Background & Aims

Small fiber neuropathy (SFN) mainly affects A-delta and C fibers responsible for pain and thermal perception1. Peripheral nerve endings terminate within the epidermis, where they are surrounded by keratinocytes that may act as primary nociceptive transducers2.We applied two approaches to gain new insights into the pathophysiology of SFN. First, for transcriptome characterization, we performed RNA sequencing of keratinocytes obtained from patients with SFN and healthy controls (Ctr). Second, we generated innervated 3D in vitro skin models entirely produced using patient-derived skin cells and induced pluripotent stem cell (iPSC)-derived sensory neurons (iSN). These personalized innervated skin models may be applied as novel in vitro tools to individually investigate the pathomechanism of nociception and denervation.

Methods

6-mm skin punch biopsies were taken from the lower leg and upper thigh of patients with SFN (n=12) and Ctr (n=5). Samples were used to isolate keratinocytes and fibroblasts. Cultured skin cells were applied for transcriptomics and for generation of skin models. For sequencing of keratinocytes, total RNA was extracted, quality controlled, and DNA libraries were prepared. Sequencing of pooled libraries was performed on the NextSeq 500 platform (Illumina). Promising candidate genes were identified using pathway analysis (Metascape). Applying an optimized protocol with collagen hydrogel, skin cell cultures were used to build 3D in vitro skin models. Patient-derived fibroblasts were reprogrammed to iPSC and differentiated to iSN for innervation of the skin models. Additionally, we generated an innervated human reconstructed epidermis model with iSN. The histological structure of the skin models were assessed using hematoxylin-eosin and immunoreactions with cell specific proteins.

Results

Primary cell cultures of keratinocytes and fibroblasts from skin punch biopsies of SFN patients and Ctr were established. Next generation sequencing identified 141 deregulated genes in keratinocytes of patients with SFN vs. Ctr. Metascape analysis revealed enriched pathways containing potassium channels (e.g. hyperpolarization activated cyclic nucleotide gated potassium and sodium channel 2, potassium voltage-gated channel modifier subfamily S member 2). We further successfully generated 3D skin models with regular human skin morphology and expression of skin cell specific proteins such as cytokeratin 14, cytokeratin 10, and vimentin. Further, we also succeeded in generating human reconstructed epidermis models innervated with iSN that showed the expression of sensory neuron and keratinocyte specific proteins (peripherin and desmoplakin, respectively). Hematoxylin-eosin reaction displayed a histological structure comparable to native epidermis.

Conclusions

With two distinct approaches using patient-derived skin cells, we substantially contribute to in depth pathophysiology research in SFN. 1) Distinct keratinocyte transcriptome signatures between patients with SFN and Ctr may pioneer insights into pathomechanisms of small fiber sensitization in SFN patients and lay the basis for advanced diagnostics. 2) Innervation of entirely human 3D in vitro skin model with patient-derived iSN opens new avenues for pathophysiological investigation of SFN. We provide a new in vitro tool that may also improve the pre-clinical assessment of novel analgesic and neuroprotective compounds.

References

1 Üçeyler N. Small fiber pathology—a culprit for many painful disorders?
PAIN 2016;157(suppl 1):S60–66.
2 Talagas M, Lebonvallet N, Leschiera R, Sinquin G, Elies P, Haftek M, Pennec JP, Ressnikoff D, La Padula V, Le Garrec R, L’Herondelle K, Mignen O, Le Pottier L, Kerfant N, Reux A, Marcorelles P, Misery L. Keratinocytes communicate with sensory neurons via synaptic-like contacts. Ann Neurol 2020;88:1205–19.

Presenting Author

Franziska Karl-Schöller

Poster Authors

Franziska Karl-Schöller

PhD

1Department of Neurology, University Hospital of Würzburg, Josef-Schneider-Str. 11, 97080 Würzburg

Lead Author

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Maximiliane Wußmann

Translational Center 'Regenerative Therapies (TLC-RT), Fraunhofer-Institute for Silicate Research

Lead Author

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Thorsten Bischler

Unit SysMed, University of Würzburg, Josef-Schneider-Str. 2, 97082 Würzburg

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Luisa Kreß

Dr. med.

University Hospital Würzburg

Lead Author

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Florian Groeber-Becker

Translational Center 'Regenerative Therapies (TLC-RT), Fraunhofer-Institute for Silicate Research

Lead Author

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Topics

  • Specific Pain Conditions/Pain in Specific Populations: Neuropathic Pain - Peripheral