Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 40
Filter
Add more filters










Publication year range
1.
Front Physiol ; 9: 547, 2018.
Article in English | MEDLINE | ID: mdl-29892229

ABSTRACT

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.

2.
J Tissue Eng Regen Med ; 12(2): e774-e785, 2018 02.
Article in English | MEDLINE | ID: mdl-27943583

ABSTRACT

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1+ /c-Kit+ /CD34+ DPSCs, expressing P75NTR , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1+ /c-Kit+ /CD34+ hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.


Subject(s)
Antigens, CD34/metabolism , Biomarkers/metabolism , Dental Pulp/cytology , Nerve Regeneration , Peripheral Nerves/physiology , Proto-Oncogene Proteins c-kit/metabolism , Stem Cells/metabolism , Adult , Animals , Cell Differentiation , Cellular Microenvironment , Humans , Multipotent Stem Cells/cytology , Myelin Sheath/metabolism , Nerve Growth Factors/metabolism , Neural Crest/metabolism , Phenotype , Rats, Sprague-Dawley , Recovery of Function , Sciatic Nerve/pathology , Sciatic Nerve/physiopathology , Sciatic Nerve/surgery , Young Adult
3.
Sci Rep ; 7(1): 14419, 2017 10 31.
Article in English | MEDLINE | ID: mdl-29089545

ABSTRACT

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4+, CD8+ T-cells and in CD56+ NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.


Subject(s)
Cholangiocarcinoma/metabolism , Fas Ligand Protein/physiology , fas Receptor/physiology , Aged , Aged, 80 and over , Apoptosis , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/physiology , CD8-Positive T-Lymphocytes/metabolism , Caspases/metabolism , Cholangiocarcinoma/genetics , Cholangiocarcinoma/pathology , Coculture Techniques , Female , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Natural Killer T-Cells , Primary Cell Culture , fas Receptor/metabolism
4.
Biopreserv Biobank ; 14(2): 138-48, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26828565

ABSTRACT

Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.


Subject(s)
Biological Specimen Banks , Bone Marrow , Cryopreservation/methods , Hematopoietic Stem Cells , Apoptosis , Cell Proliferation , Cells, Cultured , Cellular Senescence , Culture Media , Humans , Immunophenotyping , Male
5.
Stem Cell Res Ther ; 6: 156, 2015 Aug 28.
Article in English | MEDLINE | ID: mdl-26316011

ABSTRACT

INTRODUCTION: Duchenne muscular dystrophy (DMD), caused by a lack of the functional structural protein dystrophin, leads to severe muscle degeneration where the patients are typically wheelchair-bound and die in their mid-twenties from cardiac or respiratory failure or both. The aim of this study was to investigate the potential of human dental pulp stem cells (hDPSCs) and human amniotic fluid stem cells (hAFSCs) to differentiate toward a skeletal myogenic lineage using several different protocols in order to determine the optimal conditions for achieving myogenic commitment and to subsequently evaluate their contribution in the improvement of the pathological features associated with dystrophic skeletal muscle when intramuscularly injected into mdx/SCID mice, an immune-compromised animal model of DMD. METHODS: Human DPSCs and AFSCs were differentiated toward myogenic lineage in vitro through the direct co-culture with a myogenic cell line (C2C12 cells) and through a preliminary demethylation treatment with 5-Aza-2'-deoxycytidine (5-Aza), respectively. The commitment and differentiation of both hDPSCs and hAFSCs were evaluated by immunofluorescence and Western blot analysis. Subsequently, hDPSCs and hAFSCs, preliminarily demethylated and pre-differentiated toward a myogenic lineage for 2 weeks, were injected into the dystrophic gastrocnemius muscles of mdx/SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. RESULTS: Both populations of cells engrafted within the host muscle of mdx/SCID mice and through a paracrine effect promoted angiogenesis and reduced fibrosis, which eventually led to an improvement of the histopathology of the dystrophic muscle. CONCLUSION: This study shows that hAFSCs and hDPSCs represent potential sources of stem cells for translational strategies to improve the histopathology and potentially alleviate the muscle weakness in patients with DMD.


Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Muscle Development , Muscle, Skeletal/physiology , Muscular Dystrophy, Duchenne/therapy , Amniotic Fluid/cytology , Animals , Cell Line , Dental Pulp/cytology , Humans , Mice , Mice, Inbred mdx , Mice, SCID , Muscle, Skeletal/cytology , Muscular Dystrophy, Duchenne/genetics , Regeneration
6.
BMC Dev Biol ; 15: 14, 2015 Mar 14.
Article in English | MEDLINE | ID: mdl-25879198

ABSTRACT

BACKGROUND: Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency. RESULTS: The STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs showed a slower proliferation, gradual loss of stemness, early cell senescence and apoptosis, compared to STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs. Both the subpopulations demonstrated similar abilities to differentiate towards mesoderm lineages, whereas a significant difference was observed after the neurogenic induction, with a greater commitment of STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs. Moreover, undifferentiated STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs did not show any expression of CD271 and nestin, typical neural markers, while STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs expressed both. CONCLUSIONS: These results suggest that STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs and STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs might represent two distinct stem cell populations, with different properties. These results trigger further analyses to deeply investigate the hypothesis that more than a single stem cell population resides within the dental pulp, to better define the flexibility of application of hDPSCs in regenerative medicine.


Subject(s)
Cell Differentiation , Dental Pulp/cytology , Stem Cells/cytology , Antigens, CD34/immunology , Antigens, Surface/immunology , Cell Proliferation , Cell Separation , Humans , Proto-Oncogene Proteins c-kit/metabolism , Stem Cells/immunology , Stem Cells/metabolism
7.
Mitochondrion ; 18: 76-81, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25128872

ABSTRACT

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.


Subject(s)
Protease La/metabolism , Protein Interaction Mapping , Protein Processing, Post-Translational , Sirtuin 3/metabolism , Acetylation , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Immunoprecipitation , Mitochondria/chemistry
8.
J Hepatol ; 61(5): 1097-105, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24953023

ABSTRACT

BACKGROUND & AIMS: Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures. METHODS: Fas and FasL expression were evaluated in situ and in vitro by immunofluorescence and western blotting. Co-cultures of hBTSCs with human leukocytes were used to analyze the influence of hBTSCs on lymphocytes activation and apoptosis. RESULTS: hBTSCs expressed HLA antigens and FasL in situ and in vitro. Western blot data demonstrated that hBTSCs constitutively expressed high levels of FasL that increased after co-culture with T cells. Confocal microscopy demonstrated that FasL expression was restricted to EpCAM(+)/LGR5(+) cells. FACS analysis of T cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4(+) and CD8(+) T cell populations. Moreover, the Fas receptor appears to be more expressed in T cells co-cultured with hBTSCs than in resting T cells. CONCLUSIONS: Our data suggest that hBTSCs could modulate the T cell response through the production of FasL, which influences the lymphocyte Fas/FasL pathway by inducing "premature" apoptosis in CD4(+) and CD8(+) T cells.


Subject(s)
Biliary Tract/cytology , Biliary Tract/immunology , Fas Ligand Protein/metabolism , Multipotent Stem Cells/cytology , Multipotent Stem Cells/immunology , fas Receptor/metabolism , Adult Stem Cells/cytology , Adult Stem Cells/immunology , Adult Stem Cells/metabolism , Apoptosis/immunology , Biliary Tract/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Coculture Techniques , Fetal Stem Cells/cytology , Fetal Stem Cells/immunology , Fetal Stem Cells/metabolism , Humans , Immunomodulation , Lymphocyte Activation , Multipotent Stem Cells/metabolism , Signal Transduction
9.
Cell Tissue Res ; 357(1): 1-13, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24788911

ABSTRACT

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, ß-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.


