[Preliminary research on the differentiation of human amniotic epithelial cells into female germ cells].

ABSTRACT

OBJECTIVE: To establish the method for isolation and culturation of human amniotic epithelial cells (hAECs) in vitro, and to investigate the differentiation potential of hAECs towards germ cells. METHODS: hAECs were isolated from human term placenta and identified by immunocytochemistry. hAECs were sequential induced to form germ cells in medium supplemented with 5% human follicular fluid. Morphological changes were observed by inverted microscope. The oestradiol levels in the spent media were assayed. The expression of germ cell special genes and protein were examined by Real-time PCR and Western blot separately. RESULTS: Most of the isolated hAECs were polygon and of typical alabstone-like appearance, could express cytokeratin 19, the specific marker of epithelial cells, as well as octamer-binding protein 4 (Oct-4), a specific marker of embryonic stem cells (ESCs). After induced in medium supplemented with 5% human follicular fluid for 14 days, these stem cells grew as aggregates morphologically. Aliquots of supernatant of culture were collected and were assayed for oestradiol using chemiluminescence immunoassay (CLIA), although oestradiol was absent in the control group, the levels of the induction group were increased by day 8 and reached the maxima by day 10. Our study showed that hAECs treated with human follicular fluid were able to express germ cell genes, including growth differentiation factor 9 (GDF9) and deleted in azoospermia-like (DAZL) (P < 0.05), but without synaptonemal complex protein 3 (SCP3) (P > 0.05). The DAZL protein were also examined (P < 0.05). CONCLUSION: Germ cells could be induced in vitro from hAECs by human follicular fluid.