Primate iPSC cell culture

The Human iPS cell lines WT-33, ADRC-40 and WT-126 were previously described [45], as was the method for generating iPSC cell lines for non-human primates [46]. Briefly, Fibroblasts from P. troglodytes (chimpanzees: PR00818), G. gorilla (gorilla: PR00053 and PR00075), and M. mulatta (rhesus) were from Coriell Cell Repositories (NJ). All fibroblasts were cultured in MEM (Invitrogen) supplemented with 10% FBS (HyClone Laboratories). Retroviral vectors expressing OCT4 (also known as POU5F1), MYC, KLF4 and SOX2 human cDNAs from Yamanaka’s group [47] were obtained from Addgene. Recombinant viruses were produced by transient transfection in 293T cells (ATCC - CRL-3216), as previously described [48]. Two days after infection, cells were plated on mitotically inactivated mouse embryonic fribroblasts (Chemicon) with human ES cell medium. after 2-4 weeks, iPSC cell colonies were picked manually and directly transferred to feeder-free conditions on matrigel-coated dishes (BD) using mTeSR1 (StemCell Technologies). Established iPS cell colonies were kept in feeder-free conditions indefinitely, and passed using mechanical dissociation. Embryoid-body-mediated differentiation in suspension was carried out for 10 days in the absence of growth factors.

iPSC clones continuously expressed pluripotency markers, retained undifferentiated morphology in culture, and maintained a normal karyotype. After embryoid body (EB)-mediated differentiation in vitro, clones contained tissue derivatives from the three embryonic germ layers and down-regulated expression of pluripotency markers.