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.

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics

“…However, these steps increase risks of inducing transcriptomic stress responses that overwrite the cells’ original transcriptomic signature [ 33 – 35 ]. A preventive measure is to add reagents during isolation steps to stop de novo transcription, such as dithio-bis(succinimidyl propionate) [ 36 ], CellCover reagent [ 37 ] or Actinomycin D [ 38 ]. Alternatively, cold-active proteases can be used at low temperatures to hinder ongoing transcription [ 33 ].…”

“…CellCover reagent [37] or Actinomycin D [38]. Alternatively, cold-active proteases can be used at low temperatures to hinder ongoing transcription [33].…”

Osteoarthritis year in review: genetics, genomics, epigenetics

“…A study of the transcriptional responses to the cytokines and growth factors in joint tissues would provide useful information for Transcriptomics of articular cartilage from mouse joints is technically challenging due to both contamination of dissected tissue with other cell types and the requirement for a long enzymatic digestion due to the ECM rich nature of the cartilage. Sunkara et al present a joint tissue cutting strategy where the distal section of the femur was segmented into three parts with known tissues and cell populations 41 . Importantly, treatment of the cells with the transcriptional inhibitor actinomycin-D during the ECM digestion revealed alterations in chondrocyte marker genes such as Col2a1, Sox9, and Comp, suggesting digestion itself can alter the measured chondrogenic phenotype of the studied cells.…”

“…Sunkara et al. present a joint tissue cutting strategy where the distal section of the femur was segmented into three parts with known tissues and cell populations 41 . Importantly, treatment of the cells with the transcriptional inhibitor actinomycin-D during the ECM digestion revealed alterations in chondrocyte marker genes such as Col2a1 , Sox9 , and Comp , suggesting digestion itself can alter the measured chondrogenic phenotype of the studied cells.…”

Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome

“…Again, we used ex vivo RNA of the same patients as positive reference and RNA isolated according to the 0.1%-18 h protocol as negative reference. Actinomycin D (ActD) blocks de novo synthesis of RNA transcripts and hence is reported to contribute to the preservation of the RNA transcriptome (Westendorf et al, 2014;Sunkara et al, 2021). Therefore, we added ActD in the following three digestion approaches: 1%-4h, 2%-4h, and 0.1%-18 h. Thus, in total, RNA obtained via five protocols was sequenced from each patient: ex vivo and 0.1%-18 h without ActD, and 0.1%-18h, 1%-4h, and 2%-4 h each with ActD (Figure 7A).…”