Isolation of human placental fibroblasts.

The first human embryonic stem cell lines (hESCs) were derived using mouse embryonic fibroblasts as feeder cells. In attempts to replace mouse embryonic fibroblasts with feeders of human origin, irradiated human placental fibroblasts were successfully used as feeder cells for the derivation and propagation of hESCs. Here we describe a protocol for the isolation and expansion of fibroblasts from placental villous stroma. We include a description of placental architecture to provide the background for a stepwise tissue digestion that leads to the isolation of villous stroma. Villous stroma from the first trimester tissue is different from term placenta and contains mesenchymal, fibroblast-like cells, only a few blood vessels, and a network of matrix fibers. The fibroblasts isolated from a single placenta of 6- to 8-weeks gestation proliferate rapidly and retain the ability to support hESC growth between passage doubling (PD) 8 and PD 12.