Engineering skeletal muscle tissues with advanced maturity improves synapse formation with human induced pluripotent stem cell-derived motor neurons

Abstract

To develop effective cures for neuromuscular diseases, human-relevant in vitro models of neuromuscular tissues are critically needed to probe disease mechanisms on a cellular and molecular level. However, previous attempts to co-culture motor neurons and skeletal muscle have resulted in relatively immature neuromuscular junctions (NMJs). In this study, NMJs formed by human induced pluripotent stem cell (hiPSC)-derived motor neurons were improved by optimizing the maturity of the co-cultured muscle tissue. First, muscle tissues engineered from the C2C12 mouse myoblast cell line, cryopreserved primary human myoblasts, and freshly isolated primary chick myoblasts on micromolded gelatin hydrogels were compared. After three weeks, only chick muscle tissues remained stably adhered to hydrogels and exhibited progressive increases in myogenic index and stress generation, approaching values generated by native muscle tissue. After three weeks of co-culture with hiPSC-derived motor neurons, engineered chick muscle tissues formed NMJs with increasing co-localization of pre- and postsynaptic markers as well as increased frequency and magnitude of synaptic activity, surpassing structural and functional maturity of previous in vitro models. Engineered chick muscle tissues also demonstrated increased expression of genes related to sarcomere maturation and innervation over time, revealing new insights into the molecular pathways that likely contribute to enhanced NMJ formation. These approaches for engineering advanced neuromuscular tissues with relatively mature NMJs and interrogating their structure and function have many applications in neuromuscular disease modeling and drug development.

Advanced models of human skeletal muscle differentiation, development and disease: Three-dimensional cultures, organoids and beyond

“…Regardless of the underlying platform/scaffold, the use of iPSCs makes it possible to obtain a virtually unlimited number of cells from a minimally invasive source to create isogenic (and often isochronic) multilineage tissues for disease modelling, drug development, cell therapy or tissue replacement. Nonetheless, at variance with models based upon non-human cells [ 110 ], additional work is required to enhance the maturation of human iPSC-derived platforms: this is particularly relevant to model late-onset diseases, for which the relatively immature myofibres currently generated by the majority of available protocols might not recapitulate phenotypic readouts of adult skeletal muscles with high fidelity. We foresee this problem being rapidly addressed by the field, with promising results already obtained by stimulating cultures in vitro chemically [ 66 ] or electrically [ 68 ].…”

Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

“…To characterize the morphological events characteristic of muscle development, we co-visualized nuclei and sarcomeric α-actinin in engineered iDRM tissues after 1 and 2 weeks in culture. We chose sarcomeric α-actinin because it is expressed relatively late in myogenesis, especially compared to other markers, such as desmin, myogenin, or myosin heavy chain ( White et al, 2014 ; Bettadapur et al, 2016b ; Nguyen et al, 2016 ; Denes et al, 2019b ; Santoso et al, 2021b ). Sarcomeric α-actinin is also present in the z-lines in sarcomeres and thus clearly demarcates mature myofibrils.…”

“…Of note, however, iDRMs require less time, cost, and expertise to generate compared to iPSC-derived myoblasts, which may be especially beneficial for generating patient-specific muscle tissues in time-sensitive or resource-limited settings. Extending culture time ( Santoso and McCain, 2021 ), integrating supporting cell types ( Juhas et al, 2018 ; Santosa et al, 2018 ; Santoso and McCain, 2021 ), providing electrical ( Nedachi et al, 2008 ; Chen et al, 2021 ) or mechanical stimulation ( Heher et al, 2015 ; Chang et al, 2016 ), or engineering 3-D tissues ( Madden et al, 2015 ; Uzel et al, 2016 ; Costantini et al, 2017 ; Davis et al, 2019 ; Ariyasinghe et al, 2020 ; Ebrahimi et al, 2021 ) or earlier exposure to AO could also help induce muscle maturation and proper localization of dystrophin and α-actinin.…”

“…The fabrication of micromolded gelatin hydrogels cross-linked with transglutaminase was performed as previously described ( Santoso et al, 2021a ; Gupta et al, 2021 ) and illustrated in Figure 1A . Briefly, 260-mm 2 hexagons were cut from 150-mm polystyrene dishes and masked with tape.…”

“…Micromolded gelatin hydrogels have been developed by us and others as substrates for lengthening the culture lifetime and improving the alignment and maturation of myotubes differentiated from C2C12 ( Bettadapur et al, 2016a ; Denes et al, 2019a ), primary chick ( Santoso et al, 2021a ; Gupta et al, 2021 ), and human control and DMD iPSC-derived myoblasts ( Al Tanoury et al, 2021 ). These gelatin substrates are relatively inexpensive and easy to fabricate while also matching the compliance of native muscle tissue.…”