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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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.
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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.
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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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DOI: https://doi.org/10.1038/nm1533
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