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Feeder-free growth of undifferentiated human embryonic stem cells

Abstract

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.

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Figure 1: (A–K) Morphology of feeder-free hES cells.
Figure 2: (A) Integrin expression in H1 cells (passage 46 + 7) maintained on feeders in ESM (MEF/ESM) or on Matrigel or laminin in MEF-CM for 42 days.
Figure 3: Differentiation of feeder-free hES cells.

References

  1. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  2. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).

    Article  CAS  Google Scholar 

  3. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  Google Scholar 

  4. Smith, A.G. et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690 (1988).

    Article  CAS  Google Scholar 

  5. Williams, R.L. et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687 (1988).

    Article  CAS  Google Scholar 

  6. Kleinman, H.K. et al. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21, 6188–6193 (1982).

    Article  CAS  Google Scholar 

  7. Bisell, D.M., Arenson, D.M., Maher, J.J. & Roll, F.J. Support of cultured hepatocytes by a laminin-rich gel. J. Clin. Invest. 79, 801–812 (1987).

    Article  Google Scholar 

  8. Bodnar, A.G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352 (1998).

    CAS  PubMed  Google Scholar 

  9. Cooper, A.R. & MacQueen, H.A. Subunits of laminin are differentially synthesized in mouse eggs and early embryos. Dev. Biol. 96, 467–471 (1983).

    Article  CAS  Google Scholar 

  10. Ekblom, P., Vestweber, D. & Kemler, R. Cell–matrix interactions and cell adhesion during development. Annu. Rev. Cell Biol. 2, 27–47 (1986).

    Article  CAS  Google Scholar 

  11. Hynes, R.O. Integrins: versatility, modulation and signaling in cell adhesion. Cell 69, 11–25 (1992).

    Article  CAS  Google Scholar 

  12. Hierck, B.P. et al. Variants of the α6β1 laminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin α6 subunit. Cell Adhesion Commun. 1, 33–53 (1993).

    Article  CAS  Google Scholar 

  13. Cooper, H.M., Tamura, R.N. & Quaranta, V. The major laminin receptor of mouse embryonic stem cells is a novel isoform of the α6β1 integrin. J. Cell Biol. 115, 843–850 (1991).

    Article  CAS  Google Scholar 

  14. Kim, N.Y. et al. Specific association of human telomerase activity with immortal cell lines and cancer. Science 266, 2011–2015 (1994).

    Article  CAS  Google Scholar 

  15. Weinrich, S.L. et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 17, 498–502 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Mohammad Hassanipour for technical assistance, Dr. Ram Mandalam for evaluating frozen CM, Drs. Choy-Pik Chiu, Calvin Harley, and Jane Lebkowski for insightful discussions and critical review of the manuscript, Dr. Peter Andrews (University of Sheffield, UK) for Tra 1-81 and Tra 1-60 antibodies, and Hybridoma Bank (Iowa City, IA) for SSEA-1 and SSEA-4 antibodies.

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Correspondence to Melissa K. Carpenter.

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Xu, C., Inokuma, M., Denham, J. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19, 971–974 (2001). https://doi.org/10.1038/nbt1001-971

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