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.
Highlights
Neuromuscular diseases cause progressive muscular atrophy and motor system impairment and affect 160 per 100 000 people worldwide.[1]
Patient-specific modeling of neuromuscular diseases in vitro has been limited by the stunted maturation of neuromuscular junctions (NMJs) formed by hiPSCderived motor neurons and engineered muscle tissues.[16,17,18,21]
We improved the structure and function of NMJs formed by hiPSCderived motor neurons by optimizing the maturity of the engineered muscle tissue
Summary
Neuromuscular diseases cause progressive muscular atrophy and motor system impairment and affect 160 per 100 000 people worldwide.[1]. Most neuromuscular diseases remain incurable[5] due largely to the complexity of these diseases, which is compounded by a lack of suitable model systems for efficiently and reproducibly investigating human disease mechanisms across multiple spatial scales. To investigate neuromuscular tissues at this spatial scale, embryonic rat spinal cord and dorsal root ganglia have been explanted onto cultured primary human muscle tissues to form relatively mature NMJs that are stable in vitro for several weeks or longer.[9,10,11] spinal cord explants consist of multiple cell types, cannot be expanded in vitro, and cannot be practically isolated from humans, which limits their reproducibility, scalability, and human relevance, respectively. There is a pressing need for new patient-specific model systems that enable rigorous examination of neuromuscular disease mechanisms on the microscale
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
