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Genetically engineered human cortical spheroid models of tuberous sclerosis


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.


  1. 1.

    van Slegtenhorst, M. et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808 (1997).

    Article  Google Scholar 

  2. 2.

    European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315 (1993).

  3. 3.

    Crino, P. B., Nathanson, K. L. & Henske, E. P. The tuberous sclerosis complex. N. Engl. J. Med. 355, 1345–1356 (2006).

    CAS  Article  Google Scholar 

  4. 4.

    Thiele, E. A. Managing and understanding epilepsy in tuberous sclerosis complex. Epilepsia 51(Suppl 1), 90–91 (2010).

    Article  Google Scholar 

  5. 5.

    Curatolo, P., Moavero, R. & de Vries, P. J. Neurological and neuropsychiatric aspects of tuberous sclerosis complex. Lancet Neurol. 14, 733–745 (2015).

    Article  Google Scholar 

  6. 6.

    Crino, P. B. Evolving neurobiology of tuberous sclerosis complex. Acta Neuropathol. 125, 317–332 (2013).

    Article  Google Scholar 

  7. 7.

    Mohamed, A. R. et al. Intrinsic epileptogenicity of cortical tubers revealed by intracranial EEG monitoring. Neurology 79, 2249–2257 (2012).

    Article  Google Scholar 

  8. 8.

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Jansen, F. E. et al. Cognitive impairment in tuberous sclerosis complex is a multifactorial condition. Neurology 70, 916–923 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    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).

    CAS  Article  Google Scholar 

  11. 11.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    CAS  Article  Google Scholar 

  14. 14.

    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).

    CAS  Article  Google Scholar 

  15. 15.

    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).

    CAS  Article  Google Scholar 

  16. 16.

    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).

    CAS  Article  Google Scholar 

  17. 17.

    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).

    CAS  Article  Google Scholar 

  18. 18.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Dehay, C. & Kennedy, H. Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    Florio, M. & Huttner, W. B. Neural progenitors, neurogenesis and the evolution of the neocortex. Development 141, 2182–2194 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Lui, J. H., Hansen, D. V. & Kriegstein, A. R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    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).

    CAS  Article  Google Scholar 

  23. 23.

    Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ma, X. M. & Blenis, J. Molecular mechanisms of mTOR-mediated translational control. Nat. Rev. Mol. Cell Biol. 10, 307–318 (2009).

    Article  CAS  Google Scholar 

  25. 25.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    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).

    CAS  Article  Google Scholar 

  27. 27.

    Huang, J. & Manning, B. D. The TSC1–TSC2 complex: a molecular switchboard controlling cell growth. Biochem J. 412, 179–190 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Crino, P. B. The mTOR signalling cascade: paving new roads to cure neurological disease. Nat. Rev. Neurol. 12, 379–392 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Magri, L. & Galli, R. mTOR signaling in neural stem cells: from basic biology to disease. Cell. Mol. Life Sci. 70, 2887–2898 (2013).

    CAS  Article  Google Scholar 

  30. 30.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Sepp, T., Yates, J. R. & Green, A. J. Loss of heterozygosity in tuberous sclerosis hamartomas. J. Med. Genet. 33, 962–964 (1996).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    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).

    CAS  Article  Google Scholar 

  35. 35.

    Crino, P. B., Aronica, E., Baltuch, G. & Nathanson, K. L. Biallelic TSC gene inactivation in tuberous sclerosis complex. Neurology 74, 1716–1723 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Martin, K. R. et al. The genomic landscape of tuberous sclerosis complex. Nat. Commun. 8, 15816 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    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).

    Article  CAS  Google Scholar 

  39. 39.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Thoreen, C. C. et al. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109–113 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Paşca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Dibble, C. C. et al. TBC1D7 is a third subunit of the TSC1–TSC2 complex upstream of mTORC1. Mol. Cell 47, 535–546 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Miller, F. D. & Gauthier, A. S. Timing is everything: making neurons versus glia in the developing cortex. Neuron 54, 357–369 (2007).

    CAS  Article  Google Scholar 

  46. 46.

    Bonni, A. et al. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 278, 477–483 (1997).

    CAS  Article  Google Scholar 

  47. 47.

    Cloëtta, D. et al. Inactivation of mTORC1 in the developing brain causes microcephaly and affects gliogenesis. J. Neurosci. 33, 7799–7810 (2013).

    Article  CAS  Google Scholar 

  48. 48.

    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).

    CAS  Article  Google Scholar 

  49. 49.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Mizuguchi, M. & Takashima, S. Neuropathology of tuberous sclerosis. Brain Dev. 23, 508–515 (2001).

    CAS  Article  Google Scholar 

  51. 51.

    Sosunov, A. A. et al. Tuberous sclerosis: a primary pathology of astrocytes? Epilepsia 49(Suppl 2), 53–62 (2008).

    Article  Google Scholar 

  52. 52.

    Grabole, N. et al. Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model. Genome Med. 8, 94 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    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).

    CAS  Article  Google Scholar 

  54. 54.

    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).

    CAS  Article  Google Scholar 

  55. 55.

    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).

    Article  Google Scholar 

  56. 56.

    Boer, K. et al. Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex. Epilepsy Res. 78, 7–21 (2008).

    CAS  Article  Google Scholar 

  57. 57.

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Lengner, C. J. et al. Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141, 872–883 (2010).

    CAS  Article  Google Scholar 

  60. 60.

    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).

    Google Scholar 

  61. 61.

    Mandal, P. K. & Rossi, D. J. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat. Protoc. 8, 568–582 (2013).

    Article  CAS  Google Scholar 

  62. 62.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    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).

    CAS  Article  Google Scholar 

  64. 64.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  65. 65.

    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).

    CAS  Article  Google Scholar 

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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.

Author information




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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Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1, 3 4 and 5

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Supplementary Table 2

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).

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