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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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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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Authors and Affiliations

  1. Lillehei Heart Institute, Department of Medicine, University of Minnesota, 4-124 Nils Hasselmo Hall, 312 Church St. S.E., Minneapolis, 55455, MN, USA

    Radbod Darabi, Weihong Pan, June Baik & Rita C. R. Perlingeiro

  2. Lillehei Heart Institute, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

    Darko Bosnakovski & Michael Kyba

Authors
  1. Radbod Darabi
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  2. Weihong Pan
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  3. Darko Bosnakovski
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  4. June Baik
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  6. Rita C. R. Perlingeiro
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Corresponding author

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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