Priming Of Mesenchymal Stem Cells Research Articles

Mesenchymal Stem Cell Priming: Potential Benefits of Administration of Molecular Hydrogen

Stem cell therapy has emerged as a promising avenue for regenerative medicine, offering the potential to treat a wide range of debilitating diseases and injuries. Among the various types of stem cells, mesenchymal stem cells (MSCs) have garnered significant attention due to their unique properties and therapeutic potential. In recent years, researchers have been exploring novel approaches to enhance the effectiveness of MSC-based therapies. One such approach that has gained traction is the priming of MSCs with molecular hydrogen (H2). This article delves into the fascinating world of mesenchymal stem cell priming with molecular hydrogen and the potential benefits it holds for regenerative medicine.

Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation

BackgroundMesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs.MethodsThis work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models.ResultsOur results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization.ConclusionPGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.

Recombinant Human Bone Morphogenetic Protein-2 Priming of Mesenchymal Stem Cells Ameliorate Acute Lung Injury by Inducing Regulatory T Cells.

Mesenchymal stromal/stem cells (MSCs) possess immunoregulatory properties and their regulatory functions represent a potential therapy for acute lung injury (ALI). However, uncertainties remain with respect to defining MSCs-derived immunomodulatory pathways. Therefore, this study aimed to investigate the mechanism underlying the enhanced effect of human recombinant bone morphogenic protein-2 (rhBMP-2) primed ES-MSCs (MSCBMP2) in promoting Tregs in ALI mice. MSC were preconditioned with 100 ng/ml rhBMP-2 for 24 h, and then administrated to mice by intravenous injection after intratracheal injection of 1 mg/kg LPS. Treating MSCs with rhBMP-2 significantly increased cellular proliferation and migration, and cytokines array reveled that cytokines release by MSCBMP2 were associated with migration and growth. MSCBMP2 ameliorated LPS induced lung injury and reduced myeloperoxidase activity and permeability in mice exposed to LPS. Levels of inducible nitric oxide synthase were decreased while levels of total glutathione and superoxide dismutase activity were further increased via inhibition of phosphorylated STAT1 in ALI mice treated with MSCBMP2. MSCBMP2 treatment increased the protein level of IDO1, indicating an increase in Treg cells, and Foxp3+CD25+ Treg of CD4+ cells were further increased in ALI mice treated with MSCBMP2. In co-culture assays with MSCs and RAW264.7 cells, the protein level of IDO1 was further induced in MSCBMP2. Additionally, cytokine release of IL-10 was enhanced while both IL-6 and TNF-α were further inhibited. In conclusion, these findings suggest that MSCBMP2 has therapeutic potential to reduce massive inflammation of respiratory diseases by promoting Treg cells.

Methods and Limitations of Augmenting Mesenchymal Stem Cells for Therapeutic Applications.

Significance: Given their capacity for self-renewal, multilineage differentiation, and immunomodulatory potential, mesenchymal stem cells (MSCs) represent a promising modality of clinical therapy for both regenerative medicine and immune diseases. In this study, we review the key approaches and popular methods utilized to boost potency and modify functions of MSCs for clinical purposes as well as their associated limitations. Recent Advances: Several major domains of cell modification strategies are currently employed by investigators to overcome these deficits and augment the therapeutic potential of MSCs. Priming MSCs with soluble factors or pharmacologic agents as well as manipulating oxygen availability in culture have been demonstrated to be effective biochemical methods to augment MSC potential. Distinct genetic and epigenetic methods have emerged in recent years to modify the genetic expression of target proteins and factors thereby modulating MSCs capacity for differentiation, migration, and proliferation. Physical methods utilizing three-dimensional culture methods and alternative cell delivery systems and scaffolds can be used to recapitulate the native MSC niche and augment their engraftment and viability for in vivo models. Critical Issues: Unmodified MSCs have demonstrated only modest benefits in many preclinical and clinical studies due to issues with cell engraftment, viability, heterogeneity, and immunocompatibility between donor and recipient. Furthermore, unmodified MSCs can have low inherent therapeutic potential for which intensive research over the past few decades has been dedicated to improving cell functionality and potency.

