Modern-day regenerative medicine is constantly exploring the development and application of stem cells for cell-based therapies in many conditions and diseases. One of such conditions is deafness, which affects over 360 million people worldwide, frequently caused by irreversible damage to the sensory cells. Therapies which could replace dead or damaged spiral ganglion neurons in deafness are sought as alternatives to the currently limited approaches. In this regard, stem cells from human dental pulp cells (hDPCs) are proposed to be intrinsically neurogenic due to their neural crest origin. Nevertheless, hDPCs have been poorly investigated in the context of auditory nerve regeneration. In the present work, we aimed to obtain a neurogenic population from hDPCs which could be differentiated into auditory neurons. We included mouse incisor dental pulp cells in our experiments, which allowed us to identify conditions that promote a neurogenic phenotype. We established hDPC cultures in serum-free, defined media as a basal condition for growth and propagation, providing pre-clinical advantages to the standard condition containing foetal bovine serum. The specific activity in an auditory regeneration system was tested co-culturing the hDPCs together with denervated cochlear explants ex-vivo. Our results have shown that dental pulp cells cultures can be established in serum-free neurogenic conditions; expressing basal levels of markers related to neural progenitor-, neural- and neural crest cells. Particularly, growing hDPCs as sphere aggregates showed and enhanced neural crest molecular signature and showed differentiation into spiral ganglion-like neurons when co-cultured with cochlear explants. Therefore, we present a comprehensive characterisation of human dental pulp cells under standard and defined culture conditions, their ability to express a neural crest stem signature and provided evidence, for the first time, of their potential to differentiate into spiral ganglion-like neurons in an ex-vivo model of auditory nerve regeneration.

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The focus of disease research often surrounds therapeutic pathway identification and the subsequent investigation of proteins or compounds that potentially interfere with disease mechanisms. However, finding targets and effective therapeutic domains often overshadows another important aspect of drug delivery and efficacy; the method of domain conjugation. Unfortunately the combination of good therapeutic components and good therapeutic design can often be amiss, with differing skills and groups needed to marry the two together. In recognition of this, there are new techniques emerging that aim to not only address old problems of stable conjugation, but incorporating new knowledge of drug design. This research takes a reverse approach to drug design in order to better comprehend requirements which are often an afterthought; first looking at conjugation technique and then how it can best be exploited. Thereby, exploring how different conjugation methods can be used to complement existing therapeutic and internalisation strategies. Here, two methods of conjugation were investigated through attachment of bacterial toxin domains derived from botulinum A and diphtheria, with the intention of targeting neuroblastoma cells using a variety of specific and non-specific pathways. Both conjugation techniques proved to be stable in vitro and capable of binding a variety of domains consistently. The data here also emphasized the importance of endosomal escape proteins to increase the translocation of catalytic domains into the cytosol. It was demonstrated that the conjugates formed were able to facilitate the binding between targeting and catalytic domains, enabling specific neuroblastoma internalisation. Moreover, that three domains were able to conjugate together using a linking method and could form a co-operative triple-functioning complex.

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