The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome—a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel—interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

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We thank J. Y. Park, K. Sabatini, T. Khan, S. Yoon, H. Gai and L. Elahi at Stanford University, R.E. Dolmetsch at Novartis Institutes for Biomedical Research, and N. Bansal and J. Fan at BD Genomics for advice and support. This work was supported by grants from NIH/National Institute of Mental Health (NIMH) grants R01MH100900 and R01MH100900-02S1, NIMH BRAINS Award R01MH107800, the California Institute of Regenerative Medicine (CIRM), the MQ Fellow Award, the Donald E. and Delia B. Baxter Foundation Faculty Award, the Kwan Research Fund and Stanford Start-up Funds (to S.P.P.), Child Research Health Institute Postdoctoral Fellowship (CHRI) (to F.B., N.H.), Walter V. and Idun Berry Postdoctoral Fellowship (to J.A.) and the Stanford Medicine Dean’s Fellowship (to F.B., J.A., N.H.), the American Epilepsy Society and Wishes for Elliott Foundation Fellowship (to C.D.M.), NIH 5P01HG00020526 (to L.M.S.), the UCSF Program for Breakthrough Biomedical Research and Sandler Foundation (to G.P.).

Author information

Author notes

    • Fikri Birey
    • , Jimena Andersen
    •  & Christopher D. Makinson

    These authors contributed equally to this work.


  1. Department of Psychiatry and Behavioral Sciences, Center for Sleep Sciences and Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    • Fikri Birey
    • , Jimena Andersen
    • , Nina Huber
    • , Nicholas Thom
    • , Nancy A. O’Rourke
    • , Joachim Hallmayer
    •  & Sergiu P. Paşca
  2. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305, USA

    • Christopher D. Makinson
    •  & John R. Huguenard
  3. Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA

    • Saiful Islam
    • , Wu Wei
    •  & Lars M. Steinmetz
  4. Stanford Genome Technology Center, Stanford University, Palo Alto, California 94304, USA

    • Wu Wei
    •  & Lars M. Steinmetz
  5. BD Genomics, Menlo Park, California 94025, USA

    • H. Christina Fan
    •  & Kimberly R. Cordes Metzler
  6. Department of Biochemistry and Biophysics, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California 94143, USA

    • Georgia Panagiotakos
  7. European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany

    • Lars M. Steinmetz
  8. Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA

    • Jonathan A. Bernstein


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F.B. and J.A. developed the differentiation platform and assays. C.D.M. and J.R.H. designed, conducted and analysed the electrophysiological experiments. F.B., J.A., N.H. and N.T. contributed to neural differentiation, live cell imaging experiments and analysis. F.B. and J.A. were able to replicate the differentiation and spheroid assembly independently. S.I., W.W., H.C.F., K.R.C.M. and L.M.S. contributed to the single-cell experiments. N.A.O. performed the array tomography. J.A.B. and J.H. recruited and characterized the subjects. G.P., J.A. and F.B. performed or analysed the electroporation experiments. F.B, J.A. and S.P.P. conceived the project, designed experiments and wrote the paper with input from all authors. S.P.P. supervised all aspects of the work.

Competing interests

Stanford University has filed a provisional patent application that covers the generation of region-specific neural spheroids and their assembly for studying human development and disease (US patent application number 62/477,858). H.C.F. and K.R.C.M. were employees of BD Genomics during this study.

Corresponding author

Correspondence to Sergiu P. Paşca.

Reviewer Information Nature thanks G. Fishell, A. Goffinet and B. Treutlein for their contribution to the peer review of this work.

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

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  1. 1.

    Live imaging (14.2 hrs) showing migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS (day 12 after fusion).

    hCS is located in the upper side part of the fused spheroids. Yellow arrowheads point to cells undergoing saltatory migration steps.

  2. 2.

    Live imaging showing migration of Dlx2i1/2b::eGFP+ cells in a hSS plated on a coverslip (example #1: 22.7 hrs; example #1: 6.7 hrs).

    Arrows indicate representative Dlx2i1/2b::eGFP+ cells.

  3. 3.

    Live imaging (15.2 hrs) showing ventricle directed migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS

    A VZ-like region in the hCS has been identified by bright field microscopy and indicated with two concentrical circles.

  4. 4.

    Live imaging (18.2 hrs; low magnification) showing migration of Dlx2i1/2b::eGFP+ cells in slices of human fetal forebrain at GW20

    Human fetal slices were labeled with the reporter 5–6 days before imaging.

  5. 5.

    Live imaging (18.9 hrs) showing examples of migrating Dlx2i1/2b::eGFP+ cells in slices of human fetal forebrain at GW18 versus fused hSS-hCS

    Human fetal slices were labeled with the reporter 5–6 days before imaging.

  6. 6.

    Live imaging showing migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS before (9.6 hrs) and during exposure to the CXCR4 receptor antagonist AMD3100 (24.4 hrs total imaging time)

    Live imaging showing migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS before (9.6 hrs) and during exposure to the CXCR4 receptor antagonist AMD3100 (24.4 hrs total imaging time).

  7. 7.

    Live imaging (4.6 hrs) showing migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS derived from TS subject (lower panel) and a control subject (upper panel)

    Live imaging (4.6 hrs) showing migration of Dlx2i1/2b::eGFP+ cells in fused hSS-hCS derived from TS subject (lower panel) and a control subject (upper panel).

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