Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes

Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (α4, α7 and β5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is responsible for the differentiation of MSCs into chondrocytes

miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

Abstract Background: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions: miR-143 is as a new target for prostate cancer treatment

Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells

Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin

Targeted delivery to inflammatory monocytes for efficient RNAi-mediated immuno-intervention in auto-immune arthritis

Poster presentationInternational audienc

Développement d'Immunothérapies anti-inflammatoires de la polyarthrite rhumatoïde par ARN interférence dans un modèle murin d'arthrite

La polyarthrite rhumatoïde (PR) est le plus fréquent des rhumatismes inflammatoires et représente un problème de santé publique majeur. A l'heure actuelle, les biothérapies anti-TNF sous forme de protéines recombinantes constituent une avancée considérable dans le traitement de la polyarthrite rhumatoïde (PR). Néanmoins, il convient de développer des approches thérapeutiques alternatives pour traiter les 40% de patients non-répondeurs ainsi queceux qui échappent à plusieurs années de traitement. La recherche de nouvelles cibles thérapeutiques est indispensable pour proposer des approches alternatives à ces biothérapies. Par ailleurs, les techniques de transfert de gène offrent une alternative thérapeutique possible pour pallier aux limitations des biothérapies actuelles, à condition de les adapter aux contraintes du tissu cible de la PR, les articulations. Les projets ont consisté à développer et valider dans des modèles expérimentaux d'arthrite de nouvelles stratégies anti-inflammatoires basées sur l'utilisation de l'ARN interférence comme outil thérapeutique. En effet, la possibilité d'interférer au niveau des mécanismes responsables de l'expression des protéines,la régulation de la stabilité des ARNm et de l'efficacité de la machinerie traductionnelle, présente un intérêt thérapeutique supérieur aux biothérapies actuelles basées sur l'inhibition des protéines sécrétées (anticorps ou récepteurs solubles) mais nécessite cependant de posséder un vecteur qui transduit efficacement les cellules productrices de la molécule ciblée.Rheumatoid arthritis (RA) is the most frequent chronic inflammatory systemicautoimmune disease that remains a major medical challenge as the exact causes of the disease are not completely elucidated. The principal treatment strategies arebased on the inhibition of TNF-a, one of the major inflammatory cytokine in RA.Although risk and benefit analyses are in favour of the use of monoclonal antibodiesagainst TNF-a, the most currently used biotherapy, they are not devoid from multipleside effects. The search for new therapeutic targets is essential to proposealternative approaches to non responders to such biotherapies. The possibility to interfere in the mechanisms responsible for regulating mRNA stability andeffectiveness of the translational machinery also present a therapeutic benefitsuperior to current biologic therapies based on inhibition secreted proteins(antibodies or soluble receptors). Such approach however requires developingvectors that efficiently transduced the specific cell type producing the targeted gene.Projects of my PhD fellowship have included both the development of gene therapyvehicles for RNAi-based intervention in experimental mouse models of arthritis andevaluation of novel candidate genes for alternative anti-inflammatory therapy in RA.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

MicroRNAs in juvenile idiopathic arthritis: can we learn more about pathophysiological mechanisms?

International audienceJuvenile idiopathic arthritis (JIA) is a heterogeneous and multifactorial group of chronic arthritis with an onset before the age of 16 years. The pathogenesis of this disease is poorly understood, which makes the distinction among subtypes unclear, delays diagnosis and optimal therapeutic management. MicroRNAs (miRNAs) are small non-coding RNAs that play a critical role in the regulation of immune responses. Their expression is tightly controlled to ensure cellular homeostasis and function of innate and adaptive immune cells. Abnormal expression of miRNAs has been associated with the development of many inflammatory and autoimmune diseases. In this review, we gather results published on miRNAs expression profiles in JIA patients with the aim to identify miRNAs that can be used as diagnostic biomarkers and provide information on disease activity and progression. We also focus on miRNAs deregulated in different forms of JIA to shed light on common pathways potentially involved in disease pathophysiology

CRISPR-Cas9: A revolution in genome editing in rheumatic diseases

International audienc

Mitochondrial MicroRNAs Contribute to Macrophage Immune Functions Including Differentiation, Polarization, and Activation

International audienceA subset of microRNA (miRNA) has been shown to play an important role in mitochondrial (mt) functions and are named MitomiR. They are present within or associated with mitochondria. Most of the mitochondrial miRNAs originate from the nucleus, while a very limited number is encoded by mtDNA. Moreover, the miRNA machinery including the Dicer and Argonaute has also been detected within mitochondria. Recent, literature has established a close relationship between miRNAs and inflammation. Indeed, specific miRNA signatures are associated with macrophage differentiation, polarization and functions. Nevertheless, the regulation of macrophage inflammatory pathways governed specifically by MitomiR and their implication in immune-mediated inflammatory disorders remain poorly studied. Here, we propose a hypothesis in which MitomiR play a key role in triggering macrophage differentiation and modulating their downstream activation and immune functions. We sustain this proposition by bioinformatic data obtained from either the human monocytic THP1 cell line or the purified mitochondrial fraction of PMA-induced human macrophages. Interestingly, 22% of the 754 assayed miRNAs were detected in the mitochondrial fraction and are either exclusively or highly enriched cellular miRNA. Furthermore, the in silico analysis performed in this study, identified a specific MitomiR signature associated with macrophage differentiation that was correlated with gene targets within the mitochondria genome or with mitochondrial pathways. Overall, our hypothesis and data suggest a previously unrecognized link between MitomiR and macrophage function and fate. We also suggest that the MitomiR-dependent control could be further enhanced through the transfer of mitochondria from donor to target cells, as a new strategy for MitomiR delivery