Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

Abstract

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes.Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture.Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels.Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.

Association of the FGF4L2 retrogene with fibrocartilaginous embolic myelopathy in dogs

“…17,52 The exact mechanism relating FGF4 overexpression to premature IVDD is not defined, however recent single cell RNASeq studies defining the heterogenous cellular populations in the embryonic and adult intervertebral disc might provide a framework for potential mechanisms. [66][67][68][69] Whatever the mechanism is determined to entail, premature degeneration of the IVD in chondrodystrophic dogs does not preclude other mechanisms for FCE pathogenesis since FCE is frequently documented in older non chondrodystrophic breeds where similar age-related NP degeneration is also likely to be present. 70,71 Developmental differences in vascular anatomy of the disc, vertebra, and spinal cord in chondrodystrophic dogs might also be nonpermissive to entry of fibrocartilage, and/or subsequent passage of the material to clinically relevant areas.…”

Development and Degeneration of the Intervertebral Disc—Insights from Across Species

“…A major challenge in identifying biomarkers of the IVD is that the heterogenous cell population leads to subclusters of NP, AF, or CEP cells with differential expression patterns. Recent advances in scRNA seq enabled the identification of IVD cell subpopulations, including NP stem-like progenitor cells, AF progenitor cells, CC-like NP cells, endothelial cells (EC) in the oAF, myeloid cells, and lymphoid cells, in both healthy and diseased discs of various species [ 35 , 39 , 55 , 56 , 57 , 58 ]. Recent scRNA seq analysis makes use of statistical analysis such as T-distributed stochastic neighborhood embedding (tSNE) to characterize clusters or subclusters of NP, AF, and CEP cells based on their expression profile [ 39 , 47 , 59 , 60 ], however labeling and defining these cell groupings currently varies between research groups and experiments.…”

“…ScRNA seq of cynomolgus monkeys and immunohistochemical (IHC) analysis of monkey and rat coccygeal IVDs enabled the expression analysis of CC-like and NC-like NP cells. Similar expression of a nuclear mediator in the sonic hedgehog (SHH) pathway, glioma-associated oncogene homolog 1 (GLI1), was shared in both CC-like and articular CC cells as well as the SHH signaling mediator, smoothened (SMO), suggesting the activation of hedgehog pathways in both articular CC and a subset of NP cells [ 56 ]. Furthermore, IHC showed expression of SHH but not indian hedgehog (IHH) in vacuolated NC-like NP cells while both SHH and IHH were detected in bone marrow (BM) cells of a cynomolgus monkey femur, suggesting BM cells as a supply source for hedgehog signaling ligands activating articular CC hedgehog signaling [ 56 ].…”

“…Similar expression of a nuclear mediator in the sonic hedgehog (SHH) pathway, glioma-associated oncogene homolog 1 (GLI1), was shared in both CC-like and articular CC cells as well as the SHH signaling mediator, smoothened (SMO), suggesting the activation of hedgehog pathways in both articular CC and a subset of NP cells [ 56 ]. Furthermore, IHC showed expression of SHH but not indian hedgehog (IHH) in vacuolated NC-like NP cells while both SHH and IHH were detected in bone marrow (BM) cells of a cynomolgus monkey femur, suggesting BM cells as a supply source for hedgehog signaling ligands activating articular CC hedgehog signaling [ 56 ]. Notably, SHH was not expressed in all NC cells, however it is thought that NC cells may begin as SHH+ NC cells and lose SHH expression over time as SHH− cells were found in individuals older than ten years old [ 38 , 56 ].…”

“…Furthermore, IHC showed expression of SHH but not indian hedgehog (IHH) in vacuolated NC-like NP cells while both SHH and IHH were detected in bone marrow (BM) cells of a cynomolgus monkey femur, suggesting BM cells as a supply source for hedgehog signaling ligands activating articular CC hedgehog signaling [ 56 ]. Notably, SHH was not expressed in all NC cells, however it is thought that NC cells may begin as SHH+ NC cells and lose SHH expression over time as SHH− cells were found in individuals older than ten years old [ 38 , 56 ]. Hedgehog signaling may activate the hypoxia inducible factor 1 alpha (HIF1α) pathway in both CC-like and articular CC, based on the demonstrated increase in HIF1α protein levels with the addition of SHH to primary chondrocytes [ 56 ].…”