Expansion of human embryonic stem cells in defined serum‐free medium devoid of animal‐derived products

Abstract

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.

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

“…Although a number of feeder-free systems have been described (1,3,7,9,10,12,13,21), all hES lines to date have been established and repeatedly passaged on mouse feeders prior to transfer to a feeder free-system for cell propagation. Thus far, the feeder free protocols which we have tried (3,21), have not been compatible with the DA differentiation of hES cell lines developed in this protocol.…”

“…Currently, there are a number of established hES lines available from NIH-approved sources (Wicell, Bresagen) and a number of new lines provided by the Howard Hughes Institute of Harvard University. All of these cell lines were originally propagated on mouse feeder layers, although several have recently been transferred to feeder free support systems (1,3,7,9,12,21). While the differentiation of DA traits in some hES lines has been described previously (4,5,16,17,18,26), with the exception of one study (19) these protocols involve the prolonged use of complex media containing serum or other undefined reagents and/or cell conditioned media (CM) or co-culture with these cells (ie.…”

Toward xeno-free culture of human embryonic stem cells

“…However, most of the described culture conditions still use KSR which contains bovine serum albumin and other animalderived products. Li, Powell, Brunette, Lebkowski, and Mandalam (2005) have investigated the use of a medium previously used for hematopoietic stem cell culture which is composed solely of human derived and recombinant proteins (X-VIVO 10). Cultures of H1 cells (WA01) were originally adapted to this culture medium on Matrigel but were subsequently adapted to growth on human laminin.…”

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…It supports the growth of human embryonic stem cells in feederfree culturing conditions and is normally included in the medium for maintenance of pluripotent stem cells in their undifferentiated state (e.g. mTeSR1) [103,104]. [106].…”