解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (1 ): 34-38.doi: 10.3969/j.issn.0529-1356.2013.01.007

• 细胞和分子生物学 • 上一篇    下一篇

可利用小鼠诱导多能干细胞的建立及鉴定

冯海凉,杨振丽,张宏,刘玉琴*   

  1. 中国医学科学院基础医学研究所细胞资源中心，北京 100005
  • 收稿日期:2012-01-13 修回日期:2012-03-27 出版日期:2013-02-06 发布日期:2013-02-06
  • 通讯作者: 冯海凉 E-mail:fenghai1219@126.com

Establishment and characterization of mouse induced pluripotent stem cells as a general research resource

FENG Hai-liang,YANG Zhen-li, ZHANG Hong,LIU Yu-qin*   

  1. Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China
  • Received:2012-01-13 Revised:2012-03-27 Online:2013-02-06 Published:2013-02-06

摘要：

目的 建立小鼠诱导多能性干细胞(iPSCs)模型，为干细胞和iPSCs研究提供可利用资源。 方法 取C57BL/6J小鼠12.5d的胚胎成纤维细胞，感染含有Sox2、Oct4、Klf4和c-Myc的慢病毒进行重编程。病毒感染后12d挑取iPSCs的克隆进行扩大培养，进行碱性磷酸酶染色鉴定。体外悬滴培养检测拟胚体（EB）的形成，并皮下接种BABL/c裸鼠检测畸胎瘤形成。全反式维甲酸诱导iPSCs克隆株定向分化。PCR法进行种属鉴定并检测支原体。 结果 得到了12个碱性磷酸酶阳性的小鼠iPSCs克隆株，体外可以形成拟胚体，其中9个克隆株可以在BALB/c裸鼠体内形成畸胎瘤。全反式维甲酸可诱导iPSCs定向分化为平滑肌细胞。iPSCs克隆株种属鉴定为鼠源性，无支原体的污染，细胞资源中心入库保藏。结论 成功建立了小鼠iPSCs模型，为干细胞、iPSCs重编程机制及定向分化研究提供可利用的资源。

关键词: 诱导多能干细胞, 病毒感染, 碱性磷酸酶染色, 反转录-聚合酶链反应, 小鼠

Abstract:

Objective To establish a cell bank of mouse induced pluripotent stem cells (iPSCs), which would be a useful resource for the research of stem cells and iPSCs. Methods C57BL/6J mouse embryonic fibroblasts were transfected with lentivirus with Sox2, Oct4, Klf4 and c-Myc. Twelve days after viral infection, mouse iPSCs clones were picked and expanded, the alkaline phosphatase (AP) was stained. The iPSCs were hanging cultured in vitro and subcutaneously inoculated into BABL/c nude mice. All-trans retinoic acid (RA) was introduced to the media to induce smooth muscle cell (SMC) differentiation of iPSCs, and SMC-specific gene (smMHC and SMA) expression were measured by RT-PCR analyses. PCR method was employed for species identification and detection of mycoplasma. Results Twelve AP positive mouse iPSCs clonal strains were picked and expanded, and 9 of them formed teratomas in BALB/c nude mice. HE staining of the teratoma showed various types of tissues. RT-PCR of iPSCs in RA media showed positive expression of smMHC and SMA. The iPSCs clonal strains were identified as murine species, without mycoplasma contamination was found, and were deposited by cell resource center. Conclusion A model of mouse iPSCs was successfully established, which may provide available resources for reprogramming and directed differentiation mechanism research of ES cells and iPSCs.

Key words: Induced pluripotent stem cells, Viral infection, AP staining, RT-PCR, Mouse