Comparative transcriptome analysis of bone marrow resident versus culture-expanded mouse mesenchymal stem/stromal cells

Abstract

Background aims: Mesenchymal stem/stromal cells (MSCs) are defined as culture-expanded populations, and although these cells recapitulate many properties of bone marrow (BM) resident skeletal stem/progenitor cells, few studies have directly compared these populations to evaluate how culture adaptation and expansion impact critical quality attributes.

Methods: We analyzed by RNA sequencing Lin^-SCA1⁺ MSCs enriched from BM by immunodepletion (ID) and after subsequent culture expansion (Ex) and Lin^-LEPR⁺ MSCs sorted (S) directly from BM. Pairwise comparisons were used to identify differentially expressed genes (DEGs) between populations, and gene set enrichment analysis was employed to identify biological pathways/processes unique to each population. K-means cluster analysis resolved isolation status-dependent changes in transcription in pseudotime.

Results: Hierarchical clustering segregated populations by isolation process, and principal component analysis identified transcripts related to vasculature development, ossification and inflammatory/cytokine signaling as key drivers of population variance. Pairwise comparisons identified 3849 DEGs in ID versus S BM-MSCs mapping to Gene Ontology (GO) terms related to immune and metabolic processes and 334 DEGs in Ex versus ID BM-MSCs mapping to GO terms related to tissue development, cell growth and replication and organelle organization. K-means cluster analysis revealed significant differences in transcripts encoding stemness and differentiation markers, extracellular matrix structural constituents and remodeling enzymes and paracrine-acting factors between populations.

Conclusions: These comparative analyses reveal significant differences in gene expression signatures between BM resident and culture-expanded MSCs, thereby providing new insight into how culture adaptation/expansion endows the latter with unique quality attributes.

Keywords: RNA-Seq; flow cytometry; mesenchymal stem cells; stromal cells; transcriptome.