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Presence of a ROCK Inhibitor in Extracellular Matrix Supports More Undifferentiated Growth of Feeder-Free Human Embryonic and Induced Pluripotent Stem Cells upon Passaging

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Abstract

Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 µM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (αV, α6, and β1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.

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References

  1. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.

    Article  CAS  PubMed  Google Scholar 

  2. Yu, J., Vodyanik, M. A., Smuga-Otto, K., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318, 1917–1920.

    Article  CAS  PubMed  Google Scholar 

  3. Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.

    Article  CAS  PubMed  Google Scholar 

  4. Nishikawa, S., Goldstein, R. A., & Nierras, C. R. (2008). The promise of human induced pluripotent stem cells for research and therapy. Nature Reviews. Molecular Cell Biology, 9, 725–729.

    Article  CAS  PubMed  Google Scholar 

  5. Klimanskaya, I., Rosenthal, N., & Lanza, R. (2008). Derive and conquer: sourcing and differentiating stem cells for therapeutic applications. Nature Reviews Drug Discovery, 7, 131–142.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Sommer, C. A., Stadtfeld, M., Murphy, G. J., Hochedlinger, K., Kotton, D. N., & Mostoslavsky, G. (2009). Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells, 27, 543–549.

    Article  CAS  PubMed  Google Scholar 

  8. Carey, B. W., Markoulaki, S., Hanna, J., et al. (2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences of the United States of America, 106, 157–162.

    Article  CAS  PubMed  Google Scholar 

  9. Kim, J. B., Sebastiano, V., Wu, G., et al. (2009). Oct4-induced pluripotency in adult neural stem cells. Cell, 136, 411–419.

    Article  CAS  PubMed  Google Scholar 

  10. Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., & Woltjen, K. (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458, 771–775.

    Article  CAS  PubMed  Google Scholar 

  11. Woltjen, K., Michael, I. P., Mohseni, P., et al. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 458, 766–770.

    Article  CAS  PubMed  Google Scholar 

  12. Soldner, F., Hockemeyer, D., Beard, C., et al. (2009). Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell, 136, 964–977.

    Article  CAS  PubMed  Google Scholar 

  13. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., & Hochedlinger, K. (2008). Induced pluripotent stem cells generated without viral integration. Science, 322, 945–949.

    Article  CAS  PubMed  Google Scholar 

  14. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., & Yamanaka, S. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322, 949–953.

    Article  CAS  PubMed  Google Scholar 

  15. Judson, R. L., Babiarz, J. E., Venere, M., & Blelloch, R. (2009). Embryonic stem cell-specific microRNAs promote induced pluripotency. Nature Biotechnology, 27, 459–461.

    Article  CAS  PubMed  Google Scholar 

  16. Zhou, H., Wu, S., Joo, J. Y., et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381–384.

    Article  CAS  PubMed  Google Scholar 

  17. Totonchi, M., Taei, A., Seifinejad, A., et al. (2009). Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells. International Journal of Developmental Biology, [Epub ahead of print]. doi:10.1387/ijdb.092903mt

  18. Skottman, H., Narkilahti, S., & Hovatta, O. (2007). Challenges and approaches to the culture of pluripotent human embryonic stem cells. Regenerative Medicine, 2, 265–273.

    Article  PubMed  Google Scholar 

  19. Maherali, N., & Hochedlinger, K. (2008). Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell, 3, 595–605.

    Article  CAS  PubMed  Google Scholar 

  20. Sjogren-Jansson, E., Zetterstrom, M., Moya, K., Lindqvist, J., Strehl, R., & Eriksson, P. S. (2005). Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Developmental Dynamics, 233, 1304–1314.

    Article  PubMed  Google Scholar 

  21. Xu, C., Inokuma, M. S., Denham, J., et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology, 19, 971–974.

    Article  CAS  PubMed  Google Scholar 

  22. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., & Brivanlou, A. H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Medicine, 10, 55–63.

