title: Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In 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.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Syed Mohmad
authors.initials: SM
authors.lastname: Shah
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Neha
authors.initials: N
authors.lastname: Saini
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Syma
authors.initials: S
authors.lastname: Ashraf
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Mohammad
authors.initials: M
authors.lastname: Zandi
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Radhey Sham
authors.initials: RS
authors.lastname: Manik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Suresh Kumar
authors.initials: SK
authors.lastname: Singla
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute , Karnal 132001, Haryana, India .
authors.forename: Manmohan Singh
authors.initials: MS
authors.lastname: Chauhan
mesh_terms: D000818:Animals; D015415:Biomarkers; D001755:Blastocyst / Q000166:cytology*; D002020:Buffaloes / Q000196:embryology*; D002454:Cell Differentiation; D002460:Cell Line; D002999:Clone Cells; D058732:Embryoid Bodies; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression; D053652:Nuclear Transfer Techniques; D010312:Parthenogenesis
chemical_list: D015415:Biomarkers