The Role of COX-2 and PGE2 in the Regulation of Immunomodulation and Other Functions of Mesenchymal Stromal Cells

The ability of MSCs to modulate the inflammatory environment is well recognized, but understanding the molecular mechanisms responsible for these properties is still far from complete. Prostaglandin E2 (PGE2), a product of the cyclooxygenase 2 (COX-2) pathway, is indicated as one of the key mediators in the immunomodulatory effect of MSCs. Due to the pleiotropic effect of this molecule, determining its role in particular intercellular interactions and aspects of cell functioning is very difficult. In this article, the authors attempt to summarize the previous observations regarding the role of PGE2 and COX-2 in the immunomodulatory properties and other vital functions of MSCs. So far, the most consistent results relate to the inhibitory effect of MSC-derived PGE2 on the early maturation of dendritic cells, suppressive effect on the proliferation of activated lymphocytes, and stimulatory effect on the differentiation of macrophages into M2 phenotype. Additionally, COX-2/PGE2 plays an important role in maintaining the basic life functions of MSCs, such as the ability to proliferate, migrate and differentiate, and it also positively affects the formation of niches that are conducive to both hematopoiesis and carcinogenesis

Premature fetal tissues are possible source of valuable mesenchymal stem cells

Objectives: Comparison of the ability to inhibit alloactivated lymphocytes proliferation of human Wharton Jelly (WJ) and amniotic membrane (AM) mesenchymal stem cells (MSCs) from preterm and term pregnancies. Material and methods: Term-WJ-MSCs (n = 5) and Preterm-WJ-MSCs (n = 1) were obtained from tissue explants by adherent method. Term-AM-MSCs (n = 5) and Preterm-AM-MSCs (n = 1) were obtained by tripsin and collagenase digestion method. Term and Preterm MSCs phenotype was confirmed in vitro by flow cytometry. To evaluate the potential of fetal and adult MSCs to diminish immunological response mixed lymphocytes reaction (MLR) has been performed. Results: Term and Preterm cells were positively identified as MSCs by the expression of CD73 and CD90 and CD105 with simultaneous absence of CD11b, CD14, CD19, CD34, CD45 and HLA-DR. The mean inhibition of allostimulated lymphocytes after addition of fetal derived MSCs amounted 64.8% for term AM-MSCs and 42.1% for term WJ-MSCs (for both populations the effect was statistically significant, p < 0.01). The addition of preterm-MSCs to MLR resulted in reduction of stimulated lymphocytes proliferation by 64.9% for AM-MSCs and 86.1% for WJ-MSCs. Conclusions: Presented results suggest that preterm fetal tissues contain MSCs which posses similar immunosuppressive capacity as those from term pregnancies. In the future MSCs from the umbilical cord and amnion can be potentially used to prevent immuno-dependent injuries in premature newborns

Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 μM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs

Ibuprofen in Therapeutic Concentrations Affects the Secretion of Human Bone Marrow Mesenchymal Stromal Cells, but Not Their Proliferative and Migratory Capacity

Mesenchymal stromal cells (MSCs) are able to modulate the immune system activity and the regeneration processes mainly through the secretion of multiple soluble factors, including prostaglandin E2 (PGE2). PGE2 is produced as a result of cyclooxygenases (COX) activity. In the present study, we investigated how ibuprofen, a nonselective COX inhibitor, affects the proliferation, migration and secretion of human bone marrow MSCs (hBM-MSCs). For this purpose, six hBM-MSCs populations were treated with ibuprofen at doses which do not differ from maximum serum concentrations during standard pharmacotherapy. Ibuprofen treatment (25 or 50 µg/mL) substantially reduced the secretion of PGE2 in all tested populations. Following ibuprofen administration, MSCs were subjected to proliferation (BrdU), transwell migration, and scratch assays, while its effect on MSCs secretome was evaluated by Proteome Profiler and Luminex immunoassays. Ibuprofen did not cause statistically significant changes in the proliferation rate and migration ability of MSCs (p > 0.05). However, ibuprofen (25 µg/mL for 3 days) significantly decreased mean secretion of: CCL2 (by 44%), HGF (by 31%), IL-6 (by 22%), VEGF (by 20%) and IL-4 (by 8%) compared to secretion of control MSCs (p < 0.05). Our results indicate that ibuprofen at therapeutic concentrations may impair the pro-regenerative properties of hBM-MSCs

In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models

Introduction: Despite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment. Material and methods: In the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 μm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function. Results: Fluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices. Conclusions: The IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application

Dynamics of Acute Local Inflammatory Response after Autologous Transplantation of Muscle-Derived Cells into the Skeletal Muscle

The vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection. Inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model. The aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection. Muscle-derived cells or medium alone was injected into the intact skeletal muscles in autologous model. Tissue samples were collected 1, 3, and 7 days after the procedure. Our analysis revealed the peak increase of the gene expression of all evaluated cytokines (Il-1α, Il-1β, Il-6, Tgf-β, and Tnf-α) at day 1. The mRNA level of analyzed cytokines normalized in subsequent time points. The increase of Il-β gene expression was further confirmed at the protein level. Analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages. The inflammatory infiltration was almost completely resolved at day 7. The survived cells were able to participate in the muscle regeneration process. Presented results demonstrate that autotransplanted muscle-derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination

Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning—An Analysis by RNA Sequencing

Mesenchymal stromal cell (MSC) therapy is making its way into clinical practice, accompanied by research into strategies improving their therapeutic potential. Preconditioning MSCs with hypoxia-inducible factors-α (HIFα) stabilizers is an alternative to hypoxic priming, but there remains insufficient data evaluating its transcriptomic effect. Herein, we determined the gene expression profile of 6 human bone marrow-derived MSCs preconditioned for 6 h in 2% O2 (hypoxia) or with 40 μM Vadadustat, compared to control cells and each other. RNA-Sequencing was performed using the Illumina platform, quality control with FastQC and adapter-trimming with BBDUK2. Transcripts were mapped to the Homo_sapiens. GRCh37 genome and converted to relative expression using Salmon. Differentially expressed genes (DEGs) were generated using DESeq2 while functional enrichment was performed in GSEA and g:Profiler. Comparison of hypoxia versus control resulted in 250 DEGs, Vadadustat versus control 1071, and Vadadustat versus hypoxia 1770. The terms enriched in both phenotypes referred mainly to metabolism, in Vadadustat additionally to vesicular transport, chromatin modifications and interaction with extracellular matrix. Compared with hypoxia, Vadadustat upregulated autophagic, phospholipid metabolism, and TLR cascade genes, downregulated those of cytoskeleton and GG-NER pathway and regulated 74 secretory factor genes. Our results provide valuable insight into the transcriptomic effects of these two methods of MSCs preconditioning