    Article  CAS  PubMed  Google Scholar 

  23. Amit, M., Margulets, V., Segev, H., et al. (2003). Human feeder layers for human embryonic stem cells. Biology of Reproduction, 68, 2150–2156.

    Article  CAS  PubMed  Google Scholar 

  24. Wang, G., Zhang, H., Zhao, Y., et al. (2005). Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers. Biochemical and Biophysical Research Communications, 330, 934–942.

    Article  CAS  PubMed  Google Scholar 

  25. Levenstein, M. E., Ludwig, T. E., Xu, R. H., et al. (2006). Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells, 24, 568–574.

    Article  CAS  PubMed  Google Scholar 

  26. Ludwig, T. E., Levenstein, M. E., Jones, J. M., et al. (2006). Derivation of human embryonic stem cells in defined conditions. Nature Biotechnology, 24, 185–187.

    Article  CAS  PubMed  Google Scholar 

  27. Draper, J. S., Smith, K., Gokhale, P., et al. (2004). Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nature Biotechnology, 22, 53–54.

    Article  CAS  PubMed  Google Scholar 

  28. Watanabe, K., Ueno, M., Kamiya, D., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25, 681–686.

    Article  CAS  PubMed  Google Scholar 

  29. Baharvand, H., Ashtiani, S. K., Taee, A., et al. (2006). Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Development, Growth & Differentiation, 48, 117–128.

    Article  Google Scholar 

  30. Baharvand, H., Ashtiani, S. K., Valojerdi, M. R., Shahverdi, A., Taee, A., & Sabour, D. (2004). Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst. Differentiation, 72, 224–229.

    Article  PubMed  Google Scholar 

  31. Mollamohammadi, S., Taei, A., Pakzad, M., et al. (2009). A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Human Reproduction, 24, 2468–2476.

    Article  CAS  PubMed  Google Scholar 

  32. Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell, 110, 673–687.

    Article  CAS  PubMed  Google Scholar 

  33. Braam, S. R., Zeinstra, L., Litjens, S., et al. (2008). Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells, 26, 2257–2265.

    Article  CAS  PubMed  Google Scholar 

  34. Hayashi, Y., Furue, M. K., Okamoto, T., et al. (2007). Integrins regulate mouse embryonic stem cell self-renewal. Stem Cells, 25, 3005–3015.

    Article  CAS  PubMed  Google Scholar 

  35. Miyazaki, T., Futaki, S., Hasegawa, K., et al. (2008). Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochemical and Biophysical Research Communications, 375, 27–32.

    Article  CAS  PubMed  Google Scholar 

  36. Kleinman, H. K., McGarvey, M. L., Hassell, J. R., et al. (1986). Basement membrane complexes with biological activity. Biochemistry, 25, 312–318.

    Article  CAS  PubMed  Google Scholar 

  37. Kosako, H., Yoshida, T., Matsumura, F., Ishizaki, T., Narumiya, S., & Inagaki, M. (2000). Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow. Oncogene, 19, 6059–6064.

    Article  CAS  PubMed  Google Scholar 

  38. Niggli, V., & Rossy, J. (2008). Ezrin/radixin/moesin: versatile controllers of signaling molecules and of the cortical cytoskeleton. International Journal of Biochemistry and Cell Biology, 40, 344–349.

    Article  CAS  PubMed  Google Scholar 

  39. Amano, M., Chihara, K., Kimura, K., et al. (1997). Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science, 275, 1308–1311.

    Article  CAS  PubMed  Google Scholar 

  40. Riento, K., & Ridley, A. J. (2003). Rocks: multifunctional kinases in cell behaviour. Nature Reviews. Molecular Cell Biology, 4, 446–456.

    Article  CAS  PubMed  Google Scholar 

  41. Koga, T., Koga, T., Awai, M., Tsutsui, J., Yue, B. Y., & Tanihara, H. (2006). Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Experimental Eye Research, 82, 362–370.

