Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Kelava, I. & Lancaster, M. A. Stem cell models of human brain development. Cell Stem Cell 18, 736–748 (2016).

  2. 2.

    Sasai, Y., Eiraku, M. & Suga, H. In vitro organogenesis in three dimensions: self-organising stem cells. Development 139, 4111–4121 (2012).

  3. 3.

    Lancaster, M. A. & Knoblich, J. A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

  4. 4.

    Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

  5. 5.

    Quadrato, G., Brown, J. & Arlotta, P. The promises and challenges of human brain organoids as models of neuropsychiatric disease. Nat. Med. 22, 1220–1228 (2016).

  6. 6.

    Renner, M. et al. Self-organized developmental patterning and differentiation in cerebral organoids. EMBO J. 36, 1316–1329 (2017).

  7. 7.

    O’Leary, D. D., Chou, S. J. & Sahara, S. Area patterning of the mammalian cortex. Neuron 56, 252–269 (2007).

  8. 8.

    Sagner, A. & Briscoe, J. Morphogen interpretation: concentration, time, competence, and signaling dynamics. Wiley Interdiscip. Rev. Dev. Biol. 6, https://doi.org/10.1002/wdev.271 (2017).

  9. 9.

    Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

  10. 10.

    Suga, H. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011).

  11. 11.

    Kadoshima, T. et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc. Natl Acad. Sci. USA 110, 20284–20289 (2013).

  12. 12.

    Jo, J. et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 19, 248–257 (2016).

  13. 13.

    Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep. 10, 537–550 (2015).

  14. 14.

    Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016).

  15. 15.

    Bagley, J. A., Reumann, D., Bian, S., Levi-Strauss, J. & Knoblich, J. A. Fused cerebral organoids model interactions between brain regions. Nat. Methods 14, 743–751 (2017).

  16. 16.

    Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).

  17. 17.

    Xiang, Y. et al. Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration. Cell Stem Cell 21, 383–398.e387 (2017).

  18. 18.

    Lupo, G., Harris, W. A. & Lewis, K. E. Mechanisms of ventral patterning in the vertebrate nervous system. Nat. Rev. Neurosci. 7, 103–114 (2006).

  19. 19.

    Jessell, T. M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).

  20. 20.

    Fattahi, F. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105–109 (2016).

  21. 21.

    Gonzalez, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

  22. 22.

    Shiraishi, A., Muguruma, K. & Sasai, Y. Generation of thalamic neurons from mouse embryonic stem cells. Development 144, 1211–1220 (2017).

  23. 23.

    Merchan, P., Bardet, S. M., Puelles, L. & Ferran, J. L. Comparison of pretectal genoarchitectonic pattern between quail and chicken embryos. Front. Neuroanat. 5, 23 (2011).

  24. 24.

    Oliver, G. et al. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045–4055 (1995).

  25. 25.

    Shinya, M., Eschbach, C., Clark, M., Lehrach, H. & Furutani-Seiki, M. Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate. Mech. Dev. 98, 3–17 (2000).

  26. 26.

    Houart, C. et al. Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35, 255–265 (2002).

  27. 27.

    Kiecker, C. & Niehrs, C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development 128, 4189–4201 (2001).

  28. 28.

    Maroof, A. M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).

  29. 29.

    Blaess, S., Szabo, N., Haddad-Tovolli, R., Zhou, X. & Alvarez-Bolado, G. Sonic hedgehog signaling in the development of the mouse hypothalamus. Front. Neuroanat. 8, 156 (2014).

  30. 30.

    Merkle, F. T. et al. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells. Development 142, 633–643 (2015).

  31. 31.

    Briscoe, J., Chen, Y., Jessell, T. M. & Struhl, G. A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7, 1279–1291 (2001).

  32. 32.

    Honig, L. S. Positional signal transmission in the developing chick limb. Nature 291, 72–73 (1981).

  33. 33.

    Fan, C. M. & Tessier-Lavigne, M. Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79, 1175–1186 (1994).

  34. 34.

    Zeng, X. et al. A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411, 716–720 (2001).

  35. 35.

    Lewis, P. M. et al. Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, 599–612 (2001).

  36. 36.

    Chen, M. H., Li, Y. J., Kawakami, T., Xu, S. M. & Chuang, P. T. Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev. 18, 641–659 (2004).

  37. 37.

    Sanders, T. A., Llagostera, E. & Barna, M. Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 497, 628–632 (2013).

  38. 38.

    Harfe, B. D. et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517–528 (2004).

  39. 39.

