Establishment of human induced pluripotent stem cell lines

Shi Ying-xu; Han Yan-qiu; Xie Yin-liang; Du Hua

doi:10.3969/j.issn.2095-4344

Abstract：BACKGROUND:Induced pluripotent stem cels have a generating process similar to cancer stem cels and possess stem cel characteristics extremely close to human embryonic stem cels. Therefore, studies on induced pluripotent stem cels are in favor of an increased awareness and understanding of human development and tumor occurrence. OBJECTIVE:To master the technology of establishing induced pluripotent stem cel lines in order to build the technology platform for reprogramming specific disease cels, thus to explore the pathogenesis of the disease using reprogramming technology. METHODS: Retroviruses containing Oct4, Sox2, Klf-4, and c-Myc4 transcription factors were used to transfect human skin fibroblasts (HS27 cels), and then the cels were induced to differentiate to human embryonic stem cel-like clones in the human embryonic stem cel culture conditions. The cels were picked and further amplified. After observation of clone morphology and alkaline phosphatase activity test, immunofluorescence detection was used to detect whether human embryonic stem cel markers Oct4, Sox2, c-Myc, Klf-4 expressed, the pendant-drop method was used to detect the ability of HS27-derived clones to form teratomas and verify the differentiation into three germ layers. RESULTS AND CONCLUSION:After viral infection, embryonic stem cel-like clones formed and were negative for <br> green fluorescent proteins. These clones were similar to human embryonic stem cel clones in cel morphology. After further amplification, they were positive for alkaline phosphatase. Immunofluorescence results showed that the clones expressed Oct4, Sox2, c-Myc, Klf-4, and HS27 cels-derived clones injected into immunodeficient mice could form teratomas and had the ability to differentiate into the three germ layers under hematoxylin-eosin staining. In this experiment, induced pluripotent stem cel lines were successfuly constructed, laying a good experimental foundation for disease-specific cel reprogramming research.