Membrane particles from mesenchymal stromal cells reduce the expression of fibrotic markers on pulmonary cells

Background: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. Methods: Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-β for 48h and gene expression of major pro-fibrotic markers were analyzed. Results: About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment

Human Allogeneic Bone Marrow and Adipose Tissue Derived Mesenchymal Stromal Cells Induce CD8+ Cytotoxic T Cell Reactivity

INTRODUCTION: For clinical applications, Mesenchymal Stromal Cells (MSC) can be isolated from bone marrow and adipose tissue of autologous or allogeneic origin. Allogeneic cell usage has advantages but may harbor the risk of sensitization against foreign HLA. Therefore, we evaluated whether bone marrow and adipose tissue-derived MSC are capable of inducing HLA-specific alloreactivity. METHODS: MSC were isolated from healthy human Bone Marrow (BM-MSC) and adipose tissue (ASC) donors. Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HLA-AB mismatched BM-MSC or ASC precultured with or without IFNy. After isolation via FACS sorting, the educated CD8+ T effector populations were exposed for 4 hours to Europium labeled MSC of the same HLA make up as in the co-cultures or with different HLA. Lysis of MSC was determined by spectrophotometric measurement of Europium release. RESULTS: CD8+ T cells educated with BM-MSC were capable of HLA specific lysis of BM-MSC. The maximum lysis was 24% in an effector:target (E:T) ratio of 40:1. Exposure to IFNγ increased HLA-I expression on BM-MSC and increased lysis to 48%. Co-culturing of PBMC with IFNγ-stimulated BM-MSC further increased lysis to 76%. Surprisingly, lysis induced by ASC was significantly lower. CD8+ T cells educated with ASC induced a maximum lysis of 13% and CD8+ T cells educated with IFNγ-stimulated ASC of only 31%. CONCLUSION: Allogeneic BM-MSC, and to a lesser extend ASC, are capable of inducing HLA specific reactivity. These results should be taken into consideration when using allogeneic MSC for clinical therapy

Inflammatory conditions dictate the effect of Mesenchymal stem or Stromal cells on B cell function

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43− selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38high CD24high B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC

Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4+ and CD8+ T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14+CD16+ monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of antiinflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders

Membrane Particles Derived From Adipose Tissue Mesenchymal Stromal Cells Improve Endothelial Cell Barrier Integrity

Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium

Adipose Tissue-Derived Mesenchymal Stem Cells Have a Heterogenic Cytokine Secretion Profile

Mesenchymal stem cells derived from adipose tissue (ASC) have immune regulatory function, which makes them interesting candidates for cellular therapy. ASC cultures are however heterogeneous in phenotype. It is unclear whether all ASC contribute equally to immunomodulatory processes. ASC are also responsive to cytokine stimulation, which may affect the ratio between more and less potent ASC populations. In the present study, we determined IL-6 receptor (CD126 and CD130 subunits) and IFN-γ receptor (CD119) expression on ASC by flow cytometry. The production of IL-6 and IFN-γ was measured by ELISA and the frequency of IL-6 and IFN-γ secreting cells by ELISPOT. The results showed that ASC did not express CD126, and only 10-20% of ASC expressed CD130 on their surface, whereas 18-31% of ASC expressed CD119. ASC produced high levels of IL-6 and 100% of ASC were capable of secreting IL-6. Stimulation by IFN-γ or TGF-β had no effect on IL-6 secretion by ASC. IFN-γ was produced by only 1.4% of ASC, and TGF-β significantly increased the frequency to 2.7%. These results demonstrate that ASC cultures are heterogeneous in their cytokine secretion and receptor expression profiles. This knowledge can be employed for selection of potent, cytokine-producing, or responsive ASC subsets for cellular immunotherapy

Fate and Effect of Intravenously Infused Mesenchymal Stem Cells in a Mouse Model of Hepatic Ischemia Reperfusion Injury and Resection

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Human mesenchymal stem cells are susceptible to lysis by CD8+ T cells and NK cells

