Apolipoprotein E is a marker of all chondrocytes in the growth plate resting zone

The resting zone (RZ) in mammalian growth plates is critical for maintaining and regulating chondrocyte turnover during longitudinal bone growth as a control tower and stem cell reservoir. Although recent lineage tracing studies have identified several markers for stem cells in the RZ, these markers only partially label chondrocytes in the RZ, suggesting that the resting chondrocytes (RCs) are a heterogeneous population with different types of stem cells. Since a comprehensive marker for RCs is still lacking, the RZ is generally determined based on ambiguous histological criteria, such as small and round chondrocytes without columnar formation, which may lead to inconsistencies among researchers. Therefore, in this study, we used single-cell RNA sequencing (scRNAseq) of growth plate chondrocytes followed by validation by fluorescence in situ hybridization (FISH) to precisely annotate cell clusters in scRNAseq and search for a marker of RCs. The scRNAseq analysis revealed that apolipoprotein E (Apoe) was the top-hit gene, which was ubiquitously expressed in the RC cluster. FISH confirmed that Apoe was exclusively localized to the histologically defined RZ. In newly generated Apoe^mCherry knock-in mice, we further confirmed that mCherry expression mirrored the distribution of Apoe-expressing chondrocytes in the RZ particularly after the formation of the secondary ossification center. These mCherry⁺ RCs were slow cycling in vivo and exhibited stem cell properties in vitro. Moreover, APOE was detected in human growth plate RCs. These findings suggest that apolipoprotein E is a novel pan-RC marker in both mouse and human growth plates.