Article

Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture

  • Nature Methods volume 12, pages 671678 (2015)
  • doi:10.1038/nmeth.3415
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Abstract

The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex–like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

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Acknowledgements

We thank R. Dolmetsch, R. O'Hara, U. Francke and J. Hallmayer for valuable scientific advice and discussions, and also acknowledge E. Engleman and the Stanford Blood Flow Cytometry Center for technical advice and support, J. Ou for assistance with RNA preparation, and D. Castaneda-Castellanos for assistance with live imaging. This work was supported by a NARSAD Young Investigator Award (Behavioral and Brain Foundation), US National Institute of Mental Health (NIMH) 1R01MH100900 and 1R01MH100900-02S1, MQ Fellow Award and Startup Funds from Stanford University (to S.P.P.); NIMH R01 MH099555-03 (to B.A.B.); NIMH T32GM007365, F30MH106261 and Bio-X Predoctoral Fellowship (to or supporting S.A.S.); NIMH 5R37 MH060233 and 5R01 MH094714 (to D.H.G.); NIH R01NS075252, R21MH099797 and R01NS092474 (to S.J.S.); and the DGIST R&D Program of the Korean Ministry of Science and ICT & Future Planning, 14-BD-16 (to C.H.K.).

Author information

Author notes

    • Anca M Paşca
    •  & Steven A Sloan

    These authors contributed equally to this work.

Affiliations

  1. Department of Pediatrics, Division of Neonatology, Stanford University School of Medicine, Stanford, California, USA.

    • Anca M Paşca
  2. Department of Neurobiology, Stanford University School of Medicine, Stanford, California, USA.

    • Steven A Sloan
    • , Laura E Clarke
    •  & Ben A Barres
  3. Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

    • Yuan Tian
    •  & Daniel H Geschwind
  4. Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

    • Yuan Tian
    •  & Daniel H Geschwind
  5. Interdepartmental Ph.D. Program in Bioinformatics, University of California, Los Angeles, California, USA.

    • Yuan Tian
    •  & Daniel H Geschwind
  6. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA.

    • Christopher D Makinson
    •  & John R Huguenard
  7. Department of Psychiatry & Behavioral Sciences, Center for Sleep Sciences and Medicine, Stanford University School of Medicine, Stanford, California, USA.

    • Nina Huber
    • , Jin-Young Park
    •  & Sergiu P Paşca
  8. Department of Pharmacology, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.

    • Chul Hoon Kim
  9. BK21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

    • Chul Hoon Kim
  10. Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, California, USA.

    • Nancy A O'Rourke
    •  & Stephen J Smith
  11. Department of Pathology, Blood Center, Stanford University School of Medicine, Stanford, California, USA.

    • Khoa D Nguyen
  12. Department of Synapse Biology, Allen Institute for Brain Science, Seattle, Washington, USA.

    • Stephen J Smith

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Contributions

A.M.P., S.A.S. and S.P.P. conceived the project. A.M.P., S.A.S., L.E.C., Y.T., C.D.M., C.H.K., J.-Y.P., N.A.O'R., K.D.N., N.H., S.J.S., J.R.H., D.H.G., B.A.B. and S.P.P. planned and/or executed experiments. A.M.P., S.A.S. and S.P.P. wrote the paper with input from all authors. S.P.P. supervised all aspects of the work.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sergiu P Paşca.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–4

Videos

  1. 1.

    Live imaging showing cell division in a radial glia inside the hCS

    At day 45 in vitro, hCS were infected with a lentivirus expressing EGFP under the human GFAP promoter (LentiGFAP::EGFP). At day 52 of differentiation in vitro, hCS were sliced and VZ-like regions were imaged at 37°C with a Leica SP8 confocal microscope for up to 3 hours (maximum projection of a z-stack, one frame collected every 10 min).

  2. 2.

    Calcium imaging in hCS showing spontaneous activity

    hCS were loaded with the calcium indicator Fluo-4 for 30 min, sectioned in half and imaged with a Zeiss confocal L710 microscope.