Powell, Rebecca Alice; (2023) Using tissue and cell engineering to improve efficacy in nerve repair. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The current gold standard treatment for large peripheral nerve injuries is an autograft but this method comes with several downsides such as donor site morbidity and limited donor tissue availability. Autografts are also not always successful with patients left with long-term impacts on quality of life such as paralysis and chronic pain. A tissue engineered alternative to the autograft could greatly improve outcomes, avoid the detrimental effects of an autograft, and hopefully lead to improve quality of life and socioeconomic outcomes. The components of the tissue engineered construct developed in this project include Schwann cells or Schwann cell precursors. Repair Schwann cells are a key cell type in supporting axon regeneration and using cells expressing genes involved in the repair phenotype is likely to be beneficial. As an autograft contains nerve extracellular matrix and aligned columns of Schwann cells and endothelial cells, the Schwann cells and precursors were also combined with collagen hydrogel and formed into an aligned cylindrical construct. Schwann cells and Schwann cell precursors were successfully differentiated from two clinically relevant hiPSC lines and characterised using immunocytochemistry and RT-qPCR. This protocol was optimised to generate sufficient cell numbers for tissue engineering applications. Conditional immortalisation methods were also assessed, and those cells derived from hiPSCs containing a conditional immortalising transgene characterised. The proliferation rates of the differentiated cells in permissive and non-permissive conditions were tracked over time to determine the tumorigenic properties of the cells to be implanted. These differentiated cells were successfully incorporated into an aligned engineered nerve construct by gel aspiration-ejection. Co-culture experiments with primary rat dorsal root ganglia showed these stable constructs can support and guide neurons along the 12mm long cylindrical gel. This project provides a method for producing human Schwann cells from clinically relevant iPSC sources and demonstrates the first use in GAE.

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