No 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.

Human embryonic stem (hES) cells possess the ability to self-renew indefinitely and provide a potential source of differentiated progeny representing all three embryonic germ layers. Although hES cell lines share the expression of typical pluripotency markers, limited data is available regarding their differentiation capabilities. We have earlier reported the in-house derivation of two hES cell lines, KIND-1 and KIND-2 on human feeders. Here, we describe a comparative study carried out on both these cell lines to better understand the differentiation potential of KIND-1 and KIND-2 by gene expression analysis of representative gene transcripts reflecting pluripotency and the three germ layers viz. ectoderm, mesoderm, and endoderm. Gene expression analysis and immunolocalization studies were undertaken on (a) 7- and 14-d old embryoid bodies (EBs) (b) spontaneously differentiated cells from EBs, (c) cells derived from EBs under the influence of various growth factor treatments and (d) KIND-1 and KIND-2 cells co-cultured on mouse embryonic visceral endoderm-like feeder (END-2). Despite both the cell lines being XX, derived, passaged, and cultured similarly, KIND-1 exhibits preferential differentiation towards endodermal lineage whereas KIND-2 spontaneously forms beating cardiomyocytes. Perhaps the occurrence of discrete epigenetic profile in both the cell lines predisposes them to encompass different developmental potential in vitro. Our data provide evidence for existence of distinct differentiation propensity among hES cell lines and emphasizes the need to derive more hES cell lines for future regenerative medicine.