ABSTRACT
Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.
Subject(s)
Pluripotent Stem Cells , Humans , Mice , Animals , Hematopoietic Stem Cells , Colon , Organoids , MacrophagesABSTRACT
Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.
Subject(s)
Pluripotent Stem Cells , Transplants , Humans , Animals , Mice , Intestines , Intestinal Mucosa , OrganoidsABSTRACT
Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.
Subject(s)
Epithelium/growth & development , Intestines/growth & development , Organoids/growth & development , Serous Membrane/growth & development , Tissue Culture Techniques , Alginates/pharmacology , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Line , Collagen/pharmacology , Drug Combinations , Epithelium/drug effects , Hedgehog Proteins/metabolism , Humans , Intestines/ultrastructure , Laminin/pharmacology , Muscle, Smooth/cytology , Organoids/drug effects , Organoids/ultrastructure , Proteoglycans/pharmacology , Serous Membrane/drug effects , Serous Membrane/ultrastructure , Signal Transduction/drug effects , Suspensions , Wnt Proteins/metabolismABSTRACT
Expression of Ikaros family transcription factor IKZF3 (Aiolos) increases during murine eosinophil lineage commitment and maturation. Herein, we investigated Aiolos expression and function in mature human and murine eosinophils. Murine eosinophils deficient in Aiolos demonstrated gene expression changes in pathways associated with granulocyte-mediated immunity, chemotaxis, degranulation, ERK/MAPK signaling, and extracellular matrix organization; these genes had ATAC peaks within 1 kB of the TSS that were enriched for Aiolos-binding motifs. Global Aiolos deficiency reduced eosinophil frequency within peripheral tissues during homeostasis; a chimeric mouse model demonstrated dependence on intrinsic Aiolos expression by eosinophils. Aiolos deficiency reduced eosinophil CCR3 surface expression, intracellular ERK1/2 signaling, and CCL11-induced actin polymerization, emphasizing an impaired functional response. Aiolos-deficient eosinophils had reduced tissue accumulation in chemokine-, antigen-, and IL-13-driven inflammatory experimental models, all of which at least partially depend on CCR3 signaling. Human Aiolos expression was associated with active chromatin marks enriched for IKZF3, PU.1, and GATA-1-binding motifs within eosinophil-specific histone ChIP-seq peaks. Furthermore, treating the EOL-1 human eosinophilic cell line with lenalidomide yielded a dose-dependent decrease in Aiolos. These collective data indicate that eosinophil homing during homeostatic and inflammatory allergic states is Aiolos-dependent, identifying Aiolos as a potential therapeutic target for eosinophilic disease.
Subject(s)
Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Eosinophils/immunology , Eosinophils/metabolism , Ikaros Transcription Factor/genetics , Allergens/immunology , Animals , Disease Susceptibility , Female , Gene Expression Profiling , Gene Expression Regulation , Granulocytes/immunology , Granulocytes/metabolism , Humans , Ikaros Transcription Factor/metabolism , Immunity, Innate , Immunophenotyping , Leukocyte Count , Male , Mice , Mice, Knockout , Models, Animal , Receptors, CCR3/genetics , Receptors, CCR3/metabolism , Signal TransductionABSTRACT
Dynamic gene expression is a major mechanism that directs hematopoietic lineage commitment and differentiation. Recent advances have revealed an association between chromatin signatures and functional genetic sequences such that these chromatin signatures can be used to predict regulatory elements such as enhancers that may direct lineage differentiation. Our understanding of the genetic elements that regulate eosinophil development is very limited, likely due to the technical challenges in working with a rare complex cell. Herein, we describe protocols to sort mature eosinophils from the bone marrow of mice and to prepare chromatin that can be used for ChIP studies for genome-wide mapping of histone marks and transcription factors in mature eosinophils. Comprehensive epigenomic profiling during critical stages in eosinophil development will ultimately aid in defining the gene regulatory networks necessary to regulate eosinophil production.
Subject(s)
Chromatin/genetics , Chromatin/metabolism , Eosinophils/immunology , Eosinophils/metabolism , Genome-Wide Association Study , Animals , Cell Separation/methods , Chromatin Immunoprecipitation , Epigenesis, Genetic , Flow Cytometry , Gene Expression Regulation , MiceABSTRACT
Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5 Tg /Cd300f -/- mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5 Tg /Cd300f -/- mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f -/- mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5 Tg /CD300f -/- were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.
