Assessment of different stages of parthenogenetic embryos for production of embryonic stem cell like colonies

Abstract

Parthenogenetic activation of oocytes has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. In this study, we described the production of goat parthenogenetic ES like cells from different stages of parthenogenetic embryos. We compared the source material on the pESCs outgrowth and culture. 8-16 cell stage embryos, morula, blastocysts and ICM (inner cell mass) from hatched blastocysts produced from chemical activation were cultured on goat fetal fibroblast monolayer in stem cell culture media. Colonies were passaged when signs of differentiation were visible. ESC like colonies were cultured on feeder cells in the presence of hLIF however, some ESC like colonies were also cultured in absence of hLIF for random differentiation. In our study, ESC like colony formation with ICM of hatched blastocyst was comparatively higher as compared to blastocysts while ESC like colony formation with blastocysts and ICM of hatched blastocyst was significantly higher as compared 8-16 cells and morula. It is observed that inner cell mass source is an important criterion for the ES like cells derivation in goats. Also, in comparison to the expanded blastocysts (80.10%), hatched blastocysts showed higher (86.06%) attachment rate and primary colony formation rate. ESC like colonies in absence of hLIF differentiated in vitro into epithelial like and neuronal like cells. Undifferentiated ESC like colonies stained positive for alkaline phosphatase, SSEA-3 and OCT-4.