Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation

Several different culture conditions were evaluated for culturing grade 4 embryos (containing 2-4 blastomeres and with >50% fragmentation) 68 h after fertilization to the blastocyst stage. Embryos were co-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium with or without 10% v/v synthetic serum substitute (SSS), co-cultured with BRL cells in KSOM with or without 10% SSS, or cultured in KSOM with 100 nm heparin binding epidermal growth factor. The most consistent development Was obtained when embryos were co-cultured with BRL, cells in KSOM. Rates of development to the blastocyst stage were between 27% and 40%. After reaching the blastocyst stage, continued culture of these blastocysts was only possible in a medium without serum. In a serum-deprived medium cells attached and showed initial outgrowth, but did not survive passaging. Using another approach, inner cell masses (ICMs), isolated from blastocysts with high efficiency using immunosurgery, were able to attach to a feeder layer in the presence of serum. Some ICMs differentiated whereas others could be succesfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead of well-defined colonies, the human colonies were characterized by individual cells and colonies without defined borders.