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Genetically engineered human cortical spheroid models of tuberous sclerosis

Abstract

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.

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Fig. 1: Generation of heterozygous and homozygous knockout TSC1 and TSC2 hESC lines.
Fig. 2: TSC1−/− and TSC2−/− cortical spheroids have impaired neuronal and enhanced glial differentiation.
Fig. 3: TSC1−/− and TSC2−/− cortical spheroids fail to suppress mTORC1 signaling during neuronal differentiation.
Fig. 4: Conditional inactivation of TSC2 models a second-hit mutation.
Fig. 5: Cellular hypertrophy and neuronal differentiation defects in TSC2 mutant cells can be prevented by early rapamycin treatment.

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Acknowledgements

We thank F. Lorbeer for her help with the teratoma formation assay. This work was supported by a Predoctoral Award from the American Epilepsy Society (to J.D.B.), Frederick Banting and Charles Best Canada Graduate Scholarship from Canadian Institutes for Health Research (no. 356733 to J.D.B.), Brain Research Foundation Seed Grant (no. BRFSG-2014-02 to H.S.B.), Hellman Family Faculty Fund Award (to H.S.B.), NINDS R01 (no. R01NS097823 to H.S.B.), Sloan Research Fellowship in Neuroscience (no. FR-2015-65790 to H.S.B.), and NCI R01 (no. R01CA196884 to D.H.). D.H. is a Pew-Stewart Scholar for Cancer Research supported by the Pew Charitable Trusts and the Alexander and Margaret Stewart Trust. Seed funding was provided by the Siebel Stem Cell Institute.

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J.D.B. designed and carried out the experiments, performed the data analysis, and contributed to writing the manuscript. D.H. reprogrammed the TSC patient cells into hiPSCs, advised on the design of CRISPR–Cas9 gene editing experiments and human stem cell culture, and contributed to writing the manuscript. H.S.B. oversaw the project, designed the experiments, carried out the pilot experiments, wrote the manuscript, and acquired the funding.

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Correspondence to Helen S. Bateup.

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Complete statistics and sample sizes for all experiments

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Blair, J.D., Hockemeyer, D. & Bateup, H.S. Genetically engineered human cortical spheroid models of tuberous sclerosis. Nat Med 24, 1568–1578 (2018). https://doi.org/10.1038/s41591-018-0139-y

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