背景:间充质基质细胞(MSC)是异质群体。异质性存在于同一组织内和不同组织之间。一些研究发现来自不同组织的MSCs在免疫调节功能方面存在巨大异质性。此外,免疫调节能力异质性的潜在机制尚不清楚.
方法:分离Foreskin间充质基质细胞(FSMSCs)和人脐带间充质基质细胞(HuMSCs),培养至第3代。根据国际细胞治疗协会的标准,我们确认了细胞类型.然后,将FSMSCs和HuMSCs与脂多糖(LPS)刺激的人外周血单核细胞(PBMC)体外共培养。此外,采样上清液进行酶联免疫吸附试验,以研究IL-1β的分泌,IL-6,IL-10,TNF-α,和TGF-β1。最后,我们对FSMSCs和HuMSCs进行了单细胞RNA测序(scRNA-seq)。
结果:我们成功地将FSMSCs和HuMSCs鉴定为MSCs。当与LPS预处理的PBMC共培养时,FSMSCs和HuMSCs能有效减少IL-1β和TNF-α的分泌。然而,FSMSC刺激PBMC分泌更多的IL-10、TGF-β1和IL-6。此外,从整合的scRNA-seq数据中鉴定出4个细胞亚群,包括增殖性MSCs(MKI67+,CD146低+,NG2+,PDGFRBR-),周细胞(CD146高+,PDGFRB+,MKI67-,CD31-,CD45-,CD34-),免疫MSCs(CXCL12high+,PTGIShigh+,PDGFRB+,CD146-,MKI67-)和祖细胞增殖MSCs(CXCL12low+,PTGISlow+,PDGFRB+,CD146-,MKI67-).其中,我们发现转录活性增强的免疫MSCs在分化程度上与周细胞相似。各种免疫相关基因,基因集,和调节子也在免疫MSC中富集。此外,在细胞-细胞通讯分析中,免疫MSCs与其他细胞亚群接近.最后,我们发现,当比较来自不同组织的MSCs亚群组成时,包皮组织中免疫MSCs的比例最高。
结论:FSMSCs在体外表现出比HuMSCs更好的免疫调节能力。此外,免疫MSCs可能在免疫调节特性的异质性中起着至关重要的作用。这项研究提供了新的见解,表明可以分离出免疫MSCs以发挥稳定的免疫调节功能,而不受来自不同组织的MSCs异质性的限制。
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+, CD146low+, NG2+, PDGFRB-), pericytes (CD146high+, PDGFRB+, MKI67-, CD31-, CD45-, CD34-), immune MSCs (CXCL12high+, PTGIShigh+, PDGFRB+, CD146-, MKI67-) and progenitor proliferative MSCs (CXCL12low+, PTGISlow+, 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.