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In the pleuripotent embryonic stem (ES) cells in mammalian species are derived from the inner cell mass (ICM ) of preimplantation embryos. In the current study we report the successful isolation of pleuripotent undifferentiated buffalo ES-like cells from the ICMs of in vitro fertilization (IVF), somatic cell nuclear transfer (NT)-reconstructed and parthenogenetic (PA) embryos. The ICMs were isolated from hatched blastocysts that had spread out after 3–5 days of culture on mouse embryonic fibroblast (MEF) feeder cell layer in the presence of leukemia inhibiting factor (LIF). The production of MEF and ES-like cells were the same as previously described in bovine ES cells (Kitiyanant Y et al., 2000 Science Asia 26, 81–86). The primary culture and propagation of the cell lines were performed every 2–3 days. The cell lines appeared to be normal diploid karyotype and expressed the cell specific markers of alkaline phosphatase, stage-specific embryonic antigen 3 and 4 (SSEA-3 and SSEA-4), TRA-1-60 and TRA-1-81 similar to those characterized in monkey ES cells (Thomson JA et al., 1995 PNAS 92, 7844–7848 and Kuo HC et al., 2003 Biol. Reprod. 68, 1727–1735). The buffalo ES-like cells from these three different sources were able to be cultured in vitro for more than 20 passages on the feeder cell layer without differentiation. They were aggregated to form the embryoid bodies (EBs) in suspension culture. When EBs were plated on 0.1% gelatin-coated culture dishes, the cells differentiated to be neural-, epithelial- and fibroblast-like morphologies. These results suggest success in establishment of buffalo ES-like cells from either IVF, NT or PA preimplantation embryos and also their differentiation in vitro. Buffalo ES-like cells should be a useful source of cells for gene targeting and for the studies of the mechanism of gene expression in the preimplantation embryos obtained from different methods. These results are encouraging for the feasibility of cell transplantation studies in the future. This research was funded by Thai Government and The National Center of Biotechnology and Genetic Engineering.

189ESTABLISHMENT OF BUFFALO EMBRYONIC STEM-LIKE (ES-LIKE) CELL LINES FROM DIFFERENT SOURCES OF DERIVED BLASTOCYSTS