Activity Of Mesenchymal Cells Research Articles

C-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

Mammalian target of rapamycin complex 2 (mTORC2) has been shown to regulate mTORC1/4E-BP1/eIF4E signaling and collagen I expression in mesenchymal cells (MCs) during fibrotic activation. Here we investigated the regulation of the mTORC2 binding partner mammalian stress-activated protein kinase-interacting protein 1 (mSin1) in MCs derived from human lung allografts and identified a novel role for mSin1 during fibrosis. mSin1 was identified as a common downstream target of key fibrotic pathways, and its expression was increased in MCs in response to pro-fibrotic mediators: lysophosphatidic acid (LPA), transforming growth factor β, and interleukin 13. Fibrotic MCs had higher mSin1 protein levels than nonfibrotic MCs, and siRNA-mediated silencing of mSIN1 inhibited collagen I expression and mTORC1/2 activity in these cells. Autocrine LPA signaling contributed to constitutive up-regulation of mSin1 in fibrotic MCs, and mSin1 was decreased because of LPA receptor 1 siRNA treatment. We identified c-Jun N-terminal kinase (JNK) as a key intermediary in mSin1 up-regulation by the pro-fibrotic mediators, as pharmacological and siRNA-mediated inhibition of JNK prevented the LPA-induced mSin1 increase. Proteasomal inhibition rescued mSin1 levels after JNK inhibition in LPA-treated MCs, and the decrease in mSin1 ubiquitination in response to LPA was counteracted by JNK inhibitors. Constitutive JNK1 overexpression induced mSin1 expression and could drive mTORC2 and mTORC1 activation and collagen I expression in nonfibrotic MCs, effects that were reversed by siRNA-mediated mSIN1 silencing. These results indicate that LPA stabilizes mSin1 protein expression via JNK signaling by blocking its proteasomal degradation and identify the LPA/JNK/mSin1/mTORC/collagen I pathway as critical for fibrotic activation of MCs.

Lineage-Specific Wnt Reporter Elucidates Mesenchymal Wnt Signaling during Bone Repair

β-Catenin-dependent Wnt signaling controls numerous aspects of skeletal development and postnatal bone repair. Currently available transgenic Wnt reporter mice allow for visualization of global canonical Wnt signaling activity within skeletal tissues, without delineation of cell type. This is particularly important in a bone repair context, in which the inflammatory phase can obscure the visualization of mesenchymal cell types of interest. To tackle the issue of tissue-specific Wnt signaling, we have generated and characterized a transgenic mouse strain [termed paired related homeobox 1 (Prx1)-Wnt-green fluorescent protein (GFP), by crossing a previously validated Prx1-Cre strain with a nuclear fluorescent reporter driven by T-cell factor/lymphoid enhancer factor activity (Rosa26-Tcf/Lef-LSL-H2B-GFP)]. Prx1-Wnt-GFP animals were subject to three models of long bone and membranous bone repair (displaced forelimb fracture, tibial cortical defect, and frontal bone defect). Results showed that, irrespective of bone type, locoregional mesenchymal cell activation of Wnt signaling occurs in a defined temporospatial pattern among Prx1-Wnt-GFP mice. In summary, Prx1-Wnt-GFP reporter animals allow for improved visualization, spatial discrimination, and facile quantification of Wnt-activated mesenchymal cells within models of adult bone repair.

Endothelial Cells Modulate Differentiation Potential and Mobility of Mesenchymal Stromal Cells.

We studied the effect of endothelial cells on in vitro migration and differentiation potential of multipotent mesenchymal stromal cells. Down-regulation of stemness genes OCT4, SOX2, and chondrogenic differentiation regulator SOX9 gene and upregulation of osteogenesis master-gene RUNX2 in mesenchymal stromal cells were observed in the presence of intact and TNFα-activated endothelial cells, which indicated an increase in commitment of mesenchymal stromal cells.The medium conditioned by endothelial cells stimulated migration activity of mesenchymal stromal cells; migration rate increased significantly in conditioned medium from activated cells in comparison with medium from non-activated cells. It was concluded that the interaction with endothelial cells modulated functional activity of mesenchymal stromal cells; moreover, activated endothelial cells produced more pronounced effects on differentiation potential and migration activity of mesenchymal stromal cells both in direct contact and through paracrine regulation.

