Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

Abstract

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271 + BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPR hi CD45 low BM-MSCs within the CD271 + BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.

Single-cell RNA sequencing identifies phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow

“…Enrichment strategies represent a possible approach for a detailed analysis of the transcriptional diversity of BM stromal cells, as recently reported by Wang et al (Wang et al, 2021). However, in their study, CD271 + cells from femoral shafts from old patients with osteoarthritis and osteoporosis were used, which might not reflect transcriptomic profiles of normal stroma cell from hematologically active bone marrow (Wang et al, 2021). In our study, we therefore used not only BM stroma-enriched CD45 low/-CD235acells from iliac crest aspirations but added also sorted CD271 + cells from healthy donors, which are highly and exclusively enriched for stromal stem/progenitor cells (Tormin et al, 2011).…”

“…However, due to the low frequencies of the stromal stem/progenitor cells, it has been difficult to investigate the potential heterogeneity of the human BM stromal cell compartment even when employing single cell approaches. Enrichment strategies represent a possible approach for a detailed analysis of the transcriptional diversity of BM stromal cells, as recently reported by Wang et al (Wang et al, 2021). However, in their study, CD271 + cells from femoral shafts from old patients with osteoarthritis and osteoporosis were used, which might not reflect transcriptomic profiles of normal stroma cell from hematologically active bone marrow (Wang et al, 2021).…”

“…Several recent studies using scRNAseq approaches provided first important insights into the complexity of the human BM and the potential functional roles of BM stromal cells (de Jong et al, 2021; Triana et al, 2021; Wang et al, 2021). A first overview of the cellular composition of the nonhematopoietic cells in human BM was provided by a recently published single-cell proteo-genomic reference map (Triana et al, 2021) and a single-cell transcriptomic map of non-hematopoietic cells (de Jong et al, 2021).…”

“…In contrast, important HME elements in human BM remain poorly defined, which is due to the fact that the identity and function of the stromal stem/progenitor cells have been difficult to investigate (Li et al, 2016). Nevertheless, a number of recent studies have provided the first important insights into the complexity of the human BM and the potential diverse functional roles of BM stromal cells (Chan et al, 2018; de Jong et al, 2021; Triana et al, 2021; Wang et al, 2021).…”

Multi-omics molecular biomarkers and database of osteoarthritis

“…Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that maintain skeletal tissues and differentiate into various mesodermal lineages. scRNA-seq was applied to investigate the transcriptional diversity of BM-MSCs in vivo, showing that they could be codified into distinct subpopulations corresponding to the osteogenic, chondrogenic, adipogenic differentiation trajectories and terminal-stage quiescent cells ( 86 ).…”