Subject(s)
Adipose Tissue/cytology , Eosinophils/metabolism , Interleukin-4/metabolism , Interleukin-5/metabolism , Receptors, Immunologic/metabolism , Animals , Endothelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Glucose Intolerance/metabolism , Glucose Intolerance/pathology , Ligands , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Interleukin-5/metabolism , Weight GainABSTRACT
The production of mature eosinophils (Eos) is a tightly orchestrated process with the aim to sustain normal Eos levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including Eos-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with Eos maturation (1199 genes) than with Eos-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eos-lineage-committed progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting Eos. Our analyses also delineated a 976-gene Eos-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with Eos. EoPs and Eos, but not granulocyte-monocyte progenitors or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during Eos development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and Eos with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during Eos development.
Subject(s)
DNA-Binding Proteins/genetics , Eosinophils/cytology , Hematopoiesis/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptome/genetics , Animals , Binding Sites/genetics , Cell Lineage/genetics , Cell Lineage/immunology , Cells, Cultured , Chromatin/genetics , Cytoplasmic Granules/metabolism , Eosinophils/immunology , Gene Expression Regulation/genetics , Granulocyte Precursor Cells , Hematopoiesis/immunology , Ikaros Transcription Factor , Mice , Mice, Inbred BALB C , Transcription Factors/biosynthesisABSTRACT
Eosinophils originate in the bone marrow from an eosinophil lineage-committed, IL-5Rα-positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors, and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. In this study, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand-receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor-produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.
Subject(s)
Cell Differentiation/immunology , Eosinophils/drug effects , Interleukin-5 Receptor alpha Subunit/immunology , Interleukin-5/pharmacology , Animals , Bone Marrow Cells/cytology , Cell Lineage/immunology , Cells, Cultured , Chemokine CCL3/biosynthesis , Chemokine CCL3/immunology , Eosinophilia/immunology , Eosinophils/immunology , Female , Interleukin-4/biosynthesis , Interleukin-4/immunology , Male , Mice , Mice, Inbred BALB C , Receptors, CCR1/biosynthesis , Receptors, CCR1/immunology , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/immunologyABSTRACT
Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of ß2-microglobulin (ß2M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb(-/-) mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.
Subject(s)
Cell Differentiation/immunology , Eosinophils/immunology , Interleukin-5/immunology , Receptors, Immunologic/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Apoptosis Regulatory Proteins/metabolism , Asthma/genetics , Asthma/immunology , Asthma/metabolism , Bcl-2-Like Protein 11 , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Colony-Forming Units Assay/methods , Eosinophils/cytology , Eosinophils/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Flow Cytometry , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/immunology , GRB2 Adaptor Protein/metabolism , Gene Expression/immunology , Interleukin-5/pharmacology , Male , Membrane Proteins/genetics , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/immunology , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/immunologyABSTRACT
Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33-regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti-IL-4 or anti-IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33-induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB-dependent mechanism. However, the induction of most IL-33-regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.
Subject(s)
Bone Marrow Cells/immunology , Eosinophils/immunology , Interleukin-4/immunology , Interleukins/immunology , Animals , Antibodies/immunology , Base Sequence , Cells, Cultured , Inflammation/immunology , Interleukin-33 , Interleukin-4/genetics , Interleukin-4/metabolism , Interleukins/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Phosphorylation , Receptors, Interleukin-4/immunology , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/metabolism , Sequence Analysis, RNA , Signal Transduction/immunologyABSTRACT
The cation-chloride cotransporter NKCC1 plays a fundamental role in the central and peripheral nervous systems by setting the value of intracellular chloride concentration. Following peripheral nerve injury, NKCC1 phosphorylation-induced chloride accumulation contributes to neurite regrowth of sensory neurons. However, the molecules and signaling pathways that regulate NKCC1 activity remain to be identified. Functional analysis of cotransporter activity revealed that inhibition of endogenously produced cytokine interleukin-6 (IL-6), with anti-mouse IL-6 antibody or in IL-6â»/â» mice, prevented chloride accumulation in a subset of axotomized neurons. Nerve injury upregulated the transcript and protein levels of IL-6 receptor in myelinated, TrkB-positive sensory neurons of murine lumbar dorsal root ganglia. Expression of phospho-NKCC1 was observed mainly in sensory neurons expressing IL-6 receptor and was absent from IL-6â»/â» dorsal root ganglia. The use of IL-6 receptor blocking-function antibody or soluble IL-6 receptor, together with pharmacological inhibition of Janus kinase, confirmed the role of neuronal IL-6 signaling in chloride accumulation and neurite growth of a subset of axotomized sensory neurons. Cell-specific expression of interleukin-6 receptor under pathophysiological conditions is therefore a cellular response by which IL-6 contributes to nerve regeneration through neuronal NKCC1 phosphorylation and chloride accumulation.
