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Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions

Abstract

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4–positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm2, where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

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Figure 1: Representative features of hPS cells culture systems based on passaging in cell aggregates and single-cell suspensions.
Figure 2: Transfer of hPS cells from feeder layer to LN-521 substratum at Step 15.
Figure 3: Bright-field images of living hPS cells plated on LN-521 at different densities at different time points (Step 6 or 9).
Figure 4: Flowchart of rapid passaging of hPS cell in single-cell suspensions at Step 6.
Figure 5: Routine characterization of hPS cells cultured on LN-521.

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References

  1. Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).

    Article  CAS  Google Scholar 

  2. Kucia, M. et al. A population of very small embryonic-like (VSEL) CXCR4+SSEA-1+Oct-4+ stem cells identified in adult bone marrow. Leukemia 20, 857–869 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  Google Scholar 

  5. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).

    Article  CAS  Google Scholar 

  6. Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232 (2005).

    Article  CAS  Google Scholar 

  7. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  Google Scholar 

  8. Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat. Biotechnol. 28, 581–583 (2010).

    Article  CAS  Google Scholar 

  9. Melkoumian, Z. et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat. Biotechnol. 28, 606–610 (2010).

    Article  CAS  Google Scholar 

  10. Braam, S.R. et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin. Stem Cells 26, 2257–2265 (2008).

    Article  CAS  Google Scholar 

  11. Miyazaki, T. et al. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3, 1236 (2012).

    Article  Google Scholar 

  12. Rodin, S. et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat. Commun. 5, 3195 (2014).

    Article  Google Scholar 

  13. Domogatskaya, A., Rodin, S. & Tryggvason, K. Functional diversity of laminins. Annu. Rev. Cell Dev. Biol. 28, 523–553 (2012).

    Article  CAS  Google Scholar 

  14. Dziadek, M. & Timpl, R. Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol. 111, 372–382 (1985).

    Article  CAS  Google Scholar 

  15. Klaffky, E. et al. Trophoblast-specific expression and function of the integrin α 7 subunit in the peri-implantation mouse embryo. Dev. Biol. 239, 161–175 (2001).

    Article  CAS  Google Scholar 

  16. Mahoney, Z.X., Stappenbeck, T.S. & Miner, J.H. Laminin α5 influences the architecture of the mouse small intestine mucosa. J. Cell Sci. 121, 2493–2502 (2008).

    Article  CAS  Google Scholar 

  17. Sugawara, K. et al. Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth. J. Histochem. Cytochem. 55, 43–55 (2007).

    Article  CAS  Google Scholar 

  18. Doi, M. et al. Recombinant human laminin-10 (α5β1γ1). Production, purification, and migration-promoting activity on vascular endothelial cells. J. Biol. Chem. 277, 12741–12748 (2002).

    Article  CAS  Google Scholar 

  19. Kortesmaa, J., Yurchenco, P. & Tryggvason, K. Recombinant laminin-8 (α(4)β(1)γ(1)). Production, purification, and interactions with integrins. J. Biol. Chem. 275, 14853–14859 (2000).

    Article  CAS  Google Scholar 

  20. Miyazaki, T. et al. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375, 27–32 (2008).

    Article  CAS  Google Scholar 

  21. Smirnov, S.P. et al. Contributions of the LG modules and furin processing to laminin-2 functions. J. Biol. Chem. 277, 18928–18937 (2002).

    Article  CAS  Google Scholar 

  22. Rodin, S. et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat. Biotechnol. 28, 611–615 (2010).

    Article  CAS  Google Scholar 

  23. Domogatskaya, A., Rodin, S., Boutaud, A. & Tryggvason, K. Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells 26, 2800–2809 (2008).

    Article  CAS  Google Scholar 

  24. Inzunza, J. et al. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23, 544–549 (2005).

    Article  CAS  Google Scholar 

  25. Klimanskaya, I., Chung, Y., Becker, S., Lu, S.J. & Lanza, R. Human embryonic stem cell lines derived from single blastomeres. Nature 444, 481–485 (2006).

    Article  CAS  Google Scholar 

  26. Stephenson, E. et al. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product–free environment. Nat. Protoc. 7, 1366–1381 (2012).

    Article  CAS  Google Scholar 

  27. Chen, G., Hou, Z., Gulbranson, D.R. & Thomson, J.A. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248 (2010).

    Article  CAS  Google Scholar 

  28. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  29. Silva, J. & Smith, A. Capturing pluripotency. Cell 132, 532–536 (2008).

    Article  CAS  Google Scholar 

  30. Amps, K. et al. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat. Biotechnol. 29, 1132–1144 (2011).

    Article  CAS  Google Scholar 

  31. Narva, E. et al. High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity. Nat. Biotechnol. 28, 371–377 (2010).

    Article  Google Scholar 

  32. Scheele, S. et al. Laminin α1 globular domains 4–5 induce fetal development but are not vital for embryonic basement membrane assembly. Proc. Natl. Acad. Sci. USA 102, 1502–1506 (2005).

    Article  CAS  Google Scholar 

  33. Horejs, C.M. et al. Biologically active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells. Proc. Natl. Acad. Sci. USA 111, 5908–5913 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by grants from the Swedish Research Council (to K.T. and O.H.), the Knut and Alice Wallenberg Foundation (to K.T.), the AFA Insurance Foundation (to K.T. and O.H.), Söderberg's Foundation (to K.T.), the European Commission (to O.H.) and the National Medical Research Council, Singapore (to K.T.).

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S.R. and L.A. conducted in vitro experiments with the pluripotent cells. S.R., L.A. and O.H. contributed to the planning and design of experiments and to the writing of the manuscript. K.T. planned and designed the project and contributed to the writing of the manuscript.

Corresponding author

Correspondence to Karl Tryggvason.

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Competing interests

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

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Rodin, S., Antonsson, L., Hovatta, O. et al. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nat Protoc 9, 2354–2368 (2014). https://doi.org/10.1038/nprot.2014.159

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