Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Transcriptomic analysis of lumbar and caudal intervertebral discs reveals anatomical level-dependent differences in immune regulatory pathway (#SP-6a)

MinKyu (Mark) Kim 1 , Troy Anderson 2 , Joanna Smeeton 2 3 , Nadeen O. Chahine 1 4
  1. Orthopedic Surgery, Columbia University, New York, USA
  2. Genetics and Development, Columbia University, New York, USA
  3. Rehabilitation and Regenerative Medicine, Columbia University, New York, USA
  4. Biomedical Engineering, Columbia University, New York, USA

INTRODUCTION: The complexity of intervertebral disc (IVD) degeneration (IDD) partly owes to the heterogeneous sub-tissue compartments: central nucleus pulposus (NP), encapsulating annulus fibrosus (AF), and cartilage endplate. Different genetic and injury mouse models have been developed to understand the heterogeneity of IVD tissue types (reporter mice) and disease progression (aging, puncture injury, loading) in both lumbar and caudal IVDs. However, growing evidence suggests region differences in IDD progression1 and physico-mechanical properties between lumbar and caudal IVDs2-3. Furthermore, how these differences can be resolved to model human IDD remains unclear, limiting the translational potential of mouse models. The objective of the study was to use the next generation sequencing to provide a benchmark of genetic profiles between lumbar and caudal NP and AF tissues from young healthy mice.

METHODS: NP and AF tissues from lumbar and caudal IVDs were dissected from C57BL/6 mice at 6-7 months-of-age (n=8). For each animal, IVD tissue types were pooled by anatomical region (lumbar NP, LNP; lumbar AF, LAF; caudal NP, CNP; caudal AF, CAF) and total RNA was extracted (average RIN = 8.6). For each sample, a library was prepared (STRPOLYA kit) and a minimum of 20 million reads were sequenced using Illumina NovaSeq6000. Gene count data were normalized using DESeq2. Differentially expressed genes were identified based on |log2FoldChange| ≥ 1.5 and adjusted p value (padj.) < 0.05. Analyses were performed in R to identify differences within NP or AF tissues from caudal versus lumbar regions (CNP vs. LNP and CAF vs. LAF). Gene set enrichment analysis (GSEA) was performed to assess up- or down-regulated biological processes and pathways between spinal regions (clusterProfiler package). 

RESULTS: CNP versus LNP: We identified 1144 upregulated genes and 3538 downregulated genes in the CNP compared to LNP (Fig. 1.A). When assessed for changes in known NP markers, the expression of Brachyury (T), Gpc3, Krt8, and Krt19 was significantly upregulated, while Shh and Slc2a3 expression was significantly downregulated (Fig. 1.B). GSEA showed motile cilium assembly, protein hydroxylation, regulation of cilium movement, vesicle transport, and nucleotide sugar metabolic process as top upregulated biological processes, while top downregulated biological processes included complement activation, membrane invagination, immunoglobulin immune response, phagocytosis recognition, and B cell receptor signaling (Fig. 1.C). CAF versus LAF: Among the differentially expressed genes, 25 upregulated and 25 downregulated genes were identified (Fig. 1.D). GSEA identified regulation of chondrocyte differentiation, negative regulation of cartilage development, connective tissue development, protein hydroxylation, and collagen fibril organization as top upregulated biological processes, while top downregulated biological processes included phagocytosis recognition, membrane invagination, cell surface receptor immune response, antigen receptor signaling, and B cell receptor signaling (Fig. 1.E).

DISCUSSION: Our findings establish a transcriptomic landscape outlining the transcriptomic differences in lumbar and caudal IVD tissues and provide insights to anatomical region-dependent IVD biology. Interestingly, most of the downregulated biological processes for both CNP and CAF were immune-related pathways, suggesting differentially regulated tissue immune response by anatomical regions. Defining how these molecular differences can recapitulate human IDD will help establish more clinically relevant in vivo models.65543aea6f6b0-Abstract+1+Figure_KIM.png

  1. REFERENCES: [1] Burt, K.G., et al., (2023). bioRxiv, pp.2023-08. [2] Brendler, J., et al., (2022). J. Anat., 240(1), pp.84-93. [3] Sarver, J.J. and Elliott, D.M., (2005). JOR, 23(1), pp.150-155.