    Zhang, Y. & Alvarez-Bolado, G. Differential developmental strategies by Sonic hedgehog in thalamus and hypothalamus. J. Chem. Neuroanat. 75, 20–27 (2016).

  40. 40.

    McGlinn, E. & Tabin, C. J. Mechanistic insight into how Shh patterns the vertebrate limb. Curr. Opin. Genet. Dev. 16, 426–432 (2006).

  41. 41.

    Ericson, J. et al. Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81, 747–756 (1995).

  42. 42.

    Huang, P. et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling. Cell 166, 1176–1187 e1114 (2016).

  43. 43.

    Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).

  44. 44.

    Tian, H., Jeong, J., Harfe, B. D., Tabin, C. J. & McMahon, A. P. Mouse Disp1 is required in sonic hedgehog-expressing cells for paracrine activity of the cholesterol-modified ligand. Development 132, 133–142 (2005).

  45. 45.

    Rietveld, A., Neutz, S., Simons, K. & Eaton, S. Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains. J. Biol. Chem. 274, 12049–12054 (1999).

  46. 46.

    Katanaev, V. L. et al. Reggie-1/flotillin-2 promotes secretion of the long-range signalling forms of Wingless and Hedgehog in Drosophila. EMBO J. 27, 509–521 (2008).

  47. 47.

    Panakova, D., Sprong, H., Marois, E., Thiele, C. & Eaton, S. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005).

  48. 48.

    Palm, W. et al. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition. PLoS Genet. 8, e1002828 (2012).

  49. 49.

    Gaspard, N. et al. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 455, 351–357 (2008).

  50. 50.

    Blassberg, R., Macrae, J. I., Briscoe, J. & Jacob, J. Reduced cholesterol levels impair Smoothened activation in Smith-Lemli-Opitz syndrome. Hum. Mol. Genet. 25, 693–705 (2016).

  51. 51.

    Lei, Q. et al. Wnt signaling inhibitors regulate the transcriptional response to morphogenetic Shh-Gli signaling in the neural tube. Dev. Cell 11, 325–337 (2006).

  52. 52.

    von Tresckow, B. et al. Depletion of cellular cholesterol and lipid rafts increases shedding of CD30. J. Immunol. 172, 4324–4331 (2004).

  53. 53.

    Murai, T. et al. Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration. J. Biol. Chem. 286, 1999–2007 (2011).

  54. 54.

    Kirsch, C., Eckert, G. P. & Mueller, W. E. Statin effects on cholesterol micro-domains in brain plasma membranes. Biochem. Pharmacol. 65, 843–856 (2003).

  55. 55.

    Hering, H., Lin, C. C. & Sheng, M. Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J. Neurosci. 23, 3262–3271 (2003).

  56. 56.

    Zhuang, L., Kim, J., Adam, R. M., Solomon, K. R. & Freeman, M. R. Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. J. Clin. Invest. 115, 959–968 (2005).

  57. 57.

    Therond, P. P. Release and transportation of Hedgehog molecules. Curr. Opin. Cell Biol. 24, 173–180 (2012).

  58. 58.

    Vyas, N. et al. Nanoscale organization of hedgehog is essential for long-range signaling. Cell 133, 1214–1227 (2008).

  59. 59.

    Cooper, M. K., Porter, J. A., Young, K. E. & Beachy, P. A. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280, 1603–1607 (1998).

  60. 60.

    Dierker, T., Dreier, R., Petersen, A., Bordych, C. & Grobe, K. Heparan sulfate-modulated, metalloprotease-mediated sonic hedgehog release from producing cells. J. Biol. Chem. 284, 8013–8022 (2009).

  61. 61.

    Edison, R. J. & Muenke, M. Central nervous system and limb anomalies in case reports of first-trimester statin exposure. N. Engl. J. Med. 350, 1579–1582 (2004).

  62. 62.

    Bellosta, S., Paoletti, R. & Corsini, A. Safety of statins: focus on clinical pharmacokinetics and drug interactions. Circulation 109, III50–57 (2004).

  63. 63.

    Lancaster, M. A. et al. Guided self-organization and cortical plate formation in human brain organoids. Nat. Biotechnol. 35, 659–666 (2017).

  64. 64.

    Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).

  65. 65.

    Cobos, I. et al. Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat. Neurosci. 8, 1059–1068 (2005).

  66. 66.

    Fontebasso, A. M. et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat. Genet. 46, 462–466 (2014).

  67. 67.

    Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 19, 1743–1749 (2016).

  68. 68.

    Lancaster, M. A. & Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).

  69. 69.

    Buglino, J. A. & Resh, M. D. Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog. J. Biol. Chem. 283, 22076–22088 (2008).

