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Functional Myogenic Engraftment from Mouse iPS Cells

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Abstract

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies.

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References

  1. 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  PubMed  CAS  Google Scholar 

  2. Takahashi, K., et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. (Translated from Eng) Cell (in Eng).

  3. Yu, J., et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. (Translated from Eng) Science (in Eng).

  4. Blelloch, R., Venere, M., Yen, J., & Ramalho-Santos, M. (2007). Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell, 1, 254–247.

    Article  Google Scholar 

  5. Wernig, M., et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 448, 318–324.

    Article  PubMed  CAS  Google Scholar 

  6. Park, I. H., et al. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 451, 141–146.

    Article  PubMed  CAS  Google Scholar 

  7. Byrne, J. A., et al. (2007). Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature, 450, 497–502.

    Article  PubMed  CAS  Google Scholar 

  8. Chang, C. W., et al. (2009) Polycistronic lentiviral vector for “hit and run” reprogramming of adult skin fibroblasts to induced pluripotent stem cells. (Translated from eng) Stem Cells, 27(5):1042–1049 (in eng).

    Google Scholar 

  9. Kaji, K., et al. (2009) Virus-free induction of pluripotency and subsequent excision of reprogramming factors. (Translated from eng) Nature, 458(7239):771–775 (in eng).

    Google Scholar 

  10. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., & Yamanaka, S. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. (Translated from eng) Science, 322(5903):949–953 (in eng).

    Google Scholar 

  11. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., & Hochedlinger, K. (2008) Induced pluripotent stem cells generated without viral integration. (Translated from eng) Science, 322(5903):945–949 (in eng).

  12. Yu, J., et al. (2009) Human induced pluripotent stem cells free of vector and transgene sequences. (Translated from eng) Science, 324(5928):797–801 (in eng).

    Google Scholar 

  13. Woltjen, K., et al. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. (Translated from eng) Nature, 458(7239):766–770 (in eng).

    Google Scholar 

  14. Kim, D., et al. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. (Translated from eng) Cell Stem Cell, 4(6):472–476 (in eng).

    Google Scholar 

  15. Zhou, H., et al. (2009) Generation of induced pluripotent stem cells using recombinant proteins. (Translated from eng) Cell Stem Cell 4(5):381–384 (in eng).

    Google Scholar 

  16. Warren, L., et al. (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. (Translated from eng) Cell Stem Cell, 7(5):618–630 (in eng).

    Google Scholar 

  17. Hanna, J. W. M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J. P., Beard, C., et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318, 1920–1923.

    Article  PubMed  CAS  Google Scholar 

  18. Xu, D., et al. (2009). Phenotypic correction of murine hemophilia A using an iPS cell-based therapy. Proceedings of the National Academy of Sciences of the United States of America, 106, 808–813.

    Article  PubMed  CAS  Google Scholar 

  19. Wernig, M., et al. (2008). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proceedings of the National Academy of Sciences of the United States of America, 105, 5856–5861.

    Article  PubMed  CAS  Google Scholar 

  20. Tsuji, O., et al. (2010). Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury. Proceedings of the National Academy of Sciences of the United States of America, 107, 12704–12709.

    Article  PubMed  CAS  Google Scholar 

  21. Alipio, Z., et al. (2010) Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells. (Translated from eng) Proceedings of the National Academy of Sciences of the United States of America, 107(30):13426–13431 (in eng).

  22. Espejel, S., et al. (2010) Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice. (Translated from eng) Journal of Clinical Investigation, 120(9):3120–3126 (in eng).

    Google Scholar 

  23. Mizuno, Y., et al. (2010). Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. The FASEB Journal, 24, 2245–2253.

    Article  PubMed  CAS  Google Scholar 

  24. Rudnicki, M. A., et al. (1993). Myod or myf-5 is required for the formation of skeletal muscle. Cell, 75, 1351–1359.

    Article  PubMed  CAS  Google Scholar 

  25. Tajbakhsh, S., Rocancourt, D., & Buckingham, M. (1996). Muscle progenitors cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature, 384, 266–270.

    Article  PubMed  CAS  Google Scholar 

  26. Kassar-Duchossoy, L., et al. (2005). Pax3/Pax7 mark a novel population of primitive myogenic cells during development. Genes & Development, 19, 1426–1431.

    Article  CAS  Google Scholar 

  27. Relaix, F., Rocancourt, D., Mansouri, A., & Buckingham, M. (2005). A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature, 435, 948–953.

    Article  PubMed  CAS  Google Scholar 

  28. Bajard, L., et al. (2006). A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes & Development, 20, 2450–2464.

    Article  CAS  Google Scholar 

  29. Goulding, M., Lumsden, A., & Paquette, A. J. (1994). Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development, 120, 957–971.

    PubMed  CAS  Google Scholar 

  30. Tremblay, J. P., et al. (1998). A crucial role for Pax3 in the development of the hypaxial musculature and the long-range migration of muscle precursors. Developmental Biology, 203, 49–61.

