免疫磁珠法分选小鼠iPS细胞获得原始生殖细胞的初步研究

Preliminary study of primordial germ cells derived from mouse induced pluripotent stem cells by magentic activated cell sorting system

目的 探讨经MACS系统体外分选小鼠诱导性多能干细胞（inducedpluripotentstemcells，iPS）IP14D-1得到纯度较高诱导性原始生殖细胞（inducedprimordialgermcells，iPGCs）的方法，并对其表面标志、形态进行鉴定。方法未分化IP14D-1细胞培养扩增，分化形成iEBs（inducedembryoidbodies，iEBs），以4、7、9d的iEB为研究对象，胰酶消化制成单细胞悬液，用特异性表面抗原Ssea-1（specialstageembryonicantigenI）单抗标记，间接免疫磁珠法分选得到Ssea-1阳性细胞，即为假定iPGCs。采用免疫荧光和碱性磷酸酶染色鉴定假定iPGCs，流式细胞术鉴定MACS分选得到的目的细胞群纯度。结果分选得到的Ssea-1阳性细胞表达PGC特异性表面标志Blimpl和组织非特异碱性磷酸酶（TNAP）；7d的iEB分选获得细胞经扩增后iPGC量较多（83．5％）。结论MACS分选方法可作为获得较纯化的iPGC体外分选体系。

Objective To evaluate the use of magentic activated cell sorting system for isolation ofhighly purified primordial germ ceils from iPS in vitro, and identify the target ceils by the expressions of surface markers and differentiation markers. Methods Undifferentiated IP14D-1 were proliferated, and then differentiated into iEBs （induced Embryoid bodies, iEBs） in vitro. The iEBs on day4,day7 and day9 were digested into single cell suspension, and the cells were marked by a specific surface antigen Ssea-1 （special stage embryonicantigenl） antibody. The cells with Ssea-l-positive were isolated from the iEBs by MACS, which were referred to as the assumed iPGCs. The assumed iPGCs were further identified by Immunofluorescence and alkaline phosphatase stains. Results Blimpl, a specific marker of PGCs, was positive for this Ssea-l＋/Oct-4＋ cell population, and a germ-cell-specific marker, tissue non-specific alkaline phosphatase （TNAP）, was also positive for this population. The Ssea-1＋/Oct-4＋ cell population was putative iPGCs. The proportion of iPGCs in 7-d iEBs was higher （83.5%）. Conclusion Magentic activated cell sorting system is suitable for isolation of highly purified primordial germ cells in vitro.