In vivo differentiation potential of buffalo (Bubalus bubalis) embryonic stem cell

Embryonic stem cells (ESCs) derived from inner cell mass (ICM) of mammalian blastocyst are having indefinite proliferation and differentiation capability for any type of cell lineages. In the present study, ICMs of in vitro-derived buffalo blastocysts were cultured into two different culture systems using buffalo fetal fibroblast as somatic cell support and Matrigel as synthetic support to obtain pluripotent buffalo embryonic stem cell (buESC) colonies. Pluripotency of the ESCs were characterised through pluripotency markers whereas, their differentiation capability was assessed by teratoma assay using immuno-compromised mice. Cumulus ooccyte complexes from slaughter house-derived ovaries were subjected to in vitro maturation, in vitro fertilization and in vitro culture to generate blastocysts. Total 262 blastocysts were derived through IVEP with 11.83 % (31/262) hatching rate. To generate buESCs, 15 ICMs from hatched blastocysts were cultured on mitomycin-C-treated homologous fetal fibroblast feeder layer, whereas the leftover 16 ICMs were cultured on extra-cellular matrix (Matrigel). No significant differences were observed for primary ESCs colony formation between two culture systems. Primary colonies as well as passaged ESCs were characterised by alkaline phosphatase staining, karyotyping and expression of transcription-based stem cell markers, OCT-4 and cell surface antigens SSEA-4 and TRA-1-60. Batch of ESCs found positive for pluripotency markers and showing normal karyotype after fifteenth passage were inoculated into eight immuno-compromised mice through subcutaneous and intramuscular route. Subcutaneous route of inoculation was found to be better than intramuscular route. Developed teratomas were excised surgically and subjected to histological analysis. Histological findings revealed presence of all the three germinal layer derivatives in teratoma sections. Presence of germinal layer derivatives were further confirmed by reverse transcriptase-polymerase chain reaction for the presence of differentiation markers like nerve cell adhesion molecule, fetal liver kinase-1 and alpha-feto protein for ectoderm, mesoderm and endoderm, respectively.