RESUMEN
Organoids capable of forming tissue-like structures have transformed our ability to model human development and disease. With the notable exception of the human heart, lineage-specific self-organizing organoids have been reported for all major organs. Here, we established self-organizing cardioids from human pluripotent stem cells that intrinsically specify, pattern, and morph into chamber-like structures containing a cavity. Cardioid complexity can be controlled by signaling that instructs the separation of cardiomyocyte and endothelial layers and by directing epicardial spreading, inward migration, and differentiation. We find that cavity morphogenesis is governed by a mesodermal WNT-BMP signaling axis and requires its target HAND1, a transcription factor linked to developmental heart chamber defects. Upon cryoinjury, cardioids initiated a cell-type-dependent accumulation of extracellular matrix, an early hallmark of both regeneration and heart disease. Thus, human cardioids represent a powerful platform to mechanistically dissect self-organization, congenital heart defects and serve as a foundation for future translational research.
Asunto(s)
Corazón/embriología , Organogénesis , Organoides/embriología , Activinas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Calcio/metabolismo , Línea Celular , Linaje de la Célula , Pollos , Células Endoteliales/citología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/citología , Proteína Homeótica Nkx-2.5/metabolismo , Humanos , Masculino , Mesodermo/embriología , Modelos Biológicos , Miocardio/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Wnt/metabolismoRESUMEN
The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Técnicas de Inactivación de Genes , Proteínas HSP90 de Choque Térmico/genética , Humanos , ARN Guía de Kinetoplastida/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Asunto(s)
Neoplasias de la Mama/genética , Epigénesis Genética , Factor de Transcripción GATA3/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Estrógenos/genética , Estrógenos/metabolismo , Femenino , Mutación del Sistema de Lectura , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.