Stem Cell Reviews and Reports

, Volume 8, Issue 2, pp 482–493 | Cite as

Retinoic Acid Enhances Skeletal Myogenesis in Human Embryonic Stem Cells by Expanding the Premyogenic Progenitor Population

  • Tammy Ryan
  • Jun Liu
  • Alphonse Chu
  • Lisheng Wang
  • Alexandre Blais
  • Ilona S. Skerjanc


Human embryonic stem cells (hESCs) are a potential source of material for cell therapy of muscle diseases. To date, it has proven difficult to generate skeletal muscle from hESCs in high yields and within a reasonable timeframe. Further, a hESC-derived Pax3/7-positive skeletal muscle progenitor population has not yet been described. Previous studies have shown that Pax3/7-positive progenitor cells can repopulate the satellite cell niche, indicating the importance of this population for therapy. We sought to optimize the differentiation of hESCs into skeletal muscle in order to characterize myogenesis at a molecular level and shorten the time course. We treated hESCs with retinoic acid (RA) and found an enhancement of skeletal myogenesis, and the expression of the myogenic regulatory factors (MRFs) MyoD and myogenin by day 25. Furthermore, we found that RA treatment expanded the muscle progenitor pool, which occurred as a distinct Pax3+ve population prior to MRF expression. Non-skeletal muscle tissue types were not significantly affected. Therefore, we have identified a differentiation pathway in hESCs that provides a skeletal muscle progenitor population which can undergo myogenesis more efficiently. We propose that RA could fit into a directed culture method for deriving skeletal muscle from hESCs.


Embryonic stem cells Retinoic acid Myogenesis Cell differentiation 



human embryonic stem cells


mouse embryonic stem cells

(EC) cells

P19 embryonal carcinoma


retinoic acid

(iPS) cells

induced pluripotent stem


myosin heavy hhain



We thank Anastassia Voronova for helpful comments and reading the manuscript. T.R. was supported by an OGSST award. This work was supported by a grant to I.S.S. from the Muscular Dystrophy Association (113716).

Conflicts of interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2011_9284_MOESM1_ESM.doc (44 kb)
Table 1 QPCR Primer sequences (DOC 43 kb)


