RESUMEN
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Asunto(s)
Células Madre Pluripotentes Inducidas , Organoides , Distrofias Retinianas , Pez Cebra , Animales , Humanos , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Luz/efectos adversos , Mutación , Organoides/metabolismo , Retina/metabolismo , Retina/patología , Distrofias Retinianas/terapia , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismoRESUMEN
Synucleinopathies refer to a group of disorders characterized by SNCA/α-synuclein (α-Syn)-containing cytoplasmic inclusions and neuronal cell loss in the nervous system including the cortex, a common feature being cognitive impairment. Still, the molecular pathogenesis of cognitive decline remains poorly understood, hampering the development of effective treatments. Here, we generated induced pluripotent stem cells (iPSCs) derived from familial Parkinson's disease (PD) patients carrying SNCA A53T mutation, differentiating them into cortical neurons by a direct conversion method. Patient iPSCs-derived cortical neurons harboring mutant α-Syn exhibited increased α-Syn-positive aggregates, shorter neurites, and time-dependent vulnerability. Furthermore, RNA-sequencing analysis, followed by biochemical validation, identified the activation of the ERK1/2 and JNK cascades in cortical neurons with SNCA A53T mutation. This result was consistent with a reverted phenotype of neuronal death in cortical neurons when treated with ERK1/2 and JNK inhibitors, respectively. Our findings emphasize the role of ERK1/2 and JNK cascades in the vulnerability of cortical neurons in synucleinopathies, and they could pave the way toward therapeutic advancements for synucleinopathies.
Asunto(s)
Sinucleinopatías , alfa-Sinucleína , Humanos , Sistema de Señalización de MAP Quinasas , Neuronas , NeuritasRESUMEN
The majority of the population of glial cells in the central nervous system consists of astrocytes, and impairment of astrocytes causes various disorders. It is useful to assess the multiple astrocytic properties in order to understand their complex roles in the pathophysiology. Although we can differentiate human astrocytes from induced pluripotent stem cells (iPSCs), it remains unknown how we can analyse and reveal the multiple properties of astrocytes in complexed human disease conditions. For this purpose, we tested astrocytic differentiation protocols from feeder-free iPSCs based on the previous method with some modifications. Then, we set up extra- and intracellular assessments of iPSC-derived astrocytes by testing cytokine release, calcium influx, autophagy induction and migration. The results led us to analytic methods with conditions in which iPSC-derived astrocytes behave as in vivo. Finally, we applied these methods for modelling an astrocyte-related disease, Alexander disease. An analytic system using iPSC-derived astrocytes could be used to recapture complexities in human astrocyte diseases.
Asunto(s)
Astrocitos , Células Madre Pluripotentes Inducidas , Humanos , Células Cultivadas , Neurogénesis , Citocinas , Diferenciación CelularRESUMEN
Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.
Asunto(s)
Diferenciación Celular , Cromosomas Humanos Par 12 , Células Madre Pluripotentes , Trisomía , Humanos , Diferenciación Celular/genética , Trisomía/genética , Cromosomas Humanos Par 12/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Mesodermo/citología , Endodermo/citología , Endodermo/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Línea Celular , Transducción de Señal/genéticaRESUMEN
Retinal dystrophies (RDs) lead to irreversible vision impairment with no radical treatment. Although photoreceptor cells (PRCs) differentiated from human induced pluripotent stem cells (iPSCs) are essential for the study of RDs as a scalable source, current differentiation methods for PRCs require multiple steps. To address these issues, we developed a method to generate PRCs from human iPSCs by introducing the transcription factors, CRX and NEUROD1. This approach enabled us to generate induced photoreceptor-like cells (iPRCs) expressing PRC markers. Single-cell RNA sequencing revealed the transcriptome of iPRCs in which the genes associated with phototransduction were expressed. Generated iPRCs exhibited their functional properties in calcium imaging. Furthermore, light-induced damage on iPRCs was inhibited by an antioxidant compound. This simple approach would facilitate the availability of materials for PRC-related research and provide a useful application for disease modeling and drug discovery.
RESUMEN
Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38â, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.
Asunto(s)
Diferenciación Celular , Vectores Genéticos , Desarrollo de Músculos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Virus Sendai , Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular/genética , Células Cultivadas , Reprogramación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Vectores Genéticos/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Desarrollo de Músculos/genética , Virus Sendai/genética , Temperatura , TransgenesRESUMEN
Schizophrenia (SCZ) is one of the major psychiatric disorders. The genetic factor is certainly influential in the onset of the disease but is not decisive. There is no identified molecular/cellular marker of the disease, and the pathomechanism is still unknown. In this study, we generated human induced pluripotent stem cells (iPSCs) derived from SCZ-discordant fraternal twins, and they could contribute to elucidation of the pathomechanism of SCZ.
