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1.
J Neuroinflammation ; 14(1): 85, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28427413

RESUMO

BACKGROUND: Adiponectin (APN) is a key player in energy homeostasis strictly associated with cerebrovascular and neurodegenerative diseases. Since APN also belongs to anti-inflammatory-acting adipokines and may influence both neuroinflammation and neurodegenerative processes, we decided to study the APN levels in amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. METHODS: We assessed APN levels by ELISA immunoassay in both the serum and cerebrospinal fluid of a cohort of familial and sporadic ALS patients, characterized by normal body mass index and absence of dysautonomic symptoms. The screening of serum APN levels was also performed in patients affected by other neurological disorders, including fronto-temporal dementia (FTD) patients. Means were compared using the non-parametric Wilcoxon test, and Pearson's or Spearman's rho was used to assess correlations between variables. RESULTS: In the whole ALS group, serum APN levels were not different when compared to the age- and sex-matched control group (CTR), but a gender-specific analysis enlightened a significant opposite APN trend between ALS males, characterized by lower values (ALS 9.8 ± 5.2 vs. CTR 15 ± 9.7 µg/ml), and ALS females, showing higher amounts (ALS 26.5 ± 11.6 vs. CTR 14.6 ± 5.2 µg/ml). This sex-linked difference was significantly enhanced in familial ALS cases (p ≤ 0.01). The APN levels in ALS cerebrospinal fluids were unrelated to serum values and not linked to sex and/or familiarity of the disease. Finally, the screening of serum APN levels in patients affected by other neurological disorders revealed the highest serum values in FTD patients. CONCLUSIONS: Opposite serum APN levels are gender-related in ALS and altered in several neurological disorders, with the highest values in FTD, which shares with ALS several overlapping and neuropathological features. Further investigations are needed to clarify the possible involvement of APN in neuroinflammation and neurodegeneration. Possible involvement of APN in neuroinflammatory neurodegenerative diseases.


Assuntos
Adiponectina/análise , Esclerose Lateral Amiotrófica/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
2.
Nanotechnology ; 24(24): 245603, 2013 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-23690139

RESUMO

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.


Assuntos
Materiais Biocompatíveis/farmacologia , Rastreamento de Células/métodos , Nanopartículas/química , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Fluorescência , Corantes Fluorescentes/química , Humanos , Implantes Experimentais , Imageamento por Ressonância Magnética , Camundongos , Microscopia Confocal , Nanopartículas/ultraestrutura , Coloração e Rotulagem , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Imagem com Lapso de Tempo
3.
Arch Ital Biol ; 151(3): 114-25, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24599629

RESUMO

We set out to assess the feasibility of exploiting expression of the mCherry gene, after lentiviral infection, in order visualise bone marrow-derived human mesenchymal stem cells (hMSCs) by optical imaging, and to provide proof of principle of this approach as a method for cell tracking and quantification in pre-clinical models. Commercial hMSCs were infected with a lentiviral vector carrying the mCherry gene under the control of the phosphoglycerate kinase promoter. After extensive in vitro culture, infected hMSCs were analysed for viability, morphology, differentiation capability, and maintenance of fluorescence. Thereafter, mCherry-positive cells were transplanted into unilaterally 6-hydroxy-dopamine lesioned rats (an experimental model of Parkinson's disease). Our analysis showed that hMSCs can be efficiently transduced with the lentiviral vector, retaining their biological features even in the long term. Intrastriatally transplanted mCherry-positive hMSCs can be detected ex vivo by a sensitive cooled CCD camera, both in the whole brain and in serial slices, and relatively quantified. Our protocol was found to be a reliable means of studying the viability of implanted hMSCs. mCherry labelling appears to be readily applicable in the post-transplantation tracking of stem cells and could favour the rapid development of new therapeutic targets for clinical treatments.


Assuntos
Citometria de Fluxo , Transplante de Células-Tronco Mesenquimais/métodos , Doenças Neurodegenerativas/cirurgia , Optogenética , Adrenérgicos/toxicidade , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Lentivirus/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Doenças Neurodegenerativas/induzido quimicamente , Oxidopamina/toxicidade , Ratos , Ratos Sprague-Dawley , Antígenos Thy-1/metabolismo , Fatores de Tempo , Transfecção , Proteína Vermelha Fluorescente
4.
Apoptosis ; 17(3): 289-304, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22160861

RESUMO

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Oxidopamina/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de Parkinson/terapia , Ratos
5.
Biomolecules ; 11(8)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34439885

RESUMO

The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.


