A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

Abstract

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.

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Figure 1: Strategy for the identification of peptide-substituted surfaces for hES cell adhesion and survival.
Figure 2: Surfaces displaying heparin-binding peptides support hES cell adhesion and self-renewal.
Figure 3: Synthetic surfaces support the long-term culture of pluripotent stem cells.
Figure 4: Pluripotent stem cells grown on synthetic surfaces maintain their ability to differentiate.
Figure 5: Streptavidin-coated surfaces presenting heparin-binding peptides support robust adhesion and self-renewal.

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Acknowledgements

We thank R. Derda, B.P. Orner and J.A. Thomson for helpful discussions, J.R. Torrealba for analysis and interpretation of the teratoma data and G.L. Case for help with the automated peptide synthesis. This research was supported by the US National Institutes of Health (R01 grants AI055258 and GM49975) and the University of Wisconsin Materials Research Science and Engineering Center (DMR-0520527). We thank the W.M. Keck Foundation for supporting the Center for Chemical Genomics and the WiCell Research Institute and members of the University of Wisconsin Paul P. Carbone Comprehensive Cancer Center Flow Cytometry Facility (5P30 CA014520-3S) for technical assistance.

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J.R.K., L.L., P.J.W. and L.L.K. conceived the experiments and interpreted the results. J.R.K. performed the in vitro experiments. L.L. synthesized and purified the molecules used to fabricate the surfaces. J.R.K. and M.S.P. conducted the teratoma assay, and P.J.W. conducted the directed differentiation assays. J.R.K. and L.L.K. wrote the manuscript.

Corresponding author

Correspondence to Laura L Kiessling.

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Competing interests

L.L.K. is an author on a patent on self-assembled monolayers for stem cell culture (US patent 2007/0207543). L.L.K., J.R.K. and L.L. are authors on a pending patent that describes surfaces for the long-term culture of pluripotent cells (US patent application 20100087004).

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Klim, J., Li, L., Wrighton, P. et al. A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nat Methods 7, 989–994 (2010). https://doi.org/10.1038/nmeth.1532

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