199 GERM-CELL-LIKE CELLS GENERATION FROM GOAT INDUCED PLURIPOTENT STEM CELLS

Abstract

Primordial germ cells (PGCs) generated from embryonic stem (ES) cells in different species may be an alternative approach to dealing with the worldwide problem of increasing female infertility. Reprogramming of fibroblasts into induced pluripotent stem cells has been achieved by overexpression of different transcription factors. Here, we report the generation of female goat germ cells from goat induced pluripotent stems cells (giPSC). Goat induced pluripotent stem cells (giPSC) were produced by transduction of adult female goat fibroblast cells with Oct4, Sox2, and Nanog lentiviral particles and further sub-cultured on fibroblast feeder layers. GiPSC were characterised by different methods. These iPSC were found to express alkaline phosphatase, SSEA1, SSEA4, Tra-1–81, and Tra-1–60 surface markers. However, SSEA3 was not observed in giPSC. GiPSC also expressed Oct4, Nanog, and Sox2. Along with Oct4, Nanog, and Sox2, the expression of different transcription factors such as Cdx1, Dapp5, Dax1, Ecat, Eras, Fgf4, Gata6, Lin28, Rex1, and Utf1 was confirmed by RT-PCR. GiPSC were in vitro differentiated and three germ layers were characterised by immunostaining of Gata4 for endoderm, α-Actinin for mesoderm, and β-III tubulin for ectoderm and RT-PCR analysis of GATA4, α-Actinin and BMP4. IPSCs were directed differentiated into germ cells using retinoic acid and bone morphogenetic protein 4 without the inactivation of exogenous factors as these are also required for germ cells development. Differentiated germ cells were characterised by immunostaining against VASA and Dazl proteins. RT–PCR assay was performed for Dazl, Nanog, Nanos1, PUM8, SCP3, Stella, and VASA genes expression. Quantitative PCR was also performed for detection of VASA and Dazl expression during the course of germ cell differentiation. Flow-cytometric analysis of differentiated germ cells was confirmed the presence of germ cells in population of differentiated giPSC. Oocytes/ova-like structures, which were comparable to natural goat oocytes, were observed under scanning electron microscope (SEM). Cumulus–oocyte complex like structure was observed, which was further used for SEM. The study concluded that adult female goat fibroblast cells can be reprogrammed into induced pluripotent stem cells using ectopic expression of Oct4, Nanog, and Sox2 genes and the germ-cells-like cells generated from reprogrammed giPSC could be differentiated into goat oocytes/ova-like structure which have immense applications in human and animal reproduction.