Extracellular Matrix Isolated From Foreskin Fibroblasts Supports Long-Term Xeno-Free Human Embryonic Stem Cell Culture

Abstract

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However, the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation, propagation, and cryopreservation. Unlike feeder-based cultures, which require the simultaneous growth of feeder and stem cells, resulting in mixed cell populations, stem cells grown on feeder-free systems are easily separated from the surface, presenting a pure population of cells for downstream applications. In this study, we have developed a novel method to expand hES cells in xeno-free, feeder-free conditions using 2 different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix, together with 100 ng/mL recombinant bFGF-supplemented HEScGRO Basal Medium, long-term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features, including morphology, pluripotency, stable karyotype, and expression of cell surface markers, for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore, the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore, without the possibility of contamination from exogenous sources.