RESUMEN
Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.
Asunto(s)
Astrocitos/citología , Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas/citología , Enfermedades Neurodegenerativas/terapia , Trastornos del Neurodesarrollo/terapia , Neuronas/citología , Factores de Transcripción/genética , Diferenciación Celular , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Enfermedades Neurodegenerativas/genética , Trastornos del Neurodesarrollo/genética , Neuronas/trasplante , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.
Asunto(s)
Neuronas/fisiología , Optogenética/métodos , Células Madre/fisiología , Diferenciación Celular/fisiología , Línea Celular , Silenciador del Gen , Humanos , Integrasas , Neuronas/metabolismo , Células Madre/metabolismo , TransgenesRESUMEN
Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.