Priming mesenchymal stem cells with α-synuclein enhances neuroprotective properties through induction of autophagy in Parkinsonian models

BackgroundMesenchymal stem cells (MSCs) may be one of candidates for disease-modifying therapy in Parkinsonian diseases. As knowledge regarding the therapeutic properties of MSCs accumulates, some obstacles still remain to be overcome, especially, successful clinical translation requires the development of culture systems that mimic the natural MSC niche, while allowing clinical-scale cell expansion without compromising quality and function of the cells. In recent years, priming approaches using bioactive peptide or complement components have been investigated to enhance the therapeutic potential of MSCs.MethodsWe investigated an innovative priming strategy by conditioning the MSCs with α-synuclein (α-syn). To induce priming, MSCs were treated with different concentrations of α-syn and various time course. We evaluated whether α-syn enhances stemness properties of MSCs and priming MSCs with α-syn would modulate autophagy-related gene expression profiles.ResultsTreatment of naïve MSCs with α-syn upregulated transcriptional factors responsible for regulation of stemness, which was associated with the elevated expression of genes involved in glycolysis and cell re-programming. Primed MSCs with α-syn enhanced the expression of autophagy-regulating miRNA, and exosomes derived from primed MSCs were packed with autophagy-associated miRNA. In α-syn-overexpressing neuronal cells, primed MSCs with α-syn enhanced neuronal viability relative to naïve MSCs, through the induction of autophagy and lysosome activity. Animal study using an α-syn-overexpressing mice showed that the pro-survival effect of MSCs on dopaminergic neurons was more prominent in primed MSC-treated mice compared with that in naïve MSC-treated mice.ConclusionsThe present data suggest that MSC priming with α-syn exerts neuroprotective effects through augmented stemness and possibly the enhancement of autophagy-mediated α-syn modulation in Parkinsonian models.

Inflammatory priming of mesenchymal stem cells: Focus on growth factors enhancement

Multipotent mesenchymal stromal cells (MSCs) are actively involved in reparation and inflammation processes, providing damaged tissue reparation and suppressing immune cell responses in vivo. The effects are mostly due to the production of a wide range of paracrine factors, including growth factors and immunomodulatory mediators. To induce immunosuppressive activity, MSCs are primed by inflammatory cytokines, which results in an increased production of immunomodulatory molecules. However, stimulation of reparative properties is also necessary. This viewpoint manuscript highlights the possibilities of inflammatory priming to increase the production of growth factors by MSCs.

Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing.

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.

Extracellular vesicles derived from inflammatory-educated stem cells reverse brain inflammation—implication of miRNAs

Inflammation plays a key role in the development of age-related diseases. In Alzheimer's disease, neuronal cell death is attributed to amyloidbeta oligomers that trigger microglial activation. Stem cells have shown promise as therapies for inflammatory diseases- because of their paracrine activity combined with their ability to respond to the inflammatory environment. However, the mechanisms underlying stem cell-promoted neurological recovery are poorly understood. To elucidate these mechanisms, we first primed stem cells with the secretome of lipopolysaccharide- or amyloidbeta-activated microglia. Then, we compared the immunomodulatory effects of extracellular vesicles (EVs) secreted from primed and non-primed stem cells. Our results demonstrate that EVs from primed cells are more effective in inhibiting microglia and astrocyte activation, amyloid deposition, demyelination, memory loss and motor and anxiety-like behavioral dysfunction, compared to EVs from non-primed cells. MicroRNA (miRNA) profiling revealed the upregulation of at least 19 miRNAs on primed-stem cell EVs. The miRNA targets were identified, and KEGG pathway analysis showed that the overexpressed miRNAs target key genes on the toll-like receptor-4 (TLR4) signaling pathway. Overall, our results demonstrate that priming mesenchymal stem cells (MSCs) with the secretome of activated microglia results in the release of miRNAs from EVs with enhanced immune regulatory potential able to fight neuroinflammation.

Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells.

Marrow adipose tissue (MAT) has been shown to be vital for regulating metabolism and maintaining skeletal homeostasis in the bone marrow (BM) niche. As a reflection of BM remodeling, MAT is highly responsive to nutrient fluctuations, hormonal changes, and metabolic disturbances such as obesity and diabetes mellitus. Expansion of MAT has also been strongly associated with bone loss in mice and humans. However, the regulation of BM plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling. We studied deletion of Adipsin, and its downstream effector, C3, in C57BL/6 mice as well as the bone-protected PPARγ constitutive deacetylation 2KR mice to assess BM plasticity. The mice were challenged with thiazolidinedione treatment, calorie restriction, or aging to induce bone loss and MAT expansion. Analysis of bone mineral density and marrow adiposity was performed using a μCT scanner and by RNA analysis to assess adipocyte and osteoblast markers. For in vitro studies, primary bone marrow stromal cells were isolated and subjected to osteoblastogenic or adipogenic differentiation or chemical treatment followed by morphological and molecular analyses. Clinical data was obtained from samples of a previous clinical trial of fasting and high-calorie diet in healthy human volunteers. We show that Adipsin is the most upregulated adipokine during MAT expansion in mice and humans in a PPARγ acetylation-dependent manner. Genetic ablation of Adipsin in mice specifically inhibited MAT expansion but not peripheral adipose depots, and improved bone mass during calorie restriction, thiazolidinedione treatment, and aging. These effects were mediated through its downstream effector, complement component C3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through inhibiting Wnt/β-catenin signaling. Adipsin promotes new adipocyte formation and affects skeletal remodeling in the BM niche. Our study reveals a novel mechanism whereby the BM sustains its own plasticity through paracrine and endocrine actions of a unique adipokine. This work was supported by the National Institutes of Health T32DK007328 (NA), F31DK124926 (NA), R01DK121140 (JCL), R01AR068970 (BZ), R01AR071463 (BZ), R01DK112943 (LQ), R24DK092759 (CJR), and P01HL087123 (LQ).

