Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived fromIn Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

Abstract

We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro-fertilized, somatic cell nuclear-transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro-produced blastocysts. Most of the ICMs (45-55%) resulted in formation of primary colonies that were subcultured after 8-10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture-derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture-derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.

Spontaneous differentiation of buffalo (Bubalus bubalis) embryonic stem cells towards germ cell lineage

“…Three previously established and characterized buffalo ES cell lines developed from in vitro fertilization (IVF) derived blastocysts were used in the study [16]. The primary ES cell colonies were developed from mechanically isolated inner cell masses of day 7 blastocysts.…”

“…Differentiation in floating cultures via EB formation: ES cell colonies were subjected to differentiation by static suspension culture (SSC) strategy which was earlier shown to have more potential for inducing differentiation across all the three germ layers than the hanging drop (HD) strategy [16]. ES cell colonies were allowed to differentiate in low attachment 35 mm Petri dishes in spontaneous differentiation medium, composed of KoDMEM supplemented with 2 mM L-glutamine, 1X nonessential amino acids and 50 µg/ml gentamicin sulphate.…”