Combining segmental bulk- and single-cell RNA-sequencing to define the chondrocyte gene expression signature in the murine knee joint

Abstract

Objective: Due to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new and pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem. Design:We propose a new cutting strategy of the murine femur which produces three segments with a predictable mixed cell populations, where one segment contains AC and growth plate (GP) chondrocytes, another contains GP chondrocytes, and the last segment contains only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find common and distinct genes between the segments. Then, the segment containing AC chondrocytes was digested and analysed via scRNA-seq. Results: Differential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related-and non-related to chondrocytes, including, cartilage development (adj. p-value: 3.45E-17) and endochondral bone growth (adj. p-value 1.22E-4), respectively.ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. p-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk-and scRNA-seq contained 75 genes, where all but ten genes were previously implicated in cartilage homeostasis or osteoarthritis (OA) progression. Conclusions:Our approach has the potential to detect the scarce disease phenotypes of chondrocytes in murine OA models.

Systematic Study of Single-Cell Isolation from Musculoskeletal Tissues for Single-Cell Sequencing

“…Also utilizing the single-cell platform, there were two nice reports that systematically uncovered the cell atlas and transcriptional regulation mechanisms of OA [30,31]. As for other related studies, several reports showed the single-cell pro ling results of developing limbs, including growth plate cartilage as well as articular cartilage [32][33][34][35]. The single-cell isolation methods used in the above studies were summarized in Table 1.…”