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1.
Cell ; 187(13): 3229-3230, 2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38906098

RESUMEN

Dr. Shinya Yamanaka is recognized for his discovery of the induction of pluripotent stem cells from fibroblasts by a combination of defined factors. In this interview with Cell, he discusses the progress of the field, what's next for clinical applications of iPS cells, and the state of science in Japan and the rest of the world.


Asunto(s)
Células Madre Pluripotentes Inducidas , Animales , Humanos , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Japón , Tratamiento Basado en Trasplante de Células y Tejidos , Separación Celular , Técnicas de Cultivo de Célula , Medicina Comunitaria
2.
Cell ; 187(10): 2446-2464.e22, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38582079

RESUMEN

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.


Asunto(s)
Células Madre Pluripotentes Inducidas , Neuronas , Tauopatías , Proteínas tau , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas tau/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Neuronas/metabolismo , Neuronas/patología , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Encéfalo/patología , Parálisis Supranuclear Progresiva/metabolismo , Parálisis Supranuclear Progresiva/patología , Parálisis Supranuclear Progresiva/genética , Diferenciación Celular , Mutación , Autofagia
3.
Cell ; 187(10): 2465-2484.e22, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38701782

RESUMEN

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.


Asunto(s)
Epigénesis Genética , Vaina de Mielina , Oligodendroglía , Remielinización , Animales , Vaina de Mielina/metabolismo , Humanos , Ratones , Remielinización/efectos de los fármacos , Oligodendroglía/metabolismo , Sistema Nervioso Central/metabolismo , Ratones Endogámicos C57BL , Rejuvenecimiento , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Organoides/metabolismo , Organoides/efectos de los fármacos , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/genética , Diferenciación Celular/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Masculino , Regeneración/efectos de los fármacos , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología
4.
Cell ; 185(4): 712-728.e14, 2022 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-35063084

RESUMEN

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.


Asunto(s)
Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Mapas de Interacción de Proteínas , Sinapsis/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilación , Encéfalo/metabolismo , Encéfalo/patología , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Demencia Frontotemporal/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Degeneración Nerviosa/patología , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Proteómica , Índice de Severidad de la Enfermedad , Fracciones Subcelulares/metabolismo , Tauopatías/genética , Proteínas tau/química
5.
Cell ; 185(13): 2213-2233.e25, 2022 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-35750033

RESUMEN

The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.


Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Astrocitos/metabolismo , Colesterol/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Microglía/metabolismo
6.
Cell ; 184(9): 2503-2519.e17, 2021 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-33838111

RESUMEN

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.


Asunto(s)
Sistemas CRISPR-Cas , Reprogramación Celular , Epigénesis Genética , Epigenoma , Edición Génica , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Diferenciación Celular , Islas de CpG , Metilación de ADN , Silenciador del Gen , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional
7.
Cell ; 184(8): 2084-2102.e19, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33765444

RESUMEN

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.


Asunto(s)
Evolución Biológica , Encéfalo/citología , Forma de la Célula/fisiología , Animales , Encéfalo/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Transición Epitelial-Mesenquimal/genética , Expresión Génica , Gorilla gorilla , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Organoides/citología , Organoides/metabolismo , Pan troglodytes , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo
8.
Cell ; 184(18): 4651-4668.e25, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-34450028

RESUMEN

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.


Asunto(s)
Productos Biológicos/uso terapéutico , Encéfalo/metabolismo , Enfermedades por Almacenamiento Lisosomal/terapia , Progranulinas/uso terapéutico , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Endosomas/metabolismo , Femenino , Demencia Frontotemporal/sangre , Demencia Frontotemporal/líquido cefalorraquídeo , Gliosis/complicaciones , Gliosis/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/patología , Metabolismo de los Lípidos , Lipofuscina/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Degeneración Nerviosa/patología , Fenotipo , Progranulinas/deficiencia , Progranulinas/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Transferrina/metabolismo , Distribución Tisular
9.
Cell ; 182(6): 1623-1640.e34, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32946783

RESUMEN

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.


Asunto(s)
Diferenciación Celular/genética , Organoides/citología , Organoides/metabolismo , Retina/citología , Retina/metabolismo , Análisis de la Célula Individual/métodos , Sinapsis/fisiología , Transcriptoma/genética , Técnicas de Cultivo de Célula/métodos , Línea Celular , Electrofisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Hibridación in Situ , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Electrónica , Familia de Multigenes , Naftoquinonas , Organoides/efectos de la radiación , Organoides/ultraestructura , Retina/patología , Retina/efectos de la radiación
10.
Cell ; 183(3): 636-649.e18, 2020 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-33031745

RESUMEN

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Nucleotidiltransferasas/metabolismo , Alarminas/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Degeneración Nerviosa/patología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Subunidades de Proteína/metabolismo , Transducción de Señal
11.
Cell ; 177(7): 1667-1669, 2019 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-31199910

RESUMEN

An "off-the-shelf" cell therapy derived from induced pluripotent stem cells (iPSCs) has entered clinical trials in the United States. Other companies are following suit, harnessing iPSCs' self-renewal ability to manufacture cell therapies that don't require customization for each patient. But some experts aren't sure such therapies are a good idea.


Asunto(s)
Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología
12.
Cell ; 179(3): 687-702.e18, 2019 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-31626770

RESUMEN

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.


Asunto(s)
Blastocisto/citología , Linaje de la Célula , Implantación del Embrión , Células Madre Pluripotentes Inducidas/citología , Células Madre Embrionarias de Ratones/citología , Creación de Embriones para Investigación/métodos , Animales , Blastocisto/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Técnicas de Reprogramación Celular/métodos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Embrionarias de Ratones/metabolismo , Transcriptoma
13.
Cell ; 176(4): 928-943.e22, 2019 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-30712874

RESUMEN

Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.


Asunto(s)
Reprogramación Celular/genética , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular/genética , Células Cultivadas , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/metabolismo
14.
Cell ; 179(1): 147-164.e20, 2019 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-31539493

RESUMEN

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.


Asunto(s)
Anexinas/metabolismo , Transporte Axonal/fisiología , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Anexinas/genética , Axones/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Unión Proteica , Ratas/embriología , Ratas Sprague-Dawley , Transfección , Pez Cebra
15.
Cell ; 178(3): 521-535.e23, 2019 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-31348885

RESUMEN

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.


Asunto(s)
Benzamidas/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Heptanos/farmacología , Lisosomas/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Benzamidas/química , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/uso terapéutico , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Mutación del Sistema de Lectura , Heptanos/uso terapéutico , Humanos , Receptores de Imidazolina/antagonistas & inhibidores , Receptores de Imidazolina/genética , Receptores de Imidazolina/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Lisosomas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteínas de Transporte Vesicular/química
16.
Cell ; 174(3): 636-648.e18, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-30017246

RESUMEN

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.


Asunto(s)
Plaquetas/metabolismo , Técnicas de Cultivo de Célula/métodos , Trombopoyesis/fisiología , Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Humanos , Hidrodinámica , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Megacariocitos/fisiología
17.
Cell ; 172(5): 979-992.e6, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29456084

RESUMEN

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.


Asunto(s)
Metilación de ADN/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Edición Génica , Neuronas/patología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Epigénesis Genética , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Masculino , Ratones , Neuronas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Expansión de Repetición de Trinucleótido/genética
18.
Cell ; 175(7): 1796-1810.e20, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30528432

RESUMEN

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.


Asunto(s)
Cromosomas Humanos Par 9 , Enfermedad de la Arteria Coronaria , Edición Génica , Haplotipos , Células Madre Pluripotentes Inducidas , Polimorfismo de Nucleótido Simple , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 9/genética , Cromosomas Humanos Par 9/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Femenino , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transcripción Genética
19.
Cell ; 172(5): 897-909.e21, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29474918

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Genoma Humano , Transcriptoma/genética , Empalme Alternativo/genética , Elementos Alu/genética , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Estudios de Cohortes , Familia , Femenino , Sitios Genéticos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Intrones/genética , Masculino , Repeticiones de Minisatélite/genética , Modelos Genéticos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
20.
Cell ; 173(4): 851-863.e16, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29576452

RESUMEN

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.


Asunto(s)
Adaptación Fisiológica , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Animales , Diferenciación Celular , Frío , Humanos , Células Madre Pluripotentes Inducidas/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neuronas/citología , Estrés Oxidativo , Inhibidores de Proteasas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , Sciuridae , Transcriptoma , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
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