Derivation of Human Embryonic Stem Cells

Zona pellucidae from blastocysts were removed with 0.5% pronase (Sigma P8811) and embryos were plated onto confluent feeder layers of mouse embryonic fibroblasts (mEF) and cultured for 6 days at 37 °C, 3% CO₂, 5% O₂ and 92% N₂ in ESC derivation medium. The medium consisted of DMEM/F12 (Gibco 11320-033) with 0.1 mM nonessential amino acids (Gibco 11140-050), 1mM L-glutamine (Gibco 21051-024), 0.1 mM β-mercaptoethanol (Sigma M6250), 5 ng/ml basic fibroblast growth factor (bFGF, Sigma F-0291), 10 µM ROCK inhibitor (Sigma SCM075), 10% fetal bovine serum (FBS, Hyclone Thermo Scientific SH30071.03) and 10% knockout serum replacement (KSR, Gibco 10828-028). ESC colonies were manually dissociated and replated onto fresh mEFs for further propagation and analyses. FBS and ROCK inhibitor were omitted after the first passage of ESCs and KSR was increased to 20%. All ESC lines have been authenticated by short tandem repeat (STR) genotyping, confirming their origin from the gamete donors from this study. ESC lines are available to researchers upon OHSU IRB approval and signed OHSU MTA.