Derivation and characterization of embryonic stem-like cells of Indian major carp Catla catla.

Embryonic stem (ES)-like cells were derived from mid-blastula stage embryos of a freshwater fish, catla Catla catla, under feeder-free condition and designated as CCES cells. The conditioned media was optimized with 10% foetal bovine serum (FBS), fish embryo extract (FEE) having 100 µg ml(-1) protein concentration, 15 ng ml(-1) basic fibroblast growth factor (bFGF) and basic media containing Leibovitz-15, DMEM with 4·5 g l(-1) glucose and Ham's F12 (LDF) in 2:1:1 ratio using a primary culture of CCES cells. Cells attached to gelatin-coated plates after 24 h of seeding and ES-like colonies were obtained at day 5 onwards. A stable cell culture was obtained after passage 10 and further maintained up to passage 44. These cells were characterized by their typical morphology, high alkaline phosphatase activity, positive expression of cell-surface antigen SSEA-1, transcription factor Oct4, germ cell marker vasa and consistent karyotype up to extended periods. The undifferentiated state was confirmed by their ability to form embryoid bodies and their differentiation potential.