Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process

Mika Nakayama; Hiroyuki Okada; Masahide Seki; Yutaka Suzuki; Ung-Il Chung; Shinsuke Ohba; Hironori Hojo

doi:10.1016/j.reth.2022.05.001

Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process

Regen Ther. 2022 May 18:21:9-18. doi: 10.1016/j.reth.2022.05.001. eCollection 2022 Dec.

Authors

Mika Nakayama¹, Hiroyuki Okada^{2

3}, Masahide Seki⁴, Yutaka Suzuki⁴, Ung-Il Chung^{1

2}, Shinsuke Ohba⁵, Hironori Hojo^{1

2}

Affiliations

¹ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, 113-8655, Japan.
² Laboratory of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.
³ Orthopaedic Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8655, Japan.
⁴ Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 277-8562, Japan.
⁵ Department of Cell Biology, Institute of Biomedical Sciences, Nagasaki University, Nagasaki, 852-8588, Japan.

Abstract

Introduction: Activation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood.

Methods: To better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26R ^tdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell-cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model.

Results: Lineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C-C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site.

Conclusion: Multipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

Keywords: Bone repair; Calvaria; Ccl9; Lineage tracing; Single-cell analysis; Sox9.