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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References
Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).
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).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).
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).
Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).
Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat. Biotechnol. 28, 581–583 (2010).
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).
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).
Miyazaki, T. et al. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3, 1236 (2012).
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).
Domogatskaya, A., Rodin, S. & Tryggvason, K. Functional diversity of laminins. Annu. Rev. Cell Dev. Biol. 28, 523–553 (2012).
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).
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).
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).
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).
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).
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).
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).
Smirnov, S.P. et al. Contributions of the LG modules and furin processing to laminin-2 functions. J. Biol. Chem. 277, 18928–18937 (2002).
Rodin, S. et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat. Biotechnol. 28, 611–615 (2010).
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).
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).
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).
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).
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).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Silva, J. & Smith, A. Capturing pluripotency. Cell 132, 532–536 (2008).
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).
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).
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).
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).
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.
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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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DOI: https://doi.org/10.1038/nprot.2014.159
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