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Derivation of engraftable skeletal myoblasts from human embryonic stem cells

Nature Medicine volume 13, pages 642–648 (2007)Cite this article

Abstract

Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

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Figure 1: Isolation and characterization of hESC-derived mesenchymal precursors generated under stroma-free conditions.
Figure 2: Changes in gene expression during hESC differentiation monitored by RT-PCR analysis.
Figure 3: Enrichment and characterization of hESC-derived skeletal myocytes.
Figure 4: Quantitative analysis of hESC-derived skeletal myoblast differentiation.
Figure 5: Long-term monitoring of luc+ hESC-derived myoblasts in SCID/Beige mice.

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References

  1. Trounson, A. The production and directed differentiation of human embryonic stem cells. Endocr. Rev. 27, 208–219 (2006).

    Article  Google Scholar 

  2. Barberi, T., Willis, L., Socci, N.D. & Studer, L. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med. 2, e161 (2005).

    Article  Google Scholar 

  3. Nakano, T., Kodama, H. & Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101 (1994).

    Article  CAS  Google Scholar 

  4. Walsh, F.S. & Ritter, M.A. Surface antigen differentiation during human myogenesis in culture. Nature 289, 60–64 (1981).

    Article  CAS  Google Scholar 

  5. Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M. & McKay, R.D.G. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59, 89–102 (1996).

    Article  CAS  Google Scholar 

  6. Gronthos, S. et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J. Cell Sci. 116, 1827–1835 (2003).

    Article  CAS  Google Scholar 

  7. Maitra, A. et al. Genomic alterations in cultured human embryonic stem cells. Nat. Genet. 37, 1099–1103 (2005).

    Article  CAS  Google Scholar 

  8. Litvinov, S.V., Velders, M.P., Bakker, H.A., Fleuren, G.J. & Warnaar, S.O. Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule. J. Cell Biol. 125, 437–446 (1994).

    Article  CAS  Google Scholar 

  9. Lanctot, C., Lamolet, B. & Drouin, J. The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. Development 124, 2807–2817 (1997).

    CAS  PubMed  Google Scholar 

  10. Chapman, D.L., Agulnik, I., Hancock, S., Silver, L.M. & Papaioannou, V.E. Tbx6, a mouse T-Box gene implicated in paraxial mesoderm formation at gastrulation. Dev. Biol. 180, 534–542 (1996).

    Article  CAS  Google Scholar 

  11. Minguillon, C., Del, B.J. & Logan, M.P. Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth. Dev. Cell 8, 75–84 (2005).

    Article  CAS  Google Scholar 

  12. Saito, D., Yonei-Tamura, S., Takahashi, Y. & Tamura, K. Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation. Dev. Biol. 292, 79–89 (2006).

    Article  CAS  Google Scholar 

  13. Cusella De Angelis, M.G. et al. Skeletal myogenic progenitors in the endothelium of lung and yolk sac. Exp. Cell Res. 290, 207–216 (2003).

    Article  Google Scholar 

  14. Xu, K., Ma, H., McCown, T.J., Verma, I.M. & Kafri, T. Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol. Ther. 3, 97–104 (2001).

    Article  CAS  Google Scholar 

  15. Xu, C. et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22, 972–980 (2004).

    Article  CAS  Google Scholar 

  16. Kubo, A. et al. Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1662 (2004).

    Article  CAS  Google Scholar 

  17. Tada, S. et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374 (2005).

    Article  CAS  Google Scholar 

  18. Yaffe, D. & Saxel, O. A myogenic cell line with altered serum requirements for differentiation. Differentiation 7, 159–166 (1977).

    Article  CAS  Google Scholar 

  19. Seale, P. et al. Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Dezawa, M. et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 309, 314–317 (2005).

    Article  CAS  Google Scholar 

  22. Guettier-Sigrist, S., Coupin, G., Warter, J.M. & Poindron, P. Cell types required to efficiently innervate human muscle cells in vitro. Exp. Cell Res. 259, 204–212 (2000).

    Article  CAS  Google Scholar 

  23. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

    Article  CAS  Google Scholar 

  24. Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    Article  CAS  Google Scholar 

  25. Smyth, G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. [online] 3, 3 (2004).

    Google Scholar 

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Acknowledgements

We thank S. Clairmont, J. Vider and G. Al Shamy for technical assistance, S. Desbordes (Memorial Sloan-Kettering Cancer Center, MSKCC) for providing reagents for the study, V. Ponomarev (MSKCC) for the TGL vector, and V. Tabar for advice on the in vivo studies. The work was supported in part through a nonrestricted grant from the Kinetics Foundation and by the ALS Association and Project ALS.

Author information

Author notes

  1. Tiziano Barberi

    Present address: Present address: Division of Neurosciences, Beckman Research Institute of The City of Hope, 1500 E Duarte Road, Duarte, California 91010, USA.,

  2. Michelle Bradbury and Zehra Dincer: These authors contributed equally to this work.

Authors and Affiliations

  1. Developmental Biology Program, Sloan-Kettering Institute, 1275 York Ave, New York, 10021, New York, USA

    Tiziano Barberi, Zehra Dincer, Georgia Panagiotakos & Lorenz Studer

  2. Division of Neurosurgery, Sloan-Kettering Institute, 1275 York Ave, New York, 10021, New York, USA

    Tiziano Barberi, Zehra Dincer, Georgia Panagiotakos & Lorenz Studer

  3. Department of Radiology, Sloan-Kettering Institute, 1275 York Ave, New York, 10021, New York, USA

    Michelle Bradbury

  4. Computational Biology Center, Sloan-Kettering Institute, 1275 York Ave, New York, 10021, New York, USA

    Nicholas D Socci

Authors
  1. Tiziano Barberi
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  4. Georgia Panagiotakos
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  6. Lorenz Studer
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Contributions

T.B. and L.S. designed the study, developed in vitro differentiation protocols, participated in data analysis, and wrote the manuscript; M.B. conducted the imaging studies; Z.D. contributed to both in vitro and in vivo studies and performed histological analyses; G.P. was responsible for muscle lesioning and transplantation; N.D.S. carried out analysis of the microrarray data.

Corresponding authors

Correspondence to Tiziano Barberi or Lorenz Studer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Chondrocytic differentiation of hESC-derived mesenchymal precursors. (PDF 66 kb)

Supplementary Fig. 2

Analysis of hESC-derived CD73− and CD73+ progeny. (PDF 111 kb)

Supplementary Fig. 3

BLI signal and histological analysis in animals grafted with hESC-derived myocytes at various time points after cardiotoxin exposure. (PDF 1173 kb)

Supplementary Fig. 4

Schematic representation of the stroma-free differentiation system. Time course and details are given for the sequential steps leading to the isolation of first mesenchymal precursors and then specific mesenchymal derivatives (further details in Methods). (PDF 2384 kb)

Supplementary Table 1

Primer sequences, cycle numbers, and annealing temperatures for the RT-PCR analyses performed in the current study. (PDF 45 kb)

Supplementary Video 1

Movie showing spontaneous contraction of mature myocytes and early myotubes in culture of hESC-derived skeletal muscle cells (SF2) after 3 weeks of N2 exposure. (AVI 567 kb)

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Barberi, T., Bradbury, M., Dincer, Z. et al. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 13, 642–648 (2007). https://doi.org/10.1038/nm1533

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