Single‐Cell Transcriptomics of Bone Marrow Stromal Cells in Diversity Outbred Mice: A Model for Population‐Level scRNA‐Seq Studies

Abstract

Genome‐wide association studies (GWASs) have advanced our understanding of the genetics of osteoporosis; however, the challenge has been converting associations to causal genes. Studies have utilized transcriptomics data to link disease‐associated variants to genes, but few population transcriptomics data sets have been generated on bone at the single‐cell level. To address this challenge, we profiled the transcriptomes of bone marrow–derived stromal cells (BMSCs) cultured under osteogenic conditions from five diversity outbred (DO) mice using single‐cell RNA‐seq (scRNA‐seq). The goal of the study was to determine if BMSCs could serve as a model to generate cell type–specific transcriptomic profiles of mesenchymal lineage cells from large populations of mice to inform genetic studies. By enriching for mesenchymal lineage cells in vitro, coupled with pooling of multiple samples and downstream genotype deconvolution, we demonstrate the scalability of this model for population‐level studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte‐like cells, and immune cells. Importantly, all cells were similar from a transcriptomic perspective to cells isolated in vivo. We employed scRNA‐seq analytical tools to confirm the biological identity of profiled cell types. SCENIC was used to reconstruct gene regulatory networks (GRNs), and we observed that cell types show GRNs expected of osteogenic and pre‐adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte‐like cells, and MALPs captured a significant component of bone mineral density (BMD) heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA‐seq can be used as a scalable and biologically informative model to generate cell type–specific transcriptomic profiles of mesenchymal lineage cells in large populations. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Cell type-specific network analysis in Diversity Outbred mice identifies genes potentially responsible for human bone mineral density GWAS associations

“…1A). With regards to the mesenchymal cell types, the only differences in cell clusters relative to our previous report 17 were the presence of MPCs and two mature osteoblast clusters. Upon comparing the two distinct osteoblast clusters, OB1 and OB2 (Fig.…”

“…We cultured BMSCs from a total of 80 Diversity Outbred (DO) mice, a genetically diverse outbred mouse population 19,20 (N=75 from the current study and N=5 from 17 ; N = 49 male and N = 31 females). We cultured BMSCs under osteogenic conditions and subsequently performed scRNA-seq, as described in 17 .…”

“…We cultured BMSCs from a total of 80 Diversity Outbred (DO) mice, a genetically diverse outbred mouse population 19,20 (N=75 from the current study and N=5 from 17 ; N = 49 male and N = 31 females). We cultured BMSCs under osteogenic conditions and subsequently performed scRNA-seq, as described in 17 . After stringent processing and quality control (Materials and Methods), the dataset consisted of the expression of 21,831 genes across 139,392 cells.…”

“…Based on our prior BMSC-OB scRNA-seq study 17 , we expected to identify a large proportion of mesenchymal cells and a smaller fraction of non-mesenchymal cell types. Consistent with this hypothesis, clusters associated with mesenchymal lineages accounted for 74.1% of all cells (Fig.…”

“…Additionally, using scRNA-seq data has the potential to provide context by predicting the specific cell types in which causal genes and their associated networks operate. In recent work, we demonstrated the utility of bone marrow-derived stromal cells cultured under osteogenic condition (BMSC-OB) for the generation of populationscale scRNA-seq data on bone relevant cell types 17 . The BMSC-OB model effectively enriches for mesenchymal lineage cells (e.g., mesenchymal progenitors, osteoblasts, osteocyte-like cells) that are highly relevant to the regulation of BMD.…”