  1. 1.
    Barberi, T., Bradbury, M., Dincer, Z., Panagiotakos, G., Socci, N. D., & Studer, L. (2007). Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nature Medicine, 13, 642–648.PubMedCrossRefGoogle Scholar
  2. 2.
    Barberi, T., Willis, L. M., Socci, N. D., & Studer, L. (2005). Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Medicine, 2, e161.PubMedCrossRefGoogle Scholar
  3. 3.
    Zheng, J. K., Wang, Y., Karandikar, A., et al. (2006). Skeletal myogenesis by human embryonic stem cells. Cell Research, 16, 713–722.PubMedCrossRefGoogle Scholar
  4. 4.
    Mizuno, Y., Chang, H., Umeda, K., et al. (2010). Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. FASEB Journal, 24, 2245–53.Google Scholar
  5. 5.
    Petropoulos, H., & Skerjanc, I. S. (2002). Beta-catenin is essential and sufficient for skeletal myogenesis in P19 cells. Journal of Biological Chemistry, 277, 15393–15399.PubMedCrossRefGoogle Scholar
  6. 6.
    Kennedy, K. A., Porter, T., Mehta, V., et al. (2009). Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin. BMC Biology, 7, 67.PubMedCrossRefGoogle Scholar
  7. 7.
    Montarras, D., Morgan, J., Collins, C., et al. (2005). Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309, 2064–2067.PubMedCrossRefGoogle Scholar
  8. 8.
    Kuang, S., Kuroda, K., Le Grand, F., & Rudnicki, M. A. (2007). Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell, 129, 999–1010.PubMedCrossRefGoogle Scholar
  9. 9.
    Relaix, F., Rocancourt, D., Mansouri, A., & Buckingham, M. (2005). A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature, 435, 948–953.PubMedCrossRefGoogle Scholar
  10. 10.
    Mankoo, B. S., Skuntz, S., Harrigan, I., et al. (2003). The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites. Development, 130, 4655–4664.PubMedCrossRefGoogle Scholar
  11. 11.
    Petropoulos, H., Gianakopoulos, P. J., Ridgeway, A. G., & Skerjanc, I. S. (2004). Disruption of Meox or Gli activity ablates skeletal myogenesis in P19 cells. Journal of Biological Chemistry, 279, 23874–23881.PubMedCrossRefGoogle Scholar
  12. 12.
    Ridgeway, A. G., & Skerjanc, I. S. (2001). Pax3 is essential for skeletal myogenesis and the expression of Six1 and Eya2. Journal of Biological Chemistry, 276, 19033–19039.PubMedCrossRefGoogle Scholar
  13. 13.
    Bober, E., Franz, T., Arnold, H. H., Gruss, P., & Tremblay, P. (1994). Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development, 120, 603–612.PubMedGoogle Scholar
  14. 14.
    Tremblay, P., Dietrich, S., Mericskay, M., Schubert, F. R., Li, Z., & Paulin, D. (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.PubMedCrossRefGoogle Scholar
  15. 15.
    Li, J., Liu, K. C., Jin, F., Lu, M. M., & Epstein, J. A. (1999). Transgenic rescue of congenital heart disease and spina bifida in Splotch mice. Development, 126, 2495–2503.PubMedGoogle Scholar
  16. 16.
    Jostes, B., Walther, C., & Gruss, P. (1990). The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system. Mechanisms of Development, 33, 27–37.PubMedCrossRefGoogle Scholar
  17. 17.
    Mansouri, A., Stoykova, A., Torres, M., & Gruss, P. (1996). Dysgenesis of cephalic neural crest derivatives in Pax7−/− mutant mice. Development, 122, 831–838.PubMedGoogle Scholar
  18. 18.
    Relaix, F., Montarras, D., Zaffran, S., et al. (2006). Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. The Journal of Cell Biology, 172, 91–102.PubMedCrossRefGoogle Scholar
  19. 19.
    Maroto, M., Reshef, R., Munsterberg, A. E., Koester, S., Goulding, M., & Lassar, A. B. (1997). Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell, 89, 139–148.PubMedCrossRefGoogle Scholar
  20. 20.
    Darabi, R., Gehlbach, K., Bachoo, R. M., et al. (2008). Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nature Medicine, 14, 134–143.PubMedCrossRefGoogle Scholar
  21. 21.
    Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, P., & Buckingham, M. E. (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.CrossRefGoogle Scholar
  22. 22.
    Mankoo, B. S., Collins, N. S., Ashby, P., et al. (1999). Mox2 is a component of the genetic hierarchy controlling limb muscle development. Nature, 400, 69–73.PubMedCrossRefGoogle Scholar
  23. 23.
    Williams, B. A., & Ordahl, C. P. (1994). Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. Development, 120, 785–796.PubMedGoogle Scholar
  24. 24.
    Reijntjes, S., Stricker, S., & Mankoo, B. S. (2007). A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo. International Journal of Developmental Biology, 51, 753–759.PubMedCrossRefGoogle Scholar
  25. 25.
