Derivation and Characterization of Two Genetically Unique Human Embryonic Stem Cell Lines on In-House–Derived Human Feeders
| Autor: | Deepa Bhartiya, Leena Mukadam, Lakshmi Pillai, Sadhana K. Desai, Gurvinder Kaur, Jyoti Telang, Punam Nagvenkar, Indira Hinduja, Kusum Zaveri, Vijay Mangoli, Neeraj Kumar, Shreyas Padgaonkar, Ranjana Mangoli, Chander P Puri |
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| Rok vydání: | 2009 |
| Předmět: |
Pluripotent Stem Cells
Cell Culture Techniques Mice SCID Embryoid body Biology Cell Line Mice Pregnancy Surplus embryos Animals Humans Embryonic Stem Cells Teratoma Cell Differentiation Cell Biology Hematology Fibroblasts Molecular biology Embryonic stem cell Cell biology P19 cell Cell culture Karyotyping Female Stem cell Stem Cell Transplantation Developmental Biology Adult stem cell |
| Zdroj: | Stem Cells and Development. 18:435-446 |
| ISSN: | 1557-8534 1547-3287 |
| DOI: | 10.1089/scd.2008.0234 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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