Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511

Abstract

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.

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Figure 1: Adhesion of hES cells to different coatings and expression of laminin chains in hES cells.
Figure 2: Integrin receptors on hES cell surface and their role in hES cell adhesion.
Figure 3: Representative immunostaining analysis, RT-PCR, fluorescence-activated cell sorting (FACS) analysis, real-time quantitative RT-PCR and quantitative western blot analysis of HS207 cultured on LN-511, either in O3 medium or in H3 medium free from any animal-derived components.
Figure 4: Pluripotency of HS207 cells after extensive passaging on LN-511.
Figure 5: Immunostaining analysis of different hES and iPS cells grown on LN-511.

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Acknowledgements

We thank A.-S. Nilsson and A.-M. Strömberg for excellent technical assistance, C. Cowan for collaboration in procuring the iPS cell lines, F. Holm and R. Bergström for their help with the cell cultures and D. Baker for carrying out karyotyping of hES cells. This work was supported in part by grants from the Knut and Alice Wallenberg Foundation (K.T.), the Novo Nordisk Foundation (K.T.), the Söderberg's Foundation (K.T.), the Swedish Research Council (K.T., O.H.), the Swedish Cancer Foundation (K.T.), the Harvard Stem Cell Institute (K.R.C.) and the Leducq Foundation (K.R.C.). E.M.H. is a Wenner-Gren Foundation fellow. EU: ESTOOLS (O.H.) has not been used for the derivation of new hES cell lines.

Author information

S.R. and A.D. contributed to the production and purification of human recombinant laminins, conducted all in vitro experiments with the hES cells and contributed to the planning and design of experiments and to the writing of the manuscript. O.H. established and provided the hES cell lines and contributed to manuscript writing and karyotyping. S.S. contributed to the establishment of the new hES cell lines. E.M.H. and K.R.C. contributed to the iPS cell work. J.I. carried out the teratoma experiments in nude mice. K.T. planned and designed the project and contributed to the writing of the manuscript.

Correspondence to Karl Tryggvason.

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Competing interests

K.T. and S.R. are shareholders in BioLamina.

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Supplementary Figs. 1–7 and Supplementary Tables 1,2 (PDF 2828 kb)

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Rodin, S., Domogatskaya, A., Ström, S. et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol 28, 611–615 (2010) doi:10.1038/nbt.1620

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