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.

Growth of Teratomas Derived from Human Pluripotent Stem Cells Is Influenced by the Graft Site

“…We and others have previously demonstrated the effect of Buffalo rat liver (BRL) cells, or media conditioned by such cells, on the growth of embryonic tissues in vitro (3,24,25). In particular, BRLconditioned media inhibits the differentiation of pluripotent stem cells (24) and has a beneficial effect in supporting the development of the inner cell mass in cultured blastocysts (3).…”

Derivation of Human Embryonic Stem Cells from Day‐8 Blastocysts Recovered after Three‐Step In Vitro Culture

“…The beneficial effect of this step on human blastocysts is probably due to the numerous factors produced by BRL cells: leukemia inhibitory factor (LIF), insulin-like growth factor-II, and transforming growth factor beta [17][18][19]. BRL cells or medium conditioned by BRL cells have been used successfully for the in vitro culture of bovine [12] and human embryos [20] and to maintain growth of mouse ES cells in culture [17]. Addition of purified LIF to the medium increased the hatching rate and cell number of mouse and ovine preimplantation embryos in culture (for review, see [21]), and Mitalipova et al [9] added human LIF to the medium to enhance the proliferation of human blastocysts with limited cell numbers.…”

The Ethics of Embryonic Stem Cells—Now and Forever, Cells Without End

“…[8][9][10][11] Any tissue grown from these salvaged stem cells would, of course, normally face the same risks of rejection as any other genetically mismatched transplantation. However, by transferring the nucleus from a patient's somatic cell into an enucleated human ovum, stimulating it to divide, and harvesting the resulting embryonic stem cells, new organs could be grown that would be a perfect genetic match.…”