RESUMEN
Alzheimer's disease (AD) is the most common cause of dementia, and disease mechanisms are still not fully understood. Here, we explored pathological changes in human induced pluripotent stem cell (iPSC)-derived neurons carrying the familial AD APPV717I mutation after cell injection into the mouse forebrain. APPV717I mutant iPSCs and isogenic controls were differentiated into neurons revealing enhanced Aß42 production, elevated phospho-tau, and impaired neurite outgrowth in APPV717I neurons. Two months after transplantation, APPV717I and control neural cells showed robust engraftment but at 12 months post-injection, APPV717I grafts were smaller and demonstrated impaired neurite outgrowth compared to controls, while plaque and tangle pathology were not seen. Single-nucleus RNA-sequencing of micro-dissected grafts, performed 2 months after cell injection, identified significantly altered transcriptome signatures in APPV717I iPSC-derived neurons pointing towards dysregulated synaptic function and axon guidance. Interestingly, APPV717I neurons showed an increased expression of genes, many of which are also upregulated in postmortem neurons of AD patients including the transmembrane protein LINGO2. Downregulation of LINGO2 in cultured APPV717I neurons rescued neurite outgrowth deficits and reversed key AD-associated transcriptional changes related but not limited to synaptic function, apoptosis and cellular senescence. These results provide important insights into transcriptional dysregulation in xenografted APPV717I neurons linked to synaptic function, and they indicate that LINGO2 may represent a potential therapeutic target in AD.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Células Madre Pluripotentes Inducidas , Neuronas , Transcriptoma , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Neuronas/metabolismo , Neuronas/patología , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Mutación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Sinapsis/patología , Sinapsis/metabolismo , Péptidos beta-Amiloides/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
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Enfermedad de Parkinson/metabolismo , Células Madre Pluripotentes/patología , Diferenciación Celular , Reprogramación Celular , Dopamina/metabolismo , Fibroblastos/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AßO). Notably, AßO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , MicroARNs , Ratones , Animales , Humanos , Adulto , Metiltransferasas/genética , Fosforilación/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , MicroARNs/genética , Proteínas tau/metabolismo , Mamíferos/metabolismoRESUMEN
Many patients with SARS-CoV-2 infection develop neurological signs and symptoms; although, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical centre. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit. Hospital-associated complications were common, including eight patients (20%) with deep vein thrombosis/pulmonary embolism, seven (17%) with acute kidney injury requiring dialysis and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 h of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischaemic changes in all brains, both global and focal; large and small infarcts, many of which appeared haemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, although none showed evidence of vasculitis. Eighteen patients (44%) exhibited pathologies of neurodegenerative diseases, which was not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR, RNAscope® and immunocytochemistry with primers, probes and antibodies directed against the spike and nucleocapsid regions. The PCR analysis revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in the nasal epithelia. RNAscope® and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in coronavirus disease 2019 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but more likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischaemia. Further studies are needed to define whether these pathologies, if present in patients who survive coronavirus disease 2019, might contribute to chronic neurological problems.
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Infarto Encefálico/patología , Encéfalo/patología , COVID-19/patología , Hipoxia-Isquemia Encefálica/patología , Hemorragias Intracraneales/patología , Lesión Renal Aguda/complicaciones , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/terapia , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/complicaciones , Encéfalo/metabolismo , Infarto Encefálico/complicaciones , COVID-19/complicaciones , COVID-19/fisiopatología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Femenino , Humanos , Hipoxia-Isquemia Encefálica/complicaciones , Inflamación , Unidades de Cuidados Intensivos , Hemorragias Intracraneales/complicaciones , Masculino , Microglía/patología , Persona de Mediana Edad , Neuronas/patología , Fagocitosis , Fosfoproteínas/metabolismo , Embolia Pulmonar/complicaciones , Embolia Pulmonar/fisiopatología , ARN Viral/metabolismo , Diálisis Renal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tasa de Supervivencia , Linfocitos T/patología , Trombosis de la Vena/complicaciones , Trombosis de la Vena/fisiopatologíaRESUMEN
Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.