    Candia, A. F., Hu, J., Crosby, J., et al. (1992). Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos. Development, 116, 1123–1136.PubMedGoogle Scholar
  26. 26.
    Ross, S. A., McCaffery, P. J., Drager, U. C., & De Luca, L. M. (2000). Retinoids in embryonal development. Physiological Reviews, 80, 1021–1054.PubMedGoogle Scholar
  27. 27.
    Soprano, D. R., Teets, B. W., & Soprano, K. J. (2007). Role of retinoic acid in the differentiation of embryonal carcinoma and embryonic stem cells. Vitamins and Hormones, 75, 69–95.PubMedCrossRefGoogle Scholar
  28. 28.
    Marletaz, F., Holland, L. Z., Laudet, V., & Schubert, M. (2006). Retinoic acid signaling and the evolution of chordates. International Journal of Biological Sciences, 2, 38–47.PubMedCrossRefGoogle Scholar
  29. 29.
    Schnerch, A., Cerdan, C., & Bhatia, M. (2010). Distinguishing between mouse and human pluripotent stem cell regulation: the best laid plans of mice and men. Stem Cells, 28, 419–30.Google Scholar
  30. 30.
    Xu, C., Inokuma, M. S., Denham, J., et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology, 19, 971–974.PubMedCrossRefGoogle Scholar
  31. 31.
    Savage, J., Conley, A. J., Blais, A., & Skerjanc, I. S. (2009). SOX15 and SOX7 differentially regulate the myogenic program in P19 cells. Stem Cells, 27, 1231–1243.PubMedCrossRefGoogle Scholar
  32. 32.
    Huang, H., Zhao, X., Chen, L., et al. (2006). Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture. Biochemical and Biophysical Research Communications, 351, 321–327.PubMedCrossRefGoogle Scholar
  33. 33.
    Yamashita, A., Takada, T., Narita, J., Yamamoto, G., & Torii, R. (2005). Osteoblastic differentiation of monkey embryonic stem cells in vitro. Cloning and Stem Cells, 7, 232–237.PubMedCrossRefGoogle Scholar
  34. 34.
    Niebruegge, S., Nehring, A., Bar, H., Schroeder, M., Zweigerdt, R., & Lehmann, J. (2008). Cardiomyocyte production in mass suspension culture: embryonic stem cells as a source for great amounts of functional cardiomyocytes. Tissue Engineering. Part A, 14, 1591–1601.PubMedCrossRefGoogle Scholar
  35. 35.
    Schuldiner, M., Eiges, R., Eden, A., et al. (2001). Induced neuronal differentiation of human embryonic stem cells. Brain Research, 913, 201–205.PubMedCrossRefGoogle Scholar
  36. 36.
    Kalluri, R., & Zeisberg, M. (2006). Fibroblasts in cancer. Nature Reviews. Cancer, 6, 392–401.PubMedCrossRefGoogle Scholar
  37. 37.
    Willey, S., Ayuso-Sacido, A., Zhang, H., et al. (2006). Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor. Blood, 107, 3122–3130.PubMedCrossRefGoogle Scholar
  38. 38.
    Sumi, T., Tsuneyoshi, N., Nakatsuji, N., & Suemori, H. (2008). Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling. Development, 135, 2969–2979.PubMedCrossRefGoogle Scholar
  39. 39.
    Rossant, J. (2008). Stem cells and early lineage development. Cell, 132, 527–531.PubMedCrossRefGoogle Scholar
  40. 40.
    Brons, I. G., Smithers, L. E., Trotter, M. W., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature, 448, 191–195.PubMedCrossRefGoogle Scholar
  41. 41.
    Tesar, P. J., Chenoweth, J. G., Brook, F. A., et al. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature, 448, 196–199.PubMedCrossRefGoogle Scholar
  42. 42.
    Wobus, A. M., Rohwedel, J., Maltsev, V., & Hescheler, J. (1994). In vitro differentiation of embryonic stem cells into cardiomyocytes or skeletal muscle cells is specifically modulated by retinoic acid. Roux’s Archives of Developmental Biology, 204, 36–45.CrossRefGoogle Scholar
  43. 43.
    Zhang, W., Deng, Z. L., Chen, L., et al. (2010). Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One, 5, e11917.Google Scholar
  44. 44.
    White, R. J., Nie, Q., Lander, A. D., & Schilling, T. F. (2007). Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biology, 5, e304.PubMedCrossRefGoogle Scholar
  45. 45.
    Darabi, R., Santos, F. N., Filareto, A., et al. (2011). Assessment of the myogenic stem cell compartment following transplantation of pax3/pax7-induced embryonic stem cell-derived progenitors. Stem Cells, 29, 777–790.Google Scholar
  46. 46.
    Rohwedel, J., Guan, K., & Wobus, A. M. (1999). Induction of cellular differentiation by retinoic acid in vitro. Cells, Tissues, Organs, 165, 190–202.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Tammy Ryan
    • 1
  • Jun Liu
    • 1
  • Alphonse Chu
    • 1
    • 2
  • Lisheng Wang
    • 1
  • Alexandre Blais
    • 1
    • 2
  • Ilona S. Skerjanc
    • 1
  1. 1.Department of Biochemistry, Microbiology and Immunology, Faculty of MedicineUniversity of OttawaOttawaCanada
  2. 2.Ottawa Institute of Systems BiologyOttawaCanada

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