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

Hiroki Hagizawa; Saeko Koyamatsu; Seiji Okada; Takashi Kaito; Noriyuki Tsumaki

doi:10.3389/fcell.2023.1151947

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

Front Cell Dev Biol. 2023 May 15:11:1151947. doi: 10.3389/fcell.2023.1151947. eCollection 2023.

Authors

Hiroki Hagizawa^{1

2}, Saeko Koyamatsu¹, Seiji Okada², Takashi Kaito², Noriyuki Tsumaki^{1

3

4}

Affiliations

¹ Department of Tissue Biochemistry, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.
² Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
³ Department of Clinical Application, Center for IPS Cell Research and Application, Kyoto University, Kyoto, Japan.
⁴ Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Osaka, Japan.

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.

Keywords: HIF-1α; chondrocytes; hedgehog; nucleus pulposus cells; single-cell RNA sequencing.

Grants and funding

This study was supported by JSPS KAKENHI, Grant Nos. 18H02923 and 23H03029 (to NT) and WPI Premium Research Institute for Human Metaverse Medicine (PRIMe) (to NT) from the Japan Society for Promotion of Science; the Center for Clinical Application Research on Specific Disease/Organ (type B), Grant No. 21bm0304004h0009 (to NT); the Practical Research Project for Rare/Intractable Diseases (step 1), Grant No. 21ek0109452h0002 (to NT); the Core Center for iPS Cell Research; Grant No. 20bm0104001h0008 (to NT); and the Acceleration Program for Intractable Diseases Research utilizing disease-specific iPS cells Grant No. 20bm0804006h0004 (to NT) from the Japan Agency for Medical Research and Development (AMED).