  70. 70.

    Berthiaume, L., Peseckis, S. M. & Resh, M. D. Synthesis and use of iodo-fatty acid analogs. Methods Enzymol. 250, 454–466 (1995).

  71. 71.

    Alland, L., Peseckis, S. M., Atherton, R. E., Berthiaume, L. & Resh, M. D. Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J. Biol. Chem. 269, 16701–16705 (1994).

Download references


We would like to thank S. Anderson for providing the LHX6-citrine hPSC line. M. Ross, S. Irion, M. Tomishima and members of the Studer lab for their valuable input on experimental design and feedback on manuscript. This work was supported in part through NYSTEM contract C030137 (L.S.) and through the NIH Cancer Center support grant P30 CA008748. G.C. is supported by a Ruth L. Kirschstein F30 M.D./Ph.D. pre-doctoral fellowship (F30 MH113343–01A1) and a training grant from the National Institute of General Medical Sciences (T32GM007739) to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program.

Author information


  1. The Center for Stem Cell Biology, Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA

    • Gustav Y. Cederquist
    • , Jason Tchieu
    • , Ryan M. Walsh
    • , Daniela Cornacchia
    •  & Lorenz Studer
  2. Weill-Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY, USA

    • Gustav Y. Cederquist
  3. Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    • James J. Asciolla
    •  & Marilyn D. Resh
  4. Biochemistry, Cell Biology and Molecular Biology Graduate Program, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    • James J. Asciolla
    •  & Marilyn D. Resh


  1. Search for Gustav Y. Cederquist in:

  2. Search for James J. Asciolla in:

  3. Search for Jason Tchieu in:

  4. Search for Ryan M. Walsh in:

  5. Search for Daniela Cornacchia in:

  6. Search for Marilyn D. Resh in:

  7. Search for Lorenz Studer in:


G.Y.C.: Conception and study design, Organoid protocol development, hPSC cell line engineering, differentiation and characterization assays and writing of manuscript. L.S.: Conception and study design, data analysis and interpretation, writing of manuscript. J.J.A.: SHH protein analysis. M.D.R.: SHH protein analysis. J.T. SHH protein analysis, hPSC cell line engineering. R.M.W.: Organoid protocol development and analysis. D.C.: cell line engineering.

Competing interests

The Memorial Sloan-Kettering Cancer Center has filed a provisional patent application (US PRO 62/538,350) on the methods described in the manuscript with G. Cederquist and L. Studer listed as inventors. L.S. is a scientific co-founder of Bluerock Therapeutics.

Corresponding author

Correspondence to Lorenz Studer.

Integrated supplementary information

  1. Supplementary Figure 1 Additional data related to Fig. 1.

    (a) Schematic of [I125]Iodopalmitate labeling experiment (left). SHH produced from the iSHH line is palmitoylated at levels proportional to the amount of SHH protein expressed, suggesting that overexpression does not saturate processing machinery. (b) Reproducibility of organizer plating and formation of SHH-H9 spheroids. 1,000 iSHH cells are plated in low-attachment microwells and allowed to aggregate overnight. 10,000 wildtype hPSCs are plated on top of the iSHH organizer cells. (c) Spheroids are embedded in matrigel, and the day of embedding is critical to efficient neuroepithelial growth. SHH organoids (no dox) embedded on day 5 exhibit no neuroepithelial growth (“Failed,” N = 3 organoids), while SHH organoids embedded on day 6 exhibit efficient neuroepithelial formation (–DOX N = 3 organoids; +DOX N = 4 organoids). (d) Typically, the iSHH organizer remains clustered at one pole during differentiation, though in ~ 25% of instances the organizer can split into multiple distinct clusters. Scale bars: 200 μm.

  2. Supplementary Figure 2 Quantification method for characterizing SHH-organoid topography.

    (a) SHH-organoids are quantified using a grid of regions of interest (ROIs), and each ROI is associated with an X and Y coordinate. The origin is defined for each section by calculating the “center of mass” of the organizer signal. The grid is then used to define ROIs that are positive for regional markers (e.g. PAX6). The linear distance from all positive ROIs to the origin is calculated, and these data can be plotted as a frequency histogram. (b) Organoids that did not express FOXG1 (7/16 batches), or that (c) had a split organizer (10/40 organoids) were not included in the quantification. Scale bars: 200 μm.

  3. Supplementary Figure 3 Topographic patterning in additional hPSC lines (MEL1, HUES6, HUES8) and iPSC lines (J1 and 348).