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Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Factores de Transcripción/genética , Animales , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Muerte Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Análisis por Conglomerados , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Perfilación de la Expresión Génica , Humanos , Ratones , Mutación , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Estrés Oxidativo , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/ultraestructura , Factores de Transcripción/metabolismo , Transcriptoma , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Abnormal generation of inhibitory neurons that synthesize γ-aminobutyric acid (GABAergic) is characteristic of neuropsychological disorders. We provide evidence that the extracellular matrix molecule tenascin-R (TNR) - which is predominantly expressed by a subpopulation of interneurons - plays a role in the generation of GABAergic and granule neurons in the murine dentate gyrus by regulating fate determination of neural stem or progenitor cells (NSCs). During development, absence of TNR in constitutively TNR-deficient (TNR(-/-)) mice results in increased numbers of dentate gyrus GABAergic neurons, decreased expression of its receptor ß1 integrin, increased activation of p38 MAPK and increased expression of the GABAergic specification gene Ascl1. Postnatally, increased GABAergic input to adult hippocampal NSCs in TNR(-/-) mice is associated not only with increased numbers of GABAergic and, particularly, parvalbumin-immunoreactive neurons, as seen during development, but also with increased numbers of granule neurons, thus contributing to the increased differentiation of NSCs into granule cells. These findings indicate the importance of TNR in the regulation of hippocampal neurogenesis and suggest that TNR acts through distinct direct and indirect mechanisms during development and in the adult.
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Proliferación Celular , Giro Dentado/crecimiento & desarrollo , Neurogénesis/genética , Tenascina/genética , Animales , Diferenciación Celular , Embrión de Mamíferos , Desarrollo Embrionario/genética , Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Ratones , Neuronas/metabolismo , Células Madre/metabolismo , Tenascina/metabolismoRESUMEN
Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.
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Diferenciación Celular , Células Eritroides/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Biomarcadores/metabolismo , Metilación de ADN , Epigenómica , Células Eritroides/metabolismo , Sangre Fetal/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The number of patients with neurodegenerative diseases is increasing significantly worldwide. Thus, intense research is being pursued to uncover mechanisms of disease development in an effort to identify molecular targets for therapeutic intervention. Analysis of postmortem tissue from patients has yielded important histological and biochemical markers of disease progression. However, this approach is inherently limited because it is not possible to study patient neurons prior to degeneration. As such, transgenic and knockout models of neurodegenerative diseases are commonly employed. While these animal models have yielded important insights into some molecular mechanisms of disease development, they do not provide the opportunity to study mechanisms of neurodegeneration in human neurons at risk and thus, it is often difficult or even impossible to replicate human pathogenesis with this approach. The generation of patient-specific induced pluripotent stem (iPS) cells offers a unique opportunity to overcome these obstacles. By expanding and differentiating iPS cells, it is possible to generate large numbers of functional neurons in vitro, which can then be used to study the disease of the donating patient. Here, we provide an overview of human stem cell models of neurodegeneration using iPS cells from patients with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, spinal muscular atrophy and other neurodegenerative diseases. In addition, we describe how further refinements of reprogramming technology resulted in the generation of patient-specific induced neurons, which have also been used to model neurodegenerative changes in vitro.
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Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Enfermedades Neurodegenerativas/patología , Células Cultivadas , Progresión de la Enfermedad , Humanos , Técnicas In Vitro , Mutación , Enfermedades Neurodegenerativas/genética , Neuronas/patologíaRESUMEN
Gangliogliomas are well-differentiated, mixed glio-neuronal tumors of the CNS that are most frequently localized within the temporal lobe. In a minority of cases, gangliogliomas have been described in the brain stem where they may critically impinge anatomical structures. Rarely, ganglioglioma develop in cranial nerves, almost exclusively in the optic pathway, where they usually present as singular space-occupying masses. Here, we report on an 83-year-old patient who presented with unusual symmetrical, bilateral gangliogliomas of the trigeminal nerves. These tumors showed an exophytic growth within the subarachnoid space toward the Gasserian ganglion and surprisingly appeared as isointense masses on T1- and T2-weighted MRI. Due to their bilateral appearance, we performed array-comparative genomic hybridization (aCGH) on the gangliogliomas to address the possibility of an underlying tumor syndrome in this patient. To our knowledge, this is the first case of bilateral ganglioglioma of the trigeminal nerve described so far.
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Neoplasias de los Nervios Craneales/patología , Ganglioglioma/patología , Enfermedades del Nervio Trigémino/patología , Anciano de 80 o más Años , Encéfalo/patología , Neoplasias de los Nervios Craneales/genética , Demencia/etiología , Ganglioglioma/genética , Humanos , Inmunohistoquímica , Masculino , Nervio Trigémino/patología , Enfermedades del Nervio Trigémino/genéticaRESUMEN
Alzheimer's disease (AD) is the most frequent form of dementia. It is characterized by pronounced neuronal degeneration with formation of neurofibrillary tangles and deposition of amyloid ß throughout the central nervous system. Animal models have provided important insights into the pathogenesis of AD and they have shown that different brain cell types including neurons, astrocytes and microglia have important functions in the pathogenesis of AD. However, there are difficulties in translating promising therapeutic observations in mice into clinical application in patients. Alternative models using human cells such as human induced pluripotent stem cells (iPSCs) may provide significant advantages, since they have successfully been used to model disease mechanisms in neurons and in glial cells in neurodegenerative diseases in vitro and in vivo. In this review, we summarize recent studies that describe the transplantation of human iPSC-derived neurons, astrocytes and microglial cells into the forebrain of mice to generate chimeric transplantation models of AD. We also discuss opportunities, challenges and limitations in using differentiated human iPSCs for in vivo disease modeling and their application for biomedical research.
RESUMEN
Neurodegenerative diseases are increasing in prevalence and comprise a large socioeconomic burden on patients and their caretakers. The need for effective therapies and avenues for disease prevention and monitoring is of paramount importance. Fluid biomarkers for neurodegenerative diseases have gained a variety of uses, including informing participant selection for clinical trials, lending confidence to clinical diagnosis and disease staging, determining prognosis, and monitoring therapeutic response. Their role is expected to grow as disease-modifying therapies start to be available to a broader range of patients and as prevention strategies become established. Many of the underlying molecular mechanisms of currently used biomarkers are incompletely understood. Animal models and in vitro systems using cell lines have been extensively employed but face important translatability limitations. Induced pluripotent stem cell (iPSC) technology, where a theoretically unlimited range of cell types can be reprogrammed from peripheral cells sampled from patients or healthy individuals, has gained prominence over the last decade. It is a promising avenue to study physiological and pathological biomarker function and response to experimental therapeutics. Such systems are amenable to high-throughput drug screening or multiomics readouts such as transcriptomics, lipidomics, and proteomics for biomarker discovery, investigation, and validation. The present review describes the current state of biomarkers in the clinical context of neurodegenerative diseases, with a focus on Alzheimer's disease and frontotemporal dementia. We include a discussion of how iPSC models have been used to investigate and test biomarkers such as amyloid-ß, phosphorylated tau, neurofilament light chain or complement proteins, and even nominate novel biomarkers. We discuss the limitations of current iPSC methods, mentioning alternatives such as coculture systems and three-dimensional organoids which address some of these concerns. Finally, we propose exciting prospects for stem cell transplantation paradigms using animal models as a preclinical tool to study biomarkers in the in vivo context.
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Enfermedad de Alzheimer , Biomarcadores , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/metabolismo , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Demencia Frontotemporal/diagnóstico , AnimalesRESUMEN
Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.
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Demencia Frontotemporal , Neuronas , Osteopontina , Proteínas tau , Osteopontina/metabolismo , Osteopontina/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Demencia Frontotemporal/metabolismo , Humanos , Neuronas/metabolismo , Neuronas/patología , Animales , Proteínas tau/metabolismo , Ratones , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Microglía/metabolismo , Microglía/patología , Mutación/genéticaRESUMEN
Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.
