Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Abstract

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling

“…Previous animal studies showed that this AChR subunit transition occurs in the presence of motor axon endplates and confirmed that transcription of the epsilon gene (CHRNE) is stimulated by AChR Inducing Activity (ARIA) via ErbB receptors, a nerve derived ligand of the neuregulin-1 (NRG1) family (Martinou et al, 1991). Consistently, CHRNE transcripts are detected in rodent 2D and 3D skeletal muscle fiber cultures when co-cultured with nerve cells (Bach et al, 2003;Ostrovidov et al, 2017;Smith et al, 2016;Vilmont et al, 2016). However, despite significant progress toward directing human pluripotent stem cells (PSCs) to the motor neuron lineage Lippmann et al, 2014;Maury et al, 2015;Shimojo et al, 2015;Zhang et al, 2001) and establishing electrically and chemically responsive human muscle fibers in vitro (Madden et al, 2015), the first reports of human NMJ models -2D human muscle fiber and motor neuron co-cultures -were unable to demonstrate synapse maturation via the gamma to epsilon AChR subunit switch (Guo et al, 2011;Steinbeck et al, 2016), and there are no reports of epsilon AChR protein expression or function in culture in the absence of enforced gene expression.…”

Feasibility and Biocompatibility of 3D‐Printed Photopolymerized and Laser Sintered Polymers for Neuronal, Myogenic, and Hepatic Cell Types

“…Such reductions were however not evident when cells were cultured on PA‐12 scaffolds, with comparable morphological and transcriptional phenotypes evident. This further outlines the applicability of this polymer and favorability of LS when fabricating scaffolds for musculoskeletal tissue engineering applications …”

“…Particularly when evaluating the specific AM process and polymer combination while creating physiologically representative in vitro models that are utilized within tissue engineering and or pharmaceutical screening applications. This research demonstrates cell type specific biocompatibility across a range of industry standard 3D printing techniques: SL, LS, and PolyJet, for incorporation within tissue engineering models of musculoskeletal and neurological disorders, in addition to pharmacological screening systems . In this work, we used C 2 C 12 , SH‐SY5Y, and HepG2 cell lines in order to ascertain critical evidence of cellular behavior in response to 3D‐printed candidate polymers due to their capacity to afford a range of optically transparent (Clear‐FL and VeroClear) and bioinert (PA‐12) parts from commercially available and accessible AM instrumentation.…”

“…3D‐printed biocompatible polymers hold significant importance for tissue and biochemical engineering applications, with the methods required to construct the models that examine highly complex cellular physiological processes currently being revolutionized by AM . Pertinent to this are tissue engineered in vitro models, designed to replicate the in vivo cellular and molecular mechanisms that regulate musculoskeletal and neuromuscular disorders . As such, defining the compatibility of mammalian cells frequently used within these models, C 2 C 12 murine skeletal myoblasts and SH‐SY5Y neuroblastoma human derived cell lines are of particular relevance.…”

Making Skeletal Muscle from Human Pluripotent Stem Cells

“…The interaction between myogenic cells and non-myogenic cells in muscle tissue may influence myogenesis either by interacting directly or by secretion of paracrine factors (Figure 2). Motor neurons were used to form neuro-muscular junctions [49] and further adapted to a 3D culture system [50]. The presence of motor neurons had positive effects on myotube maturation [50].…”