Background & Aims
Many promising discoveries rooted in pre-clinical models of neuropathic pain fail to be translated into effective clinical treatments (1), so a humanized investigating strategy will be necessary. Through bulk RNA sequencing and histological validation, we identified higher densities of intraneural T cells and macrophages, particularly a glucocorticoid-induced macrophage subpopulation, in human injured nerves (Morton’s neuroma) compared to control nerves (2). Here, we describe the development of a humanized in vitro microfluidic co-culture platform to investigate the temporal phenotypic changes of human peripheral blood mononuclear cells (hPBMCs) when co-cultured with healthy or injured human induced pluripotent stem cell-derived sensory neurons (hiPSCdSN). Using this microfluidic in vitro platform, we aim to monitor the temporal changes of human immune cells in the context of human peripheral nerve injury and to understand whether immune cells can modulate the excitability of hiPSCdSN.
Methods
hiPSCdSN were differentiated for 11 days from two healthy hiPSC lines according to a well-established differentiation protocol (3). Neurons were then harvested and seeded into the soma side of Geltrex-coated microfluidic devices and matured for one month before experiments. We applied structural and functional viability assays to identify the optimal co-culture condition. hPBMCs were isolated from the leukocyte cones of two healthy blood donors and added either in isolation or co-cultured at the neurite side for four days, with or without daily stimulation of soma using 50 mM KCl (two replicates per condition). hPBMCs were then harvested from the devices and stained using a 23-color panel to quantify the major lymphocyte populations. Data was acquired on a spectral flow cytometer (Aurora, Cytek) and analyzed by FlowJo. We also report the optimization of a UV laser ablation method (4) to focally injure the neurites and a jGCaMP8f calcium imaging method (5) (AAV9 transduction protocol).
Results
After 4-day culture, hPBMCs and hiPSCdSN showed comparable viability in the optimized co-culture media compared to conventional media, with optimal seeding densities of 400,000 and 60,000, respectively. TrypLE-based harvesting increased the viability of hPBMCs by 5% and improved the yield of harvested live monocytes (CD14+) by 2% compared to conventional EDTA-based harvesting. At day 4, our pilot co-culture experiment identified a significant increase (adjusted p-value = 0.048) of NKT cells within co-culture (4.7% of live lymphocytes; N=8) compared to the 4-day culture of hPBMCs alone (1.7% of live lymphocytes; N=4). The daily stimulation of hiPSCdSN with 50 mM KCl didn’t significantly change the proportions of each human lymphocyte population. UV laser ablation was used to selectively injure the neurites without affecting the viability of neuron soma 1 day after ablation. jGCaMP8f-based calcium imaging was highly sensitive and capable of revealing the spontaneous activity of iPSCdSN.
Conclusions
We have developed a humanized in vitro microfluidic neuro-immune co-culture platform with good cell viability for the assessment of immunological phenotypes and neuronal function over time. Early observation indicates an expansion of the NKT cell population within hPBMCs when co-cultured with sensory neurons. UV laser ablation and live cell calcium imaging methods will be used to further probe the effects of selective neuronal injury on the co-cultured immune cell populations and any resulting changes to neuronal activity.
References
1.McGonigle P, Ruggeri B. Animal models of human disease: challenges in enabling translation. Biochem Pharmacol. 2014;87(1):162-71.
2.Sandy-Hindmarch OP, Chang P-S, Scheuren PS, Schoenmacker ID, Hubli M, Loizou C, et al. The Molecular Signature of Neuropathic Pain in a Human Model System. medRxiv. 2024:2024.01.04.23299847.
3.Clark AJ. Establishing myelinating cocultures using human iPSC-derived sensory neurons to investigate axonal degeneration and demyelination. Axon Degeneration: Methods and Protocols. 2020:111-29.
4.Vaquie A, Sauvain A, Duman M, Nocera G, Egger B, Meyenhofer F, et al. Injured Axons Instruct Schwann Cells to Build Constricting Actin Spheres to Accelerate Axonal Disintegration. Cell Rep. 2019;27(11):3152-66 e7.
5.Zhang Y, Rozsa M, Liang Y, Bushey D, Wei Z, Zheng J, et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature. 2023;615(7954):884-91.
Presenting Author
Dr Pao-Sheng Chang
Poster Authors
Pao-Sheng Chang
PhD
Nuffield Department of Clinical Neurosciences
Lead Author
Sungwoo Song
BSc MSc (ongoing
Department of Psychiatry, University of Oxford
Lead Author
Alexander Davies
University of Oxford
Lead Author
Annina Schmid
PhD
Nuffield Department of Clinical Neurosciences
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology