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Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells |
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| Authors: | Veronika Akopian Peter W. Andrews Stephen Beil Nissim Benvenisty Jennifer Brehm Megan Christie Angela Ford Victoria Fox Paul J. Gokhale Lyn Healy Frida Holm Outi Hovatta Barbara B. Knowles Tenneille E. Ludwig Ronald D. G. McKay Takamichi Miyazaki Norio Nakatsuji Steve K. W. Oh Martin F. Pera Janet Rossant Glyn N. Stacey Hirofumi Suemori |
| Affiliation: | 1. The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA 2. Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK 3. Institute of Life Sciences, Department of Genetics, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, 91904, Israel 4. WiCell Research Institute, P.O. Box?7365, Madison, WI, 53707-7365, USA 5. UK Stem Cell Bank, Division of Cell Biology and Imaging, National Institute for Biological Standards and Control-Health Protection Agency, South Mimms, Herts, EN6 3QG, UK 6. Karolinska Institutet, Department of Clinical Science, Technology and Intervention, Karolinska University Hospital, SE 141 86, Stockholm, Sweden 7. Institute of Medical Biology 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Republic of Singapore 8. NIH Stem Cell Unit, National Institutes of Health, Bldg 35/2B-213, MSC 3703, 9000 Rockville Pike, Bethesda, MD, 20892, USA 9. Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan 10. Bioprocessing Technology Institute, Agency for Science Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Republic of Singapore 11. Developmental Biology Program, The Hospital for Sick Children, TMDT Building, Room 13-305, 101 College Street, Toronto, ON, M5G 1L7, Canada |
| Abstract: | There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. |
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