Production and Characterization of Putative ntES Cells from Handmade Cloned Goat Embryos Derived from Adult Fibroblast Donor Cells

Abstract

In the field of nuclear transfer handmade cloning (HMC) technique has been a relatively recent addition. In the present investigation attempts were made to efficiently derive stem cell from hand made cloned (HMC) embryos in goat using adult fibroblast donor cell and to characterize the derived putative ntES cell for their stemness. Oocytes were collected from slaughterhouse ovaries and matured in maturation medium containing TCM199 (HEPES modified), 1 μg/ml FSH, 10 μg/ml LH, 1 μg/ml Estradiol-17β, 3% BSA and 5% FBS. The efficiency of the donor fibroblast cell for nuclear transfer was also assessed and an overall cleavage and morula formation rate of 62.44%±3.9 & 35.30%±3.86 was obtained respectively. Genetic relationship between the cloned embryos and donor cells was established by PCR amplification of highly polymorphic goat major histocompatibility complex (MHC) class II DRB gene. The PCR products were further subjected to SSCP for confirming the genetic relationship conclusively. Stem cell colonies were successfully derived from HMC embryos obtained from fibroblast donor cell. Primary colony formation rate was 61.66%±4.62 for fibroblast donor cell derived embryos. The putative ntES colonies were positively characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The differentiating ability of the derived cells was tested by embryoid body formation. Then the derived putative ntES cells were successfully cryopreserved for further investigation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos using adult fibroblast as a donor cell. It could be concluded that the present investigation has substantial significance as patient specific ntES cells have proven their therapeutic significance.