Derivation of human embryonic stem cell lines without any exogenous growth factors.

ABSTRACT

Human embryonic stem cell (hESC) lines are traditionally derived through immunosurgery. Their maintenance in culture requires the presence of mouse embryonic fibroblasts (MEFs) as feeder cells and media supplemented with basic fibroblast growth factor (bFGF) or other growth factors-both of which might introduce animal-derived culture components. The drawbacks associated with immunosurgery, MEF co-culture, and the cost of growth factors necessitate the exploration of a xeno-free method to maintain the self-renewal capacity of hESCs. Here, we describe an isolation method for the human inner cell mass (ICM), which was then cultured in the absence of exogenous growth factors and in the presence of human foreskin fibroblasts (HFFs) as feeder cells. Three hESC lines were obtained from poor-quality embryos by this near-xeno-free protocol. After culturing for more than 10 months, the hESCs retained normal morphology, expressed all expected cell surface markers, could differentiate to embryoid bodies upon culture in vitro, and formed teratomas in vivo. Furthermore, secretion of bFGF by HFFs was observed. In conclusion, this is the first study to describe an inexpensive, xeno-free culture system for the isolation and maintenance of hESCs that does not require bFGF supplementation.