    Article  PubMed  CAS  Google Scholar 

  31. Ordahl, C. P., & Le Douarin, N. M. (1992). Two myogenic lineages within the developing somite. Development, 114, 339–353.

    PubMed  CAS  Google Scholar 

  32. Tajbakhsh, S. (2003). Stem cells to tissue: molecular, cellular and anatomical heterogeneity in skeletal muscle. Current Opinion in Genetics & Development, 13, 412–422.

    Article  Google Scholar 

  33. Conboy, I. M., & Rando, T. A. (2002). The regulation of Notch signaling controls satellite cell activation and cell fate determination in posnatal myogenesis. Developmental Cell, 3, 397–409.

    Article  PubMed  CAS  Google Scholar 

  34. Darabi, R., et al. (2008). Functional skeletal muscle regeneration from differentiating embryonic stem cells. Natural Medicines, 14, 134–143.

    Article  CAS  Google Scholar 

  35. Darabi, R., et al. (2011) Assessment of the myogenic stem cell compartment following transplantation of Pax3/Pax7-induced embryonic stem cell-derived progenitors. Stem Cells, “in press”.

  36. Iacovino, M., et al. (2009). A conserved role for Hox paralog group 4 in regulation of hematopoietic progenitors. Stem Cells and Development, 18, 783–792.

    Article  PubMed  CAS  Google Scholar 

  37. Bosnakovski, D., et al. (2008) An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies. (Translated from eng) EMBO Journal, 27(20):2766–2779 (in eng).

    Google Scholar 

  38. Hochedlinger, K., Yamada, Y., Beard, C., & Jaenisch, R. (2005). Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell, 121(3), 465–477.

    Article  PubMed  CAS  Google Scholar 

  39. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., & Nishimune, Y. (1997). ‘Green mice’ as a source of ubiquitous green cells. FEBS Letters, 407, 313–319.

    Article  PubMed  CAS  Google Scholar 

  40. Yan Z, et al. (2003) Highly coordinated gene regulation in mouse skeletal muscle regeneration. (Translated from eng) Journal of Biological Chemistry, 278(10):8826–8836 (in eng).

    Google Scholar 

  41. Cerletti, M., et al. (2008). Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.+. Cell, 134, 37–47.

    Article  PubMed  CAS  Google Scholar 

  42. Couteaux, R., Mira, J.-C., & d’Albis. (1988). Regeneration of muscles after cardiotoxin injury I. Cytological aspects. Biology of the Cell, 62, 171–182.

    Article  PubMed  CAS  Google Scholar 

  43. Harris, J. B., & Johnson, M. A. (1978). Further observations on the responses of rat skeletal muscle to single subcutaneous injection of a toxin isolated from the venom of the Astralian tiger snake Notechis scutatos scutatus. Clinical and Experimental Pharmacology and Physiology, 5, 587–600.

    Article  PubMed  CAS  Google Scholar 

  44. Harris, J. B. (2003). Myotoxic phospholipases A2 and the regeneration of skeletal muscles. Toxicon, 42, 933–945.

    Article  PubMed  CAS  Google Scholar 

  45. Chretien, F., et al. (2005) In vivo fusion of circulating fluorescent cells with dystrophin-deficient myofibers results in extensive sarcoplasmic fluorescence expression but limited dystrophin sarcolemmal expression. (Translated from eng) American Journal of Pathology, 166(6):1741–1748 (in eng).

    Google Scholar 

  46. Auda-Boucher, G., et al. (2007) Fetal muscle-derived cells can repair dystrophic muscles in mdx mice. (Translated from eng) Experimental Cell Research, 313(5):997–1007 (in eng).

    Google Scholar 

  47. Asakura, A., Seale, P., Girgis-Gabardo, A., & Rudnicki, M. A. (2002). Myogenic specification of side population cells in skeletal muscle. The Journal of Cell Biology, 159, 123–134.

    Article  PubMed  CAS  Google Scholar 

  48. Bachrach, E., et al. (2006). Muscle engraftment of myogenic progenitor cells following intraarterial transplantation. Muscle & Nerve, 34, 44–52.

    Article  Google Scholar 

  49. Rando, T. A., & Blau, H. M. (1994). Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. The Journal of Cell Biology, 125(6), 1275–1287.

    Article  PubMed  CAS  Google Scholar 

  50. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., & Blau, H. M. (2008). Self-renewal and expansion of single transplanted muscle stem cells. Nature, 456, 502–506.

    Article  PubMed  CAS  Google Scholar 

  51. Lee, J. Y., et al. (2000). Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. The Journal of Cell Biology, 150, 1085–1000.

