Transplantation of PSC-derived myogenic progenitors counteracts disease phenotypes in FSHD mice

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a genetically dominant progressive myopathy caused by improper silencing of the DUX4 gene, leading to fibrosis, muscle atrophy, and fatty replacement. Approaches focused on muscle regeneration through the delivery of stem cells represent an attractive therapeutic option for muscular dystrophies. To investigate the potential for cell transplantation in FSHD, we have used the doxycycline-regulated iDUX4pA-HSA mouse model in which low-level DUX4 can be induced in skeletal muscle. We find that mouse pluripotent stem cell (PSC)-derived myogenic progenitors engraft in muscle actively undergoing DUX4-mediated degeneration. Donor-derived muscle tissue displayed reduced fibrosis and importantly, engrafted muscles showed improved contractile specific force compared to non-transplanted controls. These data demonstrate the feasibility of replacement of diseased muscle with PSC-derived myogenic progenitors in a mouse model for FSHD, and highlight the potential for the clinical benefit of such a cell therapy approach.

Fig. 1. In vivo regenerative potential of Pax3-induced myogenic progenitors in iDUX4pA-HSA mice.

a Outline of experimental design. b Representative images show immunostaining for RFP (in red) and Dys (in gray) in non-injured (−CTX) and CTX-injured (+CTX) TA muscles from iDUX4pA-HSA mice that had been transplanted with Pax3-induced mouse ESC-derived myogenic progenitors. Images from PBS-injected TA muscle are shown in the upper panel (-CTX). DAPI stains nuclei (in blue). Scale bar is 50 µm. c Representative images showing immunostaining for RFP (in red), PCM1 (in green) and Dys (in gray) in TA muscles of iDUX4pA-HSA mice that had been injected with PBS (left panel) or cells (right panel) from the +CTX group. DAPI stained nuclei in blue. Arrowheads show RFP +/PCM1 + nuclei. Scale bar is 50 µm. d Representative confocal images from +CTX group show a nucleus positive for RFP and PCM1. Scale bar is 10 µm. e Engraftment quantification based on the number of RFP + /Dys+ and RFP + /Dys + /PCM1 + myofibers. Data are shown as mean ± SEM (n = 6 for PBS, n = 6 for -CTX and n = 4 for +CTX). *p < 0.05 by the Student’s t test.

Fig. 2. Engraftment is persistent even in the context of bursts of DUX4 expression.

a Outline of experimental design. b Representative images, capturing the complete engrafted area, show immunostaining for RFP (in red), Dys (in gray), and DAPI (in blue) of the 7 + 7 cohort. Scale bar is 200 µm. c Representative images show the same staining as b for the 3 experimental groups depicted in a: 7 + 0, 7 + 7, and 7 + 14. Upper panel shows PBS-injected control. Scale bar is 50 µm. d Graph shows quantification of engraftment (from c) as shown by the number of donor-derived RFP + /Dys+ myofibers. Data are shown as mean ± SEM (n = 7 for PBS, n = 13 for 7 + 0, n = 18 for 7 + 7 and n = 6 for 7 + 14). **p < 0.01, ***p < 0.001 by the Student’s t test. e Distribution of the number of RFP + /Dys+ myofibers along the TA muscle. Data are shown as mean ± SEM (n = 13 for 7 + 0, n = 18 for 7 + 7, and n = 6 for 7 + 14).

Fig. 3. Testing environment vs. cell autonomous effect of dox post-transplant.

a Representative images show immunostaining for RFP (in red) and Dys (in gray) in TA muscles of BL6 mice that had been injected with PBS or Pax3-induced myogenic progenitors, and later fed with dox in a similar fashion as iDUX4pA-HSA 7 + 0 and 7 + 14 cohorts (Fig. 2a). DAPI stained nuclei in blue. Scale bar is 50 µm. b Graph shows engraftment quantification from a. Data are shown as mean ± SEM (n = 7 for 7 + 0 and n = 7 for 7 + 14). c Representative images show immunostaining for tdTomato (in red), and Dys (in gray) in TA muscles of iDUX4pA-HSA mice that had been injected with PBS or 5000 satellite cells isolated from a mTmG mouse and fed with dox following the 7 + 0 and 7 + 14 dox regimen. DAPI stained nuclei in blue. Scale bar is 50 µm. d Graph shows engraftment quantification from c based on the number tdTomato + /Dys+ myofibers. Data are shown as mean ± SEM (n = 6 for 7 + 0 and n = 5 for 7 + 14). p = 0.061 by the Student’s t test.

Fig. 4. Transplantation of myogenic progenitors counteracts the fibrosis induced by DUX4 expression in iDUX4pA-HSA mice.

a Representative images show Masson’s trichrome staining for the 3 experimental cohorts of iDUX4pA-HSA mice: 7 + 0, 7 + 7, and 7 + 14. Right panels show transplanted TA muscles, while left panels display respective contralateral PBS controls. Scale bar is 100 µm. b Quantification of the percentage of collagen staining in the total muscle section. Data are shown as mean ± SEM (n = 13 for 7 + 0, n = 18 for 7 + 7 and n = 6 for 7 + 14). *p < 0.05 by the Student’s t test. c Representative images of a whole muscle section show immunostaining for RFP (in red), Lam (in green), and Col VI (in purple; staining performed on the consecutive slide) in transplanted TA muscle from iDUX4pA-HSA mice (7 + 14). Engrafted area has been highlighted in red to denote the engraftment location on the Col VI staining. DAPI in blue. Scale bar is 500 µm. d Images show higher magnification of negative and positive RFP areas from c (from white dashed lines used to depict RFP- and RFP + areas). Scale bar is 50 µm.

Fig. 5. Effect of cell transplantation on the muscle contractile properties of iDUX4pA-HSA mice.

a Representative example of force tracing upon tetanic stimulation in TA muscles from iDUX4pA-HSA mice. Red and white lines show force tracing from muscles that had received cell transplantation or PBS, respectively. b, c Effect of cell transplantation on b absolute (F₀) and c specific (sF₀: F₀ normalized to CSA) force. Control BL6 mice were used as reference. Data are shown as mean ± SEM (n = 20 for BL6, n = 6 for PBS and cells). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by the Student’s t test. d Average CSA of analyzed muscles (from b, c). Data are shown as mean ± SEM. **p < 0.01 by the Student’s t test.