Derivation of Human Embryonic Stem Cell Lines from Poor Quality Embryos

Abstract

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.

Heterogeneity in Human Embryonic Stem Cells May Prevent Endodermal Guided Differentiation

“…In contrast, culture in a commercially available BEGM (with tri-iodothyronin and retinoic acid but without BSA) promoted differentiation to predominantly bronchiolar alveolar cell (i.e., Clara cell) phenotype. Lung lineage-specific cell differentiation was achieved in a relatively shorter span of time (22 days) in contrast to other reported lung lineage culture conditions [22][23][24][25][26][27][28][29][30]. These culture media, normally used to maintain and grow mature cells, could successfully induce differentiation of pluripotent embryonic stem cells into three types of mature lung lineage-specific non-ciliated cells.…”

“…Going on the same hypothesis, we embarked upon inducing directed guided differentiation of two cell lines BJNhem 19 and 20 generated in a JNCSAR lab [30][31][32][33][34]. We used similar coaxing of the ESC as in our work with H7.…”

“…These cells were derived from the inner cell mass (ICM) of grade III poor quality blastocysts that were not suitable for in vitro fertility treatment. Both the lines as claimed by JNCASR are pluripotent and have been extensively characterized and cultured continuously for over 250 passages [28][29][30][31][32][33][34][35].…”

“…The cellular response to heat shock includes the transcriptional up-regulation of genes encoding heat shock proteins (HSPs) as part of the cell's internal repair mechanism [30]. They are also called stress-proteins [31] and respond to heat, cold and oxygen deprivation by activating several cascade pathways [32][33][34][35].…”

“…Previous work [22][23][24][25][26][27][28][29][30] has demonstrated differentiation steps to AEII cells from murine ES cells and the hES H7 cell line.…”