Subject(s)
Amniotic Fluid/cytology , Neurons/cytology , Stem Cells/cytology , Amniotic Fluid/metabolism , Animals , Cell Differentiation/physiology , Humans , In Vitro Techniques , Neurons/metabolism , Rats , Regenerative Medicine , Stem Cells/metabolism
10.
Dig Liver Dis ; 45(8): 669-76, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23643565

ABSTRACT

AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic ß-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.


Subject(s)
Amniotic Fluid/metabolism , Dental Pulp/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Stem Cells/metabolism , Cell Differentiation , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Humans , In Vitro Techniques , Insulin Secretion
11.
Stem Cell Res Ther ; 4(3): 53, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23688855

ABSTRACT

INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells and amniotic fluid stem cells, combined with collagen scaffold to reconstruct critical-size cranial bone defects in an animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks, cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all of the samples but the most relevant differences in defect correction were shown by stem cell-collagen samples 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSION: These data confirmed the strong potential of bioengineered constructs of stem cell-collagen scaffold for correcting large cranial defects in an animal model and highlighting the role of stem cells in neovascularization during skeletal defect reconstruction.


Subject(s)
Amniotic Fluid/cytology , Collagen/chemistry , Dental Pulp/cytology , Stem Cells/cytology , Tissue Scaffolds , Animals , Bone Diseases/pathology , Bone Diseases/therapy , Bone and Bones/diagnostic imaging , Bone and Bones/metabolism , Bone and Bones/pathology , Cell Differentiation , Cells, Cultured , Humans , Immunohistochemistry , Male , Neovascularization, Physiologic , Osteogenesis , Radiography , Rats , Regenerative Medicine , Stem Cell Transplantation , Transplantation, Heterologous
12.
PLoS One ; 7(11): e50542, 2012.
Article in English | MEDLINE | ID: mdl-23209773

ABSTRACT

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.


Subject(s)
Cell Differentiation/physiology , Dental Pulp/cytology , Osteogenesis/physiology , Serum/metabolism , Stem Cells/cytology , Animals , Apoptosis/physiology , Bone and Bones/cytology , Cell Proliferation , Cells, Cultured , Cellular Senescence/physiology , Flow Cytometry , Humans , Male , Rats , Rats, Sprague-Dawley , Regeneration , Stem Cell Transplantation , Tissue Engineering
13.
AIDS ; 26(16): 2017-26, 2012 Oct 23.
Article in English | MEDLINE | ID: mdl-22948272

ABSTRACT

BACKGROUND: The association between HAART and lipodystrophy is well established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. METHODS: SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10-200 µmol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2 were studied by flow cytometry and multispectral imaging flow cytometry. RESULTS: In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. CONCLUSION: Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.


Subject(s)
Adipocytes/metabolism , Anti-HIV Agents/pharmacology , Autophagy/drug effects , HIV Infections/metabolism , HIV Protease Inhibitors/pharmacology , HIV-Associated Lipodystrophy Syndrome/metabolism , Mitophagy/drug effects , Oligopeptides/pharmacology , Pyridines/pharmacology , Adipocytes/drug effects , Antiretroviral Therapy, Highly Active , Atazanavir Sulfate , Blotting, Western , Cells, Cultured , Dose-Response Relationship, Drug , Female , Flow Cytometry , Green Fluorescent Proteins/metabolism , HIV Infections/drug therapy , HIV-Associated Lipodystrophy Syndrome/drug therapy , Humans , Male , Microscopy, Electron
14.
Tissue Eng Part A ; 18(9-10): 1006-13, 2012 May.
Article in English | MEDLINE | ID: mdl-22166080

