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Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize \"danger\" signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol. Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1. Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies.

Human mesenchymal stromal cells (MSCs) have served as a major cellular resource for cell-based immunomodulatory and regenerative therapies. However, genomic instability may accumulate during ex vivo expansion of MSCs, thereby increasing the potential of malignant transformation. Here, we performed whole genome sequencing of two peripheral blood-derived MSC lines (MSC1 and MSC2) at various passages (passage 1 [P1] to P9). The majority of single-nucleotide variations (SNVs) occurred in later passages; specifically, 90% and 70% of all SNVs in MSC1 and MSC2 were observed in P9 and P7/P9, respectively. These late-occurring SNVs were enriched with C > A transversions and were overrepresented in intronic regions compared to intergenic regions, suggesting that the mutational forces are not constant across the passages. Clonality analyses also distinguished early-occurring, subclonal SNVs from late-occurring, clonally fixed SNVs. In addition, MSCs were largely devoid of copy number alterations (CNAs) (i.e., 0–2 CNAs per passage), with one exception (MSC2-P3) harboring 29 passage-specific CNAs. Our findings suggest that the SNVs found to be abundant at later passages likely resulted from the accumulation of replication stress, which can be associated with proliferation activity. Thus, the genomic instability associated with proliferation records should be considered for clinical applications of MSCs.

Mesenchymal stem cells (MSCs) show potential for treating septic arthritis in aged populations, but their efficacy and safety in aged patients remain unclear. The objective of this study is to evaluate the therapeutic potential and safety profiles of bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) in aged murine model of septic arthritis. MSCs were isolated, characterized, and labeled for in vivo tracking. The experiment consisted of a total of 36 mice, which included 9 subgroups with four replicates per treated group: control group, treated groups (BM-MSC1, BM-MSC2, AD-MSC1, and AD-MSC2), and untreated groups (Un-BM1, Un-BM2, Un-AD1, and Un-AD2). The treated groups received MSC therapy following the induction of septic arthritis via intra-articular injection of Staphylococcus aureus. The results showed that BM-MSC1 significantly performed higher than AD-MSCs in reducing inflammation, promoting cartilage repair, and modulating immune responses. BM-MSC1 showed significant upregulation of regenerative markers such as interleukin-10 (IL-10) and collagen type II alpha 1 chain (COL2A1) and downregulation of pro-inflammatory markers such as tumor necrosis factor-alpha (TNF-a) and matrix metalloproteinase-13 (MMP-13). Imaging confirmed superior retention, engraftment, and host tissue interaction for BM-MSCs. AD-MSCs showed slightly lower efficacy and safety, highlighting the need for optimization. Untreated groups experienced severe inflammation, tissue degradation, and systemic organ damage, emphasizing the significance of intervention. The study had identified BM-MSC1 as a superior therapeutic option for septic arthritis in elderly populations and suggested AD-MSCs as an alternative in cases where extraction is not feasible. Future research can optimize MSC therapies, explore alternative sources, and conduct translational studies. Targeted preconditioning, combinatory approaches, and advanced molecular analyses are crucial for maximizing therapeutic outcomes.

Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of numerous diseases. Increasingly, however, there is a concern over the use of MSCs because they home to tumors and can support tumor growth and metastasis. For instance, we established that MSCs in the ovarian tumor microenvironment promoted tumor growth and favored angiogenesis. In parallel studies, we also developed a new approach to induce the conventional mixed pool of MSCs into two uniform but distinct phenotypes we termed MSC1 and MSC2. Here we tested the in vitro and in vivo stability of MSC1 and MSC2 phenotypes as well as their effects on tumor growth and spread. In vitro co-culture of MSC1 with various cancer cells diminished growth in colony forming units and tumor spheroid assays, while conventional MSCs or MSC2 co-culture had the opposite effect in these assays. Co-culture of MSC1 and cancer cells also distinctly affected their migration and invasion potential when compared to MSCs or MSC2 treated samples. The expression of bioactive molecules also differed dramatically among these samples. MSC1-based treatment of established tumors in an immune competent model attenuated tumor growth and metastasis in contrast to MSCs- and MSC2-treated animals in which tumor growth and spread was increased. Also, in contrast to these groups, MSC1-therapy led to less ascites accumulation, increased CD45+leukocytes, decreased collagen deposition, and mast cell degranulation. These observations indicate that the MSC1 and MSC2 phenotypes may be convenient tools for the discovery of critical components of the tumor stroma. The continued investigation of these cells may help ensure that cell based-therapy is used safely and effectively in human disease.

