Wang Jun-long, Yang Shi, Tian Ru-hui, Zhu Zi-jue, Guo Ying, Yuan Qing-qing, He Zu-ping, Li Zheng
Zhonghua Nan Ke Xue. 2015 Mar;21(3):208-13.
To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.
We detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.
The isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.
CD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.
分离、鉴定和培养人精原干细胞（SSC），进而获得纯化和富集的人SSC用于研究和应用。
采用免疫荧光技术检测人睾丸中CD90的表达，通过两步酶消化法分离人睾丸生精细胞，随后进行差异贴壁培养以及以CD90作为SSC标志物的磁珠分选（MACS）。然后通过逆转录聚合酶链反应（RT-PCR）和免疫细胞化学鉴定分离出的CD90阳性生精细胞，同时在体外将其与支持细胞在SG培养基中共培养。
分离出的CD90阳性细胞在大小和形态上表现出相对均一的特征，并且表达人SSC特异性基因，胶质细胞源性神经营养因子受体α1（GFRA1）、G蛋白偶联受体125（GPR125）和泛素羧基末端水解酶L1（UCHL1）的表达率较高（90.5%）。在SG培养基中与支持细胞共培养2周后，分离出的CD90阳性细胞保持良好的活性。
CD90可被视为人类SSC的特异性标志物，用于通过差异贴壁培养和MACS获得高度富集的人SSC。此外，分离出的人SSC可在体外SG培养基中培养。
