Lead supervisor: A. Lampert, Co-Supervisor: L. De Laporte, Junior supervisor: C. Rösseler Uniklinik RWTH Aachen, Institute of Physiology
Hypothesis: Dysfunctional free nerve endings in the epidermis of patients with altered nociception are more sensitive to mechanical stress than those of healthy probands.
Human keratinocytes (green) were co-cultured side by side with mouse sensory neurons (white). After one day neurites grow on the keratinocyte layer. Cell nuclei were stained with DAPI (blue). Data generated by Melanie Hilgers.
Background: Mechanical pain is transduced in the epidermis by crosstalk between keratinocytes and thin nerve fiber endings both of which are in very close contact. Nociceptive free nerve endings degenerate in the chronic neuropathic pain syndrome small fiber neuropathy (SFN). The affected patients suffer from intense, burning neuropathic pain. SFN occurs sporadically and can be linked to inheritable sodium channel mutations72. The nociceptive nerve endings in the skin are hard to assess experimentally as they are too small and too closely associated with the surrounding cells to be amenable to techniques such as the patch clamp technique, Ca++ imaging or multi-electrode arrays (MEAs). Alternative approaches are needed to better examine the pathophysiology of human sensory nerve endings. iPSC-derived sensory neurons from healthy individuals and SFN patients offer the required improvement73. Aims: The aim of this project is to build a 3D innervated skin model using iPSC-derived sensory neurons, epidermal equivalents and defined hydrogel-based ECMs to assess sensory nerve ending function in an environment mimicking the complex in vivo situation. We use cutting-edge hydrogel engineering (D1) and laser-induced mechanical actuation of these gels to study motion-induced neuronal responses. The model will help to understand (i) how mechanical signals within the epidermis are transduced into electrical signals in the nerve endings, and (ii) how this transduction changes under chronic pain conditions due to mutations of genes expressed in nociceptors. Approach: The established fibrin-based Anisogel with magnetically oriented microgels (D1)74 is used as a dermal ECM equivalent in the innervated skin model (body of LEGO-brick). Small pillars containing poly-NIPAAm gel from DWI are printed on top of the fibrin-based Anisogel (with D2; studs of the LEGO brick). The poly-NIPAAm gel contains gold nanorods, which can be heated in a reversible and dynamic manner (down to ms range) by exposure to near-infrared light. This results in rapid collapse and reswelling of the gel, thus inducing hydrogel actuation. Keratinocytes will be seeded around the studs, and differentiation into epidermal equivalents are induced by calcium shift and lifting to the air-liquid interface (MOCA and BioTex). The poly-NIPAAm pillars are designed to have the same height as or to be higher than the epidermal equivalent, leaving an optical window for actuation and imaging of the epidermal equivalent. Murine sensory neurons are seeded underneath the fibrin-based Anisogel and their neurite outgrowth are directed towards the top. Combining the hydrogel containing the sensory neurons with the artificial epidermis facilitates keratinocyte nerve interaction. When actuated, the poly-NIPAAm pillars leads to mechanical movement of the dermal equivalent. Ca++ or Na+ imaging are performed to assess neuronal responses to the mechanical deformation induced by poly-NIPAAm pillar collapse. Once the model is established using murine sensory neurons, we adapt it to human iPSC-derived sensory neurons and keratinocytes of healthy individuals and SFN patients with known mutations.
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