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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
van Slegtenhorst, M. et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808 (1997).
European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315 (1993).
Crino, P. B., Nathanson, K. L. & Henske, E. P. The tuberous sclerosis complex. N. Engl. J. Med. 355, 1345–1356 (2006).
Thiele, E. A. Managing and understanding epilepsy in tuberous sclerosis complex. Epilepsia 51(Suppl 1), 90–91 (2010).
Curatolo, P., Moavero, R. & de Vries, P. J. Neurological and neuropsychiatric aspects of tuberous sclerosis complex. Lancet Neurol. 14, 733–745 (2015).
Crino, P. B. Evolving neurobiology of tuberous sclerosis complex. Acta Neuropathol. 125, 317–332 (2013).
Mohamed, A. R. et al. Intrinsic epileptogenicity of cortical tubers revealed by intracranial EEG monitoring. Neurology 79, 2249–2257 (2012).
Sosunov, A. A. et al. Epileptogenic but MRI-normal perituberal tissue in Tuberous Sclerosis Complex contains tuber-specific abnormalities. Acta Neuropathol. Commun. 3, 17 (2015).
Jansen, F. E. et al. Cognitive impairment in tuberous sclerosis complex is a multifactorial condition. Neurology 70, 916–923 (2008).
Magri, L. et al. Sustained activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous sclerosis complex-associated lesions. Cell Stem Cell. 9, 447–462 (2011).
Feliciano, D. M., Su, T., Lopez, J., Platel, J. C. & Bordey, A. Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice. J. Clin. Invest. 121, 1596–1607 (2011).
Way, S. W. et al. Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse. Hum. Mol. Genet. 18, 1252–1265 (2009).
Onda, H. et al. Tsc2 null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway. Mol. Cell. Neurosci. 21, 561–574 (2002).
Carson, R. P., Van Nielen, D. L., Winzenburger, P. A. & Ess, K. C. Neuronal and glia abnormalities in Tsc1-deficient forebrain and partial rescue by rapamycin. Neurobiol. Dis. 45, 369–380 (2012).
Goto, J. et al. Regulable neural progenitor-specific Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex. Proc. Natl Acad. Sci. USA 108, E1070–1079 (2011).
Meikle, L. et al. A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J. Neurosci. 27, 5546–5558 (2007).
Zeng, L. H. et al. Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Hum. Mol. Genet. 20, 445–454 (2011).
Silbereis, J. C., Pochareddy, S., Zhu, Y., Li, M. & Sestan, N. The cellular and molecular landscapes of the developing human central nervous system. Neuron 89, 248–268 (2016).
Dehay, C. & Kennedy, H. Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450 (2007).
Florio, M. & Huttner, W. B. Neural progenitors, neurogenesis and the evolution of the neocortex. Development 141, 2182–2194 (2014).
Lui, J. H., Hansen, D. V. & Kriegstein, A. R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).
Tee, A. R. et al. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc. Natl Acad. Sci. USA 99, 13571–13576 (2002).
Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).
Ma, X. M. & Blenis, J. Molecular mechanisms of mTOR-mediated translational control. Nat. Rev. Mol. Cell Biol. 10, 307–318 (2009).
Inoki, K., Li, Y., Xu, T. & Guan, K. L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003).
Chong-Kopera, H. et al. TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. J. Biol. Chem. 281, 8313–8316 (2006).
Huang, J. & Manning, B. D. The TSC1–TSC2 complex: a molecular switchboard controlling cell growth. Biochem J. 412, 179–190 (2008).
Crino, P. B. The mTOR signalling cascade: paving new roads to cure neurological disease. Nat. Rev. Neurol. 12, 379–392 (2016).
Magri, L. & Galli, R. mTOR signaling in neural stem cells: from basic biology to disease. Cell. Mol. Life Sci. 70, 2887–2898 (2013).
Au, K. S., Hebert, A. A., Roach, E. S. & Northrup, H. Complete inactivation of the TSC2 gene leads to formation of hamartomas. Am. J. Hum. Genet. 65, 1790–1795 (1999).
Henske, E. P. et al. Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am. J. Hum. Genet. 59, 400–406 (1996).
Smolarek, T. A. et al. Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis. Am. J. Hum. Genet. 62, 810–815 (1998).
