RESUMEN
One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Corteza Motora/embriología , Neurogénesis/fisiología , Células Piramidales/trasplante , Animales , Encéfalo/embriología , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Corteza Motora/citología , Células Piramidales/citología , Trasplante HeterólogoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Citometría de Flujo/métodos , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Adulto , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Astrocitos/patología , Astrocitos/fisiología , Axones/patología , Axones/fisiología , Supervivencia Celular , Células Cultivadas , Niño , Técnicas de Cocultivo , Genes Reporteros , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Lentivirus , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Mutación , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
By providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Lisosomas/metabolismo , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/fisiopatología , Neuronas/citología , Acetilglucosaminidasa/genética , Acetilglucosaminidasa/metabolismo , Proliferación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/metabolismo , Lisosomas/enzimología , Masculino , Modelos Biológicos , Mucopolisacaridosis III/enzimología , Mucopolisacaridosis III/genética , Mutación , Neuronas/enzimología , Neuronas/metabolismoRESUMEN
Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.
Asunto(s)
Azoospermia , Células Madre Pluripotentes Inducidas , Azoospermia/genética , Azoospermia/metabolismo , Diferenciación Celular/genética , Eritroblastos , Células Germinativas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
The use of mesenchymal stem cells (MSC) derived from several sources has been suggested as a major anti-inflammation strategy during the recent outbreak of coronavirus-19 (COVID-19). As the virus enters the target cells through the receptor ACE2, it is important to determine if the MSC population transfused to patients could also be a target for the virus entry. We report here that ACE2 is highly expressed in adult bone marrow, adipose tissue, or umbilical cord-derived MSC. On the other hand, placenta-derived MSC express low levels of ACE2 but only in early passages of cultures. MSC derived from human embryonic stem cell or human induced pluripotent stem cells express also very low levels of ACE2. The transcriptome analysis of the MSCs with lowest expression of ACE2 in fetal-like MSCs is found to be associated in particularly with an anti-inflammatory signature. These results are of major interest for designing future clinical MSC-based stem cell therapies for severe COVID-19 infections.
Asunto(s)
Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Mesenquimatosas , SARS-CoV-2/inmunología , Transcriptoma/inmunología , Adulto , Femenino , Humanos , Recién Nacido , Masculino , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , Células Madre Mesenquimatosas/virología , Especificidad de Órganos/inmunologíaRESUMEN
A multicentric retrospective study was carried out on 29 operations (28 patients) to evaluate the long-term results of the treatment of popliteal artery aneurysms by transposition of the superficial femoral artery (SFA). This treatment consisted in proximal and distal ligation and bypass grafting or endoaneurysmorrhaphy followed by reconstruction of the popliteal artery. This surgery was always performed when the homolateral SFA could be used. After surgery, every patient was prescribed a long-term antiplatelet treatment. Mean follow-up was 39.2+/-28 months. Actuarial primary patency was 100% at 1 year and 92% at 3 years. No patients presented with either aneurysmal evolution of arterial graft or septic complication of prosthetic bypass. SFA can be used to treat isolated popliteal aneurysms with satisfying long-term results. This technique is an alternative to the use of autologous saphenous vein.
Asunto(s)
Aneurisma/cirugía , Implantación de Prótesis Vascular , Arteria Femoral/cirugía , Arteria Poplítea/cirugía , Anciano , Anciano de 80 o más Años , Aneurisma/diagnóstico , Implantación de Prótesis Vascular/efectos adversos , Femenino , Francia , Humanos , Ligadura , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/uso terapéutico , Arteria Poplítea/diagnóstico por imagen , Estudios Retrospectivos , Factores de Tiempo , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Ultrasonografía Doppler Dúplex , Grado de Desobstrucción VascularRESUMEN
Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.
Asunto(s)
Sistema Nervioso Central/fisiopatología , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/genética , Células Madre Pluripotentes/metabolismo , Receptores de Superficie Celular/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adolescente , Empalme Alternativo , Animales , Apoptosis , Encéfalo/diagnóstico por imagen , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Preescolar , Eliminación de Gen , Variación Genética , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/fisiología , Receptores de Superficie Celular/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiologíaRESUMEN
Mucopolysaccharidoses type III (MPSIII, Sanfilippo syndrome) are genetic diseases due to deficient heparan sulfate (HS) saccharide digestion by lysosomal exoglycanases. Progressive accumulation of undigested saccharides causes early-onset behavioural and cognitive symptoms. The precise role of these saccharides in the pathophysiological cascade is still unclear. We showed that exposure of wild-type neural cells to exogenous soluble HS fragments of at least eight saccharides activated integrin-based focal adhesions (FAs), which attach cells to the extracellular matrix. FAs were constitutively activated in MPSIII type B astrocytes or neural stem cells unless undigested saccharides were cleared by exogenous supply of the missing exoglycanase. Defective cell polarisation and oriented migration in response to focal extracellular stimuli in affected cells suggest improper sensing of the environment. We consistently observed abnormal organisation of the rostral migratory stream in the brain of adult mice with MPSIII type B. These results suggest that cell polarisation and oriented migration defects participate to the neurological disorders associated with Sanfilippo syndrome.
Asunto(s)
Astrocitos/metabolismo , Adhesiones Focales/metabolismo , Heparitina Sulfato/farmacología , Mucopolisacaridosis III/patología , Células-Madre Neurales/metabolismo , Animales , Astrocitos/citología , Encéfalo/patología , Movimiento Celular/genética , Polaridad Celular/genética , Células Cultivadas , Activación Enzimática , Quinasa 1 de Adhesión Focal/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/citología , Paxillin/biosíntesis , Paxillin/genética , Fosforilación , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , PorcinosRESUMEN
Over the course of cortical neurogenesis, the transition of progenitors from proliferation to differentiation requires a precise regulation of involved gene networks under varying environmental conditions. In order to identify such regulatory mechanisms, we analyzed microRNA (miRNA) target networks in progenitors during early and late stages of neurogenesis. We found that cyclin D1 is a network hub whose expression is miRNA-dosage sensitive. Experimental validation revealed a feedback regulation between cyclin D1 and its regulating miRNAs miR-20a, miR-20b, and miR-23a. Cyclin D1 induces expression of miR-20a and miR-20b, whereas it represses miR-23a. Inhibition of any of these miRNAs increases the developmental stage-specific mean and dynamic expression range (variance) of cyclin D1 protein in progenitors, leading to reduced neuronal differentiation. Thus, miRNAs establish robustness and stage-specific adaptability to a critical dosage-sensitive gene network during cortical neurogenesis. Understanding such network regulatory mechanisms for key developmental events can provide insights into individual susceptibilities for genetically complex neuropsychiatric disorders.