Fibroblast and myeloid cells with high mitochondrial RNA content represent biologically significant populations
Wesley Tran1, Garth Blackler1, Rebecca Luo1
1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada; Bone and Joint Institute, University of Western Ontario, London, ON, Canada.
Objectives:
Standard scRNA-seq QC often excludes cells with high mitochondrial RNA content (pMT), assuming they reflect low viability or dissociation-induced stress. Emerging evidence suggests high-pMT cells may represent disease-relevant cell populations. This study investigates how exclusion of high-pMT cells affects the transcriptional landscape of knee OA synovium and explores their potential role in disease pathobiology.
Design:
Seven published human and mouse scRNA-seq datasets were reanalyzed using QC thresholds and quantile-based filtering to include high-pMT cells. Analyses included pMT distribution, dissociation-induced stress scores, cell death-related and mitochondrial apoptotic gene signatures. Dissociation-induced stress was assessed using a gene set from three published studies, while cell death and mitochondrial signatures were derived from established GSEA gene sets. Focusing on a single human knee OA synovial dataset stratified by pain (GSE248453), differential gene expression and pathway enrichment were compared between conventionally filtered and high-pMT inclusive pipelines.
Results:
Across all datasets, pMT showed no correlation with dissociation-induced stress scores (R= -0.036). High-pMT cells showed no association with apoptosis pathways, suggesting that they are not actively undergoing cell death. High-pMT cells primarily localized to fibroblast and myeloid subsets. High-pMT synovial fibroblasts were enriched in ECM remodeling processes, while high-pMT myeloid cells were linked to inflammatory signaling and immune activation.
Conclusions:
These findings suggest high-pMT cells are viable and potentially disease relevant, with their exclusion possibly obscuring key aspects of OA pathophysiology. This highlights the necessity of context-specific QC strategies in OA research and further exploration of high-pMT fibroblast and myeloid populations as potential disease drivers.
Insights
High mitochondrial RNA content (pMT) cells in osteoarthritis (OA) synovium are viable and disease-relevant, not stressed or dying. Excluding these cells may obscure key OA pathobiology, necessitating context-specific quality control strategies.
Area of Science:
- Single-cell genomics
- Immunology
- Rheumatology
Background:
- Standard single-cell RNA sequencing (scRNA-seq) quality control (QC) often removes cells with high mitochondrial RNA content (pMT).
- This exclusion is typically based on the assumption that high-pMT cells indicate poor cell viability or stress during dissociation.
- However, emerging evidence suggests high-pMT cells may represent biologically significant cell populations.
Purpose of the Study:
- To investigate the impact of excluding high-pMT cells on the transcriptional landscape of knee osteoarthritis (OA) synovium.
- To explore the potential role of high-pMT cells in OA disease pathobiology.
Main Methods:
- Reanalyzed seven human and mouse scRNA-seq datasets using inclusive QC thresholds for high-pMT cells.
- Assessed pMT distribution, dissociation-induced stress, cell death, and mitochondrial apoptotic gene signatures.
- Compared differential gene expression and pathway enrichment in a human knee OA synovial dataset (GSE248453) between conventional and high-pMT inclusive pipelines.
Main Results:
- High-pMT cells showed no correlation with dissociation-induced stress or apoptosis pathways, indicating viability.
- High-pMT cells were predominantly found in fibroblast and myeloid subsets.
- High-pMT synovial fibroblasts were enriched in extracellular matrix (ECM) remodeling pathways.
- High-pMT myeloid cells were associated with inflammatory signaling and immune activation.
Conclusions:
- High-pMT cells in OA synovium are viable and potentially disease-relevant.
- Exclusion of these cells may obscure critical aspects of OA pathophysiology.
- Context-specific QC strategies are essential for OA research.
- High-pMT fibroblast and myeloid populations warrant further investigation as potential OA drivers.
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