Direct conversion and microfluidic chambers as tools to study the in-vivo sketch of human sensory neurons
- Autores: Simona Giorgi
- Directores de la Tesis: Antonio Vicente Ferrer Montiel (dir. tes.), Asia Fernández Carvajal (codir. tes.)
- Lectura: En la Universidad Miguel Hernández de Elche ( España ) en 2024
- Idioma: español
- Tribunal Calificador de la Tesis: M. Victoria Moreno Manzano (presid.), Isabel Devesa Giner (secret.), Karolina Pircs (voc.)
- Materias:
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- Resumen
Sensory neurons are the primary cells responsible for detecting temperature, mechanical forces, pain, pruritus, and many other somatosensations. These cells, primarily located in the dorsal root ganglia (DRGs) or trigeminal ganglia (TG), exhibit a distinctive morphology, with axons extending up to one meter in length in humans. The cellular and molecular features of sensory neurons, both in physiological and pathological states, are commonly studied in vitro using rodent models, which examine both the cell body and its extensions in the same environment. However, the limited applicability of animal models to the human somatosensory system and the differences between cell bodies and neurites have emerged as significant challenges in sensory neuron research.
To address these issues, this work aims to develop a human-based model of sensory neurons through the direct conversion of fibroblasts, a process also known as transdifferentiation. Fibroblasts obtained from foetal lungs, neonatal dermis, or adult dermis were transduced to express Brn3a and Ngn1 using lentiviral vectors. Additionally, the cultured cells were subjected to conversion media containing small molecules that either inhibited or induced intracellular pathways crucial for neuronal development. The effectiveness of the conversion protocols was primarily assessed using calcium imaging, to quantify the functionality of putative induced sensory neurons (iSN). The most promising conversion protocols were further characterized with molecular (qPCR, immunocytochemistry) or functional studies (MEA CMOS, patch clamp). As a result, at least one promising conversion protocol was found for every fibroblast type. Foetal lung fibroblasts presented sensory neurons morphology and some neuronal markers after few days of conversion. However, we believe these protocols need to be refined due to their cytotoxicity. Neonatal dermal fibroblasts conversion was more challenging, however finally the addition of the small molecule DAPT seemed to increase the conversion protocol efficiency. However, the same protocol was proven to be inefficient in adult dermal fibroblasts. Therefore, Ascl1 and Brn2 induction was added to the protocol as well as REST inhibition. The addition of these genetic modifications and changing media composition led to a significant increase in the conversion efficiency and in generating putative induced sensory neurons cultured in vitro up to 50 days, which was the end point of our experiment.
10 Importantly, electrophysiological assays should be performed to confirm that the converted cells are functional neurons.
Moreover, to recreate the in vivo architecture of sensory neurons, the culture of mouse DRGs in microfluidic chambers (MFC) was optimized. MFCs enabled the physical separation of cell bodies and neurites, allowing for targeted stimulation of terminals and the recording of activity in the cell body. The optimization of this compartmentalized culture allowed to co-culture sensory nerve endings with keratinocytes, paving the way to co-culturing with other cell types. Furthermore, nerve endings were treated with inflammatory mediators or a chemotherapeutic agent, demonstrating peripheral sensitization through these agents.
This work paves the way to optimizing several conversion protocols and further investigating the already optimized protocol to understand the role of the different components used during the conversion. Moreover, future experiments may be focused on setting up human sensory neurons' compartmentalized culture, which would be a valuable tool for studying human peripheral signalling.
