Background & Aims

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells of healthy individuals and neuropathic pain patients offer a unique opportunity to obtain sensory neurons harboring the donor’s genotype in vitro. IPSC-derived sensory neurons have been successfully used to model phenotypes of small-fiber-neuropathy (SFN) and erythromelalgia (IEM) patients with mutations in the voltage-gated sodium channels Nav1.8 or Nav1.7. Despite expressing mRNA for the tetrodotoxin-resistant (TTXr) voltage-gated sodium channels Nav1.8 and Nav1.9, the functional presence of these channels in iPSC-derived sensory neurons remains incompletely characterized. However, the functional presence of Nav1.8 and Nav1.9 is a hallmark of nociceptor neuron identity. Here, we investigated the presence and composition of the TTXr currents in iPSC-derived sensory neurons.

Methods

IPSCs from healthy donors and two neuropathic pain patients with a p.T66S variant in Nav1.9 were differentiated using three distinct methods: 1) a small molecule-driven approach [1,2], 2) a TALEN-mediated insertion of an inducible transcription factor cassette into the AAVS1-locus harboring NGN1, BRN3A and ISL1 [3], or 3) a virally-induced overexpression of NGN1 [4]; with a minimum differentiation period of 8 weeks. Voltage Clamp experiments were performed with 500nM TTX in the bath and mouse dorsal root ganglia (mDRG) served as control. To assess the presence of a functional phenotype, current clamp was performed on iPSC-derived sensory neurons from patients carrying the Nav1.9 variant p.T66S and a healthy individual.

Results

Our findings reveal that 95% of measured iPSC-derived sensory neurons exhibited TTXr currents, indicating their nociceptive identity. The overall TTXr current amplitude was slightly larger in mDRGs than in iPSC-derived sensory neurons. Still, the fast TTXr current component was observed in both cell types. A distinct TTXr current component with a much slower decay was mainly observed in mDRGs, and hardly detectable in iPSC-derived sensory neurons. We did not observe a phenotypic difference in current clamp between iPSC-derived sensory neurons with and without a p.T66S Nav1.9 variant so far.

Conclusions

The presence of TTXr currents is in line with a nociceptor identity of the iPSC-derived sensory neurons. Our study provides initial insights into the composition of the TTXr currents in iPSC-derived sensory neurons, suggesting the presence of Nav1.8, while functional expression of Nav1.9 is not confirmed. Further research is warranted to elucidate the precise composition of ion channels and support proteins in both dorsal root ganglia and iPSC-derived sensory neurons.

References

[1] Chambers SM, Qi Y, Mica Y, Lee G, Zhang XJ, Niu L, et al. (2012) Nat. Biotechnol. 30(7)
[2] Neureiter A, Eberhardt E and Lampert A (2022) Methods Mol. Biol. 2429
[3] Pascal Röderer, Oliver Brüstle unpublished data.
[4] Schrenk-Siemens K, Pohle J, Rostock C,Abd El Hay M, Lam R M, Szczot M, Lu S, Chesler A T, Siemens J, (2022) Cells 2905 (11)

Presenting Author

Fiona Maria Roll

Poster Authors

Fiona Maria Roll

MSc

Universityklinik Aachen

Lead Author

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Anika Neueiter PhD

Institute of Neurophysiology, University Hospital RWTH Aachen

Lead Author

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Marlene Menke MSc

Institute of Neurophysiology, University Hospital RWTH Aachen

Lead Author

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Aylin Kesdo?an

Institute of Neurophysiology, University Hospital RWTH Aachen

Lead Author

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Sofie Rämisch

Institute of Neurophysiology, University Hospital RWTH Aachen

Lead Author

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Andelain Erickson PhD

Institute of Neurophysiology, University Hospital RWTH Aachen

Lead Author

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Pascal Röderer PhD

Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital B

Lead Author

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Oliver Brüstle Professor MD

Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital B

Lead Author

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Esther Eberhardt MD

Institute of Neurophysiology, University Hospital RWTH Aachen; Scientific Center for Neuropathic Pai

Lead Author

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Angelika Lampert Professor MD

Institute of Neurophysiology, University Hospital RWTH Aachen; Scientific Center for Neuropathic Pai

Lead Author

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Topics

  • Models: Chronic Pain - Neuropathic