Potential of allogeneic feeders to support the development of buffalo embryonic stem cells

5-Title: Potential of allogeneic feeders to support the development of buffalo embryonic stem cells

Authors: Manjinder Sharma, Rajesh Kumar and G Taru Sharma

Source: Ruminant Science (2015)-4(1):21-27.

How to cite this manuscript: Sharma Manjinder, Kumar Rajesh and Sharma G Taru (2015). Potential of allogeneic feeders to support the development of buffalo embryonic stem cells. Ruminant Science 4(1):21-27.

Abstract

The present study was undertaken to evaluate the development of embryonic stem cells (ESCs) derived from in vitro produced buffalo blastocysts over allogeneic feeders with three different concentrations (10 ng, 20 ng and 30 ng per ml) of leukemia inhibitory factor (LIF) in the culture media. Three different feeders viz. fetal fibroblast, granulosa cell and oviductal cell feeder layers were prepared from the same species. Inner cell masses (ICMs) of hatched blastocysts were separated out mechanically from TE cells and seeded over mitomycin-C inactivated allogeneic feeders for the development of primary colonies of pluripotent stem cells for 7-9 days at 38.5 oC and 5% CO2 in air with 95% relative humidity. Developmental potential of ESCs was evaluated based upon the time of attachment, formation of undifferentiated primary colonies and their characterization. Results revealed that ICMs seeded onto fetal fibroblast and granulosa cell monolayer showed non-significant difference (P>0.05) for the attachment time at all concentrations of LIF. Developmental efficiency was found numerically higher for ESCs cultured on fetal fibroblast and granulosa feeder layers (83.33%) at 30 ng LIF. Oviductal feeder was found to be least efficient showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, none of the feeder layer could support the development of primary colonies at 10 ng LIF. The primary ESC colonies developed onto the allogeneic feeders were characterized by stem cell specific markers. AP stain positive colonies were found expressing SSEA-4, TRA-1-60 and Oct-4. In conclusion, both the allogeneic feeders; fetal fibroblast and granulosa cell feeders were found to be equally efficient to support the undifferentiated growth and maintenance of pluripotency in buffalo ESCs with 30 ng LIF.

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