Assuntos
Esclerose Lateral Amiotrófica/sangue , Exossomos/metabolismo , MicroRNAs/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto
6.
J Neurol Sci ; 265(1-2): 116-21, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17619025

RESUMO

Stem cell therapy is considered a promising strategy aiming at neuronal and glial cell replacement or neuroprotection in neurological diseases affecting the brain and spinal cord. Multiple Sclerosis (MS), characterized by inflammation-induced destruction of the myelin sheath surrounding axons leading to conduction deficits and variability of clinical signs, is not an exception. MS is considered an autoimmune disease and, in the last few years, an intense immunodepletion followed by autologous hematopoietic-stem-cell transplant (HSCT) is being assessed as potential therapeutical strategy for severe patients unresponsive to the immunomodulatory and immunosuppressive treatment. Partially supported by evidence in animal models and by anecdotal reports on the beneficial effects on MS patients with concomitant malignant diseases, HSCT programs for MS have been initiated worldwide and follow-up data are accumulating. A Consensus Meeting has been held in Milano (1998) providing a document that defined criteria for patient selection, transplantation procedures, and outcome evaluations. Nowadays the high number of patients already treated allows us to draw initial conclusions related to clinical efficacy. After careful monitoring of the available data and improvement of the procedure, safety seems not to be anymore an issue. Ethics of HSCT deserve, on the contrary, a profound evaluation: the procedure is a multistep process with manifold options, each step with different ethical implications. Even more difficult appears the definition of the MS patient selection criteria for HSCT. The informed consensus needs to be exhaustive for the full comprehension of a complex procedure. In conclusion, although HCST is today an established therapeutical option for MS patients, safety and ethical issues need to be further clarified.


Assuntos
Esclerose Múltipla/cirurgia , Transplante de Células-Tronco/ética , Transplante de Células-Tronco/métodos , Humanos
7.
Cell Transplant ; 16(1): 41-55, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17436854

RESUMO

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFR(p75)) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Fatores de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/terapia , Adulto , Animais , Encéfalo/cirurgia , Diferenciação Celular , Imunofluorescência , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neurotrofina 3/metabolismo , Técnicas de Cultura de Órgãos , Transplante Heterólogo
8.
Sci Rep ; 7(1): 15808, 2017 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-29150616

RESUMO

Cell metabolism is a key determinant factor for the pluripotency and fate commitment of Stem Cells (SCs) during development, ageing, pathological onset and progression. We derived and cultured selected subpopulations of rodent fetal, postnatal, adult Neural SCs (NSCs) and postnatal glial progenitors, Olfactory Ensheathing Cells (OECs), respectively from the subventricular zone (SVZ) and the olfactory bulb (OB). Cell lysates were analyzed by proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy leading to metabolites identification and quantitation. Subsequent multivariate analysis of NMR data by Principal Component Analysis (PCA), and Partial Least Square Discriminant Analysis (PLS-DA) allowed data reduction and cluster analysis. This strategy ensures the definition of specific features in the metabolic content of phenotypically similar SCs sharing a common developmental origin. The metabolic fingerprints for selective metabolites or for the whole spectra demonstrated enhanced peculiarities among cell types. The key result of our work is a neat divergence between OECs and the remaining NSC cells. We also show that statistically significant differences for selective metabolites characterizes NSCs of different ages. Finally, the retrived metabolome in cell cultures correlates to the physiological SC features, thus allowing an integrated bioengineering approach for biologic fingerprints able to dissect the (neural) SC molecular specificities.


Assuntos
Metabolômica , Células-Tronco Neurais/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Animais , Análise Discriminante , Análise dos Mínimos Quadrados , Metaboloma , Camundongos , Análise Multivariada , Análise de Componente Principal
9.
Cell Res ; 16(4): 329-36, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16617328

RESUMO

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.


Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Células-Tronco Pluripotentes , Adipogenia , Adulto , Biomarcadores/análise , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Desenvolvimento Muscular , Osteogênese , Fenótipo , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/metabolismo
10.
Stem Cell Rev Rep ; 12(2): 224-34, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26553037

RESUMO

Olfactory Ensheathing Cells (OECs), exhibiting phenotypic characteristics of both astrocytes and Schwann Cells, show peculiar plasticity. In vitro, OECs promote axonal growth, while in vivo they promote remyelination of damaged axons. We decided to further investigate OEC potential for regeneration and functional recovery of the damaged Central Nervous System (CNS). To study OEC antigen modulation, OECs prepared from postnatal mouse olfactory bulbs were grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs) and analyzed for different neural specific markers. OEC functional characterizations were also achieved. Resistance of OECs to the neurotoxin 6-hydroxydopamine (6-OHDA) was analyzed by evaluating apoptosis and death. OEC neuroprotective properties were investigated by in vitro co-cultures or by addition of OEC conditioned medium to the neuroblastoma SH-SY5Y cells exposed to 6-OHDA. We observed: 1) modification of OEC morphology, reduced cell survival and marker expression in serum-free medium; 2) GF addition to serum-free medium condition influenced positively survival and restored basal marker expression; 3) no OEC apoptosis after a prolonged exposition to 6-OHDA; 4) a clear OEC neuroprotective tendency, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them potential clinical agents able to support injured CNS.


