435 STEM CELLS AND DEVELOPMENT Volume 18, Number 3, 2009 © Mary Ann Liebert, Inc. DOI: 10.1089/scd.2008.0234 Derivation and Characterization of Two Genetically Unique Human Embryonic Stem Cell Lines on In-House–Derived Human Feeders Neeraj Kumar, 1 Indira Hinduja, 2 Punam Nagvenkar, 1 Lakshmi Pillai, 1 Kusum Zaveri, 2 Leena Mukadam, 2 Jyoti Telang, 1 Sadhana Desai, 3 Vijay Mangoli, 3 Ranjana Mangoli, 3 Shreyas Padgaonkar, 4 Gurvinder Kaur, 5 Chander Puri, 1 and Deepa Bhartiya 1 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 expan- sion 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 phospha- tase 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 confrmed 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 immunodefcient mice revealed cells of all the three embryonic germ layers. Comparison of the STR and human leukocyte antigen profles 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. Introduction A fter the successful derivation of human embryonic stem (hES) cell lines by Thomson et al. [1], the past few years have witnessed a marked increase in number of hES cell lines available worldwide. At present ~414 hES cell lines have been derived across the world, of which 78 hES cell lines are registered with National Institutes of Health (NIH) [2]. Only 22 lines of the 78 registered with NIH are available for research purposes [3]. All the cell lines registered at NIH were grown on mouse embryonic feeder (MEF) layers. Such cell lines run the risk of contaminating hES cell colonies with animal retroviruses and other nonhuman pathogens that may get transplanted to humans during cell therapy and may be immunogenic in nature. A conscious effort is thus evident in the published literature on the part of sci- entists to derive hES cell lines under xeno-free conditions. Various approaches have been tried viz., use of extracellu- lar matrices (matrigel, laminin, collagen, fbronectin) along with conditioned medium from MEFs, use of human feed- ers (fetal muscle, skin, foreskin, adult fallopian tube epithe- lial cells) and use of feeder and serum free system. Two hES cell lines derived under feeder and serum free conditions were reported to have abnormal karyotype [4]. The culture condition driven genetic abnormalities in these cell lines emphasize the importance of feeder layers which secrete “yet not fully identifed” factors that support proliferation 1 Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India. 2 INKUS IVF Clinic, Mumbai, India. 3 Fertility Clinic, Mumbai, India. 4 Shreyas Infertility and IVF Center, Mumbai, India. 5 Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India.