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

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
611–615
Year published:
DOI:
doi:10.1038/nbt.1620
Received
Accepted
Published online

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.

At a glance

Figures

  1. Adhesion of hES cells to different coatings and expression of laminin chains in hES cells.
    Figure 1: Adhesion of hES cells to different coatings and expression of laminin chains in hES cells.

    (a) Crystal violet staining of hES cells adherent to LN-511 and LN-111. Scale bars, 220 μm (insets, 55 μm). (b) Contact area of ES cells with different adhesive substrata. Bars represent average relative contact area (compared with the cells plated on poly-D-lysine). Statistical significance (P) was calculated by Student's t-test. Error bars show s.e.m.; number inside each bar indicates number of independent measurements. LN, laminins; MG, Matrigel; PL, poly-D-lysine. (c) RT-PCR analysis of total RNA isolated from HS420 cells. Primer sets for all known laminin chains were used. bp, base pairs.

  2. Integrin receptors on hES cell surface and their role in hES cell adhesion.
    Figure 2: Integrin receptors on hES cell surface and their role in hES cell adhesion.

    (a) Adhesion-blocking experiment: inhibition of hES cell adhesion to LN-511 by different integrin antibodies. Bars represent inhibition by antibodies to chains indicated below graph (all from Millipore). IgG was used as a control for uninhibited cell adhesion. Error bars show s.e.m. (n = 4). Statistical significance (P) was calculated by Student's t-test. (b) Adhesion of hES cells to surfaces coated by different integrin antibodies. Bars represent adhesion with antibodies to chains indicated below. Error bars show s.e.m. (n = 4). P calculated by Student's t-test is shown; **P < 0.01. (c) Immunofluorescence: integrin α6 coexpression with β1 integrin subunit in pluripotent (SOX2-positive) hES cells cultured on LN-511. Scale bars, 37 μm.

  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 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.

    (a) Growth curves for hES cells cultured in O3 medium on LN-511 and Matrigel. The cells were passaged as described in the Online Methods for the long-term experiment. After each TrypLE Express treatment and subsequent washing, one-third of the cells were plated in clumps on fresh LN-511– or Matrigel-coated dishes. The rest were dissociated into single-cell suspension and counted. Two independent duplicate experiments were performed for each coating. After the fifth passage, a portion of the cells were fixed and analyzed by immunofluorescence staining, confirming that the majority of the cells still expressed Nanog, a marker of pluripotency. (b) Immunostaining of HS207 cells with antibodies to Nanog, SOX2 and OCT4 after 20 passages (6 months) on LN-511 in O3 medium. Right panels show nuclear 4,6-diamidino-2-phenylindole (DAPI) staining. Scale bars, 0.15 mm. (c) RT-PCR analysis of total RNA isolated from H207 cells grown on feeder cells (Feeders), on Matrigel after 7 passages in O3 medium (MG, p.7 in O3), on LN-511 after 8 passages in H3 medium (LN-511, p.8 in H3) and on LN-511 after 27 passages in O3 medium (LN-511, p.27 in O3). Primer sets were designed for pluripotency markers OCT4 and NANOG, along with differentiation markers Brachyury, α-fetoprotein (AFP), SOX1 and PAX6, and for a housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (d) Real-time quantitative RT-PCR analysis was used to measure numbers of mRNA transcripts of the pluripotency markers OCT4 and NANOG at different time points in HS207 cells cultured on LN-511 and on Matrigel; values shown are normalized to OCT4 and NANOG expression levels in control HS207 cells cultured on feeder layer (Feeders). Number of passages, adhesion surface and medium are denoted as in c. Error bars show 95% confidence intervals. (e) Expression of pluripotency markers OCT4 and SOX2 in HS207 cells cultured on feeder cells, Matrigel and LN-511 at different time points and in different media (denoted as in c) was measured by western blotting and quantified by densitometry. Error bars represent range. (f) FACS analysis of HS207 cells after 25 passages on LN-511 in O3 medium for OCT4, a marker of pluripotency. The percentage of positive cells is listed in parentheses.

  4. Pluripotency of HS207 cells after extensive passaging on LN-511.
    Figure 4: Pluripotency of HS207 cells after extensive passaging on LN-511.

    Teratomas containing components of the three germ layers were formed after HS207 cells that had been cultured for 15 passages on LN-511 were injected subcutaneously into SCID mice. (a) Cartilage, stained with hematoxylin and eosin (HE). Magnification, ×100. (b) Developing neural tissue and intestinal endoderm, with HE and periodic acid-Schiff (HE-PAS) staining. Goblet cells are shown in red. Magnification, ×400. (c) Developing kidney glomerulus, HE staining. Magnification, ×400. (d) Retinal pigment epithelium, HE staining. Magnification, ×400. (e) Immunostaining of embryoid bodies formed from HS207 cells after 20 passages on LN-511 revealed expression of markers for the three embryonic cell layers: smooth-muscle (SM) actin, nestin, MAP-2 and AFP. Scale bars, 55 μm.

