將羊水細胞分離出幹細胞(pluripotency of human amniotic fluid stem cells (hAFSCs)[含stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA)]
將幹細胞與豬濾泡液共同培養7-10天, 約2%幹細胞可形成類似原始卵細胞

Biol Reprod. 2014 Apr 3;90(4):73. doi: 10.1095/biolreprod.113.112920. Print 2014 Apr.

Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro.

Yu X1, Wang N, Qiang R, Wan Q, Qin M, Chen S, Wang H.

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Abstract

Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117(+)/CD44(+) hAFSC line that showed fibroblastoid morphology and intrinsically expressed bothstem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cellaggregates were collected and replated in oocyte culture medium for an additional 7-10 days. OLCs ranging from 50 to 120 μm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.