    (a) The iSHH organizer can induce distinct regional domains that emerge in the anatomically correct topographic order. However, the size of domains and overall growth rate of organoids may differ between lines. Without doxycycline all lines are predominantly PAX6 and FOXG1 positive. Sparse induction of NKX2.1 and NKX2.2 was observed in MEL1 and 348 lines. +Doxycycline, N = 8 organoids, 2 batches for all lines; - Doxycycline, N = 4 organoids, 1 batch for all lines. (b) Pluripotency analysis for 348 iPSC line. Scale bars: 200 μm (a); 100 μm (b).

  4. Supplementary Figure 4 Additional data related to Fig. 2.

    (a) iSHH organizer cells (red) at least partially express NKX2.1 (green) and are negative for FOXG1, suggesting hypothalamic identity. N = 8 organoids, 2 batches. (b) SHH-dependent topographic patterning can be achieved without matrigel embedding. N = 8 organoids, 2 batches. (c) MGE-like neuroepithelium (FOXG1+/NKX2.1+) acquires a circular, rosette-like structure suggesting a radial organization. N = 6 organoids, 2 batches. Scale bars: 50 μm (a), 200 μm (b, c).

  5. Supplementary Figure 5 Characterization of maintenance of SHH-organoid topography.

    (a) OTX2 and FOXG1 regions remain largely distinct over time, and appear to retain their orientation with respect to the organizer. In some instances, the organizer tissue disperses throughout the organoid, making it difficult to determine orientation. –Dox N = 4 organoids, 1 batch; +Dox N = 8 organoids, 3 batches. (b, c) Cerebral cortex-like tissue consisting of radially organized bands of PAX6, TBR2, and TBR1 emerge in regions distal to the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 5 organoids, 2 batches. (d) Striatum-like tissue expressing DARPP32 emerges in regions distal to the organizer. LHX6 + cells are found more proximal to the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 6 organoids, 2 batches. (e, f) Hypothalamic-like cells, which express OTP, POMC, and TH, are found in the immediate vicinity of the organizer. –Dox N = 4 organoids, 1 batch; +Dox N = 3 organoids, 2 batches. Scale bars: 50μm (high magnification), 200μm (low magnification).

  6. Supplementary Figure 6 Characterization of interneuron diversity in SHH-organoids using LHX6-citrine line.

    (a) LHX6 + cells emerge in regions proximal to the organizer, and co-express NKX2.1. N = 1 organoid. (b) A subset of LHX6 + cells expresses FOXG1, consistent with striatal or cortical interneuron identity. N = 6 organoids, 2 batches. (c) Some FOXG1+/LHX6 + cells have a leading process morphology, characteristic of migrating cortical interneurons. (df) Diverse interneuron populations expressing somatostatin N = 3 organoids, 2 batches (d), parvalbumin N = 3 organoids, 2 batches (e), and calretinin N = 6 organoids, 2 batches (f) are observed. Parvalbumin + cells do not express LHX6, suggesting a non-MGE source of these cells. Scale bars: 50 μm (high magnification), 100 μM (intermediate magnification), 200 μm (low magnification).

  7. Supplementary Figure 7 Additional data related to Fig. 4.

    (a) AY9944 and lovastatin treated organoids retain NKX2.1 and OTX2 expression within the organizer at day 20. No Drug N = 12 organoids, 3 batches; Lova N = 11 organoids, 3 batches; AY9944 N = 12 organoids, 3 batches; Cyclopamine N = 8 organoids, 2 batches. (b) Absolute and relative quantification of organizer size in drug treated organoids at day 20. Graphs depict mean ± S.D., dots represent individual organoids. One-way ANOVA with Dunnett Test. **P=0.0038, ****P = 0.0001. No drug N = 14 organoids; Lova N = 13 organoids; AY9944 N = 16 organoids; Cyclopamine N = 14 organoids. All samples from 3 batches. (c) AY9944 strongly inhibits induction of NKX2.2 in day 6 organoids at all concentrations tested. Graph depicts mean ± S.D., dots represent individual organoids. No Drug N = 18 organoids, 3 batches; AY9944 0.31μM N = 14 organoids, 2 batches; AY9944 0.62μM N = 16 organoids, 2 batches; AY9944 1.25μM N = 15 organoids, 2 batches; Lova 2μM, 5μM, 20μM, N = 12 organoids, 2 batches each. (d) Western blot for SHH protein from day 4 hPSC-derived neural differentiations shows appropriate processing of SHH peptide length. Representative image of N = 3 experiments. Scale bars: 100 μM.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1 and 2

  2. Reporting Summary

About this article

Publication history




Issue Date