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Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.
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Criopreservação , Feto , Transplante de Rim , Rim , Organoides , Animais , Criopreservação/métodos , Suínos , Rim/citologia , Organoides/citologia , Organoides/transplante , Camundongos , Transplante de Rim/métodos , Feto/citologia , Feminino , Transplante Heterólogo/métodos , Preservação de Órgãos/métodosRESUMO
The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs.
Assuntos
Rim , Néfrons , Humanos , Camundongos , Animais , Células-Tronco Embrionárias , Diferenciação Celular , Camundongos Transgênicos , OrganoidesRESUMO
BACKGROUND: Several genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS. METHODS: We enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS. RESULTS: Three novel loci were significantly associated with the survival of patients with sporadic ALS-FGF1 at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10-9), THSD7A at 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10-8) and LRP1 at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10-8). FGF1 and THSD7A variants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression of FGF1 and THSD7A was partially disrupted. The rs60565245 was not associated with LRP1 mRNA expression. CONCLUSIONS: We identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression of FGF1 and THSD7A and the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Estudo de Associação Genômica Ampla , População do Leste Asiático , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Neurônios Motores/patologiaRESUMO
Peripheral artery disease (PAD) is commonly caused by atherosclerosis and has an unfavorable prognosis. Complete revascularization (CR) of the coronary artery reduces the risk of major adverse cardiovascular event (MACE) in patients with coronary artery disease (CAD). However, the impact of CR in patients with PAD has not been established to date. Therefore, we evaluated the impact of CR of CAD on the five-year clinical outcomes in patients with PAD. This study was based on a prospective, multicenter, observational registry in Japan. We enrolled 366 patients with PAD undergoing endovascular treatment. The primary endpoint was MACE, defined as a composite of all-cause death, non-fatal myocardial infarction, and non-fatal stroke. After excluding ineligible patients, 96 and 68 patients received complete revascularization of the coronary artery (CR group) and incomplete revascularization of the coronary artery (ICR group), respectively. Freedom from MACE in the CR group was significantly higher than in the ICR group at 5 years (66.7% vs 46.0%, p < 0.01). Multivariate analysis revealed that CR emerged as an independent predictor of MACE (Hazard ratio: 0.56, 95% confidential interval: 0.34-0.94, p = 0.03). CR of CAD was significantly associated with improved clinical outcomes in patients with PAD undergoing endovascular treatment.
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Doença da Artéria Coronariana , Doença Arterial Periférica , Humanos , Estudos Prospectivos , Doença da Artéria Coronariana/cirurgia , Doença da Artéria Coronariana/complicações , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/cirurgia , Doença Arterial Periférica/complicações , Sistema de Registros , Fatores de Risco , Resultado do TratamentoRESUMO
The neuropathological hallmarks of Parkinson's disease (PD) include the appearance of α-synuclein (α-SYN)-positive Lewy bodies (LBs) and the loss of catecholaminergic neurons. Thus, a potential mechanism promoting the uptake of extracellular α-SYN may exist in susceptible neurons. Of the various differentially expressed proteins, we are interested in flotillin (FLOT)-1 because this protein is highly expressed in the brainstem catecholaminergic neurons and is strikingly up-regulated in PD brains. In this study, we found that extracellular monomeric and fibrillar α-SYN can potentiate FLOT1-dopamine transporter (DAT) binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity. Moreover, we demonstrated that α-SYN itself exploited the DAT endocytic process to enter dopaminergic neuron-like cells, and both FLOT1 and DAT were found to be the components of LBs. Altogether, these findings revealed a novel role of extracellular α-SYN on cellular trafficking of DAT and may provide a rationale for the cell type-specific, functional, and pathologic alterations in PD.-Kobayashi, J., Hasegawa, T., Sugeno, N., Yoshida, S., Akiyama, T., Fujimori, K., Hatakeyama, H., Miki, Y., Tomiyama, A., Kawata, Y., Fukuda, M., Kawahata, I., Yamakuni, T., Ezura, M., Kikuchi, A., Baba, T., Takeda, A., Kanzaki, M., Wakabayashi, K., Okano, H., Aoki, M. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.
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Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Corpos de Lewy/patologia , Proteínas de Membrana/metabolismo , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Endocitose , Humanos , Corpos de Lewy/metabolismo , Proteínas de Membrana/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transporte Proteico , alfa-Sinucleína/genéticaRESUMO
The extracellular L-glutamate (L-Glu) concentration is elevated in neuroinflammation, thereby causing excitotoxicity. One of the mechanisms is down-regulation of astrocyte L-Glu transporters. Some antidepressants have anti-inflammatory effects. We therefore investigated effects of various antidepressants on the down-regulation of astrocyte L-Glu transporters in the in vitro neuroinflammation model. Among these antidepressants, only paroxetine was effective. We previously demonstrated that the down-regulation of astrocyte L-Glu transporters was caused by L-Glu released from activated microglia. We here clarified that only paroxetine inhibited L-Glu release from microglia. This is the novel action of paroxetine, which may bring advantages on the therapy of neuroinflammation.
