Priming Of Mesenchymal Stem Cells Research Articles

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.

Mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis.

Objectives: Mesenchymal stem cells (MSCs) release extracellular vesicles (EVs) that display a therapeutic effect in inflammatory disease models. Although MSCs can prevent arthritis, the role of MSCs-derived EVs has never been reported in rheumatoid arthritis. This prompted us to compare the function of exosomes (Exos) and microparticles (MPs) isolated from MSCs and investigate their immunomodulatory function in arthritis.Methods: MSCs-derived Exos and MPs were isolated by differential ultracentrifugation. Immunosuppressive effects of MPs or Exos were investigated on T and B lymphocytes in vitro and in the Delayed-Type Hypersensitivity (DTH) and Collagen-Induced Arthritis (CIA) models.Results: Exos and MPs from MSCs inhibited T lymphocyte proliferation in a dose-dependent manner and decreased the percentage of CD4+ and CD8+ T cell subsets. Interestingly, Exos increased Treg cell populations while parental MSCs did not. Conversely, plasmablast differentiation was reduced to a similar extent by MSCs, Exos or MPs. IFN-γ priming of MSCs before vesicles isolation did not influence the immunomodulatory function of isolated Exos or MPs. In DTH, we observed a dose-dependent anti-inflammatory effect of MPs and Exos, while in the CIA model, Exos efficiently decreased clinical signs of inflammation. The beneficial effect of Exos was associated with fewer plasmablasts and more Breg-like cells in lymph nodes.Conclusions: Both MSCs-derived MPs and Exos exerted an anti-inflammatory role on T and B lymphocytes independently of MSCs priming. However, Exos were more efficient in suppressing inflammation in vivo. Our work is the first demonstration of the therapeutic potential of MSCs-derived EVs in inflammatory arthritis.

Multi-channel biphasic calcium phosphate granules as cell carrier capable of supporting osteogenic priming of mesenchymal stem cells

Advances in bone tissue engineering include versatile and intricate biomaterial scaffolds in combination with stem cells for enhanced bone regeneration. In this study, a unique scaffold with multi-channels designed to allow cell infiltration within its pores was investigated for its capability to serve as a stable platform for adhesion and osteogenic priming of mesenchymal stem cells. The biphasic calcium phosphate multi-channel granule consisted of 60% hydroxyapatite and 40% β-tricalcium phosphate. Successful loading and retention of isolated and expanded rat bone marrow-derived mesenchymal stem cells (rBMSCs) were observed. The cells proliferated within the micro-channels starting from the surface then into the channels. The multi-channel granules were also able to support osteogenic priming of rBMSCs in 2D culture without the aid of a growth factor. Alkaline phosphatase, type I collagen, and runt-related transcription factor 2 expressions were detected with high osteopontin marker expression in as early as 7days, which persisted for 14days of culture under osteogenic condition. Results confirmed commitment towards osteogenic lineage of rBMSCs that have attached and grown onto the surface of the multi-channel granules and thus have high potential as a cell-scaffold based approach in bone regenerative medicine.

Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue.

The use of mesenchymal stem cells (MSC) as a therapeutic tool in cardiovascular diseases is promising. Since androgens exert some beneficial actions on the cardiovascular system, we tested our hypothesis that this hormone could promote MSC-mediated repair processes, also. Cultured MSCs isolated from Wharton's jelly were exposed to 30 nM dihydrotestosterone (DHT) for 1 or 4 days and the effects of the hormone on their growth/migration/adhesion and the underlying mechanisms were assessed. Results were obtained by real-time cell impedance measurements, and DNA quantification showed that DHT increased MSC proliferation by ~30%. As determined by xCELLigence system, DHT augmented (~2 folds) the migration of MSC toward cardiac tissue slices (at 12 h), and this effect was blocked by flutamide, an androgen receptor (AR) antagonist. Exposure of cells to DHT, upregulated the gene and protein expression of AR, EMMPRIN and MMP-9 and downregulated the expression of MMP-2 DHT significantly induced the release of nitric oxide by MSC (≥2-fold) and flutamide blocked this effect. When MSCs were co-cultured with cardiac slices, immunohistochemical analysis and qRT-PCR showed that the integration of DHT-stimulated MSC was significantly higher than that of in controls. In conclusion, our findings provide the first evidence that DHT promotes MSC growth, migration and integration into the cardiac slices. The modulating effects of DHT were associated with upregulation of ARs and of key molecules known to promote tissue remodeling and angiogenesis. Our findings suggest that priming of MSC with DHT may potentially increase their capability to regenerate cardiac tissue; in vivo studies are needed to confirm our in vitro findings.

