Single-cell transcriptome analysis defines mesenchymal stromal cells in the mouse incisor dental pulp

The potential for human adipose-derived stromal cells (hASCs) to be used as a therapeutic product is being assessed in multiple clinical trials. However, much is still to be learned about these cells before they can be used with confidence in the clinical setting. An inherent characteristic of hASCs that is not well understood is their heterogeneity. The aim of this exploratory study was to characterize the heterogeneity of freshly isolated hASCs after two population doublings (P2) using single-cell transcriptome analysis. A minimum of two subpopulations were identified at P2. A major subpopulation was identified as contractile cells which, based on gene expression patterns, are likely to be pericytes and/or vascular smooth muscle cells (vSMCs).

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Single Cell Transcriptome Sequencing Reveals the Potential Mechanism of Heterogeneity in Immunoregulatory Function Between Mesenchymal Stromal Cells

10.21203/rs.3.rs-823639/v1 ◽

2021 ◽

Author(s):

Siyu Cai ◽

Chuiqin Fan ◽

Lichun Xie ◽

Huifeng Zhong ◽

Aijia Li ◽

...

Keyword(s):

Single Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Transcriptome Sequencing ◽

Enzyme Linked Immunosorbent Assay ◽

Human Umbilical Cord ◽

Tnf Α ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Immunomodulatory Function

Abstract Background: Mesenchymal stromal cells (MSCs) could be applied for the treatment of immune-related diseases. However, some studies have found there is enormous heterogeneity in immunomodulatory function of MSCs isolated from different tissue. At present, the underlying mechanism of heterogeneity in immunoregulatory function is still unclear. Methods: In this study, the foreskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured to the 3rd passage. Cell types were confirmed according to the standard of International Association for Cell Therapy. Then, FSMSCs and HuMSCs were co-cultured with human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharides (LPS) in viro respectively. And the supernatant was sampled for enzyme-linked immunosorbent assay to investigate the secretion of IL-1β, IL-6, IL-10, TNF-α and TGF-β. Finally, single cell transcriptome sequencing was performed in order to elucidate the mechanism for the difference of immunomodulatory function.Results: FSMSCs and HuMSCs are successfully identified as MSCs. When co-cultured with LPS pre-treated PBMC, FSMSCs and HuMSCs could effectively reduce the secretion of IL-1β and TNF-α. But FSMSCs were able to stimulate the PBMC to secrete more IL-10, TGF-β and IL-6. Furthermore, 4 MSCs subsets in integrated data were identified, including Proliferative MSCs , Pericyte, Immune MSCs and Progenitor Proliferative MSCs. Among them, the proportions of Immune MSCs in FSMSCs and HUMSCs were 56% and 10% respectively. Varieties of immune-related genes, gene sets and regulons were enriched in Immune MSCs. And Immune MSCs with powful transcriptional activity were found to be near to Pericyte at the degree of differentiation and closed to other cell subsets. Finally, the foreskin tissue might be an ideal source of isolating Immune MSCs when comparing the subset composition of MSCs derived from adipose tissue and bone marrow from public database. Conclusions: Immune MSCs may play a key role in the heterogeneity of immunoregulatory function. It is a new insight that Immune MSCs could be isolated and better applied for the treatment of immune-related diseases without being limited by the heterogeneity of immunomodulatory function derived from different tissues.

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Strategies to Identify Mesenchymal Stromal Cells in Minimally Manipulated Human Bone Marrow Aspirate Concentrate Lack Consensus

The American Journal of Sports Medicine ◽

10.1177/0363546521993788 ◽

2021 ◽

pp. 036354652199378

Author(s):

Severin Ruoss ◽

J. Todd Walker ◽

Chanond A. Nasamran ◽

Kathleen M. Fisch ◽

Conner J. Paez ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Bone Marrow Aspirate ◽

Transcriptional Profile ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Background: There is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC. Purpose: To establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies. Study Design: Descriptive laboratory study. Methods: Five published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile. Results: ISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells. Conclusion: Currently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences. Clinical Relevance: This study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.

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