Subject(s)
Chlorides/physiology , Interleukin-6/physiology , Sensory Receptor Cells/metabolism , Sodium-Potassium-Chloride Symporters/metabolism , Animals , Axotomy/methods , Cells, Cultured , Chlorides/metabolism , Enzyme Inhibitors/pharmacology , Female , Ganglia, Spinal/metabolism , Interleukin-6/genetics , Janus Kinases/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Regeneration/drug effects , Nerve Regeneration/physiology , Neurites/drug effects , Neurites/physiology , Patch-Clamp Techniques , Phosphorylation , Receptors, Interleukin-6/biosynthesis , Receptors, Interleukin-6/physiology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiology , Sodium-Potassium-Chloride Symporters/physiology , Solute Carrier Family 12, Member 2 , Up-RegulationABSTRACT
OBJECTIVES: Mesenchymal stromal cells (MSC) are characterised by their capacity to suppress immune reactions. This function was reported to be shared in vitro by fibroblasts but their role has been poorly investigated in vivo. This study explored whether fibroblasts isolated from skin may suppress the host immune response in a model of autoimmune disorder. METHODS AND RESULTS: It was first confirmed that skin fibroblasts lack the capacity to differentiate into osteoblasts or chondrocytes but possess the capacity to inhibit in vitro the proliferation of T lymphocytes. Fibroblasts also secrete modulatory molecules, in particular prostaglandin E2 and nitric oxide, similar to MSC. To assess their role in vivo, the collagen-induced arthritis model was used, and showed that similar to MSC the intravenous injection of fibroblasts efficiently suppress clinical signs of arthritis and delay disease onset. This effect was associated with reduced inflammation as reflected by biological parameters and increased levels of IL-5, IL-10 and IL-13 in the spleens of treated mice. To characterise the mechanism of immunosuppression further, phenotypic analyses were performed and could not detect any induction of CD4 CD25 Foxp3(+) regulatory T (Treg) cells. A population of CD4 IL-10(+) T cells was, however, detected that was slightly increased after fibroblast injection and significantly upregulated after MSC administration. CONCLUSIONS: This study gives the first evidence for an immunosuppressive role of fibroblasts in vivo, and strongly suggests that fibroblasts induce a T-helper type 2 immune profile, although the possibility that IL-10-secreting Treg cells may be generated cannot be excluded.
Subject(s)
Arthritis, Experimental/prevention & control , Autoimmune Diseases/prevention & control , Fibroblasts/transplantation , Immunosuppression Therapy/methods , Skin/immunology , Animals , Arthritis, Experimental/immunology , Autoimmune Diseases/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cells, Cultured , Fibroblasts/immunology , Immune Tolerance , Immunophenotyping , Interleukin-10/biosynthesis , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Mice , Mice, Inbred DBA , Spleen/immunologyABSTRACT
BACKGROUND: Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis. PRINCIPAL FINDINGS: In the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response. SIGNIFICANCE: Our data indicate that mscs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile.
Subject(s)
Arthritis, Experimental/metabolism , Dinoprostone/metabolism , Interleukin-6/metabolism , Mesenchymal Stem Cells/cytology , Animals , Arthritis/metabolism , Cell Differentiation , Collagen/metabolism , Immunosuppressive Agents/chemistry , Inflammation , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Nitric Oxide Synthase Type II/metabolism , Phenotype , Th2 Cells/metabolismABSTRACT
Cartilage engineering using mesenchymal stem cells (MSC) will require the use of a scaffold which will act as a support for cell adhesion keeping the cells in the cartilage defect. Optimally, a tissue engineered construct should allow sustained delivery of bioactive factors capable of inducing MSC differentiation into chondrocytes and should be easily injected inside the cartilage lesions to avoid surgical operations. We therefore developed pharmacologically active microcarriers (PAM) made of poly-lactic-co-glycolic acid (PLGA) produced using an oil-in-water (o/w) emulsion method. The microspheres were coated with a biomimetic surface of fibronectin (FN) and engineered to release TGF-beta3 as a chondrogenic differentiation factor. When human MSCs were incubated in vitro with TGF-beta3 releasing FN-coated PAMs in chondrogenic medium, they firmly adhered onto the surface of PAMs rapidly forming cell aggregates. After 3 weeks, strong up-regulation of cartilage-specific markers was observed both at the mRNA and protein level whereas osteogenic or adipogenic genes could not be detected. Importantly, implantation of MSC/TGF-beta3 releasing PAM complexes in SCID mice resulted in the formation of histologically resembling cartilage which stained positive for chondrocyte markers, collagen II and aggrecan. The present study demonstrated that functionalized PLGA-based microparticles can provide an appropriate environment for chondrogenic differentiation of MSCs and should contribute to injectable biomedical device development improving in vivo cartilage engineering.
