Derivation of human embryonic stem cell lines from poor quality embryos.

A serious shortcoming in the derivation of human embryonic stem cell (hESC) lines has been the availability of human embryos. About 60% of human embryos generated by in vitro fertilization (IVF) fail to develop normally and are unusable for fertility treatment. Such embryos often retain sufficient pluripotent cells that can generate genetically normal, pluripotent hESC lines with stable phenotype. We describe here a simple protocol for isolating pluripotent stem cells from abnormally developed grade III human embryos that are an unutilized byproduct of in vitro fertility treatment. Embryos that progress to the blastocyst stage are subjected to immunosurgery or mechanical surgery to isolate the inner cell mass (ICM). Isolated cells are plated on to fibroblast feeders in hESC derivation media. Pluripotent cells that grow from the ICM are isolated mechanically and cultured to obtain a stable hESC line. In this way, we derived two sibling hESC lines BJNhem19 and BJNhem20 that represent the Indian ethnic background and show stable phenotype upon long-term continuous culture of over 225 passages.