Tissue engineering strategies for the development of a human 3D model of the neuromuscular junction

In vitro models represent an important tool in the regenerative medicine research area. Great importance is given to utilising a representative system, which closely mimics the in vivo environment. Developing a model of the neuromuscular junction (NMJ) is a promising step which will allow scientists to investigate the physiology and pathophysiology of the interface between skeletal muscles (SkM) and peripheral motor neurons (MNs). These cell types can be affected by degenerative disease such as amyotrophic lateral sclerosis (ALS), that leads to progressive paralysis and for which no treatment is available to this date. A human model of the NMJ could be used to study potential therapeutic agents and reduce the use of animals in research. The main aim of this project was to humanise currently existing models of the NMJ using tissue engineered constructs. In the first part of the work, cell lines were used to establish a chimeric co-culture. Immunofluorescent staining, gene expression analysis and the evaluation of morphological features were used. As a result, the co-culture conditions for C2C12s and SH-SY5Ys were optimised, the expression of pre- and post-synaptic proteins was verified and agrin was not included in the medium for 3D cultures. Fibrin and collagen constructs allowed for the alingnment of C2C12 fibres, and the addition of SH-SY5Ys to the culture did not affect matrix remodeling or myotube width. However, information on the cells distribution, training on the use of tissue engineered constructs and observations on the alignment of SH-SY5Ys were gathered. Subsequently, iPSC-derived MN progenitors were differentiated for 35 days and showed typical MN morphology, as well as cholinergic markers expression. The culture of SkM on gelatin proved to be more effective for its differentiation, and donor-to-donor variability was observed when generating the co-culture with MNs. Despite the expression of NMJ markers and the use of 3D constructs, gene expression was depleted when the neurons were present in the culture, the distribution of the cells within collagen/Matrigel® gels did not allow for an increase in force generation and it was not possible to verify the presence of a NMJ. The co-culture of human muscle and nerve in a 3D environment is a promising model which allows researchers to understand the mechanisms underpinning NMJ development and formation. Tissue engineered constructs are suitable systems as they allow for the cells to grow and interact in a matrix which mimics the in vivo conditions, while mechanically or electrically stimulate the cells. The work carried out for this thesis led to a great amount of future developments using a novel co-culture system, which is an improvement compared to exhisting animal or embryonic models. This can be further optimised for applications such as drug screening, personalised medicine testing, or disease modeling.