title: Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells.
abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.
authors: Reproductive Physiology Laboratory, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, UP, India. gts553@gmail.com
authors: G
authors: G
authors: Taru Sharma
authors: Pawan K
authors: PK
authors: Dubey
authors: Om Prakash
authors: OP
authors: Verma
authors: M D
authors: MD
authors: Pratheesh
authors: Amar
authors: A
authors: Nath
authors: G
authors: G
authors: Sai Kumar
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002020:Buffaloes; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D019070:Cell Lineage; D024141:Collagen Type IV / Q000494:pharmacology*; D004475:Ectoderm / Q000166:cytology; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology*; D004707:Endoderm / Q000166:cytology; D005109:Extracellular Matrix / Q000378:metabolism; D005854:Germ Cells / Q000166:cytology; D008648:Mesoderm / Q000166:cytology
chemical_list: D024141:Collagen Type IV; D000469:Alkaline Phosphatase