    Article  PubMed  CAS  Google Scholar 

  52. Sampaolesi, M., et al. (2003). Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science, 301, 487–492.

    Article  PubMed  CAS  Google Scholar 

  53. Sampaolesi, M., et al. (2006). Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature, 444, 574–579.

    Article  PubMed  CAS  Google Scholar 

  54. Montarras, D., et al. (2005). Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309(5743), 2064–2067.

    Article  PubMed  CAS  Google Scholar 

  55. Gálvez, B. G., et al. (2009). Human cardiac mesoangioblasts isolated from hypertrophic cardiomyopathies are greatly reduced in proliferation and differentiation potency. Cardiovascular Research, 83, 707–716.

    Article  PubMed  Google Scholar 

  56. Hanna, J., et al. (2009). Direct cell reprogramming is a stochastic process amenable to acceleration. Nature, 462, 595–601.

    Article  PubMed  CAS  Google Scholar 

  57. Maherali, N., et al. (2008). A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell, 3, 340–345.

    Article  PubMed  CAS  Google Scholar 

  58. Yu, J., et al. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science, 324, 797–801.

    Article  PubMed  CAS  Google Scholar 

  59. Kaji, K., et al. (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458, 771–775.

    Article  PubMed  CAS  Google Scholar 

  60. Moretti, A., et al. (2010) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. New England Journal of Medicine. Jul 21. [Epub ahead of print].

  61. Carvajal-Vergara, X., et al. (2010) Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature, 465.

  62. Agarwal, S., et al. (2010). Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature, 464, 292–296.

    Article  PubMed  CAS  Google Scholar 

  63. Maehr, R., et al. (2009). Generation of pluripotent stem cells from patients with type 1 diabetes. Proceedings of the National Academy of Sciences of the United States of America, 106, 15768–15773.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The project was supported by NIH grants RC1AR058118 to RCRP and R01 HL081186 to MK and by the Dr. Bob and Jean Smith Foundation. The monoclonal antibody to MHC was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.

Authorship Statement Contribution

R.D. designed and conducted experiments, performed final analysis of the data and contributed to writing the paper. W.P. generated iPS cells and assisted R.D. with experiments. D.B. and M.K. generated the A2Lox.cre ES cells, the ICE mice and derivative ICE iPS cells. J.B. conducted the breeding to generated appropriate strains. R.C.R.P. supervised the overall project, designed experiments, analyzed the data and wrote the paper.

Conflict of Interest

The authors declare no potential conflicts of interest.

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Correspondence to Rita C. R. Perlingeiro.

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Supplemental Fig. 1

Characterization of iPS clone—ICE7. (A) Morphology of iPax7-iPS clone. (B) FACS analysis for SSEA-1 expression. (C) Immunofluorescent staining for Nanog. (D) Staining for alkaline phosphatase (AP). (E) Morphology of iPS-derived embryoid bodies (EBs) (F) H&E staining of teratomas derived from Rag2−/−/GammaC−/− immunodeficient mice injected with iPax7 ICE7 iPS cells. (PPT 5526 kb)

Supplemental Fig. 2

Fusion index and gene expression analyses for the 5 iPax7 TTF and ICE clones investigated. (A) Fusion index of studied clones following terminal differentiation in vitro. (B) Real time RT-PCR expression analysis for myogenic markers in iPax7 iPS- (TTF vs. ICE) PDGFαR+Flk-1-derived cells under proliferation and differentiation conditions. Transcripts are normalized to GAPDH. Data are mean of 2 independent experiments. (PPT 157 kb)

Supplemental Fig. 3

FACS profile of iPax7-iPS derived myogenic progenitors. FACS characterization of TTF2 and ICE7 iPax7-iPS-derived myogenic progenitors obtained following purification for PDGFαR+Flk-1 cell fraction. Monolayers were maintained under proliferation conditions. Representative plots show isotype or secondary control staining profile (gray line) versus specific antibody staining profile (red line). Percentages represent the fraction of cells that express a given surface antigen. (PPT 255 kb)

Supplemental Fig. 4

Engraftment and specific force in muscles transplanted with TTF1, ICE3, and ICE15 iPS clones. (A) Immunofluorescent staining shows lower levels of dystrophin expression (red), as confirmed by quantification of Dystrophin+ myofibers (B). (C) Effect of cell transplantation on specific force (sF0: F0 normalized to CSA). Values shown are the results of experiments on seven animals per group ±SEM. Karyotype analysis later on performed which indicated the presence of chromosomal abnormalities in these clones. (D) Single myofiber staining of iPax7 iPS treated muscle shows the presence of GFP+ donor derived satellite cells expressing Pax7 and M-cadherin. (PPT 3623 kb)

Supplemental Table 1

Characterization of iPax7 iPS clones. (DOC 70 kb)

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Darabi, R., Pan, W., Bosnakovski, D. et al. Functional Myogenic Engraftment from Mouse iPS Cells. Stem Cell Rev and Rep 7, 948–957 (2011). https://doi.org/10.1007/s12015-011-9258-2

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