c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究  

Infl uence of c-Myc on Induction Effi ciency and Pluripotency of Induced Pluripotent Stem Cells

作　　者：魏小元[1] 徐雍羽[1] 王敏[1] 张儒[1] 

机构地区：[1]上海市信号转导与疾病研究重点实验室,同济大学生命科学与技术学院,上海200092

出　　处：《中国细胞生物学学报》2014年第4期430-437,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31071227)资助的课题~~

摘　　要：该研究探讨转录因子c-Myc对多能诱导干细胞(induced pluripotent stem cells,iPS)诱导效率及形成的iPS克隆全能性的影响。将Yamanaka四因子(Oct4、Sox2、Klf4和c-Myc,OSKM)和不含c-Myc的三因子(OSK)病毒分别感染OG2小鼠成纤维细胞(mouse embryo fi broblast,MEF),诱导成为iPS细胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白(GFP)阳性比例,比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶(AP)染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示:在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除c-Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。This work aimed to investigate the influence of transcription factor c-Myc on induction efficiency and pluripotency of induced pluripotent stem cells （iPS cells）. We generated mouse iPS cells from OG2 mouse em- bryo fibroblasts （MEF） using retroviral virus delivered 4 factors （Oct-4, Sox-2, Klf-4 and c-Myc, OSKM） or 3 fac- tors （Oct-4, Sox-2 and Klf-4, OSK）. The number of iPS clones was counted under the microscope. The percentage of green fluorescent protein （GFP）-positive iPS cells was analyzed using flow cytometry. The induction efficiency of iPS was compared between OSKM and OSK. Reprogrammed colonies were picked and expanded into cell lines, which were further subjected to the sternness characterization： pluripotent marker expression, teratoma formation, chimera formation and tetraploid compensation. In our current induction system, compared to OSKM, our results showed that OSK induced iPS clones more efficiently and led to higher proportion of GFP-positive cells. Randomly picked iPS clones generated by OSKM or 0SK possessed similar ES-like morphology, alkaline phosphatase （AP） positive staining, expression of pluripotent genes and ability to differentiate into three germ layers. However, the pro- portion of chimera generated by OSK clones was higher than OSKM clones and only OSK clones were able to pro- duce chimeric mice with germline transmission and were capable of generating viable, live-born progeny by tetraploid complementation. Thus the present study demonstrated that in our current induction system, omitting c-Myc from the OSKM combination enhanced the reprogramming efficiency and improved pluripotency of generated iPS cells.

关 键 词：诱导多能干细胞 c—Myc 嵌合体小鼠 四倍体补偿小鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]