Immortalised human skin fibroblast feeder cells support growth and maintenance of human embryonic stem cells and induced pluripotent stem cells

摘要

We established an immortal human foreskin fibroblast line to use as feeder cells for human embryonic stem cells (hESC), as well as induced pluripotent stem (iPS) cells that were generated by genetic engineering from the same parental fibroblasts. The fibroblasts were conditionally immortalised by lentiviral vector (LV) mediated transduction of Bmi-1 and telomerase (hTERT) genes and further modified by LV-mediated transduction of a secreted form of the basic fibroblast growth factor (bFGF) gene product. Four hESC lines derived in three laboratories were used to test the established feeder cells. Immortalised fibroblast cells secreting stable amounts of bFGF supported growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least ten passages. The feeders were tested up to passage 50 and they were still supportive. We then generated human iPS cells from parental fibroblasts by LV-mediated transduction with Oct 4, Sox2, Nanog, and Lin 28 genes, using their own immortalised bFGF-transduced descendants as feeders. At passage seven these iPS cells expressed pluripotency markers, and had hESC morphology. In vitro differentiation within embryoid bodies resulted in tissue components of the three embryonic germ layers. These immortalised bFGF-producing foreskin fibroblasts can thus be used as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cells.