Subject(s)
Cartilage/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Transforming Growth Factor beta3/administration & dosage , Animals , Cell Adhesion , Cells, Cultured , Coated Materials, Biocompatible/chemistry , Fibronectins/chemistry , Humans , Lactic Acid/chemistry , Mice , Mice, SCID , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Prostheses and ImplantsABSTRACT
Mesenchymal stem cells (MSCs) are multipotential nonhematopoietic progenitor cells that are isolated from many adult tissues, in particular from the bone marrow and adipose tissue. Along with their capacity for differentiating into cells of mesodermal lineage, such as adipocytes, osteoblasts and chondrocytes, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough came with the finding that they are able to induce peripheral tolerance, suggesting they may be used as therapeutic tools in immune-mediated disorders. The present review aims at discussing the current knowledge on the targets and mechanisms of MSC-mediated immunosuppression as well as the potential use of MSCs as modulators of immune responses in a variety of diseases related to alloreactive immunity or autoimmunity.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Peripheral Tolerance/immunology , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/therapy , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Graft vs Host Disease/immunology , Graft vs Host Disease/therapy , Humans , Immunosuppression Therapy , Mesenchymal Stem Cells/metabolism , Mice , Neutrophils/immunology , Transplantation Immunology/immunologyABSTRACT
OBJECTIVE: RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis. METHODS: We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay. RESULTS: After a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically. CONCLUSION: Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.
Subject(s)
Arthritis, Experimental/therapy , RNA Interference/physiology , RNA, Small Interfering/administration & dosage , Tumor Necrosis Factor-alpha/genetics , Animals , Dependovirus , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Gene Silencing/physiology , Genetic Vectors , Green Fluorescent Proteins/genetics , Immunohistochemistry , Injections, Intra-Articular , Mice , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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.
Subject(s)
Cartilage Diseases/therapy , Cartilage/metabolism , Chondrocytes/metabolism , Mesenchymal Stem Cell Transplantation , Tissue Engineering , Animals , Biocompatible Materials/therapeutic use , Cartilage/pathology , Cartilage Diseases/pathology , Cell Differentiation , Chondrocytes/pathology , Chondrocytes/transplantation , Guided Tissue Regeneration , Hematopoietic Stem Cell Mobilization , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Stem Cell Niche , Tissue ScaffoldsABSTRACT
Mesenchymal stem cells (MSCs), or multipotent mesenchymal stromal cells as they are also known, have been identified in bone marrow as well as in other tissues of the joint, including adipose, synovium, periosteum, perichondrium, and cartilage. These cells are characterized by their phenotype and their ability to differentiate into three lineages: chondrocytes, osteoblasts and adipocytes. Importantly, MSCs also potently modulate immune responses, exhibit healing capacities, improve angiogenesis and prevent fibrosis. These properties might be explained at least in part by the trophic effects of MSCs through the secretion of a number of cytokines and growth factors. However, the mechanisms involved in the differentiation potential of MSCs, and their immunomodulatory and paracrine properties, are currently being extensively studied. These unique properties of MSCs confer on them the potential to be used for therapeutic applications in rheumatic diseases, including rheumatoid arthritis, osteoarthritis, genetic bone and cartilage disorders as well as bone metastasis.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Multipotent Stem Cells/immunology , Rheumatic Diseases/therapy , Animals , Bone Marrow Cells/immunology , Cell Differentiation/immunology , Disease Models, Animal , Humans , Multipotent Stem Cells/transplantation , Rheumatic Diseases/immunology , Stromal Cells/immunology , Stromal Cells/transplantationABSTRACT
Multipotent mesenchymal stromal cells (MSCs) have raised interest mainly because of cartilage/bone differentiation potential which is now partly eclipsed by their capacity to counteract inflammation and suppress host immune responses as well as to prevent fibrosis. MSCs have been identified within joint tissues including synovium, cartilage, subchondral bone, periosteum or adipose tissue. They are characterized by their phenotype and their ability to differentiate into three lineages, chondrocytes, osteoblasts and adipocytes. MSCs have also paracrine effects through the secretion of a number of cytokines and growth factors. This may explain the trophic effects that may be of therapeutic value for rheumatic diseases including OA and RA. On the other hand, MSCs have been associated with tumour growth. MSCs migrate to the tumour stroma, express chemokines involved in the attraction of carcinoma cells in metastasis. Indeed, the aim of this review is not only to focus on new potential therapeutic applications in osteo-articular diseases, but also to assess the potential risk of MSC-based cell therapy.
Subject(s)
Arthritis, Rheumatoid/pathology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/pathology , Multipotent Stem Cells/pathology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/therapy , Autoimmune Diseases/pathology , Autoimmune Diseases/therapy , Humans , Immune Tolerance , Multipotent Stem Cells/immunology , Multipotent Stem Cells/transplantationABSTRACT
Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.