Sepp, T., Yates, J. R. & Green, A. J. Loss of heterozygosity in tuberous sclerosis hamartomas. J. Med. Genet. 33, 962–964 (1996).
Chan, J. A. et al. Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J. Neuropathol. Exp. Neurol. 63, 1236–1242 (2004).
Crino, P. B., Aronica, E., Baltuch, G. & Nathanson, K. L. Biallelic TSC gene inactivation in tuberous sclerosis complex. Neurology 74, 1716–1723 (2010).
Qin, W. et al. Analysis of TSC cortical tubers by deep sequencing of TSC1, TSC2 and KRAS demonstrates that small second-hit mutations in these genes are rare events. Brain Pathol. 20, 1096–1105 (2010).
Martin, K. R. et al. The genomic landscape of tuberous sclerosis complex. Nat. Commun. 8, 15816 (2017).
de Vries, P. J. & Howe, C. J. The tuberous sclerosis complex proteins—a GRIPP on cognition and neurodevelopment. Trends Mol. Med. 13, 319–326 (2007).
Blair, J. D., Hockemeyer, D., Doudna, J. A., Bateup, H. S. & Floor, S. N. Widespread translational remodeling during human neuronal differentiation. Cell Rep. 21, 2005–2016 (2017).
Thoreen, C. C. et al. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109–113 (2012).
Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).
Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).
Paşca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).
Dibble, C. C. et al. TBC1D7 is a third subunit of the TSC1–TSC2 complex upstream of mTORC1. Mol. Cell 47, 535–546 (2012).
Miller, F. D. & Gauthier, A. S. Timing is everything: making neurons versus glia in the developing cortex. Neuron 54, 357–369 (2007).
Bonni, A. et al. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 278, 477–483 (1997).
Cloëtta, D. et al. Inactivation of mTORC1 in the developing brain causes microcephaly and affects gliogenesis. J. Neurosci. 33, 7799–7810 (2013).
Au, K. S. et al. Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States. Genet. Med. 9, 88–100 (2007).
Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).
Mizuguchi, M. & Takashima, S. Neuropathology of tuberous sclerosis. Brain Dev. 23, 508–515 (2001).
Sosunov, A. A. et al. Tuberous sclerosis: a primary pathology of astrocytes? Epilepsia 49(Suppl 2), 53–62 (2008).
Grabole, N. et al. Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model. Genome Med. 8, 94 (2016).
Costa, V. et al. mTORC1 inhibition corrects neurodevelopmental and synaptic alterations in a human stem cell model of tuberous sclerosis. Cell Rep. 15, 86–95 (2016).
Li, Y. et al. Abnormal neural progenitor cells differentiated from induced pluripotent stem cells partially mimicked development of TSC2 neurological abnormalities. Stem Cell Rep 8, 883–893 (2017).
Tsai, P. T. et al. Prenatal rapamycin results in early and late behavioral abnormalities in wildtype C57BL/6 mice. Behav. Genet. 43, 51–59 (2013).
Boer, K. et al. Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex. Epilepsy Res. 78, 7–21 (2008).
D’Gama, A. M. et al. Somatic mutations activating the mTOR pathway in dorsal telencephalic progenitors cause a continuum of cortical dysplasias. Cell Rep. 21, 3754–3766 (2017).
Tyburczy, M. E. et al. Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet. 11, e1005637 (2015).
Lengner, C. J. et al. Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141, 872–883 (2010).
Blair, J. D., Bateup, H. S. & Hockemeyer, D. F. Establishment of genome-edited human pluripotent stem cell lines: from targeting to isolation. J. Vis. Exp 108, e53583 (2016).
Mandal, P. K. & Rossi, D. J. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat. Protoc. 8, 568–582 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Chambers, S. M., Mica, Y., Studer, L. & Tomishima, M. J. Converting human pluripotent stem cells to neural tissue and neurons to model neurodegeneration. Methods Mol. Biol. 793, 87–97 (2011).
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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
Cell Death & Differentiation (2021)
SCN2A channelopathies in the autism spectrum of neuropsychiatric disorders: a role for pluripotent stem cells?
Molecular Autism (2020)
Molecular Autism (2020)
TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling
Molecular Autism (2020)
Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells
Nature Biotechnology (2020)