Abstract
Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.
Highlights
Significant advances have been made in studying the molecular causes of DM1, there is still no effective treatment for patients
By performing Fluorescence in situ hybridization (FISH) and immunofluorescence analyses, we demonstrated the presence of CUGexp-RNA foci and their colocalization with MBNL1, confirming that these cells retain DM1-associated molecular characteristics and are suitable for generating in vitro 3D models for the disease
We described a detailed protocol for fabricating 3D skeletal muscle microtissues that can be maintained in long-lasting cultures
Summary
Significant advances have been made in studying the molecular causes of DM1, there is still no effective treatment for patients. Specific blocking of miR-218 and miR-23b has resulted in increased MBNL protein levels and rescue of mis-splicing events in DM1 human myoblasts [46] and HSALR DM1 model mice, where low toxicity, high efficacy, and long-lasting biological effects were observed [46, 47]. While these are encouraging results, the effectiveness of treatments for DM1 still needs to be investigated in clinical phases. We describe a method to fabricate a bioengineered 3D DM1 skeletal muscle tissue model using transdifferentiated myoblasts from patient-derived fibroblasts These cells were encapsulated in micropatterned GelMA-carboxymethyl cellulose methacrylate (CMCMA) hydrogels on top of functionalized glass coverslips. The developed fabrication method for DM1 could be translated for drug development and other studies on muscular dystrophies
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