Article

Cell diversity and network dynamics in photosensitive human brain organoids

  • Nature volume 545, pages 4853 (04 May 2017)
  • doi:10.1038/nature22047
  • Download Citation
Received:
Accepted:
Published:

Abstract

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.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

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

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    & Stem cell models of human brain development. Cell Stem Cell 18, 736–748 (2016)

  2. 2.

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

  3. 3.

    et al. The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534, 267–271 (2016)

  4. 4.

    et al. Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell 19, 258–265 (2016)

  5. 5.

    et al. Zika virus impairs growth in human neurospheres and brain organoids. Science 352, 816–818 (2016)

  6. 6.

    et al. Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem cells. Cell Stem Cell 18, 591–596 (2016)

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    & Generation of cerebral organoids from human pluripotent stem cells. Nat. Protocols 9, 2329–2340 (2014)

  11. 11.

    et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015)

  12. 12.

    , , , & Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015)

  13. 13.

    et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat. Biotechnol. 31, 545–552 (2013)

  14. 14.

    & Visualizing data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605 (2008)

  15. 15.

    et al. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207–221 (2005)

  16. 16.

    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)

  17. 17.

    et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl Acad. Sci. USA 112, 15672–15677 (2015)

  18. 18.

    et al. A survey of human brain transcriptome diversity at the single cell level. Proc. Natl Acad. Sci. USA 112, 7285–7290 (2015)

  19. 19.

    et al. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science 352, 1586–1590 (2016)

  20. 20.

    et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016)

  21. 21.

    et al. Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons. J. Neurosci. 29, 12343–12354 (2009)

  22. 22.

    et al. Molecular identity of human outer radial glia during cortical development. Cell 163, 55–67 (2015)

  23. 23.

    et al. Transcriptional code and disease map for adult retinal cell types. Nat. Neurosci. 15, 487–495 (2012)

  24. 24.

    et al. Transcriptome analysis and molecular signature of human retinal pigment epithelium. Hum. Mol. Genet. 19, 2468–2486 (2010)

  25. 25.

    et al. Transcriptional comparison of human induced and primary midbrain dopaminergic neurons. Sci. Rep. 6, 20270 (2016)

  26. 26.

    et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014)

  27. 27.

    et al. Purification and Characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron 89, 37–53 (2016)

  28. 28.

    , & Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91, 531–541 (1997)

  29. 29.

    , , & Recoverin in pineal organs and retinae of various vertebrate species including man. Brain Res. 595, 57–66 (1992)

  30. 30.

    et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, 3 (2003)

  31. 31.

    & Regional differences in synaptogenesis in human cerebral cortex. J. Comp. Neurol. 387, 167–178 (1997)

  32. 32.

    et al. Close-packed silicon microelectrodes for scalable spatially oversampled neural recording. IEEE Trans. Biomed. Eng. 63, 120–130 (2016)

  33. 33.

    , , & Behavior-dependent short-term assembly dynamics in the medial prefrontal cortex. Nat. Neurosci. 11, 823–833 (2008)

  34. 34.

    & Unbiased estimation of precise temporal correlations between spike trains. J. Neurosci. Methods 179, 90–100 (2009)

  35. 35.

    & Pharmacological characterization of non-NMDA subtypes of glutamate receptor in the neonatal rat hemisected spinal cord in vitro. Br. J. Pharmacol. 106, 367–372 (1992)

  36. 36.

    et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat. Commun. 5, 4047 (2014)

  37. 37.

    et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011)

  38. 38.

    et al. Nuclear factor of activated T cells (NFATc4) is required for BDNF-dependent survival of adult-born neurons and spatial memory formation in the hippocampus. Proc. Natl Acad. Sci. USA 109, E1499–E1508 (2012)

  39. 39.

    et al. Long-term culture and electrophysiological characterization of human brain organoids. Protoc. Exch. (2017)

  40. 40.

    & A smart local moving algorithm for large-scale modularity-based community detection. Eur. Phys. J. B 86, 471 (2013)

  41. 41.

    & Statistical significance of variables driving systematic variation in high-dimensional data. Bioinformatics 31, 545–554 (2015)

  42. 42.

    et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015)

  43. 43.

    et al. Spike sorting for large, dense electrode arrays. Nat. Neurosci. 19, 634–641 (2016)

  44. 44.

    & Drawing inferences from Fano factor calculations. J. Neurosci. Methods 190, 149–152 (2010)

Download references

Acknowledgements

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

Affiliations

  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

Authors

  1. Search for Giorgia Quadrato in:

  2. Search for Tuan Nguyen in:

  3. Search for Evan Z. Macosko in:

  4. Search for John L. Sherwood in:

  5. Search for Sung Min Yang in:

  6. Search for Daniel R. Berger in:

  7. Search for Natalie Maria in:

  8. Search for Jorg Scholvin in:

  9. Search for Melissa Goldman in:

  10. Search for Justin P. Kinney in:

  11. Search for Edward S. Boyden in:

  12. Search for Jeff W. Lichtman in:

  13. Search for Ziv M. Williams in:

  14. Search for Steven A. McCarroll in:

  15. Search for Paola Arlotta in:

Contributions

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).

Videos

  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.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.