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

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.

Progressive Recruitment of Mesenchymal Progenitors Reveals a Time-Dependent Process of Cell Fate Acquisition in Mouse and Human Nephrogenesis.

Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis.

Fabrication of Micromolded Gelatin Hydrogels for Long-Term Culture of Aligned Skeletal Myotubes.

Cultured skeletal myotubes are a powerful in vitro system for identifying mechanisms of skeletal muscle development and disease. However, skeletal myotubes routinely delaminate from conventional culture substrates after approximately 1 week, which significantly hampers their utility for in vitro disease modeling and drug screening. To address this problem, we fabricated micromolded gelatin hydrogels as culture substrates that are more biomimetic than conventional substrates. On micromolded gelatin hydrogels, C2C12 skeletal myoblasts align and differentiate into skeletal myotubes that are stable in culture for multiple weeks. With this protocol, we detail three key steps: (1) Fabrication of micromolded gelatin hydrogels; (2) Culture of mouse C2C12 myoblasts and differentiation into myotubes; and (3) Quantification of myotube morphology. These substrates have many applications for skeletal muscle disease modeling and drug screening over longer time scales.

Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.

In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (µmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (µprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-µprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS µprinted with FN, myogenic index, myotube width, and myotube length on µmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on µmolded gelatin hydrogels compared to FN-µprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.