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

TLDR

The study reports deriving two human embryonic stem cell lines from surplus frozen and fresh embryos using in‑house human feeder layers. Derivation involved whole‑embryo culture and inner cell mass expansion, with characterization by immunocytochemistry for SSEA‑3/4 and TRA‑1‑60/81, alkaline phosphatase staining, and RT‑PCR of pluripotency genes. Both lines display typical embryonic stem cell features, including Oct‑4, TERT, Nanog, Rex1, Sox2 expression, a normal female karyotype after 40 passages, pluripotency confirmed in vitro and in vivo through embryoid bodies expressing ectoderm, mesoderm, and endoderm markers and teratoma formation, and are genetically distinct from existing lines, thereby expanding the global hES cell repertoire.

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.