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development.

Mesenchymal stem cells are increasingly studied for their use as drug‐carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well‐known for its antioxidant and anti‐inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl‐beta‐cyclodextrin (HPβCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle‐derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration‐dependent and not time‐dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.

Priming mesenchymal stem cells with uric acid enhances neuroprotective properties in parkinsonian models

Mesenchymal stem cells (MSCs) are a potential source of cell-based disease-modifying therapy in Parkinsonian disorders. A promising approach to develop in vitro culture methods that mimic natural MSC niche is cell priming. Uric acid (UA), a powerful antioxidant, scavenges reactive oxygen species, which has a vital role in maintaining self-renewal and differentiation potential of MSCs. Here, we demonstrated that UA treatment in naïve MSCs stimulated glycolysis and upregulated transcriptional factors responsible for regulation of stemness, leading to increase in the expression levels of osteogenesis-, adipogenesis-, and chondrogenesis-related genes. UA-primed MSCs had more enhanced neuroprotective properties in cellular and parkinsonian animal models compared to naïve MSCs by inhibiting apoptotic signaling pathways. Additionally, expression of miR-137 and miR-145 was decreased in UA-treated MSCs. Our data demonstrated that priming MSCs with UA augment neuroprotective properties through enhanced self-renewal and differentiation potential, suggesting a practical strategy for improving the application of MSCs in parkinsonian disorders.

Abstract 15843: A Novel Role for Telomerase in Calcific Aortic Valve Disease

Introduction: Calcific aortic valve disease (CAVD) is a disorder characterized by the slow, but a progressive thickening of the aortic valve leaflet that develops into severe calcification. The only current therapy is aortic valve replacement. Telomerase is an enzymatic complex best known for its telomere-extending activities on the ends of chromosomes yet, the catalytic subunit (TERT) has been implicated in multiple non-canonical transcriptional and epigenetic activities, including priming of mesenchymal stem cells (MSCs) to differentiate into osteoblasts, and transcriptional regulation of inflammatory genes. Hypothesis: We hypothesize that non-canonical TERT activity contributes to the progression of CAVD. Methods: We performed biochemical assays to study the role of TERT in the calcification process using primary tissues and valve interstitial cells (VICs) from control and CAVD patients, smooth muscle cells (SMCs) from WT and Tert knockout mice, and mesenchymal stem cells (MSCs). Results: We found that TERT protein is highly expressed in calcified aortic valves and VICs isolated from patients with calcified valves, compared to healthy valves. VICs can be induced to calcify under osteogenic differentiation conditions, and we found that TERT accumulated after fourteen days in this culture, with no effect on either telomere length, proliferation, or senescence. We expanded the scope of our approach by evaluating TERT's influence on calcification of mice aortic smooth muscle cells (mSMCs). We found WT mSMCs readily calcified in vitro, but mSMCs from Tert knockout mice did not, and Tert deletion also reduced valve calcification in a Ldlr / Tert double knockout mice model compared to Ldlr knockout alone. In VICs, shRNA mediated TERT downregulation reduced expression of RUNX2 . Finally, we found that inflammatory signals intensify in vitro calcification, induce TERT expression, and we show evidence that TERT interacts with STAT5. Conclusion: Our data suggest that TERT is required for valve calcification by stimulating transcriptional pathways promoting the osteogenic transition of quiescent VICs into calcifying VICs in the aortic valve. These results indicate that TERT is an active contributor to the calcification process of valve tissues.

'Primed' Mesenchymal Stem Cells: a Potential Novel Therapeutic for COVID19 Patients.

