RESUMEN
Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.
Asunto(s)
Sistemas CRISPR-Cas , Descubrimiento de Drogas/métodos , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Terapia Molecular Dirigida , Riñón Poliquístico Autosómico Dominante/genética , Células A549 , Animales , Diferenciación Celular , Células Cultivadas , Doxiciclina/farmacología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Riñón/citología , Organoides/efectos de los fármacos , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , ARN Guía de Kinetoplastida/genética , Porcinos , Canales Catiónicos TRPP/genéticaRESUMEN
Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation.
