Protocol

Human Embryonic Stem Cell Protocols

Volume 331 of the series Methods In Molecular Biology pp 91-104

Culture of Human Embryonic Stem Cells on Human and Mouse Feeder Cells

  • Gautam DravidAffiliated withInstitute for Cell Engineering, Johns Hopkins University School of Medicine
  • , Holly HammondAffiliated withInstitute for Cell Engineering, Johns Hopkins University School of Medicine
  • , Linzhao ChengAffiliated withInstitute for Cell Engineering, Johns Hopkins University School of Medicine

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Abstract

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.

Key Words

Embryonic stem cells human stem cells mesenchymal stem cells marrow stromal cells MSCs feeder cells fibroblasts immortalization self-renewal pluripotency teratoma