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
  • Download Citation


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.

  • Subscribe to Nature Methods for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


Primary accessions

Gene Expression Omnibus


  1. 1.

    & The human brain in a dish: the promise of iPSC-derived neurons. Cell 145, 831–834 (2011).

  2. 2.

    & Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

  3. 3.

    & Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nat. Rev. Genet. 15, 82–92 (2014).

  4. 4.

    , , & Modeling psychiatric disorders at the cellular and network levels. Mol. Psychiatry 17, 1239–1253 (2012).

  5. 5.

    , & Generating human neurons in vitro and using them to understand neuropsychiatric disease. Annu. Rev. Neurosci. 37, 479–501 (2014).

  6. 6.

    et al. Modeling human cortical development in vitro using induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 109, 12770–12775 (2012).

  7. 7.

    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).

  8. 8.

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

  9. 9.

    & Modeling psychiatric disorders through reprogramming. Dis. Model. Mech. 5, 26–32 (2012).

  10. 10.

    et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

  11. 11.

    , & Neuronal nuclear antigen (NeuN): a marker of neuronal maturation in early human fetal nervous system. Brain Dev. 20, 88–94 (1998).

  12. 12.

    et al. A quantitative framework to evaluate modeling of cortical development by neural stem cells. Neuron 83, 69–86 (2014).

  13. 13.

    et al. Spatio-temporal transcriptome of the human brain. Nature 478, 483–489 (2011).

  14. 14.

    et al. Transcriptional landscape of the prenatal human brain. Nature 508, 199–206 (2014).

  15. 15.

    et al. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J. Neurosci. 25, 247–251 (2005).

  16. 16.

    , , & Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010).

  17. 17.

    , , , & Neurogenic radial glial cells in reptile, rodent and human: from mitosis to migration. Cereb. Cortex 13, 550–559 (2003).

  18. 18.

    & Neural progenitor nuclei IN motion. Neuron 67, 906–914 (2010).

  19. 19.

    & Prenatal development of reelin-immunoreactive neurons in the human neocortex. J. Comp. Neurol. 397, 29–40 (1998).

  20. 20.

    et al. Neocortical layer formation of human developing brains and lissencephalies: consideration of layer-specific marker expression. Cereb. Cortex 21, 588–596 (2011).

  21. 21.

    et al. Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57, 364–377 (2008).

  22. 22.

    et al. Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron 57, 378–392 (2008).

  23. 23.

    et al. Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons. Genes Dev. 16, 1760–1765 (2002).

  24. 24.

    et al. Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures. Cell 149, 483–496 (2012).

  25. 25.

    et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9, 743–751 (2006).

  26. 26.

    & Neocortical Development (Raven Press, New York, 1991).

  27. 27.

    & Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

  28. 28.

    et al. Development of a method for the purification and culture of rodent astrocytes. Neuron 71, 799–811 (2011).

  29. 29.

    & Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890–902 (1980).

  30. 30.

    et al. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012).

  31. 31.

    & Astrocyte glycogen and brain energy metabolism. Glia 55, 1263–1271 (2007).

  32. 32.

    et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat. Med. 17, 1657–1662 (2011).

  33. 33.

    et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473, 221–225 (2011).

  34. 34.

    et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010).

  35. 35.

    & Synaptic efficacy enhanced by glial cells in vitro. Science 277, 1684–1687 (1997).

  36. 36.

    , , & Control of synapse number by glia. Science 291, 657–661 (2001).

  37. 37.

    , , , & Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat. Biotechnol. 29, 528–534 (2011).

  38. 38.

    et al. Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proc. Natl. Acad. Sci. USA 111, 829–832 (2014).

  39. 39.

    et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012).

  40. 40.

    et al. Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139, 380–392 (2009).

  41. 41.

    et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471, 230–234 (2011).

  42. 42.

    et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).

  43. 43.

    , , , & Functional genomic analysis of oligodendrocyte differentiation. J. Neurosci. 26, 10967–10983 (2006).

  44. 44.

    , , , & Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010).

Download references


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.


  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


  1. Search for Anca M Paşca in:

  2. Search for Steven A Sloan in:

  3. Search for Laura E Clarke in:

  4. Search for Yuan Tian in:

  5. Search for Christopher D Makinson in:

  6. Search for Nina Huber in:

  7. Search for Chul Hoon Kim in:

  8. Search for Jin-Young Park in:

  9. Search for Nancy A O'Rourke in:

  10. Search for Khoa D Nguyen in:

  11. Search for Stephen J Smith in:

  12. Search for John R Huguenard in:

  13. Search for Daniel H Geschwind in:

  14. Search for Ben A Barres in:

  15. Search for Sergiu P Paşca in:


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


  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.