3D culture platform of human iPSCs-derived nociceptors for peripheral nerve modeling and tissue innervation

Afonso Malheiro,Paul Wieringa,Paul G A Volders,Gonda F Konings,Joyce Bernardi,Lorenzo Moroni,Andrea Romano,Adrián Seijas-Gamardo,Carlos Mota,Abhishek Harichandan

doi:10.1088/1758-5090/ac36bf

Abstract

Functional humanized in vitro nerve models are coveted as an alternative to animal models due to their ease of access, lower cost, clinical relevance and no need for recurrent animal sacrifice. To this end, we developed a sensory nerve model using induced pluripotent stem cells-derived nociceptors that are electrically active and exhibit a functional response to noxious stimuli. The differentiated neurons were co-cultured with primary Schwann cells on an aligned microfibrous scaffold to produce biomimetic peripheral nerve tissue. Compared to glass coverslips, our scaffold enhances tissue development and stabilization. Using this model, we demonstrate that myelin damage can be induced from hyperglycemia exposure (glucose at 45 mM) and mitigated by epalrestat (1 µM) supplementation. Through fibrin embedding of the platform, we were able to create 3D anisotropic myelinated tissue, reaching over 6.5 mm in length. Finally, as a proof-of-concept, we incorporated pancreatic pseudoislets and endometrial organoids into our nerve platform, to demonstrate the potential in generating nociceptor innervation models. In summary, we propose here an improved tool for neurobiology research with potential applications in pathology modeling, drug screening and target tissue innervation.

Highlights

The ability to detect external noxious stimuli and internal organ dysfunction signals is essential to maintain physical integrity and homeostasis[1]
Human induced pluripotent stem cells (iPSCs) displaying a pluripotency phenotype (Fig. S1) were seeded on a 400 μm agarose microwell mold (200 cells / microwell; 1580 microwells) and the cells synchronized via dimethyl sulfoxide (DMSO) treatment for 3 days
We followed a differentiation protocol adapted from Chambers et al.[19], in which human iPSCs were driven towards nociceptors, via a neural crest intermediate, in a quick and efficient manner

Summary

Introduction

The ability to detect external noxious stimuli and internal organ dysfunction signals is essential to maintain physical integrity and homeostasis[1]. This process is mediated by nociceptors, which recognize this input via specialized receptors, such as the transient receptor potential vanilloid 1 (TRPV1), and conduct the information to the central nervous system (CNS), whilst locally releasing neuropeptides, e.g. substance P, at the site of stimulus[2,3]. Most cases of PN damage arise from diabetes type II pathophysiological imbalances[6], which has prompted significant research into prevention and mitigation of diabetes-related disorders as well as strategies for neural tissue repair[78]. Women with endometriosis, a benign condition affecting up to 10% of reproductive-aged women, experience nociceptive pain because of infiltrating nociceptors within endometriotic implants and inflammatory sensitization of peripheral nociceptors[12]

Methods

Results

Conclusion