ABSTRACT

The main aim of this study was the comparative evaluation of fibroin scaffolds combined with human stem cells, such as dental pulp stem cells (hDPSCs) and amniotic fluid stem cells (hAFSCs), used to repair critical-size cranial bone defects in immunocompromised rats. Two symmetric full-thickness cranial defects on each parietal region of rats have been replenished with silk fibroin scaffolds with or without preseeded stem cells addressed toward osteogenic lineage in vitro. Animals were euthanized after 4 weeks postoperatively and cranial tissue samples were taken for histological analysis. The presence of human cells in the new-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Fibroin scaffolds induced mature bone formation and defect correction, with higher bone amount produced by hAFSC-seeded scaffolds. Our findings demonstrated the strong potential of stem cells/fibroin bioengineered constructs for correcting large cranial defects in animal model and is likely a promising approach for the reconstruction of human large skeletal defects in craniofacial surgery.


Subject(s)
Amniotic Fluid/cytology , Bone Diseases/therapy , Dental Pulp/cytology , Fibroins/chemistry , Stem Cells/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cells, Cultured , Humans , Male , Microscopy, Electron, Scanning , Rats , Rats, Sprague-Dawley , Stem Cells/physiology , Wound Healing/physiology
15.
Tissue Eng Part A ; 17(21-22): 2833-43, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21864161

ABSTRACT

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.


Subject(s)
Amniotic Fluid/cytology , Fibroins/chemistry , Silk/chemistry , Stem Cells/cytology , Tissue Scaffolds/chemistry , Animals , Blotting, Western , Bombyx , Cell Differentiation/physiology , Cells, Cultured , Collagen/chemistry , Core Binding Factor Alpha 1 Subunit/metabolism , Fluorescent Antibody Technique , Humans , Microscopy, Atomic Force , Microscopy, Confocal , Osteocalcin/metabolism , Osteogenesis/physiology , Osteopontin/metabolism , Sp7 Transcription Factor , Transcription Factors/metabolism
16.
Neurotoxicology ; 32(2): 180-7, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21215776

ABSTRACT

Organic and inorganic selenium compounds were used to examine whether low selenium concentration is able to trigger apoptotic degeneration in a human neuron cell line in vitro and to explore changes in reactive oxygen and nitrogen species and antioxidant protein content during the apoptotic processes. The results indicated that: (1) SKNBE neuroblastoma cells treated with sodium selenite, sodium selenate and seleno-methionine (0.1, 0.5 and 0.5 µM, respectively) for 24h exhibited a viability decrease, unlike kidney or prostatic cells; (2) the PARP (poly-ADP-ribose-polymerase) degradation and caspase activation detected by Western blot and flow cytometry fluorimetric examination showed induction of apoptosis; (3) during selenium treatment, a ROS/RNS increase occurred despite the GSH increment, as revealed by fluorimetric analysis; (4) the RNS production could be blocked by a peroxynitrite scavenger; (5) after exposure to selenium compounds, the concentration of nitric oxide synthase, manganese superoxide dismutase (SOD2), P-NF-kB (phospho nuclear factor kB), glutathione reductase and glutathione peroxidase increased, whereas that of P-ERK (phospho extracellular signal-regulated kinase) decreased; (6) selenium presence induced copper/zinc superoxide dismutase (SOD1) translocation into mitochondria, in a way similar to what is observed in amyotrophic lateral sclerosis (ALS). This study supports epidemiologic studies showing the possibility that excess environmental exposure to Se represents a risk factor for a devastating human neurodegenerative disease.


Subject(s)
Apoptosis/physiology , Neurons/metabolism , Reactive Oxygen Species/metabolism , Selenium Compounds/administration & dosage , Selenium Compounds/toxicity , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Humans , Nerve Degeneration/chemically induced , Nerve Degeneration/metabolism , Neurons/drug effects
17.
Cancer Lett ; 279(2): 221-9, 2009 Jul 08.
Article in English | MEDLINE | ID: mdl-19268443

