Single-cell RNA sequencing reveals the potential mechanism of heterogeneity of immunomodulatory properties of foreskin and umbilical cord mesenchymal stromal cells

Abstract

Background: Mesenchymal stromal cells (MSCs) are heterogeneous populations. Heterogeneity exists within the same tissue and between different tissues. Some studies have found enormous heterogeneity in immunomodulatory function among MSCs derived from different tissues. Moreover, the underlying mechanism of heterogeneity in immunomodulatory abilities is still unclear.

Methods: Foreskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured until the third passage. According to the International Association for Cell Therapy standard, we confirmed the cell type. Then, FSMSCs and HuMSCs were cocultured with human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) in vitro. Furthermore, the supernatant was sampled for an enzyme-linked immunosorbent assay to investigate the secretion of IL-1β, IL-6, IL-10, TNF-α, and TGF-β1. Finally, we performed single-cell RNA sequencing (scRNA-seq) of FSMSCs and HuMSCs.

Results: We successfully identified FSMSCs and HuMSCs as MSCs. When cocultured with LPS pretreated PBMCs, FSMSCs and HuMSCs could effectively reduced the secretion of IL-1β and TNF-α. However, FSMSCs stimulated the PBMCs to secrete more IL-10, TGF-β1, and IL-6. Furthermore, 4 cell subsets were identified from integrated scRNA-seq data, including proliferative MSCs (MKI67⁺, CD146^low+, NG2⁺, PDGFRB^-), pericytes (CD146^high+, PDGFRB⁺, MKI67^-, CD31^-, CD45^-, CD34^-), immune MSCs (CXCL12^high+, PTGIS^high+, PDGFRB⁺, CD146^-, MKI67^-) and progenitor proliferative MSCs (CXCL12^low+, PTGIS^low+, PDGFRB⁺, CD146^-, MKI67^-). Among them, we found that immune MSCs with strengthened transcriptional activity were similar to pericytes with regard to the degree of differentiated. Various of immune-related genes, gene sets, and regulons were also enriched in immune MSCs. Moreover, immune MSCs were determined to be close to other cell subsets in cell-cell communication analysis. Finally, we found that the proportion of immune MSCs in foreskin tissue was highest when comparing the subset compositions of MSCs derived from different tissues.

Conclusions: FSMSCs show better immunomodulatory capacity than HuMSCs in vitro. Moreover, immune MSCs may play a vital role in the heterogeneity of immunoregulatory properties. This study provides new insights suggesting that immune MSCs can be isolated to exert stable immunoregulatory functions without being limited by the heterogeneity of MSCs derived from different tissues.

Keywords: Foreskin mesenchymal stromal cells; Heterogeneity of immunomodulatory function; Human umbilical cord mesenchymal stromal cells; Mesenchymal stromal cells; Single-cell RNA sequencing.