Mitochondrial activity is central to tissue homeostasis. Mitochondria dysfunction constitutes a hallmark of many genetic diseases and plays a key role in tumor progression. The essential role of mitochondria, added to their recently documented capacity to transfer from cell to cell, obviously contributes to their current interest. However, determining the proper role of mitochondria in defined biological contexts was hampered by the lack of suitable experimental tools. We designed a protocol (MitoCeption) to directly and quantitatively transfer mitochondria, isolated from cell type A, to recipient cell type B. We validated and quantified the effective mitochondria transfer by imaging, fluorescence-activated cell sorting (FACS) and mitochondrial DNA analysis. We show that the transfer of minute amounts of mesenchymal stem/stromal cell (MSC) mitochondria to cancer cells, a process otherwise occurring naturally in coculture, results in cancer cell enhanced oxidative phosphorylation (OXPHOS) activity and favors cancer cell proliferation and invasion. The MitoCeption technique, which can be applied to different cell systems, will therefore be a method of choice to analyze the metabolic modifications induced by exogenous mitochondria in host cells.
The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or “grooves” were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed for cytology and biomarkers. At 10 weeks post-surgery, horses were euthanized for macroscopic and histologic joint evaluation. Arthroscopic grooving allowed precise and identical damage to the cartilage of all animals. Under the controlled exercise regime, this osteoarthritis groove model displayed significant radiographic, macroscopic, and microscopic degenerative and reactive changes. Histology demonstrated consistent surgically induced grooves limited to non-calcified cartilage and accompanied by secondary adjacent cartilage lesions, chondrocyte necrosis, chondrocyte clusters, cartilage matrix softening, fissuring, mild subchondral bone inflammation, edema, and osteoblastic margination. Synovial fluid biochemistry and cytology demonstrated significantly elevated total protein without an increase in prostaglandin E2, neutrophils, or chondrocytes. This equine metacarpophalangeal groove model demonstrated that standardized non-calcified cartilage damage accompanied by exercise triggered altered osteochondral morphology and cartilage degeneration with minimal or inefficient repair and little inflammatory response. This model, if validated, would allow for assessment of disease processes and the effects of therapy.
Application of mesenchymal stem/stromal cells (MSCs) in treating different disorders, in particular osteo-articular diseases, is currently under investigation. We have already documented the safety of administrating human adipose tissue-derived stromal MSCs (hASCs) in immunodeficient mice. In the present study, we investigated whether the persistence of MSC is affected by the degree of inflammation and related to the therapeutic effect in two inflammatory models of arthritis.
We used C57BL/6 or DBA/1 mice to develop collagenase-induced osteoarthritis (CIOA) or collagen-induced arthritis (CIA), respectively. Normal and diseased mice were administered 2.5×105 hASCs in the knee joints (IA) or 106 in the tail vein (IV). For CIA, clinical scores were monitored during the time course of the disease while for CIOA, OA scores were assessed by histology at euthanasia. Thirteen tissues were recovered at different time points and processed for real-time PCR and Alu sequence detection. Immunological analyses were performed at euthanasia. After IV infusion, no significant difference in the percentage of hASCs was quantified in the lungs of normal and CIA mice at day 1 while no cell was detected at day 10 taking into account the sensitivity of the assay, indicating that a high level of inflammation did not affect the persistence of cells. In CIOA mice, we reported the therapeutic efficacy of hASCs at reducing OA clinical scores at day 42 when hASCs were not detected in the joints. However, the percentage and distribution of hASCs were similar in osteoarthritic and normal mice at day 1 and 10 after implantation indicating that moderate inflammation does not alter hASC persistence in vivo.
While inflammatory signals are required for the immunosuppressive function of MSCs, they do not enhance their capacity to survive in vivo, as evaluated in two xenogeneic inflammatory pre-clinical models of arthritis.
Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.
Col-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.
Col-Treg clones are characterized by their specific cytokine profile (IL-10highIL-4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.
Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.
Although biologic therapies have changed the course of rheumatoid arthritis (RA), today's major challenge remains to identify biomarkers to target treatments to selected patient groups. Circulating micro(mi)RNAs represent a novel class of molecular biomarkers whose expression is altered in RA. Our study aimed at quantifying miR-125b in blood and serum samples from RA patients, comparing healthy controls and patients with other forms of rheumatic diseases and arthritis, and evaluating its predictive value as biomarker for response to rituximab. Detectable levels of miR-125b were measured in total blood and serum samples and were significantly elevated in RA patients compared to osteoarthritic and healthy donors. The increase was however also found in patients with other forms of chronic inflammatory arthritis. Importantly, high serum levels of miR-125b at disease flare were associated with good clinical response to treatment with rituximab three months later (P = 0.002). This predictive value was not limited to RA as it was also found in patients with B lymphomas. Our results identify circulating miR-125b as a novel miRNA over expressed in RA and suggest that serum level of miR-125b is potential predictive biomarker of response to rituximab treatment.
Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).
We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.
p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.
We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.
Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and bone tissue engineering given their ability to differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question about the identification of regulatory pathways determining the differentiation fate of MSCs into chondrocyte or osteoblast.
Chondrogenesis, osteoblastogenesis, and adipogenesis of human and mouse MSC were induced by using specific inductive culture conditions. Expression of promyelocytic leukemia zinc-finger (PLZF) or differentiation markers in MSCs was determined by RT-qPCR. PLZF-expressing MSC were implanted in a mouse osteochondral defect model and the neotissue was analyzed by routine histology and microcomputed tomography.
We found out that PLZF is not expressed in MSCs and its expression at early stages of MSC differentiation is the mark of their commitment toward the three main lineages. PLZF acts as an upstream regulator of both Sox9 and Runx2, and its overexpression in MSC enhances chondrogenesis and osteogenesis while it inhibits adipogenesis. In vivo, implantation of PLZF-expressing MSC in mice with full-thickness osteochondral defects resulted in the formation of a reparative tissue resembling cartilage and bone.
Our findings demonstrate that absence of PLZF is required for stemness maintenance and its expression is an early event at the onset of MSC commitment during the differentiation processes of the three main lineages.
Matching sets of human primary fibroblasts cocultured with placenta explants are used to compare tissue capacities to support trophoblast invasion. Substituting endometrium with dermis dramatically reduces EVCT interstitial invasion, a phenomenon related to the ECM fibronectin content, FN alternative splicing, and expression of the SR protein SRSF1.
Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA–) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.
Multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) are adult stem cells exhibiting functional properties that have opened the way for cell-based clinical therapies. MSCs have been reported to exhibit immunosuppressive as well as healing properties, improving angiogenesis and preventing apoptosis or fibrosis through the secretion of paracrine mediators. This review summarizes recent progress on the clinical application of stem cells therapy in some inflammatory and degenerative rheumatic diseases. To date, most of the available data have been obtained in preclinical models and clinical efficacy needs to be evaluated through controlled randomized double-blind trials.
mesenchymal stem cells; rheumatic diseases; osteoarthritis; rheumatoid arthritis; autoimmune diseases; immune tolerance
Mesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8+ cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4+ cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages.
MSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4+ T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the ‘conditioned’ subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4+ T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry.
MSCs were able to suppress the proliferation, activation and differentiation of CD4+ T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4+CD25+Foxp3+ regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4+CD25+Foxp3+ T lymphocytes when administrated at early stages of the disease.
This study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.
Mesenchymal stem cells; Th1; Th17; Immunosuppression; CD4+CD25+Foxp3+ T cells
Duane retraction syndrome (DRS) is a rare congenital strabismus condition with genetic heterogeneity. DRS associated with intellectual disability or developmental delay is observed in several genetic diseases: syndromes such as Goldenhar or Wildervanck syndrome and chromosomal anomalies such as 12q12 deletion. We report on the case of a patient with DRS, developmental delay and particular facial features (horizontal and flared eyebrows, long and smooth philtrum, thin upper lip, full lower lip and full cheeks). We identified a duplication of the long arm of chromosome 8 (8q12) with SNP-array. This is the third case of a patient with common clinical features and 8q12 duplication described in the literature. The minimal critical region is 1.2 Mb and encompasses four genes: CA8, RAB2, RLBP1L1 and CHD7. To our knowledge, no information is available in the literature regarding pathological effects caused by to overexpression of these genes. However, loss of function of the CHD7 gene leads to CHARGE syndrome, suggesting a possible role of the overexpression of this gene in the phenotype observed in 8q12 duplication patients. We have observed that patients with 8q12 duplication share a common recognizable phenotype characterized by DRS, developmental delay and facial features. Such data combined to the literature strongly suggest that this entity may define a novel syndrome. We hypothesize that CHD7 duplication is responsible for a part of the features observed in 8q12.2 duplication.
duane retraction syndrome; intellectual disability; 8q12 duplication; CHD7 gene; common facial features
The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.
Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies) in patients with rheumatoid arthritis have been described in literature.
We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient.
Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis.
Granulomatosis with polyangiitis; Rheumatoid arthritis; ANCA associated vasculitis; Rheumatoid vasculitis
MSC display potent suppressive properties initially described a decade ago. More recently, MSC suppressive activities on T-cell effector pathways have been investigated. MSC modulate CD4 differentiation through different mechanisms depending on culture conditions and display disparate activities on T cells according to their differentiation status. A significant amount of evidence for MSC effects on Th17 cells revealed that MSC could be suppressive under diverse circumstances but also enhance Th17 cell activity under other conditions. In the present study, we investigated the suppressive effects of MSC on Th1 and Th17 subsets of T cells using T cells undergoing Th1 and Th17 polarization or mature Th1 and Th17 cells. MSC inhibited the proliferation of T cells during their differentiation toward Th1 cells and mature Th1 cells. This suppressive effect was maintained in a transwell cell culture insert demonstrating the major role played by soluble factors. Using the transwell cell separation barrier, we observed that MSC decrease the number of T cells undergoing Th17 differentiation whereas they did not affect IL-17 production by mature Th17, demonstrating the need for cell contact for suppressing Th17 cell function. Moreover, we reported that PD-L1 is highly expressed on MSC co-cultured with differentiating or polarized Th1 and Th17 cells. Using neutralizing antibodies specific for PD-L1 and PD-1 we showed that the mechanisms by which MSC mediate Th17 cell repolarization depend on PD-L1 expression on MSC. Taken together our results demonstrated a cell-to-cell contact depend mechanism in the selective immunosuppression of MSC on mature Th17 cells through up-regulation of PD-L1.
Growing knowledge on the biology of mesenchymal stem cells (MSCs) has provided new insights into their potential clinical applications, particularly for rheumatologic disorders. Historically, their potential to differentiate into cells of the bone and cartilage lineages has led to a variety of experimental strategies to investigate whether MSCs can be used for tissue engineering approaches. Beyond this potential, MSCs also display immunosuppressive properties, which have prompted research on their capacity to suppress local inflammation and tissue damage in a variety of inflammatory autoimmune diseases and, in particular, in rheumatoid arthritis. Currently, an emerging field of research comes from the possibility that these cells, through their trophic/regenerative potential, may also influence the course of chronic degenerative disorders and prevent cartilage degradation in osteoarthritis. This review focuses on these advances, specifically on the biological properties of MSCs, including their immunoregulatory characteristics, differentiation capacity and trophic potential, as well as the relevance of MSC-based therapies for rheumatic diseases.
Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, the lack of efficient modalities of treatment for large chondral defects has prompted research on tissue engineering combining chondrogenic cells, scaffold materials and environmental factors. The aim of this review is to focus on the recent advances made in exploiting the potentials of cell therapy for cartilage engineering. These include: 1) defining the best cell candidates between chondrocytes or multipotent progenitor cells, such as multipotent mesenchymal stromal cells (MSC), in terms of readily available sources for isolation, expansion and repair potential; 2) engineering biocompatible and biodegradable natural or artificial matrix scaffolds as cell carriers, chondrogenic factors releasing factories and supports for defect filling, 3) identifying more specific growth factors and the appropriate scheme of application that will promote both chondrogenic differentiation and then maintain the differentiated phenotype overtime and 4) evaluating the optimal combinations that will answer to the functional demand placed upon cartilage tissue replacement in animal models and in clinics. Finally, some of the major obstacles generally encountered in cartilage engineering are discussed as well as future trends to overcome these limiting issues for clinical applications.
Animals; Biocompatible Materials; therapeutic use; Cartilage; metabolism; pathology; Cartilage Diseases; pathology; therapy; Cell Differentiation; Chondrocytes; metabolism; pathology; transplantation; Guided Tissue Regeneration; Hematopoietic Stem Cell Mobilization; Humans; Intercellular Signaling Peptides and Proteins; metabolism; Mesenchymal Stem Cell Transplantation; Stem Cell Niche; Tissue Engineering; Tissue Scaffolds
Genostem (acronym for “Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side”) has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
Animals; Bone Marrow Cells; cytology; Cell Culture Techniques; standards; Cell Differentiation; Cell Proliferation; Humans; Mesenchymal Stem Cells; cytology; Tissue Engineering; differentiation; stem cell; bone; cartilage; tendon; smooth muscle; regenerative medicine
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.