载体重编程人脐带血CD34^+细胞形成诱导多能干细胞的研究

Two efficient reprogramming methods of human cord blood CD34^+ cells induced pluripotent stem cells with non-integrating plasmid system

目的探索两种非整合载体重编程人脐血来源的CD34^+细胞形成诱导性多能干细胞(iPSCs)的方法。方法利用细胞核转染仪分别将两组非整合质粒转入短暂培养后的脐带血CD34^+细胞中,使其进行重编程形成iPSCs,14d后两种方法均可观察到2×10~6个脐带血CD34^+细胞中约有900个类似ES细胞特征的克隆出现,并对产生的iPSCs进行体内外多能性鉴定及细胞核型检测。结果两种方法重编程效率基本相同,重编程形成的hiPSCs多能性基因OCT4、SOX2、NANOG的表达量与hES细胞系H1比较接近(P>0.05),而与CD34+细胞差别较大(P<0.05),且具有体内分化成三胚层的全能性。结论两种非整合质粒重编程人脐带血来源的CD34^+细胞形成iPSCs的方法,效率基本相同,均无外源基因插入,为建立相对安全的iPSCs提供了有效途径,为临床科研、药物筛选和再生医学研究等提供了较好的方法。

Objective To establish two episomal vector reprogramming methods to reprogram iPSCs from human cord blood （CB） CD34＋ cells. Methods Two sets of non-integrating plasmids were respectively transported into two groups of short-term cultured CB CD34＋ ceils by using transfector nucles, to reprogram iPSCs from CB CD34＋ cells. Within 14 days of transfection by two sets of non-integrating plasmids, up to 900 iPSC-like colonies per 2 million transfected CB CD34＋ cells were generated. And the pluripotency and karyotypes of iPSCs were tested in vitro. Results The repro- gramming efficiency of two methods was basically the same. The pluripotency genes OCT4, SOX2 and NANOG expression levels of the hiPSCs ：were close to the hES cell line HI cells （P 〉 0.05）, but different from the CD34＋ cells （P 〈 0.05）. Furthermore, the hiPSCs formed teratomas with three embryonic germ layers. Conclusion Two efficient reprogramming methods have basically the same efficiency, and no vector integration is found in iPSCs. It is concluded that the non-integrating plasmid system to generate human iPSCs from CB CD34＋ cells is reliable and can provide new ways for clinical research, drug screening and regenerative medicine research.