Induced Pluripotent Stem Cells derived Muscle Progenitors Effectively Mitigate Muscular Dystrophy through Restoring the Dystrophin Distribution

Abstract

Background: Duchenne Muscular Dystrophy (DMD) is a recessive form of muscular disorder, resulting from the dystrophin gene mutations in X-chromosome. Application of embryonic stem cells or adult stem cells has demonstrated the therapeutic effects on DMD through both cell-based and non-cell based mechanisms. In this study, we proposed that Myogenic Progenitor Cells from Induced Pluripotent Stem Cells (iPSC-MPCs) would be more effective in repairing muscle damage caused by muscular dystrophy.

Methods and results: Mouse iPSCs were cultured in myogenic differentiation culture medium and the MPCs were characterized using Reverse Transcription Polymerase Chain Reaction (RT-PCR) and flow cytometry. iPSCs were successfully converted into MPCs, as evidenced by the distinct expression of myogenic genes and cell surface markers. The muscle injury was induced in tibialis muscle of mdx mouse by cardiotoxin injection, and the iPSC-MPCs were then engrafted into the damage site. Firefly luciferase expression vector was transduced into iPSC-MPCs and the in vivo bioluminescence imaging analysis revealed that these progenitor cells survived even at 30-days post transplantation. Importantly, histological analysis revealed that the central nuclei percentage, as well as fibrosis, was significantly reduced in the iPSC-MPCs treated muscle. In addition,the transplantation of progenitor cells restored the distributions of dystrophin and nicotinic acetylcholine receptors together with up-regulation of pair box protein 7(Pax7), a myogenic transcription factor.

Conclusion: iPSCs-derived MPCs exert strong therapeutic effects on muscular dystrophy by restoring dystrophin expression and acetylcholine receptor distribution.

Keywords: Duchenne muscular dystrophy; Dystrophin; Muscular progenitors; iPSCs.

Figure 1

Characterization of mouse fibroblast-derived iPSC. (A) iPSCs colonies exhibited typical stem cell morphology (left) and showed high alkaline phosphatase (AP) activity (right). (B) iPSCs express SSEA1, Oct4 and Nanog as shown by immune-fluorescent staining.

Figure 2

Generation of dystrophin overexpressing iPSC-derived myocyte progenitor cell (MPC) (A) Embryoid body of iPSC in undifferentiated and differentiated conditions. (B) Myogenic genes expression was assessed by PCR. Representative electrophoresis illustrates the expression levels of Pax-3, Pax-7, MyoD, Myf5, Myogenin, and GAPDH. (C) Flow cytometry was performed to determine the distribution of CD34, CD56, SM/C-2.6, M-cadherin, and integrin- α7.

Figure 3

Myogenic progenitor cells were purified from iPSC-derived cells. (A) CD29⁺CXCR4⁺ cell population was identified as MPCs, which was sorted from iPSC-derived cells by flow cytometer. (B) Expressions of Pax-7, MyoD and MEF-2C in sorted-MPCs were analyzed by flow cytometer. (C) Flowcytometry analysis indicated the expressions of alpha-actin, Myh6 and troponin I in MPCs-differentiated cells.

Figure 4

In vivo tracking of iPSC-derived MPC with BLI and x-ray overlay post transplantation. (A) A lentiviral vector carrying a double fusion (DF) reporter gene with the ubiquity promoter driving both firefly luciferase (Fluc) and EGFP was constructed and transduced into iPSC-derived MPC. (B) Bioluminescent imaging (BLI) in iPSC-MPCs plates. (C) Linear correlation of cell numbers and BLI signals (photons/s/cm2 per steridian) (D) An example of in vivo BLI is shown for mice implanted with iPSC-MPCs without reporter gene as background control (E,F) Mice implanted with iPSC-MPCs that transduced with firefly luciferase reporter gene.

Figure 5

Central nuclei and fibrosis in mdx mice. (A) The histological sections of MDX mice anterior muscle stained with hematoxylin and eosin (H&E), picrosirus red. Nuclei centralization and fibrosis were investigated using light and polarized microscope. (B, C) Quantitative analysis indicated that treatment with dystrophin overexpressing iPSC-derived MPC improved pathologies associated with cardiotoxin injury, as evidence of reduced percentage of central nuclei muscle fibers (B), but significantly decreased tissue-fibrosis in anterior muscle. (n=5 each group, *P<0.05 vs. Model group).

Figure 6

Expressions of dystrophin and acetylcholine receptor in the tibialis anterior muscle of MDX mice. (A) Acetylcholine receptors (AchR) were stained with AlexaFluo488-conjugated bungarotoxin (green) and the dystrophin was stained by TRITC-conjugated antibody (red). (B,C) quantitative analysis indicated that treatment with dystrophin overexpressing iPSC-derived MPC enhanced the expression levels of dystrophin (B) and AchR (C) in mdx mouse skeletal muscle post cardiotoxin injury. (n=5 each group, *P<0.05 vs. Model group).

Figure 7

Treatment with dystrophin overexpressing iPSC-derived MPC promotes the expression of Pax-7 in the skeletal muscle. (A) Pax-7 and dystrophin in tibialis anterior muscle were stained with specific antibody and shown in green (FITC) and red (TRITC) fluorescence, respectively. The co-localization of green and DAPI (blue fluorescence) was identified as the pax-7 positive nuclear, which was indicated by white arrows. (B) Quantitative result of Pax-7 positive nuclei. (n=5 each group, *P<0.05 vs. Model group).