Derivation and Characterization of Two Genetically Unique Human Embryonic Stem Cell Lines on In-House–Derived Human Feeders

Abstract

This study describes the successful derivation of two human embryonic stem (hES) cell lines using 53 frozen and 18 fresh "slow-growing" surplus embryos, obtained from collaborating in vitro fertilization clinics, on in-house-derived human feeder layers. The cell lines have been derived by whole embryo culture followed by further expansion of manually dissected inner cell mass from the surrounding trophoectodermal cells. Immunocytochemical localization of cell surface markers like SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, staining for alkaline phosphatase and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of pluripotency state markers viz. Oct-4, TERT, Nanog, Rex1, and Sox2 indicate that both cell lines possess typical features of embryonic stem cells. Both cell lines exhibit normal female karyotype after 40 passages in culture. Pluripotent nature of the cell lines was confirmed both in vitro and in vivo. Embryoid bodies, formed in suspension culture, express markers for all three lineages as indicated by RT-PCR analysis for SOX 1 (ectoderm), HAND 1 (mesoderm), AFP (endoderm), and CDX2 (trophoectoderm). Teratoma formed in vivo in severe combined immunodeficient mice revealed cells of all the three embryonic germ layers. Comparison of the STR and human leukocyte antigen profiles of these cell lines with the existing human ES cell lines indicate that they are genetically distinct. The addition of our hES cell lines contributes usefully to the globally restricted repertoire of human ES cell lines.

Retraction: Detection, Characterization, and Spontaneous Differentiation In Vitro of Very Small Embryonic-Like Putative Stem Cells in Adult Mammalian Ovary

“…Appropriate positive control, i.e., in-housederived human ES cells [26] and adult human testicular tissue (collected from prostate cancer patients as part of their clinical management) were also processed similarly.…”

Evaluating differentiation propensity of in-house derived human embryonic stem cell lines KIND-1 and KIND-2

“…Two human embryonic stem (hES) cell lines KIND-1 and KIND-2 derived at the Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India were used in this study (Kumar et al 2009). Briefly, both the cell lines were expanded and maintained on mitomycin C-treated human fetal fibroblast feeder at 37°C, in a humid 5% CO 2 environment.…”

“…Besides spare human blastocysts, hES cell lines have also been derived from morula-stage embryos (Strelchenko et al 2004), abnormally developing and arrested embryos (Zhang et al 2006), single blastomeres of eight-cell-stage embryos (Klimanskaya et al 2007) and four-cell-stage embryos (Feki et al 2008;Geens et al 2009). Seven well-characterized cell lines derived using spare human embryos have recently been published from the Indian subcontinent (Mandal et al 2006;Inamdar et al 2009;Kumar et al 2009;Mandal et al 2010), of which two from our group were derived and propagated on human feeders by manual cut and paste method (Kumar et al 2009). hES stem cell lines are similar with respect to selfrenewal and expression of pluripotency markers however published literature suggests that they exhibit differences in their differentiation ability under identical culture conditions (Oh et al 2005;Denning et al 2006;Osafune et al 2008;Pal et al 2009;Tavakoli et al 2009).…”

Role of TGF beta and myofibroblasts in supporting the propagation of human embryonic stem cells in vitro

“…Human fetal fibroblast culture, with prior permission from Institute Ethics Committee, was established as described earlier (Kumar et al, 2009).…”

“…An earlier report from our laboratory has shown that feeder layers derived from 13.5dpc CF-1 mouse embryo and human fetal fibroblasts (HFF) are supportive for hES cells culture (Kumar et al 2009) whereas the feeder layers derived from 18.5dpc mouse embryo exhibit massive differentiation in hES colonies beyond 1-2 passages. Thus in an effort to identify potential candidates having role in self renewal of hES cells, we studied the transcriptome of supportive and non-supportive feeder fibroblasts by microarray analysis and correlated with earlier reported proteomics data of factors detected in the conditioned medium of supportive feeders.…”