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Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inflamação/metabolismo , Microglia/efeitos dos fármacos , Paroxetina/farmacologia , Antidepressivos/farmacologia , Astrócitos/metabolismo , Células Cultivadas , Ácido Glutâmico/metabolismo , Humanos , Inflamação/induzido quimicamente , Lipopolissacarídeos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
The presence of residual undifferentiated pluripotent stem cells (PSCs) in PSC-derived cell therapy products (CTPs) is a major safety issue for their clinical application, due to the potential risk of PSC-derived tumor formation. An international multidisciplinary multisite study to evaluate a droplet digital PCR (ddPCR) approach to detect residual undifferentiated PSCs in PSC-derived CTPs was conducted as part of the Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee. To evaluate the use of ddPCR in quantifying residual iPSCs in a cell sample, different quantities of induced pluripotent stem cells (iPSCs) were spiked into a background of iPSC-derived cardiomyocytes (CMs) to mimic different concentrations of residual iPSCs. A one step reverse transcription ddPCR (RT-ddPCR) was performed to measure mRNA levels of several iPSC-specific markers and to evaluate the assay performance (precision, sensitivity, and specificity) between and within laboratories. The RT-ddPCR assay variability was initially assessed by measuring the same RNA samples across all participating facilities. Subsequently, each facility independently conducted the entire process, incorporating the spiking step, to discern the parameters influencing potential variability. Our results show that a RT-ddPCR assay targeting ESRG, LINC00678, and LIN28A genes offers a highly sensitive and robust detection of impurities of iPSC-derived CMs and that the main contribution to variability between laboratories is the iPSC-spiking procedure, and not the RT-ddPCR. The RT-ddPCR assay would be generally applicable for tumorigenicity evaluation of PSC-derived CTPs with appropriate marker genes suitable for each CTP.
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BACKGROUND: Lower limb artery disease (LEAD) is accompanied by multiple comorbidities; however, the effect of hyperpolypharmacy on patients with LEAD has not been established. This study investigated the associations between hyperpolypharmacy, medication class, and adverse clinical outcomes in patients with LEAD. METHODS: This study used data from a prospective multicenter observational Japanese registry. A total of 366 patients who underwent endovascular treatment (EVT) for LEAD were enrolled in this study. The primary endpoints were major adverse cardiac events (MACE), including myocardial infarction, stroke, and all-cause death. RESULTS: Of 366 patients with LEAD, 12 with missing medication information were excluded. Of the 354 remaining patients, 166 had hyperpolypharmacy (≥10 medications, 46.9â¯%), 162 had polypharmacy (5-9 medications, 45.8â¯%), and 26 had nonpolypharmacy (<5 medications, 7.3â¯%). Over a 4.7-year median follow-up period, patients in the hyperpolypharmacy group showed worse outcomes than those in the other two groups (log-rank test, pâ¯<â¯0.001). Multivariate analysis revealed that the total number of medications was significantly associated with an increased risk of MACE (hazard ratio per medication increase 1.07, 95â¯% confidence interval 1.02-1.13 pâ¯=â¯0.012). Although an increased number of non-cardiovascular medications was associated with an elevated risk of MACE, the increase in cardiovascular medications was not statistically significant (log-rank test, pâ¯=â¯0.002 and 0.35, respectively). CONCLUSIONS: Hyperpolypharmacy due to non-cardiovascular medications was significantly associated with adverse outcomes in patients with LEAD who underwent EVT, suggesting the importance of medication reviews, including non-cardiovascular medications.
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iPSC-based drug discovery led to a phase 1/2a trial of ropinirole in ALS. 20 participants with sporadic ALS received ropinirole or placebo for 24 weeks in the double-blind period to evaluate safety, tolerability, and therapeutic effects. Adverse events were similar in both groups. During the double-blind period, muscle strength and daily activity were maintained, but a decline in the ALSFRS-R, which assesses the functional status of ALS patients, was not different from that in the placebo group. However, in the open-label extension period, the ropinirole group showed significant suppression of ALSFRS-R decline and an additional 27.9 weeks of disease-progression-free survival. iPSC-derived motor neurons from participants showed dopamine D2 receptor expression and a potential involvement of the SREBP2-cholesterol pathway in therapeutic effects. Lipid peroxide represents a clinical surrogate marker to assess disease progression and drug efficacy. Limitations include small sample sizes and high attrition rates in the open-label extension period, requiring further validation.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Indóis/efeitos adversos , Indóis/farmacologia , Neurônios MotoresRESUMO
BACKGROUND: In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown. METHODS: We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 µM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model. RESULTS: Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST. CONCLUSIONS: Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.
