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

Abstract

Several different culture conditions were evaluated for culturing grade 4embryos (containing 2–4 blastomeres and with >50%fragmentation) 68 h after fertilization to the blastocyst stage. Embryos wereco-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium withor without 10% v/v synthetic serum substitute (SSS), co-culturedwith BRL cells in KSOM with or without 10% SSS, or cultured in KSOMwith 100 nM heparin binding epidermal growth factor. The most consistentdevelopment was obtained when embryos were co-cultured with BRL cells in KSOM.Rates of development to the blastocyst stage were between 27% and40%. After reaching the blastocyst stage, continued culture of theseblastocysts was only possible in a medium without serum. In a serum-deprivedmedium cells attached and showed initial outgrowth, but did not survivepassaging. Using another approach, inner cell masses (ICMs), isolated fromblastocysts with high efficiency using immunosurgery, were able to attach to afeeder layer in the presence of serum. Some ICMs differentiated whereas otherscould be succesfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead ofwell-defined colonies, the human colonies were characterized by individualcells and colonies without defined borders.