This article includes supplementary material available via the Internet at http://www.interscience.com/jpages/0006-3592/suppmat.
Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products†
Article first published online: 21 JUN 2005
Copyright © 2005 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 91, Issue 6, pages 688–698, 20 September 2005
How to Cite
Li, Y., Powell, S., Brunette, E., Lebkowski, J. and Mandalam, R. (2005), Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol. Bioeng., 91: 688–698. doi: 10.1002/bit.20536
- Issue published online: 17 AUG 2005
- Article first published online: 21 JUN 2005
- Manuscript Accepted: 15 MAR 2005
- Manuscript Received: 6 OCT 2004
- human embryonic stem cells;
- defined serum-free medium;
Human embryonic stem cells (hESCs) can serve as an unlimited cell source for cellular transplantation and tissue engineering due to their prolonged proliferation capacity and their unique ability to differentiate into derivatives of all three-germ layers. In order to reliably and safely produce hESCs, use of reagents that are defined, qualified, and preferably derived from a non-animal source is desirable. Traditionally, mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture undifferentiated hESCs. We recently reported a scalable feeder-free culture system using medium conditioned by MEFs. The base and conditioned medium (CM) still contain unknown bovine and murine-derived components, respectively. In this study, we report the development of a hESC culture system that utilizes a commercially available serum-free medium (SFM) containing human sourced and recombinant proteins supplemented with recombinant growth factor(s) and does not require conditioning with feeder cells. In this system, which employs human laminin coated surface and high concentration of hbFGF, the hESCs maintained undifferentiated hESC morphology and had a twofold increase in expansion compared to hESCs grown in MEF-CM. The hESCs also expressed surface markers SSEA-4 and Tra-1-60 and maintained expression of hTERT, Oct4, and Cripto genes similar to cells cultured in MEF-CM. In addition, hESCs maintained in this culture system were able to differentiate in vitro and in vivo into cells of all three-germ layers. The cells maintained a normal karyotype after prolonged culture in SFM. In summary, this study demonstrates that the hESCs cultured in defined non-conditioned serum-free medium (NC-SFM) supplemented with growth factor(s) retain the characteristics and replicative potential of hESCs. The use of defined culture system with NC-SFM on human laminin simplifies scale-up and allows for reproducible generation of hESCs under defined and controlled conditions that would serve as a starting material for production of hESC derived cells for therapeutic use. © 2005 Wiley Periodicals, Inc.