RESUMEN
Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A-deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A-containing SWI/SNF/BAF complexes to mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.
Asunto(s)
Diferenciación Celular , Proteínas de Microfilamentos , Células-Madre Neurales , Animales , Ratones , Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Metabolismo Energético , Mitocondrias/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Microfilamentos/metabolismo , Células-Madre Neurales/citologíaRESUMEN
Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.
Asunto(s)
Técnicas de Cultivo de Célula , Organoides , Encéfalo , Humanos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodosRESUMEN
For decades, Waddington's concept of the 'epigenetic landscape' has served as an educative hierarchical model to illustrate the progressive restriction of cell differentiation potential during normal development. While still being highly valuable in the context of normal development, the Waddington model falls short of accommodating recent breakthroughs in cell programming. The advent of induced pluripotent stem (iPS) cells and advances in direct cell fate conversion (also known as transdifferentiation) suggest that somatic and pluripotent cell fates can be interconverted without transiting through distinct hierarchies. We propose a non-hierarchical 'epigenetic disc' model to explain such cell fate transitions, which provides an alternative landscape for modelling cell programming and reprogramming.
Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular/genética , Epigénesis Genética , Células Madre Pluripotentes/metabolismo , Animales , Linaje de la Célula/genética , Transdiferenciación Celular/genética , Humanos , Modelos Genéticos , Células Madre Pluripotentes/citologíaRESUMEN
For decades, Waddington's concept of the 'epigenetic landscape' has served as an educative hierarchical model to illustrate the progressive restriction of cell differentiation potential during normal development. While still being highly valuable in the context of normal development, the Waddington model falls short of accommodating recent breakthroughs in cell programming. The advent of induced pluripotent stem (iPS) cells and advances in direct cell fate conversion (also known as transdifferentiation) suggest that somatic and pluripotent cell fates can be interconverted without transiting through distinct hierarchies. We propose a non-hierarchical 'epigenetic disc' model to explain such cell fate transitions, which provides an alternative landscape for modelling cell programming and reprogramming.
Asunto(s)
Diferenciación Celular , Epigénesis Genética , Modelos Genéticos , Células Madre/fisiología , Animales , Linaje de la Célula , Ectodermo/citología , Endodermo/citología , Humanos , Mesodermo/citologíaRESUMEN
Induced pluripotent stem cells (iPSCs) and their derivatives have been described to display epigenetic memory of their founder cells, as well as de novo reprogramming-associated alterations. In order to selectively explore changes due to the reprogramming process and not to heterologous somatic memory, we devised a circular reprogramming approach where somatic stem cells are used to generate iPSCs, which are subsequently re-differentiated into their original fate. As somatic founder cells, we employed human embryonic stem cell-derived neural stem cells (NSCs) and compared them to iPSC-derived NSCs derived thereof. Global transcription profiling of this isogenic circular system revealed remarkably similar transcriptomes of both NSC populations, with the exception of 36 transcripts. Amongst these we detected a disproportionately large fraction of X chromosomal genes, all of which were upregulated in iPSC-NSCs. Concurrently, we detected differential methylation of X chromosomal sites spatially coinciding with regions harboring differentially expressed genes. While our data point to a pronounced overall reinstallation of autosomal transcriptomic and methylation signatures when a defined somatic lineage is propagated through pluripotency, they also indicate that X chromosomal genes may partially escape this reinstallation process. Considering the broad application of iPSCs in disease modeling and regenerative approaches, such reprogramming-associated alterations in X chromosomal gene expression and DNA methylation deserve particular attention.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Humanos , Metilación de ADN , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Epigénesis Genética , Reprogramación Celular/genéticaRESUMEN
Recreating human cell and organ systems in vitro has tremendous potential for disease modeling, drug discovery and regenerative medicine. The aim of this short overview is to recapitulate the impressive progress that has been made in the fast-developing field of cell programming during the past years, to illuminate the advantages and limitations of the various cell programming technologies for addressing nervous system disorders and to gauge their impact for perinatal medicine.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Medicina RegenerativaRESUMEN
BACKGROUND: NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. METHODS: Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5'-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. RESULTS: In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5'-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. CONCLUSIONS: Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract.
