title: Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.
abstract: This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.
authors.affiliation: Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, India.
authors.forename: Musharifa
authors.initials: M
authors.lastname: Muzaffar
authors.forename: Naresh L
authors.initials: NL
authors.lastname: Selokar
authors.forename: Karn P
authors.initials: KP
authors.lastname: Singh
authors.forename: Mohammad
authors.initials: M
authors.lastname: Zandi
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Singh
authors.forename: Riaz A
authors.initials: RA
authors.lastname: Shah
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Radheysham
authors.initials: R
authors.lastname: Manik
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002020:Buffaloes / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods* / Q000662:veterinary; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D005260:Female; D005306:Fertilization / Q000235:genetics / Q000502:physiology*; D005307:Fertilization in Vitro / Q000662:veterinary; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D010312:Parthenogenesis / Q000235:genetics / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D011247:Pregnancy
chemical_list: D015415:Biomarkers