Assuntos
Astrócitos/metabolismo , Regulação para Baixo/fisiologia , Transportador 1 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Microglia/metabolismo , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Astrócitos/efeitos dos fármacos , Benzopiranos/farmacologia , Carbenoxolona/farmacologia , Morte Celular , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/antagonistas & inibidores , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/farmacologia , L-Lactato Desidrogenase , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Sais de Tetrazólio , TiazóisRESUMO
We investigated the prognostic effects of hyperuricemia and high or low body mass index (BMI) in peripheral artery disease (PAD) after endovascular therapy (EVT). Between July 2015-2016, 357 consecutive patients with PAD who underwent EVT were enrolled. Patients were divided into 2 groups: BMI < 25 kg/m2 (low BMI) and ≥ 25 kg/m2 (high BMI); they were also divided into 2 more groups based on the presence/absence of hyperuricemia. The primary and secondary endpoints were major adverse cardiovascular and limb events (MACLE), and all-cause death at 3 years post-EVT. Patients with hyperuricemia had significantly lower freedom from MACLE than patients without hyperuricemia at 3 years (57.0 vs 71.9%, p = .0068). The overall survival of patients with hyperuricemia was significantly lower than that of patients without hyperuricemia (63.9 vs 81.7%, p = .0012). Patients with hyperuricemia who had low BMI experienced significantly lower freedom from MACLE than those without hyperuricemia who had low BMI (48.2 vs 69.9%, p = .002). The overall survival of patients with hyperuricemia who had low BMI was significantly lower than that of patients without hyperuricemia who had low BMI (55.2 vs 77.1%, p = .003). Patients with hyperuricemia had significantly more MACLE and a lower survival at 3 years than patients without hyperuricemia, even if they had a low BMI.
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Procedimentos Endovasculares , Hiperuricemia , Doença Arterial Periférica , Índice de Massa Corporal , Procedimentos Endovasculares/efeitos adversos , Humanos , Sistema de Registros , Fatores de Risco , Resultado do Tratamento , Redução de PesoRESUMO
Induced pluripotent stem cells (iPSCs) are increasingly used in the study of disease mechanisms and the development of effective disease-modifying therapies for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Recently, three candidate anti-ALS drugs - ropinirole (ROPI), retigabine, and bosutinib - have been identified in iPSC-based drug screens and are now being evaluated in clinical trials for safety and effectiveness. We review the preclinical data, clinical research design, and rationale for ROPI as an anti-ALS drug candidate compared with those of the other two drugs. We also discuss the use of iPSCs for understanding and monitoring treatment response as well as for new insights into the development of new drugs and therapeutic interventions for major neurodegenerative diseases.
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Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Preparações Farmacêuticas , Esclerose Lateral Amiotrófica/tratamento farmacológico , Humanos , Indóis/farmacologiaRESUMO
Bipolar disorder (BP) and schizophrenia (SCZ) are major psychiatric disorders, but the molecular mechanisms underlying the complicated pathologies of these disorders remain unclear. It is difficult to establish adequate in vitro models for pathological analysis because of the heterogeneity of these disorders. In the present study, to recapitulate the pathologies of these disorders in vitro, we established in vitro models by differentiating mature neurons from human induced pluripotent stem cells (hiPSCs) derived from BP and SCZ patient with contributive copy number variations, as follows: two BP patients with PCDH15 deletion and one SCZ patient with RELN deletion. Glutamatergic neurons and GABAergic neurons were induced from hiPSCs under optimized conditions. Both types of induced neurons from both hiPSCs exhibited similar phenotypes of MAP2 (microtubule-associated protein 2)-positive dendrite shortening and decreasing synapse numbers. Additionally, we analyzed isogenic PCDH15- or RELN-deleted cells. The dendrite and synapse phenotypes of isogenic neurons were partially similar to those of patient-derived neurons. These results suggest that the observed phenotypes are general phenotypes of psychiatric disorders, and our in vitro models using hiPSC-based technology may be suitable for analysis of the pathologies of psychiatric disorders.
Assuntos
Transtorno Bipolar/patologia , Técnicas de Cultura de Células/métodos , Neurônios/patologia , Células-Tronco Pluripotentes , Esquizofrenia/patologia , Adulto , Transtorno Bipolar/genética , Proteínas Relacionadas a Caderinas , Caderinas/genética , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Variações do Número de Cópias de DNA , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteína Reelina , Esquizofrenia/genética , Serina Endopeptidases/genéticaRESUMO
BACKGROUND: The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. METHOD: Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma (FUS) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. FINDINGS: We identified aberrant increasing of axon branchings in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS. INTERPRETATION: Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. FUND: Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and "Inochi-no-Iro" ALS research grant.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Axônios/metabolismo , Axônios/patologia , Diferenciação Celular/genética , Linhagem Celular , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Neurônios Motores/metabolismo , Mutação , Neurogênese/genética , Fenótipo , RNA Interferente Pequeno/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients' full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.
