Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Abstract

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.

Keywords: disease modeling; iPS cells; muscular dystrophy; myogenic differentiation; organoids; pluripotent stem cells; skeletal muscle; tissue engineering.

Graphical abstract

Figure 1

Differentiation of Multiple hPSC Lines into Skeletal Myotubes and Remodeling of Fibrin Hydrogels upon 3D Culture (A) Immunofluorescence analysis for myosin heavy chain (MyHC) in standard monolayer cultures of differentiated hESC- and hiPSC-derived cells (DMD, Duchenne muscular dystrophy; LGMD2D, limb-girdle muscular dystrophy type 2D; LMNA, skeletal muscle laminopathies, specific mutations listed). (B) Side-view of the 3D culture platform with freshly polymerized gels at day 0 (left) and day 10 (right) of culture containing hiPSC-derived myogenic cells. (C) Representative phase contrast images of cellularized hydrogels after polymerization (day 0) and after 10 days in culture. Gels undergo remodeling, shorten, and thin in culture (graph). Mean ± SD, N = 2–4 per time point. (D) H&E staining of a transverse hydrogel sections after 10 days of differentiation. Magnification: centronucleated myofibers. Scale bars: (A and D) 100 μm; (C) 1 mm.

Figure 2

3D Artificial Skeletal Muscle Constructs Derived from Healthy and Dystrophic hPSCs (A) Whole-mount immunofluorescence for myosin heavy chain (MyHC) on muscle constructs derived from hESCs, WT hiPSCs (transgene-based and transgene-free differentiation protocols) and dystrophic hiPSCs (DMD, LGMD2D, and skeletal muscle LMNA) differentiated in 3D for 10 days. Nuclei are counterstained with Hoechst. Arrowheads: multinucleated myotubes. (B) Graph quantifying the proportion of MyHC⁺ cells using z stack confocal microscopy of three hPSC lines shown in (A). (C) Immunolabeling for LAMININ (extracellular matrix), MyHC, and nuclei (Hoechst) on DMD artificial muscles. (D) Western blot for MyHC (250 kDa) in undifferentiated and 3D differentiated iPSC-derived, inducible myogenic cells. β-tubulin: loading control (50 kDa). (E) qRT-PCR analysis of artificial muscles for myogenic markers. DYSTROPHIN (DYS) is absent from DMD-derived artificial muscles. N = 3 for all lines apart from LMNA mutant and LGMD2D hiPSCs, whose error bars represent intra-experimental replicates (n = 3). Values are normalized on GAPDH expression; ΔΔCt is calculated on the corresponding expression values of undifferentiated cells. (F) Immunohistochemistry for sarcomeric actin in DMD artificial muscles after 10 days of differentiation. (G) Transmitted electron microscopy images of DMD iPSC-derived artificial muscle showing sarcomeres (white arrowheads: z lines). (H) Immunofluorescence showing PAX7⁺ nuclei adjacent to DESMIN⁺ myofibers following transgene-free commitment and differentiation of hiPSCs in 3D for 14 days. The graph quantifies the percentage of PAX7⁺ nuclei within the hydrogels (a total of 5,341 nuclei across 10 random fields). (I) Bright-field image of a tibialis anterior (TA) muscle 1 week after implantation of artificial muscles generated using GFP⁺ myogenic cells. Dashed rectangle: grafted area. (J) Immunofluorescence showing engrafted human nuclei (LAMIN A/C⁺, left) corresponding to an area in a serial section with embryonic MyHC⁺ (eMyHC) fibers in transverse sections of a TA muscle 1 week after implantation. Right graphs show quantification of human nuclei from three healthy or dystrophic cell lines; N = 6, 2 mice/cell type; mean ± SD: hESCs 92 ± 30, hiPSCs 59 ± 19, DMD hiPSCs 1,068 ± 132. (K) Immunofluorescence of systemically delivered 594-conjugated IB4 isolectin (red) labeling endothelial cells within the implanted human artificial muscle (LAMIN A/C: human nuclei). Error bars: mean ± SD. Scale bars: (A) top 250 μm, bottom 25 μm; (C, F, and K) 100 μm; (G) 1 μm; (H) 20 μm; (I) 1 mm; (J) 200 μm. For additional information, see Figures S1 and S2.