Assuntos
Astrócitos/citologia , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Bulbo Olfatório/citologia , Células de Schwann/citologia , Animais , Apoptose/fisiologia , Astrócitos/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Técnicas de Cocultura/métodos , Meios de Cultivo Condicionados/metabolismo , Meios de Cultura Livres de Soro/metabolismo , Camundongos , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Oxidopamina/farmacologia , Células de Schwann/metabolismo
11.
Lancet ; 364(9429): 200-2, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15246734

RESUMO

CONTEXT: With the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), and compelling preclinical data, stem-cell research has highlighted this disease as a candidate for stem-cell treatment. Stem-cell transplantation is an attractive strategy for neurological diseases and early successes in animal models of neurodegnerative disease generated optimism about restoring function or delaying degeneration in human beings. The restricted potential of adult stem cells has been challenged over the past 5 years by reports on their ability to acquire new unexpected fates beyond their embryonic lineage (transdifferentiation). Therefore, autologous or allogeneic stem cells, undifferentiated or transdifferentiated and manipulated epigenetically or genetically, could be a candidate source for local or systemic cell-therapies in ALS. STARTING POINT: Albert Clement and colleagues (Science 2003; 302: 113-17) showed that in SOD1G93A chimeric mice, motorneuron degeneration requires damage from mutant SOD1 acting in non-neuronal cells. Wild-type non-neuronal (glial) cells could delay degeneration and extend survival of mutant-expressing motorneurons. Letizia Mazzini and colleagues (Amyotroph Lateral Scler Other Motor Neuron Disord 2003; 4: 158-61) injected autologous bone-marrow-derived stem cells into the spinal cord of seven ALS patients. These investigators reported that the procedure had a reasonable margin of clinical safety. WHERE NEXT? The success of cell-replacement therapy in ALS will depend a lot on preclinical evidence, because of the complexity and precision of the pattern of connectivity that needs to be restored in degenerating motoneurons. Stem-cell therapy will need to be used with other drugs or treatments, such as antioxidants and/or infusion of trophic molecules.


Assuntos
Esclerose Lateral Amiotrófica/terapia , Transplante de Células-Tronco , Adulto , Animais , Embrião de Mamíferos/citologia , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Neurônios/citologia , Medula Espinal
12.
Stem Cells Dev ; 13(1): 121-31, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15068700

RESUMO

In the past few years research on stem cells has exploded as a tool to develop potential therapies to treat incurable neurodegenerative diseases. Stem cell transplantation has been effective in several animal models, but the underlying restorative mechanisms are still unknown. Several events such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation (adult cell acquisition of new unexpected identities) may explain therapeutic success, in addition to replacement of lost cells. This issue needs to be clarified further to maximize the potential for effective therapies. Preliminary stem transplantation trials have already been performed for some neurodegenerative diseases. There is no effective pharmacological treatment for amyotrophic lateral sclerosis, but recent preliminary data both in experimental and clinical settings have targeted it as an ideal candidate disease for the development of stem cell therapy in humans. This review summarizes recent advances gained in stem cell research applied to neurodegenerative diseases with a special emphasis to the criticisms put forward.


Assuntos
Diferenciação Celular , Doenças Neurodegenerativas/terapia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Divisão Celular , Fusão Celular , Humanos , Fatores de Crescimento Neural/metabolismo
13.
Brain Res ; 925(2): 213-21, 2002 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-11792370

RESUMO

Neural stem cells can be derived from the adult/embryonic nervous system as well as from more primitive embryonic stem cells but, because of the lack of specific markers, only their differentiated progeny can be characterized. We here report the presence of several endothelial and hematopoietic receptors (at protein and mRNA level) on the surface of embryonic human neural stem cells, which are partially maintained during differentiation. This suggests that neural stem cells have a greater potential than previously thought, which involves the ability to respond to different and so far unconsidered environmental signals and may be responsible for the recently discovered process of stem cell-fate conversion.


Assuntos
Antígenos de Diferenciação/biossíntese , Neurônios/metabolismo , Células-Tronco/metabolismo , Antígenos CD34/análise , Antígenos CD34/biossíntese , Antígenos de Diferenciação/análise , Antígenos de Diferenciação/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Neurônios/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptor TIE-2 , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Células-Tronco/química , Células-Tronco/citologia
15.
Methods Mol Biol ; 1052: 13-28, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23640251

RESUMO

The recently developed near-infrared (NIR) light imaging technology combines low background noise with deep tissue penetration and readily allows imaging and tracking of NIR-labeled cells, following transplantation in small animal model of diseases. The real-time and longitudinal detection of grafted cells in vivo, as well as their rapid ex vivo localization, may further clarify graft interactions with the surrounding, in target and nontarget organs throughout the body, over time. The present chapter describes a protocol for (1) the efficient labeling of human mesenchymal stem cells (hMSCs) using a membrane intercalating dye, emitting in the NIR 815 nm spectrum; (2) the stereotaxic transplantation of NIR 815-hMSCs in rodent model of Parkinson's disease; and (3) the longitudinal in vivo detection of the grafted cells and the subsequent ex vivo imaging in selected tissues.