Asunto(s)
Células Madre Pluripotentes , Isoformas de ARN , Tetraciclina , Diferenciación Celular , Humanos , Isoenzimas/genética , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células Madre Pluripotentes/citología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The incidence of Alzheimer's disease is increasing with the aging population, and it has become one of the main health concerns of modern society. The dissection of the underlying pathogenic mechanisms and the development of effective therapies remain extremely challenging, also because available animal and cell culture models do not fully recapitulate the whole spectrum of pathological changes. The advent of human pluripotent stem cells and cell reprogramming has provided new prospects for tackling these challenges in a human and even patient-specific setting. Yet, experimental modeling of non-cell autonomous and extracellular disease-related alterations has remained largely inaccessible. These limitations are about to be overcome by advances in the development of 3D cell culture systems including organoids, neurospheroids and matrix-embedded 3D cultures, which have been shown to recapitulate extracellular pathologies such as plaque formation in vitro. Recent xenograft studies have even taken human stem cell-based disease modeling to an in vivo scenario where grafted neurons are probed in a disease background in the context of a rodent brain. Here, we review the latest developments in this emerging field along with their advantages, challenges, and future prospects.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Medicina de Precisión/métodos , Cultivo Primario de Células/métodos , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Edición Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Trasplante Heterólogo/métodosRESUMEN
Recent studies show an association of Parkin RBR E3 ubiquitin protein ligase (PARK2) copy number variations (CNVs) with attention deficit hyperactivity disorder (ADHD). The aim of our pilot study to investigate gene expression associated with PARK2 CNVs in human-derived cellular models. We investigated gene expression in fibroblasts, hiPSC and dopaminergic neurons (DNs) of ADHD PARK2 deletion and duplication carriers by qRT PCR compared with healthy and ADHD cell lines without PARK2 CNVs. The selected 10 genes of interest were associated with oxidative stress response (TP53, NQO1, and NFE2L2), ubiquitin pathway (UBE3A, UBB, UBC, and ATXN3) and with a function in mitochondrial quality control (PINK1, MFN2, and ATG5). Additionally, an exploratory RNA bulk sequencing analysis in DNs was conducted. Nutrient deprivation as a supplementary deprivation stress paradigm was used to enhance potential genotype effects. At baseline, in fibroblasts, hiPSC, and DNs, there was no significant difference in gene expression after correction for multiple testing. After nutrient deprivation in fibroblasts NAD(P)H-quinone-dehydrogenase 1 (NQO1) expression was significantly increased in PARK2 CNV carriers. In a multivariate analysis, ubiquitin C (UBC) was significantly upregulated in fibroblasts of PARK2 CNV carriers. RNA sequencing analysis of DNs showed the strongest significant differential regulation in Neurontin (NNAT) at baseline and after nutrient deprivation. Our preliminary results suggest differential gene expression in pathways associated with oxidative stress, ubiquitine-proteasome, immunity, inflammation, cell growth, and differentiation, excitation/inhibition modulation, and energy metabolism in PARK2 CNV carriers compared to wildtype healthy controls and ADHD patients.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad , Variaciones en el Número de Copia de ADN , Ubiquitina-Proteína Ligasas , Trastorno por Déficit de Atención con Hiperactividad/genética , Línea Celular , Variaciones en el Número de Copia de ADN/genética , Expresión Génica , Humanos , Proyectos Piloto , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33ΔE2 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-ß1-42 and bacterial particles were increased after knockout of CD33 or CD33ΔE2 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-ß1-42 or bacterial particles was increased after CD33 knockout but not in CD33ΔE2 -expressing microglia. In summary, deletion of CD33 or expression of CD33ΔE2 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid ß1-42 , but potentially detrimental oxidative burst and inflammation, which was absent in CD33ΔE2 -expressing microglia.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Estudio de Asociación del Genoma Completo , Humanos , Inflamación , Microglía , Fenotipo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on myeloid-derived cell types. The extracellular immunoglobulin-like domain of TREM2 binds anionic ligands including Apolipoprotein E and Amyloid-ß. The transmembrane domain interacts with its adaptor protein DAP12/TYROBP that is responsible for propagation of downstream signaling upon ligand interaction. Several sequence variants of TREM2 have been linked to different neurodegenerative diseases including Alzheimer's disease. Here, we generated HEK 293 Flp-In cell lines stably expressing human TREM2 and DAP12 using a bicistronic construct with a T2A linker sequence allowing initial expression of both proteins in stoichiometric amounts. Cell biological and biochemical analyses revealed transport of TREM2 to the cell surface, and canonical sequential proteolytic processing and shedding of TREM2 (sTREM2). The functionality of this cell system was demonstrated by detection of phosphorylated spleen tyrosine kinase (SYK) upon stimulation of TREM2 with the anionic membrane lipid phosphatidylserine or anti-TREM2 antibodies. Using this cell model, we demonstrated impaired signaling of disease associated TREM2 variants. We also identified a monoclonal antibody against the stalk region of TREM2 with agonistic activity. Activation of TREM2-DAP12 signaling with the monoclonal antibody and the partial loss of function of disease associated variants were recapitulated in induced pluripotent stem cell derived microglia. Thus, this reporter cell model represents a suitable experimental system to investigate signaling of TREM2 variants, and for the identification of ligands and compounds that modulate TREM2-DAP12 signaling. MAIN POINTS: Disease associated variants impair the signaling activity of TREM2 by distinct mechanisms. Targeting the stalk region of TREM2 with bivalent antibodies activates TREM2 signaling.
Asunto(s)
Enfermedad de Alzheimer , Microglía , Anticuerpos Monoclonales , Proteínas Portadoras , Células HEK293 , Humanos , Ligandos , Glicoproteínas de Membrana/genética , Células Mieloides , Receptores Inmunológicos/genéticaRESUMEN
Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased ß2-transferrin levels in patient CSF Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/metabolismo , Neuronas/metabolismo , Receptor ErbB-4/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Humanos , Transporte de Proteínas , Ratas , Receptor ErbB-4/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440.
Asunto(s)
Encéfalo/citología , Encéfalo/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Humanos , Neuronas/citología , Neuronas/metabolismo , Rombencéfalo/citología , Rombencéfalo/metabolismoRESUMEN
Recent advances in cell reprogramming have enabled assessment of disease-related cellular traits in patient-derived somatic cells, thus providing a versatile platform for disease modeling and drug development. Given the limited access to vital human brain cells, this technology is especially relevant for neurodegenerative disorders such as Parkinson's disease (PD) as a tool to decipher underlying pathomechanisms. Importantly, recent progress in genome-editing technologies has provided an ability to analyze isogenic induced pluripotent stem cell (iPSC) pairs that differ only in a single genetic change, thus allowing a thorough assessment of the molecular and cellular phenotypes that result from monogenetic risk factors. In this review, we summarize the current state of iPSC-based modeling of PD with a focus on leucine-rich repeat kinase 2 (LRRK2), one of the most prominent monogenetic risk factors for PD linked to both familial and idiopathic forms. The LRRK2 protein is a primarily cytosolic multi-domain protein contributing to regulation of several pathways including autophagy, mitochondrial function, vesicle transport, nuclear architecture and cell morphology. We summarize iPSC-based studies that contributed to improving our understanding of the function of LRRK2 and its variants in the context of PD etiopathology. These data, along with results obtained in our own studies, underscore the multifaceted role of LRRK2 in regulating cellular homeostasis on several levels, including proteostasis, mitochondrial dynamics and regulation of the cytoskeleton. Finally, we expound advantages and limitations of reprogramming technologies for disease modeling and drug development and provide an outlook on future challenges and expectations offered by this exciting technology.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mitofagia , Modelos Neurológicos , Enfermedad de Parkinson , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapiaRESUMEN
The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Cresta Neural/citología , Células Madre Pluripotentes/citología , Adipocitos/citología , Adipocitos/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Células Madre Embrionarias/citología , Humanos , Células Madre Mesenquimatosas/citología , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Células Madre Pluripotentes/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factores de Transcripción SOXE/metabolismoRESUMEN
Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.
Asunto(s)
Lesiones Encefálicas/cirugía , Potenciales Evocados Somatosensoriales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Sinapsis/fisiología , Potenciales de Acción , Vías Aferentes/fisiología , Animales , Encéfalo/citología , Encéfalo/ultraestructura , Lesiones Encefálicas/etiología , Línea Celular Transformada , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Modelos Animales de Enfermedad , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Neuronas/fisiología , Neuronas/ultraestructura , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Accidente Cerebrovascular/complicaciones , Sinapsis/ultraestructura , Núcleos Talámicos Ventrales/citologíaRESUMEN
Despite extensive efforts, half of patients with rare movement disorders such as hereditary spastic paraplegias and cerebellar ataxias remain genetically unexplained, implicating novel genes and unrecognized mutations in known genes. Non-coding DNA variants are suspected to account for a substantial part of undiscovered causes of rare diseases. Here we identified mutations located deep in introns of POLR3A to be a frequent cause of hereditary spastic paraplegia and cerebellar ataxia. First, whole-exome sequencing findings in a recessive spastic ataxia family turned our attention to intronic variants in POLR3A, a gene previously associated with hypomyelinating leukodystrophy type 7. Next, we screened a cohort of hereditary spastic paraplegia and cerebellar ataxia cases (n = 618) for mutations in POLR3A and identified compound heterozygous POLR3A mutations in â¼3.1% of index cases. Interestingly, >80% of POLR3A mutation carriers presented the same deep-intronic mutation (c.1909+22G>A), which activates a cryptic splice site in a tissue and stage of development-specific manner and leads to a novel distinct and uniform phenotype. The phenotype is characterized by adolescent-onset progressive spastic ataxia with frequent occurrence of tremor, involvement of the central sensory tracts and dental problems (hypodontia, early onset of severe and aggressive periodontal disease). Instead of the typical hypomyelination magnetic resonance imaging pattern associated with classical POLR3A mutations, cases carrying c.1909+22G>A demonstrated hyperintensities along the superior cerebellar peduncles. These hyperintensities may represent the structural correlate to the cerebellar symptoms observed in these patients. The associated c.1909+22G>A variant was significantly enriched in 1139 cases with spastic ataxia-related phenotypes as compared to unrelated neurological and non-neurological phenotypes and healthy controls (P = 1.3 × 10-4). In this study we demonstrate that (i) autosomal-recessive mutations in POLR3A are a frequent cause of hereditary spastic ataxias, accounting for about 3% of hitherto genetically unclassified autosomal recessive and sporadic cases; and (ii) hypomyelination is frequently absent in POLR3A-related syndromes, especially when intronic mutations are present, and thus can no longer be considered as the unifying feature of POLR3A disease. Furthermore, our results demonstrate that substantial progress in revealing the causes of Mendelian diseases can be made by exploring the non-coding sequences of the human genome.
Asunto(s)
Discapacidad Intelectual/genética , Espasticidad Muscular/genética , Atrofia Óptica/genética , ARN Polimerasa III/genética , Paraplejía Espástica Hereditaria/genética , Ataxias Espinocerebelosas/genética , Anciano , Técnicas de Cultivo de Célula , Exones/genética , Femenino , Estudios de Asociación Genética , Humanos , Células Madre Pluripotentes Inducidas , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/fisiopatología , Intrones/genética , Masculino , Persona de Mediana Edad , Espasticidad Muscular/diagnóstico por imagen , Espasticidad Muscular/fisiopatología , Mutación , Atrofia Óptica/diagnóstico por imagen , Atrofia Óptica/fisiopatología , Linaje , Fenotipo , Paraplejía Espástica Hereditaria/diagnóstico por imagen , Paraplejía Espástica Hereditaria/fisiopatología , Ataxias Espinocerebelosas/diagnóstico por imagen , Ataxias Espinocerebelosas/fisiopatologíaRESUMEN
At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.
Asunto(s)
Trasplante de Células Madre/legislación & jurisprudencia , Europa (Continente) , Humanos , Italia , Seguridad del Paciente , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/normas , Células Madre , Investigación Biomédica TraslacionalRESUMEN
Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487LeX, 5750LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487LeX-, 5750LeX- and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCsFGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCsFGF-2/EGF. Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487LeX, 5750LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.
Asunto(s)
Diferenciación Celular , Glicoconjugados/fisiología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/biosíntesis , Línea Celular , Polaridad Celular , Epítopos/biosíntesis , Glicoconjugados/biosíntesis , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/inmunología , Polisacáridos/biosíntesisRESUMEN
Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.