Figure 3

hiPSC-Derived Artificial Skeletal Muscles Model Skeletal Muscle Laminopathies (A) Confocal (z stacks merge) whole-mount immunofluorescence for DESMIN (myotubes), LAMIN A/C, and EMERIN (nuclear lamina) on hiPSC-derived (healthy and LMNA mutant) artificial muscles. Hoechst: nuclei. (B) Comparison of confocal 3D nuclear reconstructions of the same iPSC lines shown in (A) differentiated as monolayer cultures (left) versus 3D artificial muscle constructs (right). Nuclei are immunolabeled for LAMIN A/C. (C) Box and whiskers graph quantifying nuclear abnormalities in hiPSC-derived artificial muscle generated from 3 patients affected by LMNA-related muscular dystrophies (red color shades) versus 3 control donors (blue color shades). LGMD2D artificial muscles are included as negative control, because they do not have nuclear abnormalities. Lower panel: representative images of 3D-reconstructed nuclei used to score laminopathy versus control muscles in the graph. ^∗∗p = 0.0022, Mann-Whitney U test. n = 6 in CTRL group and 6 in LMNA mutant group (3 cell populations per group in 2 independent experiments). A minimum of 45 nuclei/hydrogel/experiment across 8 random high-power fields were scored. Boxes, 25^th to 75^th percentiles; horizontal line inside, median; +, mean; whiskers, min to max values. (D) Scatter dot plot of the specific length of the nuclei scored in (C). ^∗∗p = 0.0022, Mann-Whitney U test. n = 6 in CTRL group and 6 in LMNA mutant group (3 cell populations per group in 2 independent experiments). Each symbol is one nucleus. Error bars: mean ± SD. (E) Distribution plot of the graph in (D). Scale bars: (A and B) 15 μm; (C) 10 μm. For additional information, see Table S1 and Videos S1 and S2.

Figure 4

Multilineage Artificial Muscles Containing Isogenic hiPSC-Derived Vascular Cells and Motor Neurons (A) Left panel: whole-mount immunofluorescence of an artificial muscle containing a self-organized isogenic network of hiPSC-derived CD31⁺ endothelial cells (arrowheads). Right panel: higher-magnification confocal image of the boxed area showing lateral z views. (B) Hyper-stack image (12 frames) processed with color-coding on CD31 staining (ImageJ) highlighting the 3D structure of the endothelial network. Frame thickness: 2 μm. (C) Confocal images of whole-mount immunolabeling of a multilineage construct containing GFP⁺ pericytes (PCs), displaying coexistence of myofibers (MyHC), ECs (CD31), and PCs. Arrowheads: CD31⁺ ECs juxtaposed to PCs. (D) Confocal image showing an additional example of a multilineage construct as in (C) with lateral z views. Arrowhead indicates a MyHC⁺ and GFP⁺ multinucleated myotube (see Discussion). (E) Quantification of confocal images of tri-lineage artificial muscles showing the average number of MyHC⁺ (muscle), GFP⁺ (PCs), and CD31⁺ (ECs) nuclei per 0.1 mm² field. Error bars: SEM. n = 10 images. (F) Confocal immunofluorescence panel of multilineage 3D artificial muscle derived from WT hiPSCs containing isogenic myofibers, vascular cells (ECs and GFP⁺ PCs), and motor neurons (SMI32). Color-coded combination enabled discrimination of the four cell types based upon the color of the merge. Arrowheads highlight two motor neurons showing multiple axon-like processes. (G) Quantification of the motor neurons (MNs) in quadruple lineage cultures shown in (F). Error bars: SEM. n = 7 images. (H) Confocal immunofluorescence of a DMD artificial muscle construct containing non-isogenic WT SMI32+ motor neurons and showing alpha-bungarotoxin (BTX)⁺ acetylcholine receptors (zoom in). (I) Confocal immunofluorescence of a sister construct of (H) with aligned, multinucleated myofibers with sarcomeric TITIN+ striations (arrowheads) and BTX⁺ acetylcholine receptors (red signal in white box). Scale bars: (A and B) 25 μm; (C–F, H, and I) 10 μm. For additional information, see Figure S3.