The COVID19 pandemic, designated as a public health crisis by the World Health Organization (WHO), is rapidly spreading around the world impacting the health and economy of almost all the countries. The data of hospitalized COVID19 patients, especially those with serious illness, indicate the involvement of immunopathological complications. As no effective treatment is currently available, we propose ‘Primed’ Mesenchymal Stem Cells (MSCs) as a therapeutic alternative to tackle devastating epidemic. The individual response to MSCs treatment is heterogeneous. During the treatment of infectious pathology, the effectiveness of the treatment may vary based on the disease scenario. Interestingly, when transplanted in vivo, MSCs are governed by the locally regulated microenvironment, suggesting that the restorative variability could be tailored by choosing a priming regimen to specifically correct a given pathology. Therefore, in our opinion, the priming of MSCs could be a novel approach to improve the responses of COVID19 patients.

Activation of the SOX-5, SOX-6, and SOX-9 Trio of Transcription Factors Using a Gene-Activated Scaffold Stimulates Mesenchymal Stromal Cell Chondrogenesis and Inhibits Endochondral Ossification.

Current treatments for articular cartilage defects relieve symptoms but often only delay cartilage degeneration. Mesenchymal stem cells (MSCs) have shown chondrogenic potential but tend to undergo endochondral ossification when implanted in vivo. Harnessing factors governing joint development to functionalize biomaterial scaffolds, termed developmental engineering, might allow to prime host MSCs to regenerate mature articular cartilage in situ without requiring cell isolation or ex vivo expansion. Therefore, the aim of this study is to develop a gene-activated scaffold capable of delivering developmental cues to host MSCs, thus priming MSCs for articular cartilage differentiation and inhibiting endochondral ossification. It is shown that delivery of the SOX-Trio induced MSCs to over-express COL2A1 and ACAN and deposit a sulfated and collagen type II rich extracellular matrix while hypertrophic gene expression and collagen type X deposition is inhibited. When cell-free SOX-Trio-activated scaffolds are implanted ectopically in vivo, they induced spontaneous chondrogenesis without evidence of hypertrophy. MSCs pre-cultured on SOX-Trio-activated scaffolds prior to implantation differentiate into phenotypically stable chondrocytes as evidenced by a lack of collagen X expression or vascular invasion. This SOX-trio-activated scaffold represents a potent, single treatment, developmentally inspired strategy to prime MSCs in situ for articular cartilage defect repair.

TNFα-Mediated Priming of Mesenchymal Stem Cells Enhances Their Neuroprotective Effect on Retinal Ganglion Cells.

PurposeTo determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes’ neuroprotective efficacy on retinal ganglion cells (RGCs).MethodsMSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell–derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes.ResultsMSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased Pigment epithelium-derived factor (PEDF) and VEGF-AA exosomal abundance.ConclusionsMSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.

Allo-antibody production after intraarticular administration of mesenchymal stem cells (MSCs) in an equine osteoarthritis model: effect of repeated administration, MSC inflammatory stimulation, and equine leukocyte antigen (ELA) compatibility

BackgroundAntibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor’s ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular administration of stimulated MSCs (MSC-primed) and unstimulated MSCs (MSC-naïve) in pathologic joints.MethodsFrom available stored samples from a previous in vivo study, cells from one donor and serially collected sera (five time-points) from three groups of recipients were used based on their ELA haplotypes to perform microcytotoxicity assays: Group 1 recipients mismatched with the donor that received MSC-naïve (naïve-mismatched recipients); Group 2 recipients mismatched with the donor that received MSC-primed (primed-mismatched recipients); Group 3 recipients halfmatched with the donor (sharing 1/2 haplotypes) that received MSC-primed (primed-halfmatched recipients). Sera from recipients (neat, 1:2 and 1:16 dilution) were tested against target cells from the donor (cryopreserved and expanded MSC-naïve and MSC-primed) or from one animal presenting the same ELA haplotypes than the donor (fresh peripheral blood lymphocytes as control).ResultsOne to three weeks after first MSC administration, all recipient groups produced allo-antibodies regardless of MSC received (naïve or primed) and matching degree with donor. However, secondary response after MSC re-exposure was less evident in halfmatched recipients (MSC-primed) than in mismatched ones (both MSC-naïve and MSC-primed). Recipients of MSC-primed (both mismatched and halfmatched) tended towards developing lower antibody response than MSC-naïve recipients in vivo, but MSC-primed were targeted to death in higher percentage in vitro in the microcytoxicity assay.ConclusionsAfter first intraarticular allogeneic administration, the immunomodulatory profile of MSC-primed would have led to lower antibody production, but these antibodies would target more easily MSC-primed after second injection (re-exposure), likely because of their higher MHC expression.

