Defined culture conditions of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into any tissue in the human body; therefore, they are a valuable resource for regenerative medicine, drug screening, and developmental studies. However, the clinical application of hESCs is hampered by the difficulties of eliminating animal products in the culture medium and͞or the complexity of conditions required to support hESC growth. We have developed a simple medium [termed hESC Cocktail (HESCO)] containing basic fibroblast growth factor, Wnt3a, April (a proliferation-inducing ligand)͞BAFF (B cell-activating factor belonging to TNF), albumin, cholesterol, insulin, and transferrin, which is sufficient for hESC self-renewal and proliferation. Cells grown in HESCO were maintained in an undifferentiated state as determined by using six different stem cell markers, and their genomic integrity was confirmed by karyotyping. Cells cultured in HESCO readily form embryoid bodies in tissue culture and teratomas in mice. In both cases, the cells differentiated into each of the three cell lineages, ectoderm, endoderm, and mesoderm, indicating that they maintained their pluripotency. The use of a minimal medium sufficient for hESC growth is expected to greatly facilitate clinical application and developmental studies of hESCs.April͞BAFF ͉ fibroblast growth factor ͉ serum free culture ͉ wnt

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

“…In earlier studies, DA differentiation of hES cells required incubation in culture with other cell types (PA6 mouse stromal line; human HepG2 liver line) or in cell-CM, usually containing fetal calf serum and/or other undefined components (4,5,16,17,18,22,26). In several recent studies, efforts have been made to move to a serum-free defined protocol for neuronal (9,10,13) or DA differentiation (19,22) of hES cells. With only one exception (19), however, media was supplemented with serum substitutes, such as B27, and often cells were grown on Matrigel, both of which contain undefined proprietary components and growth substances of animal origin.…”

“…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.…”

“…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. PA6 mouse stromal cells) (4,5,10,13,16,17,18,22,26). Since animal cells contain immunogenic antigens that can be incorporated into hES cells (14), they ultimately can cause immune rejection after their transplantation into the brain.…”

“…Since animal cells contain immunogenic antigens that can be incorporated into hES cells (14), they ultimately can cause immune rejection after their transplantation into the brain. Even in cases where animal sera, CM and cells have been eliminated from the hES differentiation protocol (9,10,13,23), media additives (B27, Matrigel, etc.) have been used which contain undefined, often proprietary, components as well as hormones and growth products of animal origin.…”

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

“…iPS cells are usually maintained on feeder cells that are derived from mouse embryonic fibroblasts (MEFs) (Takahashi and Yamanaka 2006;Takahashi et al 2007;Yu et al 2007). However, several feederfree conditions were developed recently especially for human embryonic stem (ES) cells and iPS cells to ensure biosafe human clinical applications (Xu et al 2001;Amit et al 2004;Rosler et al 2004;Lu et al 2006;Navarro-Alvarez et al 2008;Miyazaki et al 2008;Sun et al 2009;Kitajima and Niwa 2010;Rodin et al 2010;Hayashi et al 2010;Fukusumi et al 2013). Animal-derived components are relatively high risk for potential infections in clinical regenerative medicine.…”