    Article  CAS  PubMed  Google Scholar 

  42. Peerani, R., Rao, B. M., Bauwens, C., et al. (2007). Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO Journal, 26, 4744–4755.

    Article  CAS  PubMed  Google Scholar 

  43. Harb, N., Archer, T. K., & Sato, N. (2008). The Rho-Rock-Myosin signaling axis determines cell-cell integrity of self-renewing pluripotent stem cells. PLoS ONE, 3, e3001.

    Article  PubMed  Google Scholar 

  44. Shi, J., & Wei, L. (2007). Rho kinase in the regulation of cell death and survival. Archivum Immunologiae et Therapiae Experimentalis (Warsz), 55, 61–75.

    Article  Google Scholar 

  45. Koyanagi, M., Takahashi, J., Arakawa, Y., et al. (2008). Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors. Journal of Neuroscience Research, 86, 270–280.

    Article  CAS  PubMed  Google Scholar 

  46. Lingor, P., Tonges, L., Pieper, N., et al. (2008). ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells. Brain, 131, 250–263.

    PubMed  Google Scholar 

  47. Li, X., Meng, G., Krawetz, R., Liu, S., & Rancourt, D. E. (2008). The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells and Development, 17, 1079–1085.

    Article  CAS  PubMed  Google Scholar 

  48. Li, X., Krawetz, R., Liu, S., Meng, G., & Rancourt, D. E. (2009). ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Human Reproduction, 24, 580–589.

    Article  CAS  PubMed  Google Scholar 

  49. Martin-Ibanez, R., Unger, C., Stromberg, A., Baker, D., Canals, J. M., & Hovatta, O. (2008). Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Human Reproduction, 23, 2744–2754.

    Article  CAS  PubMed  Google Scholar 

  50. Heng, B. C., Ye, C. P., Liu, H., et al. (2006). Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis. Journal of Biomedical Science, 13, 433–445.

    Article  CAS  PubMed  Google Scholar 

  51. Krawetz, R. J., Li, X., & Rancourt, D. E. (2009). Human embryonic stem cells: caught between a ROCK inhibitor and a hard place. Bioessays, 31, 336–343.

    Article  CAS  PubMed  Google Scholar 

  52. Yanazume, T., Hasegawa, K., Wada, H., et al. (2002). Rho/ROCK pathway contributes to the activation of extracellular signal-regulated kinase/GATA-4 during myocardial cell hypertrophy. Journal of Biological Chemistry, 277, 8618–8625.

    Article  CAS  PubMed  Google Scholar 

  53. sHall, A. (1994). Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annual Review of Cell Biology, 10, 31–54.

    Article  CAS  PubMed  Google Scholar 

  54. Tominaga, T., Ishizaki, T., Narumiya, S., & Barber, D. L. (1998). p160ROCK mediates RhoA activation of Na-H exchange. Embo Journal, 17, 4712–4722.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgement

This study was funded by a grant provided from Royan Institute.

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The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran

    Mohammad Pakzad, Adeleh Taei, Ali Seifinejad, Seyedeh Nafiseh Hassani & Hossein Baharvand

  2. Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

    Mehdi Totonchi

  3. Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

    Hossein Baharvand

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  1. Mohammad Pakzad
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  2. Mehdi Totonchi
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  3. Adeleh Taei
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  4. Ali Seifinejad
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  5. Seyedeh Nafiseh Hassani
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  6. Hossein Baharvand
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Correspondence to Hossein Baharvand.

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Pakzad, M., Totonchi, M., Taei, A. et al. Presence of a ROCK Inhibitor in Extracellular Matrix Supports More Undifferentiated Growth of Feeder-Free Human Embryonic and Induced Pluripotent Stem Cells upon Passaging. Stem Cell Rev and Rep 6, 96–107 (2010). https://doi.org/10.1007/s12015-009-9103-z

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