HIGH-RESOLUTION SPATIAL TRANSCRIPTOMIC MAPPING OF THE DEVELOPING HUMAN INTERVERTEBRAL DISC

The intervertebral disc (IVD) is made up of a central nucleus pulposus (NP) and an outer fibrous ring called the annulus fibrosus (AF). The NP developmentally arises from the notochord, a rod-like structure that is derived from the axial mesoderm and later segments to form the NP. During early life, the NP is populated by large vacuolated notochordal cells, which are gradually replaced by small round chondrocyte-like cells during adolescence. This transition coincides with the onset of disc degeneration, indicating that notochordal cells are vital for maintaining intervertebral disc function. However, the specific function of human notochordal cells and their influence on the surrounding cells during development have not been fully elucidated. A detailed cellular and spatial characterisation of the developing spine is required to improve our understanding of IVD biology. To elucidate the cellular composition and spatial landscape of cells in the developing human IVD, we prepared tissue sections from fetal human spines at 5 developmental stages: 5 and 7 weeks post-conception (WPC) representing pre-notochordal segmentation and 9, 12, and 14WPC representative of post-notochordal segmentation. All sections were processed using the 10X Genomics Visium high-definition platform at a subcellular resolution of 2μm. All experiments were conducted in compliance with the approving committee's ethical guidelines and full consent was obtained from all donors. High-resolution spatial transcriptomics revealed significant differences in the cellular distribution and gene expression profiles of IVD cell populations, particularly notochordal cells, as development progressed. Notochordal cells were defined by their high expression of TBXT , KRT19 , and CHRM2. Differential expression analyses showed that 5WPC and 7WPC notochordal cells had higher expression of SHH , ALCAM , and SPP1, which were notably downregulated following notochordal segmentation at 9, 12 and 14WPC. Pre-segmentation notochordal cells had higher expression of genes regulating embryonic skeletal system development. In contrast, post-segmentation notochordal cells expressed higher levels of genes involved in structural maintenance and cell growth and survival. Interestingly, we observed two regionally distinct notochordal populations (Notochord 1 and 2) present only during segmentation at 7WPC. The Notochord 2 population had increased levels of cell migration genes, CRIP1 , CD44 , TPPP3 , TAGLN and TNXB suggesting that this cell population regulates notochordal segmentation and spine patterning. Overall, our research highlights the complex heterogeneity within the IVD, providing new insights into cellular behaviour and its impact on the spinal microenvironment throughout development.