title: 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.
authors.affiliation: Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Kumar
authors.forename: Indira
authors.initials: I
authors.lastname: Hinduja
authors.forename: Punam
authors.initials: P
authors.lastname: Nagvenkar
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Pillai
authors.forename: Kusum
authors.initials: K
authors.lastname: Zaveri
authors.forename: Leena
authors.initials: L
authors.lastname: Mukadam
authors.forename: Jyoti
authors.initials: J
authors.lastname: Telang
authors.forename: Sadhana
authors.initials: S
authors.lastname: Desai
authors.forename: Vijay
authors.initials: V
authors.lastname: Mangoli
authors.forename: Ranjana
authors.initials: R
authors.lastname: Mangoli
authors.forename: Shreyas
authors.initials: S
authors.lastname: Padgaonkar
authors.forename: Gurvinder
authors.initials: G
authors.lastname: Kaur
authors.forename: Chander
authors.initials: C
authors.lastname: Puri
authors.forename: Deepa
authors.initials: D
authors.lastname: Bhartiya
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007621:Karyotyping; D051379:Mice; D016513:Mice, SCID; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D011247:Pregnancy; D033581:Stem Cell Transplantation; D013724:Teratoma / Q000473:pathology