Asunto(s)
Células Madre Pluripotentes Inducidas , Esquizofrenia , Humanos , Esquizofrenia/genética , Gemelos DicigóticosRESUMEN
Idiopathic basal ganglia calcification (IBGC) is a rare neurodegenerative disease, characterized by abnormal calcium deposits in basal ganglia of the brain. The affected individuals exhibit movement disorders, and progressive deterioration of cognitive and psychiatric ability. The genetic cause of the disease is mutation in one of several different genes, SLC20A2, PDGFB, PDGFRB, XPR1 or MYORG, which inheritably or sporadically occurs. Here we generated an induced pluripotent stem cell (iPSC) line from an IBGC patient, which is likely be a powerful tool for revealing the pathomechanisms and exploring potential therapeutic candidates of IBGC.
Asunto(s)
Enfermedades de los Ganglios Basales , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Ganglios Basales/metabolismo , Enfermedades de los Ganglios Basales/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Enfermedades Neurodegenerativas/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Although thalidomide is highly teratogenic, it has been prescribed for treating multiple myeloma and Hansen's disease. However, its mechanism of action is not fully understood. Here, we employed a reverse transcription quantitative PCR array to measure the expression of 84 genes in human induced pluripotent stem cells (hiPSCs) and their mesodermal differentiation. Thalidomide altered the expression of undifferentiated marker genes in both cell types. Thalidomide affected more genes in the mesoderm than in the hiPSCs. Ectoderm genes were upregulated but mesendoderm genes were downregulated by thalidomide during mesoderm induction, suggesting that thalidomide altered mesoderm differentiation. We found that FABP7 (fatty acid binding protein 7) was dramatically downregulated in the hiPSCs. FABP is related to retinoic acid, which is important signaling for limb formation. Moreover, thalidomide altered the expression of the genes involved in TGF-ß signaling, limb formation, and multiple myeloma, which are related to thalidomide-induced malformations and medication. In summary, iPSCs can serve as useful tools to elucidate the mechanisms underlying thalidomide malformations in vitro.
RESUMEN
Glycogen storage disease type 1a (GSD1a) is an autosomal recessive disorder caused by mutations of the glucose-6-phosphatase (G6PC) gene. Mutations of the G6PC gene lead to excessive accumulation of glycogen in the liver, kidney, and intestinal mucosa due to the deficiency of microsomal glucose-6-phosphatase. Human induced pluripotent stem cells (iPSCs) enable the production of patient-derived hepatocytes in culture and are therefore a promising tool for modeling GSD1a. Here, we report the establishment of human iPSCs from a GSD1a patient carrying a G6PC mutation (c.648Gâ¯>â¯T; p.Leu216â¯=â¯).
Asunto(s)
Línea Celular , Enfermedad del Almacenamiento de Glucógeno Tipo I , Células Madre Pluripotentes Inducidas , Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Hepatocitos , Humanos , Hígado , MutaciónRESUMEN
Cleft lip and palate are the most common congenital abnormalities that occur early in pregnancy. The majority of cranial mesenchyme is derived from cranial neural crest cells that differentiate into odontoblasts, cartilage, craniofacial bone, and connective tissue. A subset of these cells differentiates into cranial ganglia. We have previously reported an induction protocol of cranial neural crest cell-like cells from human pluripotent stem cells. This study tested detection of the cytotoxic sensitivities of dental materials, including titanium ions, palladium ions, and hydroxyethyl methacrylate, on the cell viability of induced cranial neural crest cell-like cells (iNC-LCs) derived from Tic human induced pluripotent stem cell (hiPSC) line. Further, the sensitivity was compared with those of human fetal lung fibroblastic cell line MRC-5, which is origin of Tic hiPSC, and osteoblastic cell line MC3T3-E1 which was derived from mouse calvaria. The results suggested that this cell-based assay system using iNC-LCs is a potential method for in vitro screening as an alternative to animal testing to predict toxic effects of dental materials on early craniofacial development.
Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Cresta Neural/citología , Cráneo/citología , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Metacrilatos/farmacología , Paladio/farmacología , Titanio/farmacologíaRESUMEN
Parkinson's disease (PD) is a devastating movement disorder with an unknown etiology. Multiplications of the SNCA gene cause the autosomal dominant form of familial PD as well as missense mutations of the gene. We established and characterized a human induced pluripotent stem cell (iPSC) line from a PD patient carrying SNCA duplication. The iPSC line displayed a capacity to differentiate into midbrain dopaminergic neurons affected in PD. The iPSC line will be useful for disease modeling applications.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Neuronas Dopaminérgicas , Humanos , Mutación Missense , Enfermedad de Parkinson/genética , alfa-Sinucleína/genéticaRESUMEN
Best Disease is an inherited retinal dystrophy that results in progressive and irreversible central vision loss caused by mutations of BESTROPHIN1 (BEST1). We established human induced pluripotent stem cells (iPSCs) from a Best disease patient with mutations R218H and A357V in the BEST1 gene. The generated iPSCs showed pluripotency markers and three-germ layer differentiation ability in vitro. A genetic analysis revealed mutations of R218H and A357V in the iPSCs. This iPSC line will be useful for elucidating the pathomechanisms of and drug discovery for Best disease.
Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofia Macular Viteliforme , Bestrofinas/genética , Diferenciación Celular , Humanos , MutaciónRESUMEN
Age-related macular degeneration (AMD) is a late-onset progressive blinding disease. We established human induced pluripotent stem cells (iPSCs) from an AMD patient. The generated iPSC line showed pluripotency markers and three-germ layer differentiation ability in vitro. This iPSC line will be useful for elucidating the pathomechanisms of and drug discovery for AMD.
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Células Madre Pluripotentes Inducidas , Degeneración Macular , Diferenciación Celular , Humanos , Degeneración Macular/genéticaRESUMEN
Astrocytes play vital roles in neurological disorders. The use of human induced pluripotent stem cell (iPSC)-derived astrocytes provides a chance to explore the contributions of astrocytes in human diseases. Here we review human iPSC-based models for neurological disorders associated with human astrocytes and discuss the points of each model.
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Astrocitos/metabolismo , Diferenciación Celular , Modelos Biológicos , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Astrocitos/citología , Biomarcadores , Susceptibilidad a Enfermedades , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/terapia , Neuroglía/citología , Neuroglía/metabolismo , Fenotipo , Células Madre Pluripotentes/citologíaRESUMEN
Cranial neural crest cells (NCCs) migrate to the branchial arches and give rise to the majority of cranial mesenchyme that eventually differentiates into odontoblasts, cartilage, craniofacial bone, and connective tissue; a subset of these cells differentiate into cranial ganglia. Here we present a protocol that describes directed differentiation method of human pluripotent stem cells into cranial NCC-like cells and a cytotoxicity assay using hPSC-derived cranial NCC-like cells. This cell-based assay system allows for high-sensitive cytotoxicity detection of test chemicals. These methods can be applied to predict drug/chemical toxicity effect on early craniofacial development.
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Encéfalo/citología , Cresta Neural/citología , Células Madre Pluripotentes/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Humanos , Modelos Biológicos , Pruebas de ToxicidadRESUMEN
Mucopolysaccharidosis type I (MPS I) is a rare inherited metabolic disorder caused by defects in alpha-L-iduronidase (IDUA), a lysosomal protein encoded by IDUA gene. MPS I is a progressive multisystemic disorder with a wide range of symptoms, including skeletal abnormalities and cognitive impairment, and is characterized by a wide spectrum of severity levels caused by varied mutations in IDUA. A human iPSC line was established from an attenuated MPS I (Scheie syndrome) patient carrying an IDUA gene mutation (c.266Gâ¯>â¯A; p.R89Q). This disease-specific iPSC line will be useful for the research of MPS I.
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Línea Celular , Iduronidasa/genética , Células Madre Pluripotentes Inducidas , Mucopolisacaridosis I/genética , Femenino , Humanos , Persona de Mediana EdadRESUMEN
Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.
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Ácido Glutámico/toxicidad , Ácido Láctico/metabolismo , Mitocondrias/metabolismo , Oxígeno/metabolismo , Animales , Respiración de la Célula , Células Cultivadas , Humanos , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ácido Pirúvico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Cell morphology is recognized as an important hallmark of neural cells. During the differentiation of human pluripotent stem cells (hPSCs) into neural cells, cell morphology changes dynamically. Therefore, characterization of the morphology of cells during this period is important to improve our understanding of the differentiation and development of neural cells. General methods for the directed induction of hPSCs include the steps of multi-cellular aggregation or high-density cell culture, particularly at the early phase of neural differentiation, and therefore, the morphology of each differentiating cell is difficult to recognize. Here, we have developed a new method for the directed differentiation of neuroepithelial-like cells (NELCs) from hPSCs at a low cell density in an adherent monolayer culture, as well as an image-processing algorithm to evaluate the cell morphology of differentiating NELCs, in order to follow cell morphology during the differentiation of hPSCs into NELCs. Using these methods, the morphological transition of differentiating cells was observed in real time using phase contrast imaging and then quantified. Because cell morphology is also considered an inherent biological marker of neural cells cultured in vitro, this method is potentially useful to study the mechanisms underlying neural cell differentiation.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Células Neuroepiteliales/citología , Neurogénesis , Neuronas/citología , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Neuroepiteliales/metabolismo , Neuronas/metabolismoRESUMEN
The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.