title: Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.
abstract: In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: Karn P
authors.initials: KP
authors.lastname: Singh
authors.forename: Ramakant
authors.initials: R
authors.lastname: Kaushik
authors.forename: Veena
authors.initials: V
authors.lastname: Garg
authors.forename: Ruchi
authors.initials: R
authors.lastname: Sharma
authors.forename: Aman
authors.initials: A
authors.lastname: George
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Singh
authors.forename: Radhey S
authors.initials: RS
authors.lastname: Manik
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002020:Buffaloes / Q000196:embryology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology / Q000196:embryology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005855:Germ Layers / Q000166:cytology / Q000196:embryology; D010312:Parthenogenesis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D000943:Antigens, Differentiation; D000970:Antineoplastic Agents; D014212:Tretinoin