Survival/Adaptation of Bone Marrow-Derived Mesenchymal Stem Cells After Long-Term Starvation Through Selective Processes.

After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DNA damage resistance, proliferation kinetic, and differentiation potential. This survival mode involved a quiescent state, confirmed by increased expression of cell cycle regulators p16, p27, and p57 and decreased expression of proliferating cell nuclear antigen (PCNA), Ki-67, mTOR, and Nanog. In addition, Jak/STAT (STAT6) antiapoptotic activity selected which cells conserved stemness and that supported metabolic, bioenergetic, and scavenging requirements. We also demonstrated that hBM-MSC exploited an autophagic process which induced lipid β-oxidation as an alternative energy source. Priming MSC by concomitant starvation and culture in hypoxic conditions to induce their quiescence would be of benefit to increase MSC survival when transplanted in vivo. Stem Cells 2019;37:813-827.

Immunoregulatory potential of mesenchymal stem cells following activation by macrophage-derived soluble factors

BackgroundImmunoregulatory capacity of mesenchymal stem cells (MSC) is triggered by the inflammatory environment, which changes during tissue repair. Macrophages are essential in mediating the inflammatory response after injury and can adopt a range of functional phenotypes, exhibiting pro-inflammatory and anti-inflammatory activities. An accurate characterization of MSC activation by the inflammatory milieu is needed for improving the efficacy of regenerative therapies. In this work, we investigated the immunomodulatory functions of MSC primed with factors secreted from macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype. We focused on the role of TNF-α and IL-10, prototypic pro-inflammatory and anti-inflammatory cytokines, respectively, as priming factors for MSC.MethodsSecretion of immunoregulatory mediators from human MSC primed with media conditioned by human macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype was determined. Immunomodulatory potential of primed MSC on polarized macrophages was studied using indirect co-cultures. Involvement of TNF-α and IL-10 in priming MSC and of PGE2 in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Collagen hydrogels were used to study MSC and macrophages interactions in a more physiological environment.ResultsPriming MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages enhanced their immunomodulatory potential through increased PGE2 secretion. We identified the pro-inflammatory cytokine TNF-α as a priming factor for MSC. Notably, the anti-inflammatory IL-10, mainly produced by pro-resolving macrophages, potentiated the priming effect of TNF-α. Collagen hydrogels acted as instructive microenvironments for MSC and macrophages functions and their crosstalk. Culturing macrophages on hydrogels stimulated anti-inflammatory versus pro-inflammatory cytokine secretion. Encapsulation of MSC within hydrogels increased PGE2 secretion and potentiated immunomodulation on macrophages, attenuating macrophage pro-inflammatory state and sustaining anti-inflammatory activation. Priming with inflammatory factors conferred to MSC loaded in hydrogels greater immunomodulatory potential, promoting anti-inflammatory activity of macrophages.ConclusionsFactors secreted by pro-inflammatory and anti-inflammatory macrophages activated the immunomodulatory potential of MSC. This was partially attributed to the priming effect of TNF-α and IL-10. Immunoregulatory functions of primed MSC were enhanced after encapsulation in hydrogels. These findings may provide insight into novel strategies to enhance MSC immunoregulatory potency.

1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites.

An indispensable role of macrophages in bone repair has been well recognized. Previous data have demonstrated the copresence of M1 macrophages and mesenchymal stem cells (MSCs) during the proinflammatory stage of bone repair. However, the exact role of M1 macrophages in MSC function and bone repair is unknown. This study aimed to define the role of M1 macrophages at bone injury sites via the function of 1,25-Dihydroxyvitamin D (1,25[OH]2D) in suppressing M1 but promoting M2 differentiation. We showed that 1,25(OH)2D suppressed M1 macrophage-mediated enhancement of MSC migration. Additionally, 1,25(OH)2D inhibited M1 macrophage secretion of osteogenic proteins (i.e., Oncostatin M, TNF-α, and IL-6). Importantly, the 1,25(OH)2D-mediated suppression of osteogenic function in M1 macrophages at the proinflammatory stage was associated with 1,25(OH)2D-mediated reduction of MSC abundance, compromised osteogenic potential of MSCs, and impairment of fracture repair. Furthermore, outside the proinflammatory stage, 1,25(OH)2D treatment did not suppress fracture repair. Accordingly, our data support 2 conclusions: (a) M1 macrophages are important for the recruitment and osteogenic priming of MSCs and, hence, are necessary for fracture repair, and (b) under vitamin D-sufficient conditions, 1,25(OH)2D treatment is unnecessary and can be detrimental if provided during the proinflammatory stage of fracture healing.

Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion.

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.