Establishment and characterization of mouse embryonic germ cell lines

• Main Authors: Chen, Yin-Zhi, 陳盈之
• Other Authors: Lo, Neng-Wen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/47085620631299757944

碩士 === 東海大學 === 畜產與生物科技學系 === 99 === Primordial germ cells (PGCs) are the primitive forms of germ cells, which ultimately differentiate into male and female gametes. PGCs cannot survive under in vitro culture conditions for more than one week. However, they would transform intopluripotent embryonic germ cells (EGCs) in vitro if they are cocultured with feeder cells supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF), basic fibroblast growth factor (FGF2). The primary goal of this study was to establish mouse embryonic germ cell lines under various conditions and characterizethem. In Experiment 1, to testthe culture conditions capable of convertingPGCs into EGCs, mouse PGCs derived from 11.5 dpc (days post coitum) mouse fetus were seeded by 1,500 cells/cm2 on STO feeders in medium supplemented with various concentrations of LIF, forskolin (FK) and FGF2. EG-like colonies were observed 4 days post the initial culture and after 7 days of culture the formation rate of EG-like colonies reached0.1-0.28%. Those EG-like cells expressed pluripotency markers alkaline phosphatase (AP) and Oct4, Nanog and SSEA1 on protein levelsand were maintained to passage 3 only. In Experiment 2, GSK3 and MEK inhibitors were added into the culture medium to examine the efficiency of EG-like colony formation. Mouse PGCs were cultured in medium supplemented with LIF, FK and FGF2 for 48 h, and then in the medium containing LIF, FK, MEK inhibitor and GSK3 inhibitor (2i) for additional five days. The EG-like cells which reprogram completely had been selected by trypsin. Twelve EG-like colonies were selected by the2i culture system as opposed to one colony by the untreated control system. All the EG-like cells can be maintained over 6 passages. The EG-like cells derived from 2i treatments expressed AP, pluripotency and germ cell marker genes oct4, sox2, stat3, c-myc, klf4, mvh and stella on RNA levels ; Oct4, Sox2, Nanog, Mvh and Stella on protein levels. Furthermore, the tested clone, N2, showed the capability of forming embryoid bodies (EBs) as evident by the expression of three germ layer marker genes. Taken together, several EG-like cell lines had been established from mouse PGCs with the presence of LIF, FK, FGF2, and GSK3 and MEK inhibitors.The full potency of these EGC-line clones awaits further confirmation by testing their capability in teratoma formation and in chimera formation via germline transmission.