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1.
Cell ; 180(1): 188-204.e22, 2020 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-31883794

RESUMEN

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Glioblastoma/metabolismo , Organoides/crecimiento & desarrollo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bancos de Muestras Biológicas , Femenino , Glioblastoma/genética , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Organoides/metabolismo , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
2.
Dev Cell ; 58(9): 744-759.e11, 2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-37054704

RESUMEN

Cell competition acts as a quality-control mechanism that eliminates cells less fit than their neighbors to optimize organ development. Whether and how competitive interactions occur between neural progenitor cells (NPCs) in the developing brain remains unknown. Here, we show that endogenous cell competition occurs and intrinsically correlates with the Axin2 expression level during normal brain development. Induction of genetic mosaicism predisposes Axin2-deficient NPCs to behave as "losers" in mice and undergo apoptotic elimination, but homogeneous ablation of Axin2 does not promote cell death. Mechanistically, Axin2 suppresses the p53 signaling pathway at the post-transcriptional level to maintain cell fitness, and Axin2-deficient cell elimination requires p53-dependent signaling. Furthermore, mosaic Trp53 deletion confers a "winner" status to p53-deficient cells that outcompete their neighbors. Conditional loss of both Axin2 and Trp53 increases cortical area and thickness, suggesting that the Axin2-p53 axis may coordinate to survey cell fitness, regulate natural cell competition, and optimize brain size during neurodevelopment.


Asunto(s)
Competencia Celular , Proteína p53 Supresora de Tumor , Animales , Ratones , Proteína Axina/genética , Tamaño de los Órganos , Transducción de Señal/fisiología , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
3.
Front Immunol ; 13: 903246, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35844565

RESUMEN

Ependymoma (EPN) is a malignant glial tumor occurring throughout central nervous system, which commonly presents in children. Although recent studies have characterized EPN samples at both the bulk and single-cell level, intratumoral heterogeneity across subclones remains a confounding factor that impedes understanding of EPN biology. In this study, we generated a high-resolution single-cell dataset of pediatric ependymoma with a particular focus on the comparison of subclone differences within tumors and showed upregulation of cilium-associated genes in more highly differentiated subclone populations. As a proxy to traditional pseudotime analysis, we applied a novel trajectory scoring method to reveal cellular compositions associated with poor survival outcomes across primary and relapsed patients. Furthermore, we identified putative cell-cell communication features between relapsed and primary samples and showed upregulation of pathways associated with immune cell crosstalk. Our results revealed both inter- and intratumoral heterogeneity in EPN and provided a framework for studying transcriptomic signatures of individual subclones at single-cell resolution.


Asunto(s)
Neoplasias Encefálicas , Ependimoma , Niño , Ependimoma/genética , Ependimoma/patología , Humanos , ARN , Análisis de Secuencia de ARN , Regulación hacia Arriba
4.
Sci Adv ; 8(46): eabq2987, 2022 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-36383654

RESUMEN

The neuroendocrine system consists of a heterogeneous collection of neuropeptidergic neurons in the brain, among which hypothalamic KNDy neurons represent an indispensable cell subtype controlling puberty onset. Although neural progenitors and neuronal precursors along the cell lineage hierarchy adopt a cascade diversification strategy to generate hypothalamic neuronal heterogeneity, the cellular logic operating within the lineage to specify a subtype of neuroendocrine neurons remains unclear. As human genetic studies have recently established a link between TBX3 mutations and delayed puberty onset, we systematically studied Tbx3-derived neuronal lineage and Tbx3-dependent neuronal specification and found that Tbx3 hierarchically established and maintained the identity of KNDy neurons for triggering puberty. Apart from the well-established lineage-dependent fate determination, we uncovered rules of interlineage interaction and intralineage retention operating through neuronal differentiation in the absence of Tbx3. Moreover, we revealed that human TBX3 mutations disturbed the phase separation of encoded proteins and impaired transcriptional regulation of key neuropeptides, providing a pathological mechanism underlying TBX3-associated puberty disorders.


Asunto(s)
Neuronas , Neuropéptidos , Pubertad , Proteínas de Dominio T Box , Humanos , Linaje de la Célula , Hipotálamo/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Pubertad/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Animales , Ratones
5.
Neuron ; 109(7): 1150-1167.e6, 2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33600763

RESUMEN

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.


Asunto(s)
Hipotálamo/citología , Hipotálamo/crecimiento & desarrollo , Neuronas/fisiología , Animales , Animales Modificados Genéticamente , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/embriología , Linaje de la Célula , Ácido Glutámico/fisiología , Proteínas de Homeodominio/metabolismo , Hipotálamo/embriología , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/fisiología , Células Madre/fisiología , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Núcleo Hipotalámico Ventromedial/citología , Núcleo Hipotalámico Ventromedial/embriología , Núcleo Hipotalámico Ventromedial/metabolismo , Ácido gamma-Aminobutírico/fisiología
6.
Cell Stem Cell ; 28(9): 1657-1670.e10, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-33961804

RESUMEN

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.


Asunto(s)
Células Madre Pluripotentes Inducidas , Síndrome de Prader-Willi , Diferenciación Celular , Humanos , Hipotálamo , Organoides
7.
bioRxiv ; 2020 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-32766575

RESUMEN

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

8.
Cell Stem Cell ; 27(6): 937-950.e9, 2020 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-33010822

RESUMEN

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.


Asunto(s)
Plexo Coroideo/virología , Células-Madre Neurales/virología , Organoides/virología , Células Madre Pluripotentes/virología , SARS-CoV-2/fisiología , Tropismo Viral , Animales , Astrocitos/virología , Encéfalo/citología , Encéfalo/virología , COVID-19/genética , COVID-19/virología , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Neuronas/virología
9.
Neuron ; 97(2): 313-325.e6, 2018 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-29346752

RESUMEN

N6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system in vivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.


Asunto(s)
Adenosina/fisiología , Axones/fisiología , Epigénesis Genética/genética , Metiltransferasas/fisiología , Regeneración Nerviosa/genética , Proteínas del Tejido Nervioso/fisiología , Procesamiento Postranscripcional del ARN , Transcripción Genética , Adenosina/análogos & derivados , Animales , Ganglios Espinales/metabolismo , Ontología de Genes , Metiltransferasas/deficiencia , Ratones Noqueados , Compresión Nerviosa , Fosfohidrolasa PTEN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Nervio Ciático/lesiones , Neuropatía Ciática/genética , Neuropatía Ciática/fisiopatología , Células Receptoras Sensoriales/fisiología , Células Receptoras Sensoriales/ultraestructura
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