In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.

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We thank J.R. Brown, S. Hyman, G. Feng, Z. Fu, A. Schinder, L. Rubin, F. Rapino, former and present members of the Arlotta laboratory for insightful discussions and editing of the manuscript, A. Pollen and A. Kriegstein for sharing of human single-cell datasets and C. Cepko for sharing of antibodies, Y. Zhang for outstanding technical support, E. Zuccaro, F. Yates and S. Pavoni for helpful advice on culturing organoids. This work was supported by grants from the Stanley Center for Psychiatric Research, the Broad Institute of Harvard and MIT, and the Star Family Award of Harvard University to P.A. E.S.B. acknowledges NIH Director's Pioneer Award 1DP1NS087724. J.W.L. acknowledges support by IARPA, Conte and MURI Army Research Office. P.A. and E.S.B. are New York Stem Cell Foundation-Robertson Investigators.

Author information


  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Giorgia Quadrato
    • , Tuan Nguyen
    • , John L. Sherwood
    • , Sung Min Yang
    • , Natalie Maria
    •  & Paola Arlotta
  2. Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA

    • Giorgia Quadrato
    • , Tuan Nguyen
    • , Evan Z. Macosko
    • , John L. Sherwood
    • , Steven A. McCarroll
    •  & Paola Arlotta
  3. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Evan Z. Macosko
    • , Melissa Goldman
    •  & Steven A. McCarroll
  4. Department of Cellular and Molecular Biology and Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA

    • Daniel R. Berger
    •  & Jeff W. Lichtman
  5. Departments of Biological Engineering and Brain and Cognitive Sciences, MIT Media Lab and McGovern Institute, MIT, Cambridge, Massachusetts 02139, USA

    • Jorg Scholvin
    •  & Edward S. Boyden
  6. LeafLabs, LLC, Cambridge, Massachusetts 02139, USA

    • Justin P. Kinney
  7. Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Ziv M. Williams


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P.A. and G.Q. conceived the experiments. G.Q. developed the long-term cultures of organoids and performed all immunohistochemical characterization with help from N.M. E.Z.M., G.Q., T.N. and M.G. performed all single-cell sequencing experiments. E.Z.M., G.Q., T.N., P.A. and S.A.M. analysed and interpreted the Drop-seq data. J.L.S., S.M.Y. and Z.M.W. performed electrophysiological experiments. J.S., J.P.K. and E.S.B. developed multi-electrode probes and helped J.L.S. adapt them to organoids. D.R.B. and J.W.L. performed electron microscopy work. P.A., G.Q., E.Z.M. and S.A.M. wrote the manuscript with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Giorgia Quadrato or Paola Arlotta.

Reviewer Information Nature thanks F. Guillemot, S. Linnarsson and the other anonymous reviewer(s) 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.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary Discussion and Supplementary Table 1, a list of primary antibodies employed in this study.

Excel files

  1. 1.

    Supplementary Table 2

    Genes differentially expressed in each of the 10 main clusters relative to the rest of the data set (first tab), and differential gene expression between each subcluster (remaining tabs). Bracketed numbers at the head of each section designate the cluster or subcluster. Column headings: p_val: p-value significance of expression level; avg_diff: log average differential expression between groups; pct.1: Fraction of cells expressing gene in group 1; pct.2: Fraction of cells expressing gene in group 2 (comparison group).


  1. 1.

    Slice animation of the aligned EM image stack with reconstructed objects as color overlay

    Organoid surface towards the top of the images.

  2. 2.

    Reconstructed axons and dendrites in the same volume as video 1, shown as 3D objects

    Organoid surface towards the top.

  3. 3.

    Three spine synapses between two axons and two dendrites, two made en-passant and one on a terminal bouton

    Same as Figure 3j, with red dendrite added.

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