ABSTRACT

Chronic mucosal inflammation is considered a risk factor for colorectal cancer. Neutrophils are a major source of oxidants, whereas cyclooxygenase 2 (COX-2) and Hypoxia Inducible Factor-1alpha (HIF-1alpha) protein expression levels are increased in inflammatory and malignant lesions. The main purpose of the present study was to evaluate myeloperoxidase (MPO) positive cell infiltration, COX-2 and HIF-1alpha protein expression in colorectal carcinogenesis, especially in its early phases, using immunohistochemistry and immunofluorescence confocal microscopy techniques. MPO, COX-2 and HIF-1alpha proteins were expressed at higher rates in the normal colorectal mucosa of patients with inflammatory bowel diseases and colorectal tumours than in patients with normal colonoscopy. A gradual increase in COX-2 and HIF-1alpha protein expression was observed in dysplastic aberrant crypt foci, adenomas and carcinomas, showing a strong relation to dysplasia. In conclusion, the present study supports the hypothesis of a key role of inflammation in malignant transformation of colorectal mucosa. The evaluation of some early markers related to inflammation in the mucosa of the large bowel may serve as potential tool for prognosis and therapeutic strategies.


Subject(s)
Colorectal Neoplasms/metabolism , Cyclooxygenase 2/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Inflammatory Bowel Diseases/metabolism , Biomarkers, Tumor/biosynthesis , Colorectal Neoplasms/pathology , Humans , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Peroxidase/biosynthesis , Up-Regulation
18.
Reprod Biomed Online ; 18(2): 226-34, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19192343

ABSTRACT

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.


Subject(s)
Autoantigens/metabolism , Granulosa Cells/metabolism , Oocytes/metabolism , Theca Cells/metabolism , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Female , Granulosa Cells/cytology , Humans , Microscopy, Confocal , Mitochondria/metabolism , Mitochondrial Proteins , Nuclear Proteins , Oocytes/cytology , Ovarian Follicle/cytology , Ovarian Follicle/ultrastructure , RNA Processing, Post-Transcriptional/physiology , Theca Cells/cytology , Tissue Distribution
19.
PLoS One ; 4(2): e4404, 2009.
Article in English | MEDLINE | ID: mdl-19198660

ABSTRACT

Tollip is an interactor of the interleukin-1 receptor involved in its activation. The endosomal turnover of ubiquitylated IL-1RI is also controlled by Tollip. Furthermore, together with Tom1, Tollip has a general role in endosomal protein traffic. This work shows that Tollip is involved in the sumoylation process. Using the yeast two-hybrid technique, we have isolated new Tollip partners including two sumoylation enzymes, SUMO-1 and the transcriptional repressor Daxx. The interactions were confirmed by GST-pull down experiments and immunoprecipitation of the co-expressed recombinants. More specifically, we show that the TIR domain of the cytoplasmic region of IL-1RI is a sumoylation target of Tollip. The sumoylated and unsumoylated RanGAP-1 protein also interacts with Tollip, suggesting a possible role in RanGAP-1 modification and nuclear-cytoplasmic protein translocation. In fact, Tollip is found in the nuclear bodies of SAOS-2/IL-1RI cells where it colocalizes with SUMO-1 and the Daxx repressor. We conclude that Tollip is involved in the control of both nuclear and cytoplasmic protein traffic, through two different and often contrasting processes: ubiquitylation and sumoylation.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , SUMO-1 Protein/metabolism , Animals , Fluorescent Antibody Technique , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Rats , Rats, Sprague-Dawley , Receptors, Interleukin-1/metabolism , Two-Hybrid System Techniques
20.
J Proteome Res ; 7(11): 4727-35, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18808171

ABSTRACT

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.


Subject(s)
Cell Nucleus/genetics , Lamin Type A/genetics , Myoblasts/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Serine/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Humans , Kidney/cytology , Lamin Type A/metabolism , Mice , Phosphorylation , Proteomics/methods , Proto-Oncogene Proteins c-akt/genetics , Recombinant Proteins/metabolism , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...