Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders

被引:151
作者
Genbacev, O
Krtolica, A
Zdravkovic, PDUT
Zdravkovic, T
Brunette, E
Powell, S
Nath, A
Caceres, E
McMaster, M
McDonagh, S
Li, Y
Mandalam, R
Lebkowski, J
Fisher, SL
机构
[1] Univ Calif San Francisco, Dept Cell & Tissue Biol, Program Human Stem Cell Biol & Dev, San Francisco, CA 94143 USA
[2] Univ Calif San Francisco, Stem Cell Biol Program, San Francisco, CA USA
[3] Lawrence Berkeley Natl Lab, Div Life Sci, Berkeley, CA USA
[4] Geron Corp, Menlo Pk, CA USA
关键词
human embryonic stem cells; human placental fibroblasts; laminin; nonxenogeneic culture; serum-free medium;
D O I
10.1016/j.fertnstert.2005.01.086
中图分类号
R71 [妇产科学];
学科分类号
100211 ;
摘要
Objective: To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stern cell niche in situ. Design: We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs. Result(s): Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco's modified Eagle's medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype. were grown for more than 25 passages, expressed markers of sternness including Oct-3/4, Tra 1-60, Tra 1-80. and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to Growth on a human placental laminin substrate in a defined medium. Conclusion(s): To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders. (Fertil Steril (R) 2005;83:1517-29. (c) 2005 by American Society for Reproductive Medicine.)
引用
收藏
页码:1517 / 1529
页数:13
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