Introduction:: Current engineered human skeletal muscle tissues fail to fully capture the cellular complexity of in vivo skeletal muscle; as a result, they are limited in their capacity to model muscle physiology and disease. The incorporation of additional cell types, including motoneurons (MNs), is expected to better recapitulate the structural and functional milieu of native muscle1. MNs play critical yet understudied roles in muscle development2, maturation3, and the formation of functional neuromuscular junctions (NMJs)3-5, as well as muscle regeneration and neuromuscular disease4,6. Additionally, the potential reciprocal roles of skeletal muscle in MN development, specification, and maturation are not fully understood7. Here we have engineered an in vitro model of highly functional human neuromuscular tissue to investigate the roles of functional innervation in co-maturation of motoneurons and skeletal muscle.
Materials and Methods:: Human induced pluripotent stem cells (hiPSCs) were differentiated into MNs via staged application of small molecules and growth factors (Fig. 1A). The identity, maturity, and functionality of MNs were characterized in 2D monolayers prior to 3D co-culture with engineered human muscle tissues (myobundles)8. Innervated myobundles were fabricated using a novel compartmentalized design which mimics the anatomical structure of native muscle. Briefly, primary human myoblasts and hiPSC-derived MNs were embedded separately in a fibrin/Matrigel hydrogel and injected into their respective compartments to generate monocultured muscle (Mu)-only and cocultured Mu+MN myobundles (Fig. 1B). Tissues were cultured for four weeks after onset of muscle differentiation8 in the presence of 20ng/ml BDNF, 10ng/ml GDNF, and 10ng/ml CNTF, which are known to support MN growth. Electrical and mechanical properties of myobundles were assessed by measuring isometric contractile force (twitch and tetanus), tissue rheobase (relative tetanic force as a function of stimulus voltage), and Ca2+ transients (using a muscle-specific Ca2+ sensor, GCaMP6), as previously described9. Functional innervation was studied by recording Ca2+ transients and contractile forces during selective stimulation of MNs with and without tubocurarine, an acetylcholine receptor (AChR) antagonist. Immunostaining of tissue cross-sections and whole-myobundle mounts was performed to assess axonal ingrowth, myofiber structure, and AChR and NMJ morphology. RNA and protein were isolated separately from MN and myobundle compartments to assess muscle and MN maturation at different time points in co-culture.
Results, Conclusions, and Discussions:: Differentiated MNs expressed key transcription factors (Isl1), structural proteins (Prph, Nefl, Nefm), and functional enzymes ( >90% ChAT+ cells at D45) (not shown). Functionally, MNs demonstrated glutamate sensitivity by D20 and spontaneous spike trains by D35 (Fig. 1C,D). In 3D co-cultures, MN axonal projections grew into Mu+MN myobundles reaching ~5 mm length over 4 weeks (Fig. 1E). Compared to age-matched Mu-only tissues, Mu+MN myobundles at 3 and 4 weeks of co-culture had increased twitch and tetanus amplitudes (Fig. 1F) and specific forces, decreased rheobase (i.e., increased excitability), and faster twitch kinetics, all indicative of improved functional maturation. Consistent with functional improvements, innervated myobundles exhibited increased gene and protein expression of neonatal and adult myosin heavy chain isoforms (Myh8, Myh7, MYH1&2) and decreased expression of embryonic MYH3 (Fig. 1G,H). Furthermore, electrical-field evoked Ca2+ transient amplitudes (Fig. 1I) and expression of Ca2+ handling genes and proteins (Casq1, Atp2a1, RYR1) were increased in Mu+MN vs. Mu-only tissues, further supporting the observation that MN innervation plays a beneficial role in engineered muscle maturation. Concurrent with muscle maturation in 3D co-culture, we observed increased expression of ChAT and Mnx1 in the MN compartment, suggesting reciprocal pro-maturation effects of muscle on motoneurons (not shown). Importantly, local electrical stimulation of MNs yielded generation of Ca2+ transients and contractions in muscle fibers within Mu+Mn myobundles, which were blocked by the application of tubocurarine (Fig. 1I). MN stimulation at increasing frequencies (1-40Hz) evoked progressive fusion of twitch contractions into a tetanus (Fig. 1J), which to our knowledge is the first demonstration of a positive force-frequency relationship in innervated 3D engineered muscles induced by MN stimulation. With time of culture, we also observed increased AChR lacunarity (Fig. 1K,L), upregulated expression of AChR-e subunits, and frequent colocalizaton of pre-synaptic MN markers and AChRs, all indicative of progressive NMJ maturation.
In conclusion, by optimizing MN differentiation protocol and 3D co-culture conditions, we have generated functionally innervated (Mu+MN) human myobundles with advanced maturation of both muscle and motoneurons. In the future, we will utilize this system to study neuromuscular diseases and the roles of functional innervation in skeletal muscle injury and regeneration.
Acknowledgements (Optional): : This work was supported by NIH grants AR070543, UG3TR002142 and U01EB028901 to N.B., as well as the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE2139754 (M.P.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
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