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Single cell RNA profiles of bone marrow aspirate concentrate

ABSTRACT: 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 fifteen orthopedic patients and on one 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. 9 of 12850 BMAC cells expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells express all Jia genes, but 25 cells express at least 13 of 22. No cells express all Silva genes, but 19 cells express 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162692 | GEO | 2021/03/12

REPOSITORIES: GEO

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Project description:Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions, and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue, and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs, and suggest that low levels of endothelial cells may persist in MSC cultures established using traditional protocols. Highly purified human adipose-tissue pericytes (AT3G5Cs) were isolated based on the expression of the antigen detected by the 3G5 antibody, lack of expression of CD31, and ability to adhere to tissue-cultured plastic whithin a short time. The in vitro properties of cultured AT3G5Cs were compared to those of adipose tissue mesenchymal stromal cells (ATMSCs) obtained through traditional methods. Gene expression profiles of cultured AT3G5Cs (n = 3 different biological samples) and ATMSCs (n = 3 different biological samples) were compared to each other by statistical analyses. Gene expression profiles of cultured AT3G5Cs and ATMSCs were compared to those of other cell types by clustering analyses.

Project description:Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that MSCs promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. MSCs encounter drastic declines in oxygen tension when injected into the avascular IVD; thus, understanding how oxygen tension affects interactions between MSCs and macrophages may offer clues as to how MSCs act therapeutically. We investigated how conditioned medium (CM) derived from MSCs cultured in hypoxia altered macrophage subsets. We cultured human, bone marrow-derived STRO3+ MSCs in hypoxia or normoxia, and collected CM. We then cultured human bone marrow-derived macrophages with IFN-y, followed by either hypoxic or normoxic STRO3+ MSC CM. We also cultured macrophages in hypoxic MSC CM in the presence of IL-4 and collected cells for scRNA-seq analyses. Bioinformatic analyses confirmed multiple macrophage subpopulations. We first performed comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM. Integration of these data sets showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. Gene Ontogeny (GO) analyses revealed enrichment in IL-10, Ccl5/Ccr5, G alpha (i) Signaling, and Rho GTPases within this unique cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. Our study identifies a unique macrophage subset induced by MSCs within hypoxic conditions, and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms

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Single cell RNA profiles of bone marrow aspirate concentrate

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