There is growing interest in the use of mesenchymal stem cells (MSCs) to improve the outcome of organ transplantation. The immunogenicity of MSCs is, however, unclear and is important for the efficacy of MSC therapy and for potential sensitization against donor antigens. We investigated the susceptibility of autolo- gous and allogeneic MSCs for lysis by CD8+ T-lymphocytes and NK cells in a kidney transplant setting. MSCs were derived from adipose tissue of human kidney donors and were CD90+, CD105+, CD166+, and HLA class I+. They showed differentiation ability and immunosuppressive capacity. Lysis of MSCs by peripheral blood mononuclear cells (PBMCs), FACS-sorted CD8+ T cells, and NK cells was measured by europium release assay. Allogeneic MSCs were susceptible for lysis by cytotoxic CD8+ T cells and NK cells, while autologous MSCs were lysed by NK cells only. NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSCs. Lysis of autologous MSCs was not dependent on culturing of MSCs in FBS, and MSCs in suspension as well as adherent to plastic were lysed by NK cells. Pretransplant recipient PBMCs did not lyse donor MSCs, but PBMCs isolated 3, 6, and 12 months after transplantation showed increasing lysing ability. After 12 months, CD8+ T-cell-mediated lysis of donor MSCs persisted, indicating there was no evidence for desensitization against donor MSCs. Lysis of MSCs is important to take into account when MSCs are considered for clinical application. Our results suggest that the HLA background of MSCs and timing of MSC administration are important for the efficacy of MSC therapy

Identification of Predictive Markers for the Generation of Well-Differentiated Human Induced Pluripotent Stem Cell-Derived Kidney Organoids

Human-induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential to advance studies to kidney development and disease. However, reproducible generation of kidney organoids is a challenge. A large variability in the percentage of nephron structures and the expression of kidney-specific genes was observed among organoids, showing no association with iPSC lines. To associate the quality of kidney organoid differentiation with predictive markers, a ranking system was developed based on the ratio of nephron structure determined by histological examination. Well-differentiated organoids were defined as organoids with >30% nephron structure and vice versa. Subsequently, correlations were made with expression profiles of iPSC markers, early kidney development markers, and fibrosis markers. Higher expression of sex-determining region Y-box 2 (SOX2) during differentiation was associated with poorly differentiated kidney organoid. Furthermore, early secretion of basic fibroblast growth factor (FGF2) predicted poorly differentiated kidney organoid. Of interest, whereas cadherin-1 (CDH1) expression in kidney organoids indicates distal tubules formation, onefold higher CDH1 expression in iPSC predicted poor differentiation. High expression of the stromal progenitor marker Forkhead Box D1 (FOXD1) and significantly increased TGFβ levels were found in well-differentiated kidney organoids. These early expression profiles could predict the outcome of kidney organoid formation. This study helps to improve the robustness of kidney organoid protocols

Differential effects of heat-inactivated, secretome-deficient MSC and metabolically active MSC in sepsis and allogenic heart transplantation

Mesenchymal stem cells (MSCs) are used in various clinical and preclinical models for immunomodulation. However, it remains unclear how the immunomodulatory effect of MSC is communicated. MSC-induced immunomodulation is known to be mediated through both MSC-secreted cytokines and direct cell-cell interactions. Recently, it has been demonstrated that metabolically inactive, heat-inactivated MSCs (HI-MSCs) have similar anti-inflammatory capacities in LPS-induced sepsis compared with viable MSC. To further investigate the immunomodulatory effects of MSC, we introduced MSC and HI-MSC in two animal models with different immunological causes. In the first model, allogeneic hearts were transplanted from C57BL/6 mice to BALB/c recipients. MSC in combination with mycophenolate mofetil (MMF) significantly improved graft survival compared with MMF alone, whereas the application of HI-MSC had no effect on graft survival. We revealed that control MSC dose-dependently inhibited CD3(+) and CD8(+) T-cell proliferation in vitro, whereas HI-MSC had no effect. In the second model, sepsis was induced in mice via cecal ligation and puncture. HI-MSC treatment significantly improved the overall survival, whereas control MSCs had no effect. in vitro studies demonstrated that HI-MSCs are more effectively phagocytosed by monocytes than control MSCs and induced cell death in particular of activated CD16(+) monocytes, which may explain the immune protective effect of HI-MSC in the sepsis model. The results of our study demonstrate that MSC-mediated immunomodulation in sepsis is dependent on a passive recognition of MSC by monocytes, whereas fully functional MSCs are required for inhibition of T-cell-mediated allograft rejection