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Enfermedad de Parkinson/cirugía , Células Madre Pluripotentes/citología , Animales , Humanos , Enfermedad de Parkinson/patología , RatasRESUMEN
Alzheimer's disease (AD) is the most common cause of dementia resulting in widespread degeneration of the central nervous system with severe cognitive impairment. Despite the devastating toll of AD, the incomplete understanding of the complex molecular mechanisms hinders the expeditious development of effective cures. Emerging evidence from animal studies has shown that different brain cell types play distinct roles in the pathogenesis of AD. Glutamatergic neurons are preferentially affected in AD and pronounced gliosis contributes to the progression of AD in both a cell-autonomous and a non-cell-autonomous manner. Much has been discovered through genetically modified animal models, yet frequently failed translational attempts to clinical applications call for better disease models. Emerging evidence supports the significance of human-induced pluripotent stem cell (iPSC) derived brain cells in modeling disease development and progression, opening new avenues for the discovery of molecular mechanisms. This review summarizes the function of different cell types in the pathogenesis of AD, such as neurons, microglia, and astrocytes, and recognizes the potential of utilizing the rapidly growing iPSC technology in modeling AD.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Animales , Humanos , Enfermedad de Alzheimer/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Encéfalo/metabolismo , Astrocitos/metabolismo , Péptidos beta-Amiloides/metabolismoRESUMEN
The pathological involvement of the central nervous system in SARS-CoV2 (COVID-19) patients is established. The burden of pathology is most pronounced in the brain stem including the medulla oblongata. Hypoxic/ischemic damage is the most frequent neuropathologic abnormality. Other neuropathologic features include neuronophagia, microglial nodules, and hallmarks of neurodegenerative diseases: astrogliosis and microglial reactivity. It is still unknown if these pathologies are secondary to hypoxia versus a combination of inflammatory response combined with hypoxia. It is also unknown how astrocytes react to neuroinflammation in COVID-19, especially considering evidence supporting the neurotoxicity of certain astrocytic phenotypes. This study aims to define the link between astrocytic and microglial pathology in COVID-19 victims in the inferior olivary nucleus, which is one of the most severely affected brain regions in COVID-19, and establish whether COVID-19 pathology is driven by hypoxic damage. Here, we conducted neuropathologic assessments and multiplex-immunofluorescence studies on the medulla oblongata of 18 COVID-19, 10 pre-pandemic patients who died of acute respiratory distress syndrome (ARDS), and 7-8 control patients with no ARDS or COVID-19. The comparison of ARDS and COVID-19 allows us to identify whether the pathology in COVID-19 can be explained by hypoxia alone, which is common to both conditions. Our results showed increased olivary astrogliosis in ARDS and COVID-19. However, microglial density and microglial reactivity were increased only in COVID-19, in a region-specific manner. Also, olivary hilar astrocytes increased YKL-40 (CHI3L1) in COVID-19, but to a lesser extent than ARDS astrocytes. COVID-19 astrocytes also showed lower levels of Aquaporin-4 (AQP4), and Metallothionein-3 in subsets of COVID-19 brain regions. Cluster analysis on immunohistochemical attributes of astrocytes and microglia identified ARDS and COVID-19 clusters with correlations to clinical history and disease course. Our results indicate that olivary glial pathology and neuroinflammation in the COVID-19 cannot be explained solely by hypoxia and suggest that failure of astrocytes to upregulate the anti-inflammatory YKL-40 may contribute to the neuroinflammation. Notwithstanding the limitations of retrospective studies in establishing causality, our experimental design cannot adequately control for factors external to our design. Perturbative studies are needed to confirm the role of the above-described astrocytic phenotypes in neuroinflammation.
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Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.
