Culture of human embryonic stem cells on human and mouse feeder cells

This chapter describes the methods we use to maintain and expand undifferentiated human embryonic stem (hES) cells on human and mouse feeder cells. All of the available hES cells have been derived and propagated on primary mouse embryonic fibroblasts as feeder cells that have been mitotically inactivated. We found that hES cells can be successfully cultured on selected human feeder cells, such as marrow stromal cells derived from adult bone marrow and breast skin fibroblasts. Detailed protocols to use human and mouse feeder cells are described here, together with our method to split hES cells by trypsin/ethylenediaminetetraacetic acid-mediated dissociation. We also describe methods we use to characterize hES cells expanded on either human or mouse feeder cells, including alkaline phosphatase staining, immunostaining for cell-surface markers associated with undifferentiated hES cells, and teratoma formation in mice.