Advancing <i>in vitro</i> models of human dorsal root ganglia sensory neurons using pluripotent stem cells and 3D bioprinting technologies

<p dir="ltr">During everyday interactions with the world, the sensory nervous system plays a crucial role in detecting, relaying, and processing information from the internal and external environments of the body. First-order peripheral sensory neurons (SNs) are central to these functions, transducing physical stimuli into chemical and electrical signals to provide sensations like touch, pain, and proprioception. Our knowledge of the sensory nervous system has been largely founded by animal studies, however recent works have highlighted fundamental differences between human and animal tissues. Due to limited access to primary human tissue, human pluripotent stem cells (hPSC) present a promising alternative by allowing the <i>in vitro</i> differentiation of human SNs. Current methods for the study of hPSC-derived SNs largely rely on two-dimensional (2D) culture platforms, which do not fully recapitulate the three-dimensional (3D) dynamics of native sensory tissues. To address these limitations, this thesis explores novel tools for more advanced modelling of human SNs, from approaches for improved hPSC differentiation to the 3D bioprinting of cell-laden scaffolds.</p><p dir="ltr">We first describe the characterisation of a genetically modified hPSC line, H9^NGN2, engineered with an inducible Neurogenin-2 (NGN2) gene cassette for the rapid and efficient differentiation of hPSCs to induced SNs (iSNs). Validation of this cell line confirmed its pluripotency and capacity for tri-lineage differentiation, establishing suitability for producing neural crest cells (NCCs), the developmental progenitors to iSNs. NCCs were enriched for CD271+ expression and differentiated into functional iSNs over 21 days <i>in vitro</i> (DIV), expressing key sensory neuronal markers and demonstrating essential electrophysiological activity. This work establishes H9^NGN2 as a robust tool for generating iSNs, providing the foundation for subsequent 3D bioprinting studies.</p><p dir="ltr">Building on this advance, we describe the development of an extrusion bioprinting method for generating 3D iSN scaffolds. A GelMA-based bioink was optimised for printability and cytocompatibility with NCCs, allowing for subsequent iSN differentiation. Bioprinted constructs facilitated the differentiation of NCCs into functional iSNs, expressing sensory-specific markers and exhibiting membrane excitability properties by 21 DIV. These results highlight the potential of bioprinting technologies to generate more physiologically relevant <i>in vitro</i> models of the sensory nervous system, offering new possibilities to study neuronal development, function, and disease in 3D.</p><p dir="ltr">We then introduce a proof-of-concept 3D bioprinted model of human skin cocultured with iSNs to expand upon this work. Using the same GelMA-based bioink, primary human dermal fibroblasts, immortalised human Schwann cells, and H9^NGN2-derived NCCs were bioprinted to form 3D dermal scaffolds, with immortalised human keratinocytes seeded on the surface of scaffolds to enable epidermal formation. After 28 DIV, discrete dermal and epidermal structures were observed, with neurite outgrowth from iSNs marking a key step towards the generation of an innervated skin model. While further optimisation is needed to enhance innervation and guide neurites towards the epidermis, these findings present an important advancement towards the fabrication of complex bioprinted cocultures with a nervous system component.</p><p dir="ltr">Overall, this thesis presents significant strides in the development of advanced in vitro models of human SNs. By establishing a robust platform for the differentiation of hPSCs into functional iSNs and demonstrating the feasibility of 3D bioprinting for generating complex tissue constructs, this work addresses key limitations of current 2D culture systems. The creation of an innervated human skin model further highlights the potential for bioprinting to generate physiologically relevant coculture models for studying sensory biology. These developments pave the way for more sophisticated investigations into SN function and offer promising applications in regenerative medicine, disease modelling, and drug discovery. While optimisation challenges remain, this research establishes a strong foundation for advancing innovative new technologies to model human sensory neurons that can be applied to further study and gain knowledge about the human sensory nervous system.</p>