RESUMO
We generated an iPSC line from a patient with spastic paraplegia type 10 (SPG10) carrying the novel missense variant c.50G > A (p.R17Q) in the N-terminal motor domain of the kinesin family member 5A (KIF5A) gene. This patient-derived in vitro cell model will help to investigate the role of different KIF5A mutations in inducing neurodegeneration in spastic paraplegia and in other KIF5A-related disorders, including Charcot-Marie-Tooth type 2 (CMT2) and amyotrophic lateral sclerosis (ALS).
Assuntos
Células-Tronco Pluripotentes Induzidas , Paraplegia Espástica Hereditária , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Paraplegia Espástica Hereditária/genética , Cinesinas/genética , Mutação/genética , ParaplegiaRESUMO
The most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) is the expansion of a G4C2 hexanucleotide repeat in the C9orf72 gene. The size of the repeat expansion is highly variable and a cut-off of 30 repeats has been suggested as the lower pathological limit. Repeat size variability has been observed intergenerationally and intraindividually in tissues from different organs and within the same tissue, suggesting instability of the pathological repeat expansion. In order to study this genomic instability, we established iPSCs from five members of the same family of which four carried a C9orf72 repeat expansion and one was wild-type.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Humanos , Proteínas/genética , Proteína C9orf72/genética , Células-Tronco Pluripotentes Induzidas/patologia , Expansão das Repetições de DNA/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Demência Frontotemporal/genéticaRESUMO
Heterozygous mutations in the gene coding for progranulin (GRN) cause frontotemporal lobar degeneration (FTLD) while homozygous mutations are linked to neuronal ceroidolipofuscinosis (NCL). While both FTLD/NCL pathological hallmarks were mostly investigated in heterozygous GRN+/- brain tissue or induced pluripotent stem cell (iPSC)-derived neurons, data from homozygous GRN-/- condition are scarce, being limited to a postmortem brain tissue from a single case. Indeed, homozygous GRN-/- is an extremely rare condition reported in very few cases. Our aim was to investigate pathological phenotypes associated with FTLD and NCL in iPSC-derived cortical neurons from a GRN-/- patient affected by NCL. iPSCs were generated from peripheral blood of a GRN wt healthy donor and a GRN-/- patient and subsequently differentiated into cortical neurons. Several pathological changes were investigated, by means of immunocytochemical, biochemical and ultrastructural analyses. GRN-/- patient-derived cortical neurons displayed both TDP-43 and phospho-TDP-43 mislocalization, enlarged autofluorescent lysosomes and electron-dense vesicles containing storage material with granular, curvilinear and fingerprints profiles. In addition, different patterns in the expression of TDP-43, caspase 3 and cleaved caspase 3 were observed by biochemical analysis at different time points of cortical differentiation. At variance with previous findings, the present data highlight the existence of both FTLD- and NCL-linked pathological features in GRN-/- iPSC-derived cortical neurons from a NCL patient. They also suggest an evolution in the appearance of these features: firstly, FTLD-related TDP-43 alterations and initial NCL storage materials were detected; afterwards, mainly well-shaped NCL storage materials were present, while some FTLD features were not observed anymore.
Assuntos
Demência Frontotemporal , Degeneração Lobar Frontotemporal , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Caspase 3/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Neurônios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Progranulinas/genéticaRESUMO
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 PilotoRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases characterized by the presence of neuropathological aggregates of phosphorylated TDP-43 (P-TDP-43) protein. The RNA-binding protein TDP-43 participates also to cell stress response by forming stress granules (SG) in the cytoplasm to temporarily arrest translation. The hypothesis that TDP-43 pathology directly arises from SG has been proposed but is still under debate because only sub-lethal stress conditions have been tested experimentally so far. In this study we reproduced a mild and chronic oxidative stress by sodium arsenite to better mimic the persistent and subtle alterations occurring during the neurodegenerative process in primary fibroblasts and induced pluripotent stem cell-derived motoneurons (iPSC-MN) from ALS patients carrying mutations in TARDBP and C9ORF72 genes. We found that not only the acute sub-lethal stress usually used in literature, but also the chronic oxidative insult was able to induce SG formation in both primary fibroblasts and iPSC-MN. We also observed the recruitment of TDP-43 into SG only upon chronic stress in association to the formation of distinct cytoplasmic P-TDP-43 aggregates and a significant increase of the autophagy marker p62. A quantitative analysis revealed differences in both the number of cells forming SG in mutant ALS and healthy control fibroblasts, suggesting a specific genetic contribution to cell stress response, and in SG size, suggesting a different composition of these cytoplasmic foci in the two stress conditions. Upon removal of arsenite, the recovery from chronic stress was complete for SG and P-TDP-43 aggregates at 72 h with the exception of p62, which was reduced but still persistent, supporting the hypothesis that autophagy impairment may drive pathological TDP-43 aggregates formation. The gene-specific differences observed in fibroblasts in response to oxidative stress were not present in iPSC-MN, which showed a similar formation of SG and P-TDP-43 aggregates regardless their genotype. Our results show that SG and P-TDP-43 aggregates may be recapitulated in patient-derived neuronal and non-neuronal cells exposed to prolonged oxidative stress, which may be therefore exploited to study TDP-43 pathology and to develop individualized therapeutic strategies for ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/patologia , Neurônios Motores/patologia , Estresse Oxidativo/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes InduzidasRESUMO
Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.
Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Diferenciação Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sinapses/genética , Sinapses/fisiologia , Sinapses/ultraestrutura , TranscriptomaRESUMO
As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72-mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Proteína C9orf72/genética , Reprogramação Celular/genética , Doenças Neurodegenerativas/sangue , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Proteína C9orf72/sangue , Diferenciação Celular/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologiaRESUMO
The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics. Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons. Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Esquizofrenia/genética , Processamento Alternativo/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Éxons/genética , Humanos , Antígeno Neuro-Oncológico Ventral , Neurônios/metabolismo , Neurônios/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Esquizofrenia/patologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, mainly affecting the motor neurons (MNs) and without effective therapy. Drug screening is hampered by the lack of satisfactory experimental and pre-clinical models. Induced pluripotent stem cells (iPSCs) could help to define disease mechanisms and therapeutic strategies as they could be differentiated into MNs, otherwise inaccessible from living humans. In this study, given the seminal role of TDP-43 in ALS pathophysiology, MNs were obtained from peripheral blood mononuclear cells-derived iPSCs of an ALS patient carrying a p.A382T TARDBP mutation and a healthy donor. Venous samples were preferred to fibroblasts for their ease of collection and no requirement for time consuming extended cultures before experimentation. iPSCs were characterized for expression of specific markers, spontaneously differentiated into primary germ layers and, finally, into MNs. No differences were observed between the mutated ALS patient and the control MNs with most of the cells displaying a nuclear localization of the TDP-43 protein. In conclusion, we here demonstrated for the first time that human TARDBP mutated MNs can be successfully obtained exploiting the reprogramming and differentiation ability of peripheral blood cells, an easily accessible source from any patient.
Assuntos
Esclerose Lateral Amiotrófica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Diferenciação Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Pessoa de Meia-Idade , Neurônios Motores/citologia , MutaçãoRESUMO
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 PrincipalRESUMO
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-IdadeRESUMO
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/metabolismoRESUMO
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/citologiaRESUMO
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 RotulagemRESUMO
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 TempoRESUMO
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 TempoRESUMO
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 , RatosRESUMO
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/metabolismoRESUMO
Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.