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1.
Invest Ophthalmol Vis Sci ; 53(2): 786-93, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22232435

ABSTRACT

PURPOSE: Mesenchymal stem/progenitor cells (MSCs) have regenerative and immunomodulatory properties, exerted by cell-cell contact and in a paracrine fashion. Part of their immunosuppressive activity has been ascribed to their ability to promote the induction of CD4+CD25+FoxP3+ T lymphocytes with regulatory functions (Treg). Here the authors studied the effect of MSCs on the induction of Treg and on the development of autoimmunity, and they examined the possibility that MSC-mediated Treg induction could be attributed to the secretion of soluble factors. METHODS: The authors induced experimental autoimmune uveitis (EAU) in mice by immunization with the 1-20 peptide of the intraphotoreceptor binding protein. At the same time, some of the animals were treated intraperitoneally with syngeneic MSCs. The authors checked T-cell responses and in vitro Treg conversion by cell proliferation and blocking assays, in cell-cell contact and transwell settings. TGFß and TGFß receptor gene expression analyses were performed by real-time PCR. RESULTS: The authors found that a single intraperitoneal injection of MSCs was able to significantly attenuate EAU and that a significantly higher percentage of adaptive Treg was present in MSC-treated mice than in MSC-untreated animals. In vitro blocking of antigen presentation by major histocompatibility complex class II precluded priming and clonal expansion of antigen-specific Treg, whereas blockade of TGFß impaired the expression of FoxP3, preventing the conversion of CD4+ T cells into functionally active Treg. CONCLUSIONS: The authors demonstrated that MSCs can inhibit EAU and that their immunomodulatory function is due at least in part to the induction of antigen-specific Treg in a paracrine fashion by secreting TGFß.


Subject(s)
Autoimmune Diseases/therapy , Immunomodulation , Mesenchymal Stem Cell Transplantation/methods , T-Lymphocytes, Regulatory/immunology , Uveitis/therapy , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Disease Models, Animal , Follow-Up Studies , Mesenchymal Stem Cells , Mice , Mice, Inbred C57BL , Uveitis/immunology , Uveitis/pathology
2.
Stem Cells Dev ; 20(7): 1183-98, 2011 Jul.
Article in English | MEDLINE | ID: mdl-20964598

ABSTRACT

In several cell types, a regulated efflux of NAD(+) across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD(+) (NAD(+)(e)) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD(+) through Cx43 HC. NAD(+) efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca(2+) (5-6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD(+)(e) activates the purinergic receptor P2Y(11) and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca(2+)](i) signaling cascade, involving the opening, unique to MSC, of L-type Ca(2+) channels. Extracellular NAD(+) enhanced nuclear translocation of cAMP/Ca(2+)-dependent transcription factors. Moreover, NAD(+), either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E(2) and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD(+)(e). Controls included Cx43-siRNA transfected and/or NAD(+)-glycohydrolase-treated MSC (autocrine effects), and NAD(+)-untreated or P2Y(11)-siRNA-transfected MSC (exogenous NAD(+)). These findings suggest a potential beneficial role of NAD(+)(e) in modulating MSC functions relevant to MSC-based cell therapies.


Subject(s)
Autocrine Communication , Gap Junctions/metabolism , Mesenchymal Stem Cells/metabolism , NAD/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Adipogenesis , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Cells, Cultured , Connexin 43/metabolism , Cyclic AMP/metabolism , Cytokines/metabolism , Dinoprostone/metabolism , Flow Cytometry , Humans , Osteogenesis , RNA, Small Interfering , Second Messenger Systems
3.
J Tissue Eng Regen Med ; 4(7): 505-13, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20213628

ABSTRACT

The combination of synthetic polymers and calcium phosphates represent an improvement in the development of scaffolds for bone-tissue regeneration. Ideally, these composites provide both mechanically and architecturally enhanced performances; however, they often lack properties such as osteoconductivity and cell bioactivation. In this study we attempted to generate a composite bone substitute maximizing the available osteoconductive surface for cell adhesion and activity. Highly porous scaffolds were prepared through a particulate leaching method, combining poly-ε-caprolactone (PCL) and hydroxyapatite (HA) particles, previously coated with a sucrose layer, to minimize their embedding by the polymer solution. Composite performances were evaluated both in vitro and in vivo. In PCL-sucrose-coated HA samples, the HA particles were almost completely exposed and physically distinct from the polymer mesh, while uncoated control samples showed ceramic granules massively covered by the polymer. In vivo results revealed a significant extent of bone deposition around all sucrose-coated HA granules, while only parts of the control uncoated HA granules were surrounded by bone matrix. These findings highlight the possibility of generating enhanced osteoconductive materials, basing the scaffold design on physiological and cellular concepts.