Assuntos
Transplante de Células-Tronco Mesenquimais , Imagem de Banda Estreita/métodos , Doença de Parkinson Secundária/terapia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Terapia Baseada em Transplante de Células e Tecidos , Corantes Fluorescentes , Humanos , Masculino , Células-Tronco Mesenquimais , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem
16.
PLoS One ; 8(11): e78435, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244310

RESUMO

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.


Assuntos
Movimento Celular/efeitos dos fármacos , Compostos Férricos/efeitos adversos , Nanopartículas de Magnetita/efeitos adversos , Células-Tronco/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Compostos Férricos/farmacologia , Feto , Humanos , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/citologia
17.
J Hypertens ; 31(8): 1618-28, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23666422

RESUMO

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.


Assuntos
Encefalopatias/fisiopatologia , Neurogênese , Acidente Vascular Cerebral/fisiopatologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/patologia , Proliferação de Células , Progressão da Doença , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Edema/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Hipertensão/fisiopatologia , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Estresse Oxidativo , Proteinúria/fisiopatologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fatores de Tempo
18.
PLoS One ; 7(2): e32326, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22384217

RESUMO

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.


Assuntos
Líquido Amniótico/citologia , Esclerose Lateral Amiotrófica/genética , Encéfalo/metabolismo , Compostos Férricos/farmacologia , Células-Tronco Fetais/citologia , Nanopartículas de Magnetita/química , Animais , Bisbenzimidazol/farmacologia , Núcleo Celular/metabolismo , Proliferação de Células , Separação Celular , Sobrevivência Celular , Modelos Animais de Doenças , Citometria de Fluxo , Heterozigoto , Humanos , Luz , Imageamento por Ressonância Magnética/métodos , Magnetismo , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Fenótipo , Espalhamento de Radiação , Fatores de Tempo
19.
Stem Cells Cloning ; 3: 145-56, 2010 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-24198520

RESUMO

Neurodegenerative diseases are a growing public health challenge, and amyotrophic lateral sclerosis (ALS) remains a fatal incurable disease. The advent of stem cell therapy has opened new horizons for both researchers and ALS patients, desperately looking for a treatment. ALS must be considered a systemic disease affecting many cell phenotypes besides motor neurons, even outside the central nervous system. Cell replacement therapy needs to address the specific neurobiological issues of ALS to safely and efficiently reach clinical settings. Moreover, the enormous potential of induced pluripotent cells directly derived from patients for modeling and understanding the pathological mechanisms, in correlation with the discoveries of new genes and animal models, provides new opportunities that need to be integrated with previously described transplantation strategies. Finally, a careful evaluation of preclinical data in conjunction with wary patient choice in clinical trials needs to be established in order to generate meaningful results.

20.
J Mol Med (Berl) ; 88(6): 553-64, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20091292

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, nowadays considered as suitable candidate for autologous stem therapy with bone marrow (BM). A careful characterization of BM stem cell (SC) compartment is mandatory before its extensive application to clinic. Indeed, widespread systemic involvement has been recently advocated given that non-neuronal neighboring cells actively influence the pathological neuronal loss. We therefore investigated BM samples from 21 ALS patients and reported normal hematopoietic biological properties while an atypical behavior and impaired SC capabilities affected only the mesenchymal compartment. Moreover, by quantitative real-time approach, we observed altered Collagen IV and Metalloproteinase-9 levels in patients' derived mesenchymal stem cells (MSCs). Widespread metalloproteinase (MMPs) and their tissue inhibitor (TIMPs) alterations were established by multiplex ELISA analysis, demonstrating diffuse enzymatic variations in MSC compartment. Since MMPs act as fundamental effectors of extra-cellular matrix remodeling and stem cell mobilization, their modifications in ALS may influence reparative mechanisms effective in counteracting the pathology. In conclusion, ALS is further confirmed to be a systemic disease, not restricted to the nervous system, but affecting also the BM stromal compartment, even in sporadic cases. Therefore, therapeutic implantation of autologous BM derived SC in ALS patients needs to be carefully reevaluated.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Medula Óssea/enzimologia , Metaloproteinases da Matriz/metabolismo , Células-Tronco Mesenquimais/enzimologia , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Células Cultivadas , Colágeno Tipo IV/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Metaloproteinases da Matriz/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Pessoa de Meia-Idade , Inibidores Teciduais de Metaloproteinases/metabolismo
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