Background Mesenchymal stem cells (MSCs) have been recognized for their regenerative and anti-inflammatory capacity which makes them very attractive to cell therapy, especially those ones to treat inflammatory and autoimmune disease. Two different immune-phenotypes have been described for MSCs depending on which Toll-like receptor (TLR) is activated. MSC1 is endowed with a pro-inflammatory phenotype following TLR4 activation with LPS. On the other hand, anti-inflammatory MSC2 is induced by the activation of TLR3 with Poly(I:C). High immunoplasticity of MSCs is a matter of concern in cell-based therapies. In this study, we investigated whether a single stimulus can induce both types of MSCs through a differential activation of TLR4 with LPS. Methods MSCs were activated with LPS following a short exposure of 1-h (MSCs-LPS1h) or long-time exposure for 48 h (MSCs-LPS48h), and then, we evaluated the biological response in vitro, the immunosuppressive capacity of MSCs in vitro, and the therapeutic potential of MSCs in an experimental autoimmune encephalomyelitis (EAE) mouse model. Results Our results showed that 1-h LPS exposure induced a MSC1 phenotype. Indeed, MSCs-LPS1h expressed low levels of NO/iNOS and decreased immunosuppressive capacity in vitro without therapeutic effect in the EAE model. In contrast, MSCs-LPS48h achieved a MSC2-like phenotype with significant increase in the immunosuppressive capacity on T cell proliferation in vitro, together with an improved in the therapeutic effect and higher Treg, compared to unstimulated MSCs. Furthermore, we determine through the MSCs-TLR4KO that the expression of TLR4 receptor is essential for MSCs’ suppressive activity since TLR4 deletion was associated with a diminished suppressive effect in vitro and a loss of therapeutic effect in vivo. Conclusions We demonstrate that MSCs display a high immunoplasticity commanded by a single stimulus, where LPS exposure time regulated the MSC suppressive effect leading into either an enhanced or an impairment therapeutic activity. Our results underscore the importance of phenotype conversion probably related to the TLR4 expression and activation, in the design of future clinical protocols to treat patients with inflammatory and autoimmune diseases.

Stem cell-related genes (SCRGs) and telomere maintenance mechanism-related genes (TMMRGs) are pivotal in breast cancer (BC) pathogenesis by facilitating tumor cell proliferation and self-renewal. This study employed integrated transcriptomic and single-cell RNA sequencing (scRNA-seq) analyses to investigate SCRGs and TMMRGs as potential biomarkers for BC and to elucidate their underlying cellular mechanisms. Total RNA was extracted from eight BC tumor samples and eight matched adjacent non-tumorous tissues. Differential expression profiling, protein-protein interaction (PPI) network construction, and Molecular Complex Detection (MCODE) were conducted. Biomarker candidates were identified using the least absolute shrinkage and selection operator (LASSO) algorithm, followed by pathway enrichment and immunological analyses. Publicly available scRNA-seq datasets were utilized to delineate BC cell types, with emphasis on cellular subsets exhibiting differential biomarker expression. Heterogeneity, communication, and pseudo-temporal analyses of key cells were examined. Biomarker expression was further validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). JUN, NFKB1, and SP1 were significantly downregulated in BC, potentially modulating disease progression through mechanisms involving extracellular matrix (ECM) remodeling, intracellular signaling, oxidative stress response, and translational regulation. Activated B cells and natural killer (NK) cells demonstrated elevated infiltration levels, accompanied by increased expression of immune checkpoint molecules CD200, CD274, TIGIT, TNFRSF25, and TNFSF15. Nine distinct cellular lineages were annotated, among which mesenchymal cells exhibited pronounced biomarker expression differences and enhanced differentiation potential, designating them as key cellular mediators. Interactions between mesenchymal subpopulations (MSC1, MSC2, MSC3) and other cell types were markedly reduced in BC, despite an overall expansion in mesenchymal cell numbers during disease progression. MSC1 emerged as the predominant subtype. RT-qPCR analyses corroborated the downregulation of JUN, NFKB1, and SP1 in BC tissues. JUN, NFKB1, and SP1 were identified as potential biomarkers for BC. These findings highlight the critical role of mesenchymal cells in tumor biology and suggest potential therapeutic targets.