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Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Neuronas/trasplante , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/fisiología , Cuerpo Estriado/cirugía , Femenino , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Macaca fascicularis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neuronas/inmunología , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/cirugía , Ratas , Ratas Sprague-DawleyRESUMEN
Parkinson's disease is the second most common neurodegenerative disease, after Alzheimer's disease, and the most common movement disorder. Drug treatment and deep brain stimulation can ameliorate symptoms, but the progressive degeneration of dopaminergic neurons in the substantia nigra eventually leads to severe motor dysfunction. The transplantation of stem cells has emerged as a promising approach to replace lost neurons in order to restore dopamine levels in the striatum and reactivate functional circuits. We have generated substrate-adherent embryonic stem cell-derived neural aggregates overexpressing the neural cell adhesion molecule L1, because it has shown beneficial functions after central nervous system injury. L1 enhances neurite outgrowth and neuronal migration, differentiation and survival as well as myelination. In a previous study, L1 was shown to enhance functional recovery in a mouse model of Huntington's disease. In another study, a new differentiation protocol for murine embryonic stem cells was established allowing the transplantation of stem cell-derived neural aggregates consisting of differentiated neurons and radial glial cells into the lesioned brain. In the present study, this embryonic stem cell line was engineered to overexpress L1 constitutively at all stages of differentiation and used to generate stem cell-derived neural aggregates. These were monitored in their effects on stem cell survival and differentiation, rescue of endogenous dopaminergic neurons and ability to influence functional recovery after transplantation in an animal model of Parkinson's disease. Female C57BL/6J mice (2 months old) were treated with the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneally to deplete dopaminergic neurons selectively, followed by unilateral transplantation of stem cell-derived neural aggregates into the striatum. Mice grafted with L1 overexpressing stem cell-derived neural aggregates showed better functional recovery when compared to mice transplanted with wild-type stem cell-derived neural aggregates and vehicle-injected mice. Morphological analysis revealed increased numbers and migration of surviving transplanted cells, as well as increased numbers of dopaminergic neurons, leading to enhanced levels of dopamine in the striatum ipsilateral to the grafted side in L1 overexpressing stem cell-derived neural aggregates, when compared to wild-type stem cell-derived neural aggregates. The striatal levels of gamma-aminobutyric acid were not affected by L1 overexpressing stem cell-derived neural aggregates. Furthermore, L1 overexpressing, but not wild-type stem cell-derived neural aggregates, enhanced survival of endogenous host dopaminergic neurons after transplantation adjacent to the substantia nigra pars compacta. Thus, L1 overexpressing stem cell-derived neural aggregates enhance survival and migration of transplanted cells, differentiation into dopaminergic neurons, survival of endogenous dopaminergic neurons, and functional recovery after syngeneic transplantation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.
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Células Madre Embrionarias/trasplante , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Recuperación de la Función/fisiología , Trasplante de Células Madre , Animales , Agregación Celular/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Pollos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/cirugía , Sustancia Negra/citología , Sustancia Negra/metabolismo , Sustancia Negra/cirugíaRESUMEN
The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/ß-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dopamina/metabolismo , Células Madre Embrionarias/citología , Factor 8 de Crecimiento de Fibroblastos/farmacología , Proteínas Hedgehog/metabolismo , Neuronas/citología , Células Madre Pluripotentes/citología , Tretinoina/farmacología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Proteínas Hedgehog/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Mesencéfalo/citología , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Parkinson , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/fisiología , Proteína Wnt1/farmacologíaRESUMEN
Neurodegenerative dementias are the most common group of neurodegenerative diseases affecting more than 40 million people worldwide. One of these diseases is frontotemporal dementia (FTD), an early onset dementia and one of the leading causes of dementia in people under the age of 60. FTD is a heterogeneous group of neurodegenerative disorders with pathological accumulation of particular proteins in neurons and glial cells including the microtubule-associated protein tau, which is deposited in its hyperphosphorylated form in about half of all patients with FTD. As for other patients with dementia, there is currently no cure for patients with FTD and thus several lines of research focus on the characterization of underlying pathogenic mechanisms with the goal to identify therapeutic targets. In this review, we provide an overview of reported disease phenotypes in induced pluripotent stem cell (iPSC)-derived neurons and glial cells from patients with tau-associated FTD with the aim to highlight recent progress in this fast-moving field of iPSC disease modeling. We put a particular focus on genetic forms of the disease that are linked to mutations in the gene encoding tau and summarize mutation-associated changes in FTD patient cells related to tau splicing and tau phosphorylation, microtubule function and cell metabolism as well as calcium homeostasis and cellular stress. In addition, we discuss challenges and limitations but also opportunities using differentiated patient-derived iPSCs for disease modeling and biomedical research on neurodegenerative diseases including FTD.
RESUMEN
Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.