Feeder-free growth of undifferentiated human embryonic stem cells

• Published in: Nature biotechnology 2001-10, Vol.19 (10), p.971-974
• Main Authors: Carpenter, Melissa K, Xu, Chunhui, Inokuma, Margaret S, Denham, Jerrod, Golds, Kathaleen, Kundu, Pratima, Gold, Joseph D
• Format: Article
• Language: English
• Subjects: Agriculture; Animal cells; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cell Culture Techniques - methods; Cell Differentiation; Cell Division; Cell growth; Coculture Techniques; Collagen; Culture Media; Culture Media, Conditioned; Drug Combinations; Embryo, Mammalian - cytology; Embryo, Nonmammalian; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Extracellular matrix; Fibroblasts; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Glycosphingolipids - biosynthesis; Heparan sulfate; Karyotypes; Laminin; Life Sciences; Methods. Procedures. Technologies; Morphology; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Stage-Specific Embryonic Antigens; Stem cells; Stem Cells - cytology; technical-report; Telomerase
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt1001-971

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin α6 and β1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.