  5. Immunostaining analysis of different hES and iPS cells grown on LN-511.
    Figure 5: Immunostaining analysis of different hES and iPS cells grown on LN-511.

    (a) H1 and H9 cells after five passages (1 month) on LN-511 in O3 medium expressed pluripotency markers OCT4 (green), Nanog (green) and SOX2 (red). DAPI staining is in blue. Scale bars, 75 μm. (b) BJ#12 and LDS 1.4 iPS cells after five passages on LN-511 in mTeSR1 medium expressed Nanog (red) and OCT4 (green). Scale bars, 75 μm.

References

  1. 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, 399404 (2000).
  2. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971974 (2001).
  3. Hovatta, O. et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18, 14041409 (2003).
  4. Martin, M.J., Rayner, J.C., Gagneux, P., Barnwell, J.W. & Varki, A. Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid. Proc. Natl. Acad. Sci. USA 102, 1281912824 (2005).
  5. Aumailley, M. et al. A simplified laminin nomenclature. Matrix Biol. 24, 326332 (2005).
  6. Miner, J.H. & Yurchenco, P.D. Laminin functions in tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 20, 255284 (2004).
  7. Ekblom, P., Lonai, P. & Talts, J.F. Expression and biological role of laminin-1. Matrix Biol. 22, 3547 (2003).
  8. Kallunki, P. et al. A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J. Cell Biol. 119, 679693 (1992).
  9. Iivanainen, A. et al. Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain. J. Biol. Chem. 272, 2786227868 (1997).
  10. Miner, J.H., Lewis, R.M. & Sanes, J.R. Molecular cloning of a novel laminin chain, alpha 5, and widespread expression in adult mouse tissues. J. Biol. Chem. 270, 2852328526 (1995).
  11. Cooper, A.R. & MacQueen, H.A. Subunits of laminin are differentially synthesized in mouse eggs and early embryos. Dev. Biol. 96, 467471 (1983).
  12. Dziadek, M. & Timpl, R. Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol. 111, 372382 (1985).
  13. Klimanskaya, I. et al. Human embryonic stem cells derived without feeder cells. Lancet 365, 16361641 (2005).
  14. Ludwig, T.E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185187 (2006).
  15. Braam, S.R. et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells 26, 22572265 (2008).
  16. Miyazaki, T. et al. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375, 2732 (2008).
  17. Yurchenco, P.D. et al. The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners. Proc. Natl. Acad. Sci. USA 94, 1018910194 (1997).
  18. Doi, M. et al. Recombinant human laminin-10 (α5β1γ1). Production, purification, and migration-promoting activity on vascular endothelial cells. J. Biol. Chem. 277, 1274112748 (2002).
  19. Kortesmaa, J., Yurchenco, P. & Tryggvason, K. Recombinant laminin-8 (α4β1γ1). Production, purification, and interactions with integrins. J. Biol. Chem. 275, 1485314859 (2000).
  20. 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, 28002809 (2008).
  21. Ginis, I. et al. Differences between human and mouse embryonic stem cells. Dev. Biol. 269, 360380 (2004).
  22. Humphrey, R.K. et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. Stem Cells 22, 522530 (2004).
  23. Wondimu, Z. et al. Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (α2β1γ1), 8 (α4β1γ1), 10 (α5β1γ1). Matrix Biol. 25, 8993 (2006).
  24. Strom, S. et al. Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum. Reprod. 22, 30513058 (2007).
  25. Evseenko, D. et al. Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly. Stem Cells Dev. 18, 919928 (2009).
  26. Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340345 (2008).
  27. Klaffky, E. et al. Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo. Dev. Biol. 239, 161175 (2001).
  28. Unger, C., Skottman, H., Blomberg, P., Dilber, M.S. & Hovatta, O. Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum. Mol. Genet. 17, R48R53 (2008).
  29. 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, 544549 (2005).
  30. Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 12181221 (2008).
  31. Inzunza, J. et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol. Hum. Reprod. 10, 461466 (2004).
  32. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637646 (2006).

Download references

Author information

Affiliations

  1. Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.

    • Sergey Rodin,
    • Anna Domogatskaya &
    • Karl Tryggvason
  2. Division of Obstetrics and Gynecology, Department of Clinical Sciences, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Huddinge, Stockholm, Sweden.

    • Susanne Ström &
    • Outi Hovatta
  3. Cardiovascular Research Center, Massachusetts General Hospital, Charles River Plaza, Boston, Massachusetts, USA.

    • Emil M Hansson &
    • Kenneth R Chien
  4. Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA.

    • Kenneth R Chien
  5. Department of Biosciences and Nutrition, Karolinska Institute, Novum, Huddinge, Karolinska Hospital, Huddinge, Stockholm, Sweden.

    • José Inzunza

Contributions

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.

Competing financial interests

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

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (3M)

    Supplementary Figs. 1–7 and Supplementary Tables 1,2

Additional data