Priming Equine Bone Marrow-Derived Mesenchymal Stem Cells with Proinflammatory Cytokines: Implications in Immunomodulation-Immunogenicity Balance, Cell Viability, and Differentiation Potential.

Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template.

In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and β-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31+, vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31+) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

Stroke Serum Priming Modulates Characteristics of Mesenchymal Stromal Cells by Controlling the Expression miRNA-20a.

Transplantation of mesenchymal stem cells (MSCs) expanded with fetal bovine serum (FBS) has some limitations, including the requirement of a long culture period to obtain a sufficient amount of stem cells. Priming of MSCs with serum from patients with ischemic stroke (stroke serum) increased the proliferation rate and the neurorestorative capacity of MSCs. We hypothesized that this novel priming method increases the proliferation rate of MSCs via the regulation of microRNAs (miRs). Thus, we investigated miR profiling in stroke serum-primed MSCs and tested whether the regulation of certain miRs may affect the proliferation rate of rat MSCs. The proliferation rate of MSCs cultured with stroke serum was higher than that of MSCs cultured with normal serum or FBS. Using miR microarray analysis, we compared the miR expression profiles between MSCs cultured in FBS and in stroke serum. Among miRs associated with cell proliferation, miR-20a was most significantly increased. Similarly, miR-20a was increased in MSCs obtained from the bone marrow of stroke rats compared with MSCs from normal rats. Furthermore, the deregulation of miR-20a by the transfection of MSCs with pre-miR-20a or anti-miR-20a was significantly correlated with the increased proliferation rate of MSCs. The overexpression of miR-20a in MSCs cultured in FBS improved the proliferation rate, while the knockdown of endogenous miR-20a decreased the proliferation rate. In addition, miR-20a promoted proliferation by suppressing the expression of p21 cyclin-dependent kinase inhibitor 1 (CDKN1A). A dual-luciferase reporter assay showed that CDKN1A is a target of miR-20a. Our findings indicate that stroke serum priming upregulated the expression of miR-20a, which promoted MSC proliferation by regulating the cell cycle inhibitor p21 CDKN1A, and suggest the possible roles of priming methods in modulating the characteristics of MSCs by controlling the expression of miR in MSCs.

Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

IntroductionDuring endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo.MethodsHuman MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson’s trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo.ResultsPre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups.ConclusionsTaken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

Primed Mesenchymal Stem Cells Alter and Improve Rat Medial Collateral Ligament Healing.

Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 h using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1 μg/ml. Rat MCLs were surgically transected and administered 1 × 10(6) cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing.

Lipocalin produced by myelofibrosis cells affects the fate of both hematopoietic and marrow microenvironmental cells

AbstractMyelofibrosis (MF) is characterized by cytopenias, constitutional symptoms, splenomegaly, and marrow histopathological abnormalities (fibrosis, increased microvessel density, and osteosclerosis). The microenvironmental abnormalities are likely a consequence of the elaboration of a variety of inflammatory cytokines generated by malignant megakaryocytes and monocytes. We observed that levels of a specific inflammatory cytokine, lipocalin-2 (LCN2), were elevated in the plasmas of patients with myeloproliferative neoplasms (MF > polycythemia vera or essential thrombocythemia) and that LCN2 was elaborated by MF myeloid cells. LCN2 generates increased reactive oxygen species, leading to increased DNA strand breaks and apoptosis of normal, but not MF, CD34+ cells. Furthermore, incubation of marrow adherent cells or mesenchymal stem cells with LCN2 increased the generation of osteoblasts and fibroblasts, but not adipocytes. LCN2 priming of mesenchymal stem cells resulted in the upregulation of RUNX2 gene as well as other genes that are capable of further affecting osteoblastogenesis, angiogenesis, and the deposition of matrix proteins. These data indicate that LCN2 is an additional MF inflammatory cytokine that likely contributes to the creation of a cascade of events that results in not only a predominance of the MF clone but also a dysfunctional microenvironment.

The Osteogenic Priming of Mesenchymal Stem Cells is Impaired in Experimental Diabetes.

Diabetes mellitus encompasses a group of metabolic conditions embracing the dysfunction and failure of various tissues and organs, including bone. Sustained bone alterations seem to result from anabolic, rather than catabolic processes, and suggest a decreased osteoblastic recruitment and activity. Current knowledge on the cellular and molecular mechanisms were provided by studies performed with osteogenic populations cultured in diabetic-simulated conditions, and osteogenic-induced precursor populations harvested from diabetic animals, sustaining an impaired cellular behavior in terms of osteogenic responsiveness and function. However, the reasons leaning to this impairment remain essentially unknown, as the priming capability and functionality of undifferentiated precursors, developed within the diabetic environment, have not been addressed. Accordingly, this work aims to evaluate the functionality and osteogenic priming capability of bone marrow-derived mesenchymal stem cells (MSCs), harvested from animals with experimental diabetes, and grown in the absence of any given differentiation factor. MSCs developed within a diabetic microenvironment displayed an impaired behavior, with diminished cell viability and proliferation, altered cytoskeleton organization, impaired osteogenic priming, and increased adipogenic activation. Further, the osteogenic induction of diabetic MSCs resulted in an impaired osteogenic commitment. The modified cell phenotype may be related, at least in part, with altered activity of ERK WNT and p38 signaling pathways in diabetic-derived cultures. Specific strategies, aiming the modulation of the verified hindrances, may be of therapeutic value to enhance the functionality of diabetic MSCs and sustain an improved outcome in the metabolism and regeneration of the bone tissue in diabetic conditions.

Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix.

Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.

An in vitro bone tissue regeneration strategy combining chondrogenic and vascular priming enhances the mineralization potential of mesenchymal stem cells in vitro while also allowing for vessel formation.

Chondrogenic priming (CP) of mesenchymal stem cells (MSCs) and coculture of MSCs with human umbilical vein endothelial stem cells (HUVECs) both have been shown to significantly increase the potential for MSCs to undergo osteogenic differentiation and mineralization in vitro and in vivo. Such strategies mimic cartilage template formation or vascularization that occur during endochondral ossification during early fetal development. However, although both chondrogenesis and vascularization are crucial precursors for bone formation by endochondral ossification, no in vitro bone tissue regeneration strategy has sought to incorporate both events simultaneously. The objective of this study is to develop an in vitro bone regeneration strategy that mimics critical aspects of the endochondral ossification process, specifically (1) the formation of a cartilage template and (2) subsequent vascularization of this template. We initially prime the MSCs with chondrogenic growth factors, to ensure the production of a cartilage template, and subsequently implement a coculture strategy involving MSC and HUVECs. Three experimental groups were compared; (1) CP for 21 days with no addition of cells; (2) CP for 21 days followed by coculture of HUVECs (250,000 cells); (3) CP for 21 days followed by coculture of HUVECs and MSCs (250,000 cells) at a ratio of 1:1. Each group was cultured for a further 21 days in osteogenic media after the initial CP period. Biochemical (DNA, Alkaline Phosphatase Activity, Calcium, and Vessel Endothelial Growth Factor) and histological analyses (Alcian blue, alizarin red, CD31(+), and collagen type X) were performed 1, 2, and 3 weeks after the media switch. The results of this study show that CP provides a cartilage-like template that provides a suitable platform for HUVEC and MSC cells to attach, proliferate, and infiltrate for up to 3 weeks. More importantly we show that the use of the coculture methodology, rudimentary vessels are formed within this cartilage template and enhanced the mineralization potential of MSCs. Taken together these results indicate for the first time that the application of both chondrogenic and vascular priming of MSCs enhances the mineralization potential of MSCs in vitro while also allowing the formation of immature vessels.

Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.

Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies.

Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

Systemic arterial hypertension (SAH), a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs), which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2) increases their therapeutic effects in spontaneously hypertensive rats (SHRs). Adult female SHRs were administered an intraperitoneal injection of vehicle solution (n = 10), MSCs cultured in conventional medium (DMEM plus 10% FBS, n = 11), or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n = 10). Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days) in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH.

Dietary Calcium Deficiency and Excess Both Impact Bone Development and Mesenchymal Stem Cell Lineage Priming in Neonatal Piglets

Background: Optimizing calcium nutrition to maximize bone accretion during growth to prevent fragility fractures later in life has spurred greater interest in calcium nutrition in neonates.Objective: The aim of this study was to determine the effect of dietary calcium, from deficiency through excess, on bone growth, and the in vivo and in vitro behavior of mesenchymal stem cells (MSCs) in neonatal pigs.Methods: Twenty-four male and female piglets (24 ± 6 h old) were fed either a calcium-deficient [Ca-D; 0.6% Ca on a dry matter (DM) basis], a calcium-adequate diet (Ca-A; 0.9% Ca on a DM basis), or a calcium-excessive diet (Ca-E; 1.3% Ca on a DM basis) for 14 d to assess the impact of dietary calcium on calcium homeostasis and on the behavior of MSCs.Results: Growth rate was not affected by the Ca-E diet, although bone ash content was 16% higher (P < 0.05) and urinary calcium excretion was 5-fold higher, when normalized to creatinine, compared with the Ca-A group at trial completion. Serum parathyroid hormone (PTH) concentrations were elevated (P < 0.05) in Ca-D piglets in comparison with other groups at both 7 and 14 d. In vivo proliferation of MSCs was 30% higher (P < 0.05) in Ca-E piglets than the other groups. MSCs from both Ca-D– and Ca-E–fed piglets had greater adipogenic potential based on increased gene expression (P < 0.05) of peroxisome proliferator-activated receptor γ (Pparg) and adipocyte fatty acid–binding protein (Ap2) than MSCs from Ca-A piglets. Interestingly, only MSCs from Ca-E–fed piglets had greater (P < 0.05) gene expression of lipoprotein lipase (Lpl) during adipocytic differentiation than those from Ca-A piglets. To assess alterations in lineage allocation and priming, the most and least osteogenic (O+ and O−, respectively) and adipogenic (A+ and A−, respectively) colonies from each MSC isolation were selected on the basis of functional staining. The O+ colonies from Ca-D piglets expressed lower (P < 0.05) levels of osteocalcin (OC) mRNA than did those from other groups, whereas the O− colonies from Ca-E piglets expressed higher (P < 0.05) levels of mRNA of Pparg, Ap2, and Lpl than did those from other groups.Conclusions: Neonatal calcium deficiency appears to reduce the osteogenic priming of MSCs while enlarging a subpopulation of potentially adipogenic cells, and excess dietary calcium appears to allow greater multipotency of MSCs. These programming alterations of MSCs could have long-term consequences for bone health.

Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

BackgroundMesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.ResultsUC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.ConclusionAlthough the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0063-9) contains supplementary material, which is available to authorized users.

Leptin signaling is required for augmented therapeutic properties of mesenchymal stem cells conferred by hypoxia preconditioning.

Hypoxia preconditioning enhances the therapeutic effect of mesenchymal stem cells (MSCs). However, the mechanism underlying hypoxia-induced augmentation of the protective effect of MSCs on myocardial infarction (MI) is poorly understood. We show that hypoxia-enhanced survival, mobility, and protection of cocultured cardiomyocytes were paralleled by increased expression of leptin and cell surface receptor CXCR4. The enhanced activities were abolished by either knockdown of leptin with a selective shRNA or by genetic deficiency of leptin or its receptor in MSCs derived, respectively, from ob/ob or db/db mice. To characterize the role of leptin in the regulation of MSC functions by hypoxia and its possible contribution to enhanced therapeutic efficacy, cell therapy using MSCs derived from wild-type, ob/ob, or db/db mice was implemented in mouse models of acute MI. Augmented protection by hypoxia pretreatment was only seen with MSCs from wild-type mice. Parameters that were differentially affected by hypoxia pretreatment included MSC engraftment, c-Kit(+) cell recruitment to the infarct, vascular density, infarct size, and long-term contractile function. These data show that leptin signaling is an early and essential step for the enhanced survival, chemotaxis, and therapeutic properties of MSCs conferred by preculture under hypoxia. Leptin may play a physiological role in priming MSCs resident in the bone marrow endosteum for optimal response to systemic signaling molecules and subsequent tissue repair.

Enamel Matrix Derivative has No Effect on the Chondrogenic Differentiation of Mesenchymal Stem Cells.

Background: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation.Methods: MSCs were chondrogenically differentiated in 2.0 × 105 cell pellets in medium supplemented with TGFβ3 in the absence or presence of 1, 10, or 100 μg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed.Results: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed.Conclusion: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.