Suppressive effect of mesenchymal stromal cells on interferon-γ-producing capability of spleen cells was specifically enhanced through humoral mediator(s) from mouse oral squamous cell carcinoma Sq-1979 Cells In Vitro

Aim: The aim of this study was to compare the immunomodulatory effects in the tumor milieu of mouse oral squamous cell carcinoma (OSCC) cells harboring primary and advanced phenotypes. We established an in vitro co-culture system using mouse OSCC cells, spleen cells, and mesenchymal stromal cells. Methods: Sq-1979 is an OSCC cell line derived from C3H mice; 233-11 cells were established from a primary Sq-1979 tumor; L3–5, L5–11, and L6–8 cells were established from lymph node-metastasized Sq-1979 cells. 10T1/2 is a fibroblast line derived from C3H mice. The OSCC cells were co-cultured with anti-CD3 antibody-stimulated mouse spleen cells in the presence or absence of 10T1/2 cells, and the producing capability of interferon (IFN)-γ and interleukin (IL)-10 was evaluated using enzyme-linked immunosorbent assay. Results: The production of IFN-γ by the stimulated spleen cells was specifically enhanced in the presence of co-cultured L-cells. The production of IL-10 was conversely reduced in the co-culture with all the OSCC cell lines used. The production of IFN-γ was significantly reduced in the co-culture with directly contacted 10T1/2 cells, and further reduction was observed in the presence of Sq-1979 and 233-11 cells but remained unchanged in the presence of L-cells. The reduction of IFN-γ production was also observed by the addition of conditioned medium from the Sq-1979-1 cells. Conclusion: Sq-1979 cells specifically enhanced the immune-suppressive activity of mesenchymal stromal cells through humoral factor (s).

Impact of collagen-alginate composition from microbead morphological properties to microencapsulated canine adipose tissue-derived mesenchymal stem cell activities

The purpose of this study was to identify the effect of collagen-alginate composition on the size and shape of microbeads and the proliferation and osteogenic properties of microencapsulated canine adipose-derived mesenchymal stem cells (ASCs) in vitro. Canine ASCs were microencapsulated in mixtures of various collagen-alginate compositions using a vibrational technologic encapsulator. The size and shape of the resultant microbeads were measured using a light field microscope and the viability of the microencapsulated canine ASCs was evaluated using a live/dead viability/cytotoxicity kit. Proliferation and osteogenic potentials of microencapsulated canine ASCs were evaluated using an alamarBlue proliferation assay and an alkaline phosphatase assay, respectively. As the collagen ratio increased, the size and size variation of microbeads increased and the shape of microbeads became more irregular. Nonetheless, homogeneous microbeads were created with no significant difference in size and shape, in the range of 0.75% alginate mixed with 0.099% collagen solution in 1.2% alginate solution. There were no significant differences in viability of the ASCs in the various collagen-alginate compositions. Both proliferation and osteogenic properties, in vitro, increased with increasing collagen ratio. Microencapsulation of canine ASCs with appropriate collagen-alginate composition increases cell proliferation and osteogenic properties, in vitro, without significant effects on the shape and size of microbeads and cell viability. Microencapsulation with adequate collagen-alginate composition may produce injectable microbeads that could enhance the therapeutic efficacy of stem cells.

Enhanced cytocompatibility and osteoinductive properties of sol-gel-derived silica/zirconium dioxide coatings by metformin functionalization.

The aim of this study was to evaluate the pro-osteogenic properties of sol-gel-derived silica/zirconium dioxide coatings functionalized with 1 mM of metformin. The matrices were applied on 316L stainless steel using dip-coating technique. First of all, physicochemical properties of biomaterials were evaluated. Surface morphology and topography was determined using energy-dispersive X-ray spectroscopy and atomic force microscopy. The chemical composition was evaluated using Fourier transform infrared spectroscopy. Further, wettability and surface free energy were characterized. Cytocompatibility of biomaterials was tested invitro using model of human multipotent mesenchymal stromal cells isolated from adipose tissue. The influence of biomaterials on cells morphology and proliferation was determined. Osteogenic effect of obtained biomaterials was evaluated in terms of their influence on secretory activity of human multipotent mesenchymal stromal cells isolated from adipose tissue and matrix mineralization. Analysis was performed in relation to the control cultures i.e. maintained on pure SS316L substrate and SS316L covered with silica/zirconium dioxide. Obtained results indicate that silica/zirconium dioxide_metformin coatings ameliorated metabolic and proliferative activity of human multipotent mesenchymal stromal cells isolated from adipose tissue, as well as promoted their proper growth and adhesion. The human multipotent mesenchymal stromal cells isolated from adipose tissue cultured on biomaterials were characterized by typical fibroblast-like morphology. The addition of metformin to the silica/zirconium dioxide coatings improved functional differentiation of human multipotent mesenchymal stromal cells isolated from adipose tissue. Osteogenic cultures on silica/zirconium dioxide_metformin were characterized by formation of well-developed osteonodules rich in calcium and phosphorous. Moreover, human multipotent mesenchymal stromal cells isolated from adipose tissue cultured on silica/zirconium dioxide_metformin synthesized increased amount of alkaline phosphatase, bone morphogenetic protein 2 and osteopontin, both on messenger RNA and protein level. Obtained biomaterials modulate cellular plasticity of human multipotent mesenchymal stromal cells isolated from adipose tissue promoting their osteogenic differentiation, thus may find application in broadly defined tissue engineering.

Mesenchymal stromal cells modulate gut inflammation in experimental colitis.

Inflammatory bowel diseases (IBDs)affect millions of people worldwide and their frequencies in developed countries have increased since the twentieth century. In this context, there is an intensive search for therapies that modulate inflammation and provide tissue regeneration in IBDs. Recently, the immunomodulatory activity of adipose tissue-derived mesenchymal stromal cells (ADMSCs) has been demonstrated to play an important role on several immune cells in different conditions of inflammatory and autoimmune diseases. In this study, we explored the immunomodulatory potential of ADMSC in a classical model of DSS-induced colitis. First, we found that treatment of mice with ADMSC ameliorated the severity of DSS-induced colitis, reducing colitis pathological score and preventing colon shortening. Moreover, a prominent reduction of pro-inflammatory cytokines levels (i.e., IFN-γ, TNF-α, IL-6 and MCP-1) was observed in the colon of animals treated with ADMSC. We also observed a significant reduction in the frequencies of macrophages (F4/80+CD11b+) and dendritic cells (CD11c+CD103+) in the intestinal lamina propria of ADMSC-treated mice. Finally, we detected the up-regulation of immunoregulatory-associated molecules in intestine of mice treated with ADMSCs (i.e., elevated arginase-1 and IL-10). Thus, this present study demonstrated that ADMSC modulates the overall gut inflammation (cell activation and recruitment) in experimental colitis, providing support to the further development of new strategies in the treatment of intestinal diseases.

Morphological characterization of ckd in cats: Insights of fibrogenesis to be recognized.

Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis and its pathogenesis is associated with the activity of mesenchymal cells (fibroblasts), being essentially characterized by a process of excessive accumulation resulting from the deposition of extracellular matrix components. The aim of this study was to characterize the morphological presentation of chronic and fibrotic lesions in the glomerular, tubular, interstitial, and vascular compartments in feline CKD, as well as the possible participation of myofibroblasts in renal fibrotic processes in this species. Cat kidneys were collected and processed according to the conventional techniques for light microscopy, circular polarization, immunohistochemistry, and electron microscopy. Fibrotic alterations were present in all compartments analyzed. The main findings in the glomerular compartment were different degrees of glomerular sclerosis, synechia formation, Bowman's capsule calcification, in addition to glomerular basement membrane thickening and pericapsular fibrosis. The tubulointerstitial compartment had intense tubular degeneration and the immunostaining in tubular cells for mesenchymal cell markers demonstrated the possibility of mesenchymal epithelial transition and consequent involvement of myofibroblasts in the development of interstitial tubule damage. Infiltration of inflammatory cells, added to vessel thickening and fibrosis, demonstrated the severity and role of inflammation in the development and perpetuation of damage. Thus, we may conclude that fibrotic lesions play a relevant role in feline CKD and the mechanism of perpetuation of these lesions need further elucidation regarding the origin and participation of myofibroblasts and consequent mesenchymal epithelial transition in this species.

Role of aryl hydrocarbon receptor in mesenchymal stromal cell activation: A minireview.

Mesenchymal stromal cells (MSCs) possess great therapeutic advantages due to their ability to produce a diverse array of trophic/growth factors related to cytoprotection and immunoregulation. MSC activation via specific receptors is a crucial event for these cells to exert their immunosuppressive response. The aryl-hydrocarbon receptor (AhR) is a sensitive molecule for external signals and it is expressed in MSCs and, upon positive activation, may potentially regulate the MSC-associated immunomodulatory function. Consequently, signalling pathways linked to AhR activation can elucidate some of the molecular cascades involved in MSC-mediated immunosuppression. In this minireview, we have noted some important findings concerning MSC regulation via AhR, highlighting that its activation is associated with improvement in migration and immunoregulation, as well as an increase in pro-regenerative potential. Thus, AhR-mediated MSC activation can contribute to new perspectives on MSC-based therapies, particularly those directed at immune-associated disorders.

Targeting subchondral bone mesenchymal stem cell activities for intrinsic joint repair in osteoarthritis.

Osteoarthritis (OA) is a common age-related disease with complex pathophysiology. It is characterized by wide-ranging tissue damage and ultimate biomechanical failure of the whole joint. However, signs of tissue adaptation and attempted repair responses are evident in OA-affected osteochondral tissues. Highlighted in this review article is the role of bone-resident mesenchymal stem cells (MSCs) in these bone remodeling responses, and a proposal that targeting MSC activities in OA subchondral bone could represent a novel approach for intrinsic joint regeneration in OA. The development of these therapies will require better understanding of MSC proliferation, migration and differentiation patterns in relation to OA tissue damage and further clarification of the molecular signaling events in these MSCs during disease progression.

Pathogenesis of systemic sclerosis: recent insights of molecular and cellular mechanisms and therapeutic opportunities

Systemic sclerosis (SSc) is a complex disease characterized by early microvascular abnormalities, immune dysregulation and chronic inflammation, and subsequent fibrosis of the skin and internal organs. Excessive fibrosis, distinguishing hallmark of SSc, is the end result of a complex series of interlinked vascular injury and immune activation, and represents a maladaptive repair process. Activated vascular, epithelial, and immune cells generate pro-fibrotic cytokines, chemokines, growth factors, lipid mediators, autoantibodies, and reactive oxygen species. These paracrine and autocrine cues in turn induce activation, differentiation, and survival of mesenchymal cells, ensuing tissue fibrosis through increased collagen synthesis, matrix deposition, tissue rigidity and remodeling, and vascular rarefaction. This review features recent insights of the pathogenic process of SSc, highlighting three major characteristics of SSc, microvasculopathy, excessive fibrosis, and immune dysregulation, and sheds new light on the understanding of molecular and cellular mechanisms contributing to the pathogenesis of SSc and providing novel avenues for targeted therapies.

Mesenchymal Stromal Cells: What Is the Mechanism in Acute Graft-Versus-Host Disease?

After more than a decade of preclinical and clinical development, therapeutic infusion of mesenchymal stromal cells is now a leading investigational strategy for the treatment of acute graft-versus-host disease (GVHD). While their clinical use continues to expand, it is still unknown which of their immunomodulatory properties contributes most to their therapeutic activity. Herein we describe the proposed mechanisms, focusing on the inhibitory activity of mesenchymal stromal cells (MSCs) at immunologic checkpoints. A deeper understanding of the mechanism of action will allow us to design more effective treatment strategies.

An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles.

The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

Silicon nitride surface chemistry: A potent regulator of mesenchymal progenitor cell activity in bone formation

Polycrystalline silicon nitride (Si3N4), sintered with the addition of minor fractions of yttrium and aluminum oxides (i.e., Y2O3 and Al2O3), possesses uniquely adjustable surface chemistry that results in improved cell metabolism and enhanced bone formation. Building upon previous in vitro mineralization studies using osteosarcoma cells, this study examined interactions between various chemically modulated Si3N4 surfaces and murine mesenchymal progenitor cells (KUSA-A1). It was discovered that various pressurized thermal-treatments coupled with adiabatic and non-adiabatic cooling of sintered Si3N4 samples resulted in partial or full coverage of their surfaces with different Si–Y–O–N compounds. Full coverage by mostly yttrium silicate (β-Y2Si2O7) was obtained by non-adiabatic cooling, whereas partial coverage with N-apatite (Y10(SiO4)6N2) occurred under adiabatic conditions. These peculiar phases were found to be particularly efficient in stimulating the in vitro differentiation of KUSA-A1 cells into osteoblasts, although according to different microscopic mechanisms. The final amount of bone formation was nearly identical for both phases. Cell differentiation was monitored by assessing the concentration of the osteogenic marker γ-carboxyglutamate (i.e., Gla-osteocalcin). It was found to be ∼45% higher for Si3N4 samples possessing the N-apatite phase than for biomedical titanium alloy controls tested under exactly the same conditions. Concurrent measurements of the bone resorption marker Glu-osteocalcin (i.e., an undercarboxylated form of γ-carboxyglutamate) showed significant inhibition of osteoclastogenesis on these surface-treated Si3N4 samples as compared to the controls. Bone formation was assessed using in situ Raman microprobe spectroscopy and ex situ laser microscopy. These two independent analytical techniques consistently found an increase of ∼80% in expressed hydroxyapatite when compared to a biomedical titanium alloy. This study suggests that surface-treated Si3N4 may have a powerful anabolic, differentiating, and antiapoptotic effect on osteoblasts in vitro, and a concurrent inhibitive action on osteoclastogenesis. Given additional research, Si3N4 may represent a new therapeutic solution for bone disorders and for engineered implants that physiologically regulate bone growth processes.

Cavin-2 Functions as a Suppressive Regulator in TNF-induced Mesenchymal Stromal Cell Inflammation and Angiogenic Phenotypes.

Tumour necrosis factor (TNF)-α activation of mesenchymal stromal cells (MSC) enhances their tumour-suppressive properties and tumour-homing ability. The molecular actors involved are unknown. We found that TNF induced MSC migration and tubulogenesis which correlated with a dose-dependent increase in Cavin-1 and Cavin-3 transcript levels. TNF triggered cyclooxygenase (COX)-2 expression, whereas specific siRNA-mediated gene silencing of Cavin-2 resulted in an amplified COX-2 expression, tubulogenesis, and migratory response partially due to a rapid and sustained increase in NF-κB phosphorylation status. Our results highlight a suppressive role for the caveolar component Cavin-2 in the angiogenic and inflammatory regulation of TNF-activated MSC.

Influence of initial biomechanical environment provided by fibrous composite intramedullary nails on bone fracture healing

Long bone fractures are primarily treated with internal fixation devices, and intramedullary (IM) nails are the most commonly used. Reamed and unreamed surgical procedures are commonly used to attach IM nails to fractured bones. It is believed that the use of flexible composites contributes to successful fracture healing because of the desirable initial interfragmentary movement (IFM) provided by the material. This finite element study was conducted to understand the influence of initial IFM on the healing process by using various IM nail materials (stainless steel, carbon/epoxy composites (WSN3k [0]2nT, WSN3k [±30]nT) and glass/polyprophylene (Twintex [0]2nT) composite). Reamed and unreamed IM nails were used to investigate the effects of mesenchymal cell activities, deviatoric strain, and body fluid flow in calluses on bone healing. The results showed that unreamed IM nails promoted healing because of a 40% increase in initial cells supplied to the central callus, compared to reamed nails. The most suitable initial interfragmentary strains (IFS) (18.3% and 6.85%, reamed and unreamed) were provided by the flexible Twintex [0]2nT IM nail and this induced 38% and 40.33% healing performances by the reamed and unreamed IM nails, respectively.

310 - A novel method to asses the biological activity of mesenchymal stromal cells for kidney repair

310 - A novel method to asses the biological activity of mesenchymal stromal cells for kidney repair

Effect of Concentration of Collagen Gel on Functional Activity of Bone Marrow Mesenchymal Stromal Cells.

Collagen I gels with protein concentrations of 1, 2, and 3.5 mg/ml were prepared and embedded in a porous polylactide scaffold to reduce their contraction. Concentration of the gel did not affect its degradation. Collagen gels promoted the formation of cell networks. The cells in the collagen gel with a concentration of 1 mg/ml embedded in polylactide scaffold had elongated spindle-like shape, in contrast to flattened cells in collagen gel of the same concentration not embedded in the scaffold. Stabilization of the collagen gel in the polylactide scaffold promoted active synthesis of laminin and fibronectin by cells as soon as on day 5 of culturing in comparison with that in free collagen substrate.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

Bioengineered gold nanoparticles targeted to mesenchymal cells from patients with bronchiolitis obliterans syndrome does not rise the inflammatory response and can be safely inhaled by rodents

The use of gold nanoparticles (GNPs) as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long-term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favors epithelial–mesenchymal transition and activation of local mesenchymal cells (MCs) leading to airway fibro-obliteration. In a previous work, we engineered GNPs loaded with the mammalian target of rapamycin inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary MCs isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these GNPs (GNP-HCe) were able to specifically inhibit primary MCs without affecting the bronchial epithelial cell. In the present work, we investigated the effect of these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibrogenesis. In addition, we administered GNP-HCe by the inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. The present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are non-toxic.