RESUMO
Gliomas are malignant primary tumors of the central nervous system. Their cell-of-origin is thought to be a neural progenitor or stem cell that acquires mutations leading to oncogenic transformation. Thanks to advances in human stem cell biology, it has become possible to derive human cell types that represent putative cells-of-origin in vitro and model the gliomagenesis process by systematically introducing genetic alterations in these human cells. Here, we present methods to derive human neural stem cells (NSCs) from human embryonic stem cells (hESCs). Because these NSCs are genetically unmodified at baseline, they can be used as a cellular platform to study the effects of individual oncogenic hits in a well-controlled manner in the backdrop of a human genetic background. We also present some key applications of these NSCs, which include their transduction with lentiviral vectors, their neuroglial differentiation and xenografting methods into immunocompromised mice to assess in vivo behavior.
Assuntos
Desdiferenciação Celular , Glioma/patologia , Células-Tronco Embrionárias Humanas/citologia , Modelos Biológicos , Células-Tronco Neurais , Animais , Astrócitos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Xenoenxertos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologiaRESUMO
Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.
