Polyclonal sensory neuron derivation from iPSCs as an efficient alternative to single clone strategies for pain-relevant in vitro models

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Induced pluripotent stem cell (iPSC) workflows typically rely on monoclonal cultures, requiring multiple independent cell lines to compensate for clonal variability. This increases workload, cost, and limits scalability for translational applications. Here, we establish polyclonal reprogramming strategies using Sendai virus to generate clonally diverse iPSC cultures already during reprogramming through FACS, MACS, manual selection, or prolonged culture. Both monoclonal and FACS-derived polyclonal cultures reached pluripotency. Reprogramming transgenes were silenced, whereas Sendai virus (SeV) mRNA persisted across passages in both culture types; heat treatment at 38.5 °C markedly reduced SeV levels. Monoclonal and polyclonal cultures differentiated efficiently into neural crest-like cells and sensory neurons. As a second polyclonal strategy, monoclonal cultures were pooled at day-5 of sensory neuron differentiation to generate percentage-controlled progenitor mixtures. TRPA1 protein expression has not been shown reliably in iPS-derived sensory neurons. We hypothesized that increased clonal diversity might facilitate detection of TRPA1 protein expression. Despite rigorous antibody validation, TRPA1 protein expression was detectable only diffusely across the whole cell area. Together, our results show that polyclonal iPSC strategies enable clonally inclusive generation of patient-specific sensory neurons. These workflows reduce early workload and provide a robust foundation for scalable drug-screening and patient-in-a-dish applications.

Authors 10

1. Anika Neureiter Aachen Institute of Neurophysiology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

   Universitätsklinikum Aachen

   Affiliation as printed

   Institute of Neurophysiology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

   Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

2. Luise Ruessmann Aachen Institute of Neurophysiology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

   Universitätsklinikum Aachen

   Affiliation as printed

   Institute of Neurophysiology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

   Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

3. Petra A. I. Hautvast Aachen Institute of Neurophysiology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

   Universitätsklinikum Aachen

   Affiliation as printed

   Institute of Neurophysiology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

   Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

4. Maike Franziska Dohrn Aachen Department of Neurology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

   Universitätsklinikum Aachen

   Affiliation as printed

   Department of Neurology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

   Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

5. Noortje W.M. van den Braak Aachen Department of Neurology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

   Universitätsklinikum Aachen

   Affiliation as printed

   Department of Neurology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

   Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

6. Arzu Stoppe Aachen Department of Neurology

   Universitätsklinikum Aachen

   Affiliation as printed

   Department of Neurology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

7. Martin Häusler Aachen Division of Neuropediatrics and Social Pediatrics Department of Pediatrics

   Universitätsklinikum Aachen

   Affiliation as printed

   Division of Neuropediatrics and Social Pediatrics, Dept. Of Pediatrics, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

8. Annette Lischka Aachen Center for Human Genetics and Genomic Medicine Faculty of Medicine

   RWTH Aachen University

   Affiliation as printed

   Center for Human Genetics and Genomic Medicine, Faculty of Medicine, RWTH Aachen University, Aachen, Germany

9. Katja Eggermann Aachen Center for Human Genetics and Genomic Medicine Faculty of Medicine

   RWTH Aachen University

   Affiliation as printed

   Center for Human Genetics and Genomic Medicine, Faculty of Medicine, RWTH Aachen University, Aachen, Germany

10. Angelika Lampert corresponding Aachen Institute of Neurophysiology Scientific Center for Neuropathic Pain Aachen SCNAACHEN

    Universitätsklinikum Aachen

    Affiliation as printed

    Institute of Neurophysiology, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany

    Scientific Center for Neuropathic Pain Aachen SCNAACHEN, Uniklinik RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany