Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/β-catenin signaling

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/β-catenin signaling pathway as indicated by the increased stability and nuclear localization of β-catenin in TIMP-1–deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced β-catenin transcriptional activity, determined by Wnt/β-catenin target gene expression analysis and a luciferase-based β-catenin– activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on β-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1–mediated effects on Wnt/β-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of β-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1–depleted hMSCs and demonstrably reduced axin 2, an antagonist of β-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/β-catenin activity

OXPHOS Supercomplexes as a Hallmark of the Mitochondrial Phenotype of Adipogenic Differentiated Human MSCs

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

INTRODUCTION: Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. METHODS: Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. RESULTS: Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH(+) cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH(-) cells. GD2(+) cells showed a 3.9-fold greater capacity than GD2(-) cells. ALDH(+)/GD2(+)cells displayed 12.8-fold greater mammosphere forming ability than ALDH(-)/GD2(-) cells. In vivo, the tumor-initiating frequency of ALDH(+)/GD2(+) cells were up to 33-fold higher than that of ALDH(+) cells, with as few as 50 ALDH(+)/GD2(+) cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH(+)/GD2(+) cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH(+) or ALDH(+)/GD2(+) cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes. CONCLUSIONS: Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH(+) and ALDH(+)/GD2(+) subpopulations

Human Sclera Maintains Common Characteristics with Cartilage throughout Evolution

BACKGROUND: The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. METHODOLOGY/PRINCIPAL FINDINGS: We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-betas and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human 'sclera'-derived cells can be considered chondrocytes when cultured ex vivo. CONCLUSIONS/SIGNIFICANCE: Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia

Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy

BACKGROUND:Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called "mesenchymal stem cells" (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term "MSC" has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source. METHODOLOGY/PRINCIPAL FINDINGS:Here we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization. CONCLUSIONS/SIGNIFICANCE:Analysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached

Mycoplasma Contamination Revisited: Mesenchymal Stromal Cells Harboring Mycoplasma hyorhinis Potently Inhibit Lymphocyte Proliferation In Vitro

Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination

Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury

The thalamus and its subnuclei—a gateway to obsessive-compulsive disorder

Larger thalamic volume has been found in children with obsessive-compulsive disorder (OCD) and children with clinical-level symptoms within the general population. Particular thalamic subregions may drive these differences. The ENIGMA-OCD working group conducted mega- and meta-analyses to study thalamic subregional volume in OCD across the lifespan. Structural T-1-weighted brain magnetic resonance imaging (MRI) scans from 2649 OCD patients and 2774 healthy controls across 29 sites (50 datasets) were processed using the FreeSurfer built-in ThalamicNuclei pipeline to extract five thalamic subregions. Volume measures were harmonized for site effects using ComBat before running separate multiple linear regression models for children, adolescents, and adults to estimate volumetric group differences. All analyses were pre-registered (https://osf.io/73dvy) and adjusted for age, sex and intracranial volume. Unmedicated pediatric OCD patients (<12 years) had larger lateral (d = 0.46), pulvinar (d = 0.33), ventral (d = 0.35) and whole thalamus (d = 0.40) volumes at unadjusted p-values <0.05. Adolescent patients showed no volumetric differences. Adult OCD patients compared with controls had smaller volumes across all subregions (anterior, lateral, pulvinar, medial, and ventral) and smaller whole thalamic volume (d = -0.15 to -0.07) after multiple comparisons correction, mostly driven by medicated patients and associated with symptom severity. The anterior thalamus was also significantly smaller in patients after adjusting for thalamus size. Our results suggest that OCD-related thalamic volume differences are global and not driven by particular subregions and that the direction of effects are driven by both age and medication status

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFβ) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFβ-induced EMT is dependent on type-1 TGFβ receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair

Stem Cell Therapy: Pieces of the Puzzle

Acute ischemic injury and chronic cardiomyopathies can cause irreversible loss of cardiac tissue leading to heart failure. Cellular therapy offers a new paradigm for treatment of heart disease. Stem cell therapies in animal models show that transplantation of various cell preparations improves ventricular function after injury. The first clinical trials in patients produced some encouraging results, despite limited evidence for the long-term survival of transplanted cells. Ongoing research at the bench and the bedside aims to compare sources of donor cells, test methods of cell delivery, improve myocardial homing, bolster cell survival, and promote cardiomyocyte differentiation. This article reviews progress toward these goals