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Esclerose Lateral Amiotrófica/genética , Neurônios Motores/patologia , Degeneração Neural/genética , Agregação Patológica de Proteínas/genética , Esclerose Lateral Amiotrófica/patologia , Diferenciação Celular/genética , Humanos , Indóis/uso terapêutico , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Mutação , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Fenótipo , Superóxido Dismutase-1/genéticaRESUMO
The cerebral cortex is subdivided into distinct areas that have particular functions. The rostrocaudal (R-C) gradient of fibroblast growth factor 8 (FGF8) signaling defines this areal identity during neural development. In this study, we recapitulated cortical R-C patterning in human pluripotent stem cell (PSC) cultures. Modulation of FGF8 signaling appropriately regulated the R-C markers, and the patterns of global gene expression resembled those of the corresponding areas of human fetal brains. Furthermore, we demonstrated the utility of this culture system in modeling the area-specific forebrain phenotypes [presumptive upper motor neuron (UMN) phenotypes] of amyotrophic lateral sclerosis (ALS). We anticipate that our culture system will contribute to studies of human neurodevelopment and neurological disease modeling.
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Esclerose Lateral Amiotrófica , Padronização Corporal , Córtex Cerebral , Fator 8 de Crescimento de Fibroblasto , Células-Tronco Pluripotentes , Transdução de Sinais , Técnicas de Cultura de Células , Córtex Cerebral/embriologia , Humanos , Modelos BiológicosRESUMO
Human pluripotent stem cells (hPSCs) represent a potentially valuable cell source for applications in cell replacement therapy, drug development, and disease modeling. For all these uses, it is necessary to develop reproducible and robust protocols for differentiation into desired cell types. However, differentiation protocols remain unstable and inefficient, which makes minimizing the differentiation variance among hPSC lines and obtaining purified terminally differentiated cells extremely time consuming. Here, we report a simple treatment with three small molecules-SB431542, dorsomorphine, and CHIR99021-that enhanced hPSC differentiation into three germ layers with a chemically transitional embryoid-body-like state (CTraS). Induction of CTraS reduced the innate differentiation propensities of hPSCs (even unfavorably differentiated hPSCs) and shifted their differentiation into terminally differentiated cells, particularly neurons. In addition, CTraS induction accelerated in vitro pathological expression concurrently with neural maturation. Thus, CTraS can promote the latent potential of hPSCs for differentiation and potentially expand the utility and applicability of hPSCs.
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Diferenciação Celular , Senescência Celular , Células-Tronco Embrionárias Humanas/citologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Via de Sinalização Wnt , Benzamidas/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Células Cultivadas , Dioxóis/farmacologia , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Neurônios/citologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Patient-specific induced pluripotent stem cells (iPSCs) facilitate understanding of the etiology of diseases, discovery of new drugs and development of novel therapeutic interventions. A frequently used starting source of cells for generating iPSCs has been dermal fibroblasts (DFs) isolated from skin biopsies. However, there are also numerous repositories containing lymphoblastoid B-cell lines (LCLs) generated from a variety of patients. To date, this rich bioresource of LCLs has been underused for generating iPSCs, and its use would greatly expand the range of targeted diseases that could be studied by using patient-specific iPSCs. However, it remains unclear whether patient's LCL-derived iPSCs (LiPSCs) can function as a disease model. Therefore, we generated Parkinson's disease patient-specific LiPSCs and evaluated their utility as tools for modeling neurological diseases. We established iPSCs from two LCL clones, which were derived from a healthy donor and a patient carrying PARK2 mutations, by using existing non-integrating episomal protocols. Whole genome sequencing (WGS) and comparative genomic hybridization (CGH) analyses showed that the appearance of somatic variations in the genomes of the iPSCs did not vary substantially according to the original cell types (LCLs, T-cells and fibroblasts). Furthermore, LiPSCs could be differentiated into functional neurons by using the direct neurosphere conversion method (dNS method), and they showed several Parkinson's disease phenotypes that were similar to those of DF-iPSCs. These data indicate that the global LCL repositories can be used as a resource for generating iPSCs and disease models. Thus, LCLs are the powerful tools for generating iPSCs and modeling neurological diseases.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos/citologia , Modelos Biológicos , Doenças do Sistema Nervoso/patologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular Transformada , Derme/citologia , Fibroblastos/citologia , Herpesvirus Humano 4/fisiologia , Humanos , Mitocôndrias/metabolismo , Mutação/genética , Neurônios/citologia , Doença de Parkinson/patologia , Fenótipo , Esferoides Celulares/citologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.