Wang Peng, Suo Li-Juan, Shang Hua, Li Ying, Li Guang-Xuan, Li Qing-Wang, Hu Jian-Hong
College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
Cytotechnology. 2014 May;66(3):365-72. doi: 10.1007/s10616-013-9584-0. Epub 2013 Jun 2.
In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6-8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10(-7) mol l(-1) of RA effectively induced the SSCs into haploid male germ cells in vitro.
精原干细胞（SSCs）的体外分化有助于深入了解精子发生的机制。本研究的目的是从小鼠睾丸组织中分离出类精原干细胞，然后用视黄酸（RA）将其诱导为单倍体生殖细胞。基于SSCs和体细胞不同的贴壁速度，采用两步接种法对类精原干细胞进行纯化和富集。在M1培养基中培养3天后出现细胞集落。通过碱性磷酸酶、RT-PCR和间接免疫荧光细胞分析，证明细胞集落为SSCs。随后，将SSCs细胞集落在含RA的M2培养基中培养2天。然后将SSCs细胞集落再次在M1培养基中培养6 - 8天，选择RT-PCR和间接免疫荧光细胞分析检测单倍体雄性生殖细胞。结果表明，10(-7) mol l(-1)的RA能在体外有效诱导SSCs分化为单倍体雄性生殖细胞。
