Development and validation of an in vitro model system to study peripheral sensory neuron development and injury

Iwan Jones,Rebecca Wiberg,Leif Carlsson,Staffan Johansson,Tushar Devanand Yelhekar,Paul J. Kingham,Mikael Wiberg

doi:10.1038/s41598-018-34280-3

Abstract

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

Highlights

The human peripheral nervous system (PNS) is a complex network of functionally distinct neurons that are organised into anatomically distinct ganglia
We generated peripheral sensory neurons from human embryonic stem cells (hESCs) grown in conditioned medium by a combination of dual-SMAD inhibition and early WNT activation coupled with small-molecule inhibition of specific pathways including Notch, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) signaling (Fig. 1a)[16]
These immature cells were subsequently replated in N2 medium containing a defined neurotrophic factor cocktail including brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and ascorbic acid

Summary

Introduction

The human peripheral nervous system (PNS) is a complex network of functionally distinct neurons that are organised into anatomically distinct ganglia. To fully exploit the potential of these hESC-derived peripheral sensory neuron models they must recapitulate the diversity of neuronal modalities found in vivo and the pathophysiological changes that underlie specific PNS injuries and diseases. This can only be accomplished by improving our current knowledge as to the molecular nature of the in vitro differentiation process in combination with in-depth molecular and functional analyses of the terminally differentiated neurons produced[15]. The in vitro differentiated cells display several hallmarks of bona fide mature peripheral sensory neurons and provide an unlimited source of biological material for comparative studies that address human sensory neuron development, injury and disease

Methods

Results

Conclusion