Recent advances in human stem cell and genome engineering have enabled the generation of genetically defined human cellular models for brain disorders. These models can be established from a patient's own cells and can be genetically engineered to generate isogenic, controlled systems for mechanistic studies. Given the challenges of obtaining and working with primary human brain tissue, these models fill a critical gap in our understanding of normal and abnormal human brain development and provide an important complement to animal models. Recently, there has been major progress in modeling the neuropathophysiology of the canonical "mTORopathy" tuberous sclerosis complex (TSC) with such approaches. Studies using two- and three-dimensional cultures of human neurons and glia have provided new insights into how mutations in the TSC1 and TSC2 genes impact human neural development and function. Here we discuss recent progress in human stem cell-based modeling of TSC and highlight challenges and opportunities for further efforts in this area.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC.

Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identified mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3' UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation and a crucial role of 3' UTRs in driving cell-type-specific translation.