Toward xeno-free culture of human embryonic stem cells

Abstract

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche

“…Several attempts have been described to replace MEF feeder layers with ECM proteins and matrices such as Matrigel, ECM derived from MEFs, etc. [1][2][3]. These biologically derived materials are also very complex and are not subject to control at the molecular level.…”

Proliferation and Pluripotency of Human Embryonic Stem Cells Maintained on Type I Collagen

“…Cells grown in this manner are not ideal for transplantation into humans due to the risk of xenogenic-based rejection by the immune system [18] as well as the potential for cross-species transfer of viruses or pathogens [19]. MEF feeder layers can be replaced with human-derived cell lines to avoid hESC contact with murine cells, but the presence of additional cell lines in hESC culture inevitably contributes to variability and increased cost during the expansion and scale-up of stem cells [19][20][21]. As a result, direct coculture has been replaced by medium conditioning, where hESC are grown on an acellular support matrix in the medium that has been conditioned by a separate, supporting cell line [19,20,22,23].…”

“…Laminin and fibronectin, along with defined supplements, can sometimes support stem cell growth, but these matrices may not be as effective as growth on MAT or direct coculture with MEF [20,28,[39][40][41]. One study found that fibroblast growth factor 2 (FGF2) and Noggin would help support hESC growth on MAT, but the use of a laminin matrix was subject to significant batch-to-batch variability and an inability to support clonal hESC growth [20,39]. Subsequent work done using similar growth conditions found that another hESC line remained undifferentiated on MAT for up to 15 passages but only for 3 confirmed passages on laminin [20].…”

Chemical Biology of Pluripotent Stem Cells: Focus on Cardiomyogenesis

“…These include KnockOut Serum Replacement, a defined media, which provides www.intechopen.com Chemical Biology of Pluripotent Stem Cells: Focus on Cardiomyogenesis 53 consistent growth conditions for maintaining ES and iPS cells [15], and mTeSR, a media specifically formulated to maintain human ESCs and iPSCs in serum-free and feeder-free conditions [16]. However, both KnockOut Serum Replacement and mTeSR require the use of animal-derived matrices and recombinant proteins, which increase costs and introduce variability [14,17]. Consequently, there has been considerable interest in replacing exogenous proteins with chemically defined conditions for the long-term maintenance of human pluripotent stem cells.…”