Background & Aims

In the early stages of embryogenesis, neural crest cells develop into bipolar peripheral sensory neurons (SNs). As these neurons mature, they undergo a morphological transformation to become pseudounipolar. This specialized morphology allows collection of sensory signals in the periphery and transmission towards the central nervous system. While human induced pluripotent stem cell (iPSC) derived SNs have become an important tool in current pain research, they typically present with a multi- or bipolar morphology under standard in vitro conditions. The underlying mechanism and time scale of SN pseudounipolarization, both in vivo and in vitro, is not yet fully understood. Here we aimed to induce and visualize the process of pseudounipolarization and to evaluate the functional impact of this morphological change.

Methods

Human iPSCs were equipped with a doxycycline-inducible expression cassette encoding the fate specifying transcription factors NGN1, BRN3A and ISLET1 (‘NBI’) in the AAVS1 safe harbor locus. Forced overexpression of ‘NBI’ for seven days induced differentiation of iPSCs into SNs. The generated neurons were characterized for gene expression (e.g. RNAseq, RNAscope), as well as electrophysiological functionality (e.g. patch clamp). To induce a pseudounipolar morphology, human SNs were co-cultured with non-neuronal cells isolated from the embryonic rat dorsal root ganglion (DRG). Co-cultured SNs were visualized with a fluorescent tdTomato reporter, which allowed the identification of pseudounipolar neurons in live cultures, as well as live cell imaging of morphological changes over time. High-density multi-electrode array (HD-MEA) recordings were employed to study the intrinsic electrical activity and axonal conduction dynamics of pseudounipolar human SNs.

Results

Overexpression of NGN1, BRN3A and ISLET1 in iPSCs for seven days enabled robust generation of induced SNs (iSNs) expressing relevant markers such as PRPH, NTRK1, SCN9A and SCN10A. Transcriptome analyses after 7, 35 and 63 days in vitro revealed a time-dependent maturation, with consecutive enrichment of transcripts associated with neuronal function, reflected in Gene Ontology terms like ‘ion channel activity’. Similarly, RNAscope experiments showed an increasing co-expression of the peripheral sodium ion channels SCN9A, SCN10A and SCN11A over time. Co-culture with non-neuronal cells from rat DRGs triggered pseudounipolarization in about 20% of iSNs. Live cell imaging showed that this process can occur within 24 hours and might be reversible at early stages. Preliminary HD-MEA recordings of iSNs, and tracking of their axonal action potential propagation, also indicated pseudounipolarized electro-morphic properties in these cells. Finally, the axonal conduction velocity of iSNs was similar to the conduction velocity of unmyelinated C-fiber neurons (approx. 0.5 m/s).

Conclusions

Together our data suggests that forward programmed iSNs recapitulate key hallmarks of sensory neuron physiology and can transition into a pseudounipolar morphology. We expect our iSNs to provide further valuable insights into the mechanisms underlying peripheral sensory neuron development, as well as pathomechanisms involved in neurological and neurodevelopmental disorders.

References

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Presenting Author

Pascal Röderer

Poster Authors

Pascal Röderer PhD

PhD

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

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Ana Catarina Costa

Nerve Regeneration Group, Instituto de Biologia Molecular e Celular-IBMC and Instituto de Inovação e

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Omar Mossad

Grünenthal GmbH, Aachen, Germany

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Holger Trucks-Jansen

Grünenthal GmbH, Aachen, Germany

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Kim Le Cann

Grünenthal GmbH, Aachen, Germany

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Anil Kumar Kalia

Grünenthal GmbH, Aachen, Germany

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Ramya Balaji

Grünenthal GmbH, Aachen, Germany

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Verena Arndt

Grünenthal GmbH, Aachen, Germany

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Fiona Maria Roll

Universityklinik Aachen

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Manuel Schröter

Bioengineering Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Swit

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Angelika Lampert Professor MD

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

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Monica Sousa

Nerve Regeneration Group, Instituto de Biologia Molecular e Celular-IBMC and Instituto de Inovação e

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Oliver Brüstle Professor MD

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

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