Background & Aims
Neuropathic pain is a devastating condition and current treatments are still limited and inadequate due to our poor understanding of the pathomechanisms. The first synapse from the periphery to the central nervous system (CNS) is a critical site of modulation, and excessive input from primary sensory neurons may help initiate and maintain central sensitisation. Unfortunately, the underlying mechanisms have been difficult to study in humans. Human induced pluripotent stem cell (hiPSC) – derived sensory neurons (dSNs) have become a widely used tool to investigate the pathogenesis of pain disorders, bridging the translational gap between rodent and human models. In vivo, these sensory neurons are but the first element of a complex signalling pathway and how nociceptor pathologies affect downstream pain pathways is still unknown. We therefore aimed to establish an in vitro model of the connection between the sensory neurons and dorsal horn neurons in the spinal cord.
Methods
We first established murine dorsal horn cultures. For characterisation, we stained for different cell-type markers (GFAP, ChAT, NeuN, b-III-tubulin, Pax2, vGLUT2, PKCy) and synapses (Synaptoporin, and PSD-95). We used established protocols (Chambers et. al., 2012, Alex J. Clark 2020) to differentiate sensory neurons from hiPSCs which we seeded both onto conventional coverslips and into microfluidic chambers. We then analysed synaptic connectivity in hiPSCdSN monocultures by immunohystochemistry of synaptic markers for anatomical confirmation. For our co-cultures, we seeded mouse dorsal horn (DH) neurons either directly onto the coverslip cultures, or onto the neurite compartment of the established microfluidic cultures. To confirm connectivity between the hiPSCdSNs and primary murine dorsal horn neurons we used calcium-imaging together with immunocytochemistry.
Results
Molecular analysis showed that our long-term murine primary DH monocultures consist of varied cell types and contain a substantial number of astrocytes which stays constant over time. Interestingly, analysis of long-term hiPSCdSN monocultures showed cell-line dependent numbers of anatomical synapses in vitro.
We found that in coverslip co-cultures of hiPSCdSNs and mouse DH neurons, the level of spontaneous activity (SA) in DH neurons is increased compared to DH monocultures. The increase in SA observed in DH neurons is further increased in co-cultures with hiPSCdSNs derived from inherited erythromelalgia (IEM) patients.
We further successfully established microfluidic hiPSCdSN-murine dorsal horn co-cultures and confirmed functional and anatomical connectivity by means of calcium imaging and immunocytochemistry respectively. As a technical note, we also collected data comparing red-shifted calcium sensors and dyes to a state-of-the-art GCaMP sensor.
Conclusions
The diversity in cell types and proportion of glia in our long-term DH monocultures suggest this to be a solid in vitro model of the dorsal spinal cord.
Our observations regarding synapse formation within long-term hiPSCdSN monocultures is an important discovery to keep in mind when using these cultures as a model.
Analysis of spontaneous activity within dorsal horn neurons co-cultures with healthy or diseased hiPSCdSNs suggests that the hyperactivity observed in sensory neurons is transmitted onto the spinal cord.
Lastly, our newly developed protocol for microfluidic hiPSCdSN-murine dorsal horn co-cultures is an exciting opportunity that can be used to investigate the intersection from the periphery to the CNS. Elucidating the mechanisms and pathomechanisms of neuropathic disorders at the synaptic level could pave the way for the discovery of novel treatment approaches.
References
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Chambers SM, Qi Y, Mica Y, Lee G, Zhang XJ, Niu L, Bilsland J, Cao L, Stevens E, Whiting P, Shi SH, Studer L. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol. 2012 Jul 1;30(7):715-20. doi: 10.1038/nbt.2249. PMID: 22750882; PMCID: PMC3516136.
Clark AJ. Establishing Myelinating Cocultures Using Human iPSC-Derived Sensory Neurons to Investigate Axonal Degeneration and Demyelination. Methods Mol Biol. 2020;2143:111-129. doi: 10.1007/978-1-0716-0585-1_9. PMID: 32524476.
Burley R, Harvey JRM. Multielectrode Arrays. Methods Mol Biol. 2021;2188:109-132. doi: 10.1007/978-1-0716-0818-0_6. PMID: 33119849.
Brouwer BA, Merkies IS, Gerrits MM, Waxman SG, Hoeijmakers JG, Faber CG. Painful neuropathies: the emerging role of sodium channelopathies. J Peripher Nerv Syst. 2014 Jun;19(2):53-65. doi: 10.1111/jns5.12071. PMID: 25250524.
Presenting Author
Kira Werder
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology