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1.
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
2.
Cell ; 169(4): 736-749.e18, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-28475899

RESUMEN

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.


Asunto(s)
Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Microambiente Tumoral , Humanos , Citometría de Imagen , Tolerancia Inmunológica , Riñón/citología , Macrófagos/inmunología , Macrófagos/patología , Análisis de la Célula Individual , Linfocitos T/inmunología , Linfocitos T/patología
3.
Mol Cell ; 83(14): 2478-2492.e8, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37369201

RESUMEN

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.


Asunto(s)
Proteínas Argonautas , MicroARNs , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Unión Proteica , Células Madre/metabolismo , Mamíferos/metabolismo
4.
Blood ; 144(3): 296-307, 2024 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-38669617

RESUMEN

ABSTRACT: Patients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Neoplasia Residual , Trasplante Homólogo , Humanos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Femenino , Neoplasia Residual/diagnóstico , Adulto , Anciano , Mutación , Secuenciación de Nucleótidos de Alto Rendimiento , Recurrencia , Adulto Joven , Adolescente
5.
Nature ; 586(7828): 275-280, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33029001

RESUMEN

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.


Asunto(s)
Organoides/citología , Organoides/fisiología , Fenotipo , Regeneración/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Enterocitos/citología , Enterocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Organoides/efectos de los fármacos , Organoides/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/metabolismo , Regeneración/efectos de los fármacos , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Tretinoina/metabolismo , Vitamina A/farmacología
6.
EMBO J ; 40(12): e106818, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-33909924

RESUMEN

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.


Asunto(s)
Receptor Nuclear Huérfano DAX-1 , Histona Desacetilasas , Células Madre Embrionarias de Ratones/citología , Animales , Diferenciación Celular , Células Cultivadas , Receptor Nuclear Huérfano DAX-1/genética , Receptor Nuclear Huérfano DAX-1/metabolismo , Femenino , Factor de Transcripción GATA6/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Masculino , Ratones , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo
7.
Cell ; 141(4): 618-31, 2010 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-20478254

RESUMEN

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. We identified the voltage-dependent glutamate transporter Slc1a1 as one of the miR-183/96/182 targets in photoreceptor cells. We found that microRNAs in retinal neurons decay much faster than microRNAs in nonneuronal cells. The high turnover is also characteristic of microRNAs in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of microRNAs in neuronal cells while stimulation with glutamate accelerated it. Our results demonstrate that microRNA metabolism in neurons is higher than in most other cells types and linked to neuronal activity.


Asunto(s)
MicroARNs/metabolismo , Neuronas/metabolismo , Animales , Adaptación a la Oscuridad , Regulación hacia Abajo , Células Madre Embrionarias , Transportador 3 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Neuronas Retinianas/metabolismo , Regulación hacia Arriba
8.
Nature ; 569(7754): 136-140, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30996347

RESUMEN

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Unión Proteica
9.
Nature ; 569(7754): 66-72, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31019299

RESUMEN

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.


Asunto(s)
Intestinos/citología , Organoides/citología , Organoides/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Organoides/metabolismo , Células de Paneth/citología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análisis de la Célula Individual , Proteínas Señalizadoras YAP
10.
Mol Cell ; 68(3): 615-625.e9, 2017 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-29056324

RESUMEN

RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.


Asunto(s)
Microscopía Fluorescente , Estabilidad del ARN , ARN Mensajero/metabolismo , Imagen Individual de Molécula/métodos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Cinética , Microscopía por Video , Modelos Genéticos , ARN Mensajero/genética , Transfección
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