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Max Salick, Postdoctoral Researcher, Novartis Institutes for Biomedical Research

April 10, 2018 @ 12:00 pm - 1:00 pm

Topic: Single-Cell Transcriptomics of CRISPR-Engineered TSC2-/- Human Cerebral Organoids Reveals Cell Type Specific Phenotypes in Tuberous Sclerosis and Treatment Response


Max R. Salick, Carleton P. Goold, Jonathan D. Biag, Robert J. Ihry, Sravya Kommineni, Daniel J. Ho, Beata Henry, Tripti Kulkarni, Julie Chen, Bob Altshuler, Ranjit Randhawa, Ned Kirkpatrick, Chaoyang Ye, David Fletcher, Bilada Bilican, Katie A. Worringer, Ricardo E. Dolmetsch, Ajamete Kaykas

Tuberous sclerosis is a mosaic disease caused by a germline heterozygous loss of function mutation in the TSC1 or TSC2 gene, which occurs in in 1 out of 6,000 live births.  Symptoms arise during development when somatic mutations in the affected gene lead to loss of heterozygosity, resulting in unregulated mTOR pathway activity.  This leads to a range of symptoms in the heart, lung, kidneys, skin, and other organs; however, the most severe symptoms arise when these somatic mutations occur in the brain.  When this happens, hamartomas form in distinct regions of the cortex, along with severe epilepsy, intellectual disability, and often autism.  There are several unique hallmarks of the afflicted region, including dysmorphic neurons, balloon cells, and disrupted cortical architecture.  To model this disease in vitro we produced TSC2-/- human embryonic stem cell and induced pluripotent stem cell lines using an inducible CRISPR/Cas9 system.  We found that neurons differentiated from these stem cell lines replicated several of the diseases phenotypes, such S6 phosphorylation and neuron size, which are then corrected by RAD001, an effective mTOR inhibitor.  We then produced cerebral organoids to mimic human brain development.  Mutant and control cerebral organoids were treated with DMSO or RAD001, which was then followed by dissociation and single cell sequencing using the 10X Chromium system.  Analysis of the single cell data revealed distinct mutation effects in the neuronal and glial populations, which were largely corrected in the RAD001-treated cerebral organoids.  Network analysis revealed a disruption in telencephalon differentiation in the neurons, which was confirmed in follow-up two-dimensional differentiation studies.  Additional single-cell analysis revealed a strong inflammatory signal in the mutant astrocytes, which was particularly rescued in the RAD001-treated samples.  Lastly, we conducted an aptamer-based screen of secreted proteins from WT and TSC2-/- neurons.  Considerable differences in the secretome of these cultures suggests that the healthy tissue surrounding the hamartomas may also be involved in the disease phenotype.  This study marks the first known investigation of isogenically-controlled disease models, along with a treatment response, using single cell transcriptomics, and may play a key role in understanding the complex physiology behind tuberous sclerosis.


April 10, 2018
12:00 pm - 1:00 pm
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