PROJECT SUMMARY The goal of this study is to establish a human model for low back pain (LBP) caused by intervertebral disc (IVD) degeneration, and to answer the question, what causes a degenerative disc to become painful? LBP is a leading cause of disability and morbidity in the adult population, affecting approximately 80% of adults. Up to 40% of all LBP is attributed to IVD degeneration, which is categorized as discogenic pain. Radiographically aged IVDs do not always correlate with pathologically painful discs. Current treatments focus on pain management and the restoration of disc height, but the underlining cause for discogenic pain remains untreated. In our previously published and preliminary studies we have established an in vitro system to simulate IVD degeneration, a nociception sensing with sensory neuron differentiated from induced pluripotent stem cells (iPSC), an in vivo bioassay using a rat model and performed single cell transcriptomics of pain inducing and asymptomatic IVD tissues and developed a hypothesis for the pain induction mechanism. In this proposal we will combine these systems in Nociception-Chip to unveil the molecular and cellular mechanism of discogenic pain induction in vitro and in vivo. In Aim 1 we will identify the cellular sub-population(s) that transform asymptomatic IVDs to discogenic pain inducing ones, test the hypothesis that this process is mediated by pro-inflammatory agents and investigate its reversibility. The single cell data will be utilized to identify specific surface cell markers of the sub- populations with which we will sort the cells out. We will simulate disc degeneration model in vitro and to test the functionality of these cells, identify the critical factors that lead to development of discogenic pain and test the response of nociceptors differentiated from induced pluripotent stem cells as a readout of pain sensing. The functionality of these cells to induce LBP behaviour will be validated in a rat model. We will test the hypothesis that this process is mediated by specific inflammatory factors or the lack of anti-inflammatory factors like MIR-155 by co-injecting them to a healthy or mildly degenerated IVDs. In Aim 2 we will identify cellular sub-population(s) enriched in degenerated asymptomatic IVDs and test the hypothesis that these cells prevent the transformation of asymptomatic to painful IVDs. We will leverage the same database of single cell transcriptomics used in Aim 1 and will look at the non-painful disc prevalent celsl as potentially therapiutics cells. These cells will be isolated, characterized and co-injected with pain inducing cells or to a degenerated IVDs in a rat model that allows measurement for stractural and behavioral LBP outcomes. Overall, both aims are not interdependant and will contain hypothesis-driven, hypthesis generating and therapituc components. This study is significant because it will fill substantial gaps in our understanding of discogenic pain develope...