Background The tumorigenesis of infused umbilical cord mesenchymal stem cells (UC-MSCs) is being preclinically evaluated. Methods We observed tumor formation in NOD SCID mice after a single subcutaneous injection of hUC-MSCs and the effect of these cells on tumor growth in tumor-bearing mice. Three generations (P5, P7, and P10) of hUC-MSCs (1 × 10 7 ) from two donors (hUC-MSC1 and hUC-MSC2) were inoculated subcutaneously into NOD SCID mice. Subcutaneous transplantation models were established in NOD SCID mice with human cervical cancer HeLa cells (solid tumor) and human B cell lymphoma Raji cells (hematological tumor). Then, the animals were euthanized, gross dissection was performed, and tissues were collected. Various organs were observed microscopically to identify pathological changes and tumor metastasis. Results In the tumorigenesis experiment, no general anatomical abnormalities were observed. In the tumor promotion experiment, some animals in the HeLa groups experienced tumor rupture, and one animal died in each of the low- and medium-dose hUC-MSC groups. The results may have occurred due to the longer feeding time, and the tumor may have caused spontaneous infection and death. Pathological examination revealed no metastasis to distant organs in any group. In the Raji tumor model, some animals in each group experienced tumor rupture, and one animal in the medium-dose hUC-MSC group died, perhaps due to increased tumor malignancy. Thus, hUC-MSCs neither promoted nor inhibited tumor growth. No cancer cell metastasis was observed in the heart, liver, spleen, lungs, kidneys or other important organs, except that pulmonary venule metastasis was observed in 1 animal in the model group. Conclusions Injected hUC-MSCs were not tumorigenic and did not significantly promote or inhibit solid or hematological tumor growth or metastasis in NOD SCID mice.

The paper characterizes phenotypic features of subpopulations of human adipose tissue mesenchymal stem cells (MSC) resulting from exposure to Toll-like receptors ligands. MSC1 and MSC2 phenotypes were induced by exposure to LPS and polyinosinic-polycytidylic acid, respectively. Different exposures to Toll-like receptors ligands, short-term (3 h) and long-term (24 h), were studied. The cytokine profile of cells with the MSC2 phenotype differed depending on the duration of exposure to the inductor, while the cytokine profile of cells with the MSC1 phenotype remained stable. Morphometric features of mesenchymal stem cells with the MSC1 and MSC2 phenotypes, as well as differences in the IDO gene expression were identified. These differences can be used to distinguish between these cell types.

Nowadays, immune diseases are a large burden in healthcare. Mesenchymal stem cells (MSCs) have prominent ability in immunomodulation and have been applicated on treating many immune-related diseases. However, the clinical outcomes can be disparate and sometimes completely counterproductive beyond explanation of cell heterogeneity. The theory of immunomodulation plasticity in MSCs has then emerged to explain that MSCs can be induced into proinflammatory MSC1 or anti-inflammatory MSC2 responding to different immune environment. It would be safer and more efficient if we could induce MSCs into a certain immune phenotype, in most cases MSC2, prior to medical treatment. In this study, we screened and identified a classical FDA-approved drug, chlorzoxazone (CZ). Unlike traditional method induced by IFN-γ, CZ can induce MSC into MSC2 phenotype and enhance the immunosuppressive capacity without elevation of immunogenicity of MSCs. CZ-treated MSCs can better inhibit T cells activation and proliferation, promote expression of IDO and other immune mediators in vitro, and alleviate inflammatory infiltration and tissue damage in acute kidney injury rat model more effectively. Moreover, we discovered that CZ modulates phosphorylation of transcriptional factor forkhead box O3 (FOXO3) independent of classical AKT or ERK signaling pathways, to promote expression of downstream immune-related genes, therefore contributing to augmentation of MSCs immunosuppressive capacity. Our study established a novel and effective approach to induce MSC2, which is ready for clinical application.

Lupus nephritis is recognized as a common and severe complication of systemic lupus erythematosus, without an optimal therapeutic strategy currently available. While mesenchymal stem cells (MSCs) hold therapeutic promise, their efficacy varies substantially, likely due to their plasticity and capacity to adopt pro-inflammatory (MSC1) or anti-inflammatory (MSC2) functional states in response to different microenvironments. Here, we report for the first time that IL-27, via JAK1–STAT1 signaling, up-regulates indoleamine 2,3-dioxygenase (IDO) in MSCs, driving MSC differentiation toward an IDO-positive MSC2 phenotype with low immunogenicity. These IDO-positive MSC2 cells produce kynurenine and kynurenic acid, the metabolites of tryptophan, which bind to the intracellular aryl hydrocarbon receptor. This interaction stimulates an increase in the anti-inflammatory factor TSG-6 and induces the differentiation of regulatory T cells. Notably, IL-27-conditioned MSC2 demonstrated superior therapeutic efficacy compared to conventional MSCs in a murine lupus nephritis model. In conclusion, this study revealed that IL-27 is a critical modulator of MSC immune plasticity and presented a novel therapeutic strategy utilizing IL-27-enhanced MSC2 for autoimmune diseases.