Subject(s)
Bone Regeneration , Bone Substitutes/chemistry , Durapatite/chemistry , Mesenchymal Stem Cells/cytology , Polyesters/chemistry , Sucrose/chemistry , Animals , Cell Adhesion , Cells, Cultured , Materials Testing/methods , Mice , Mice, Mutant Strains , Sheep
4.
Tissue Eng Part A ; 16(2): 489-99, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19712045

ABSTRACT

In tissue-engineered applications bone marrow stromal cells (BMSCs) are combined with scaffolds to target bone regeneration; animal models have been devised and the cells' long-term engraftment has been widely studied. However, in regenerated bone, the cell number is severely reduced with respect to the initially seeded BMSCs. This reflects the natural low cellularity of bone but underlines the selectivity of the differentiation processes. In this respect, we evaluated the short-term survival of BMSCs, transduced with the luciferase gene, after implantation of cell-seeded scaffolds in a nude mouse model. Cell proliferation/survival was assessed by bioluminescence imaging: light production was decreased by 30% on the first day, reaching a 50% loss within 48 h. Less than 5% of the initial signal remained after 2 months in vivo. Apoptotic BMSCs were detected within the first 2 days of implantation. Interestingly, the initial frequency of clonogenic progenitors matched the percentage of in vivo surviving cells. Cytokines and inflammation may contribute to the apoptotic onset at the implant milieu. However, preculturing cells with tumor necrosis factor alpha enhanced survival, allowing detection of 8.1% of the seeded BMSCs 2 months after implantation. Thus culturing conditions may reduce the apoptotic overload of seeded osteoprogenitors, strengthening the constructs' osteogenic potential.


Subject(s)
Bone Marrow Cells/cytology , Bone Regeneration/physiology , Models, Biological , Animals , Apoptosis/drug effects , Biocompatible Materials/pharmacology , Bone Marrow Cells/drug effects , Bone Regeneration/drug effects , Cell Survival/drug effects , Cells, Cultured , Collagen/pharmacology , Durapatite/pharmacology , Imaging, Three-Dimensional , Implants, Experimental , Light , Luminescent Measurements , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mice , Mice, Nude , Sheep , Stromal Cells/cytology , Stromal Cells/drug effects , Time Factors , Tissue Scaffolds/chemistry , Tumor Necrosis Factor-alpha/pharmacology
5.
Tissue Eng Part A ; 15(1): 155-63, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18657026

ABSTRACT

In principle, three-dimensional (3D) osteoconductive grafts with a proper chemical composition, high total porosity, and fully interconnected pores are suitable carriers to provide a proper substrate for in vivo neobone tissue ingrowth. However, most porous materials carry some intrinsic limits because of their internal structure (i.e., limited macroporosity and small pore interconnection size), representing a physical constraint for a massive blood afflux and bone ingrowth and therefore for generating effective osteopermissive grafts. We therefore hypothesized that an unconventional scaffold, based on an "open-structure" concept, should not pose any limit to vascularization of grafts and consequently to the amount of bone growth. Starting from this hypothesis, we have designed and developed a 3D osteoconductive polymeric-based wide-net mesh. Polymer fibers, joining hydroxyapatite beads, were coated with a thin layer of calcium phosphate (Ca-P), coupling the osteoconductivity properties of Ca-P with the handness and bulk properties of polymers. This completely open 3D scaffold prototype was tested both in vitro and in vivo, displaying a promising in vivo blood vessel invasion and bone-forming efficiency.


Subject(s)
Bone Regeneration , Durapatite/chemistry , Polyesters/chemistry , Polymers/chemistry , Animals , Bone Marrow Cells/cytology , Bone Substitutes/chemistry , Calcium Phosphates/chemistry , Cell Adhesion , Cells, Cultured , Coated Materials, Biocompatible/chemistry , Electron Probe Microanalysis , Mice , Mice, SCID , Neovascularization, Physiologic , Sheep, Domestic , Stem Cells/cytology , Stem Cells/ultrastructure , Tissue Scaffolds/chemistry , Transplantation, Heterologous
6.
Mol Cell ; 12(5): 1201-11, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14636578

ABSTRACT

The Wnt/beta-catenin pathway rapidly induces the transcription of the cell-type-restricted transcription factor Pitx2 that is required for effective cell-specific proliferation activating growth-regulating genes. Here we report that Pitx2 mRNA displays a rapid turnover rate and that activation of the Wnt/beta-catenin pathway stabilizes Pitx2 mRNA as well as other unstable mRNAs, including c-Jun, Cyclin D1, and Cyclin D2, encoded by critical transcriptional target genes of the same pathway. Our data indicate that Pitx2 mRNA stabilization is due to a reduced interaction of Pitx2 3'UTR with the destabilizing AU-rich element (ARE) binding proteins (BPs) KSRP and TTP as well as to an increased interaction with a stabilizing ARE-BP, HuR. Pitx2 itself is a mediator of Wnt/beta-catenin-induced mRNA stabilization. Our previous and present data support the hypothesis that a single pathway can coordinately regulate sequential transcriptional and posttranscriptional events leading to an integrated functional gene regulatory network.


Subject(s)
Cytoskeletal Proteins/metabolism , Gene Expression Regulation , Homeodomain Proteins/metabolism , Nuclear Proteins , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Zebrafish Proteins , 3' Untranslated Regions , Animals , Cell Line , Homeodomain Proteins/genetics , Mice , Open Reading Frames , RNA Stability , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription, Genetic , Wnt Proteins , beta Catenin , Homeobox Protein PITX2
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