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Cell diversity and network dynamics in photosensitive human brain organoids


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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Figure 1: Large-scale, single-cell sequencing demonstrates development of a broad spectrum of cell types in human brain organoids.
Figure 2: Human brain organoids contain subclasses of forebrain and retinal cells.
Figure 3: Extended culture allows development of mature cell types including differentiated photoreceptors.
Figure 4: Brain organoids develop spontaneous networks and photosensitive neurons that can be modulated by sensory stimulation with light.

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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

Authors and Affiliations



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.

Corresponding authors

Correspondence to Giorgia Quadrato or Paola Arlotta.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks F. Guillemot, S. Linnarsson and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Time-course of expression of selected marker genes in human brain organoids.

a, Top, expression of the hypoxia marker HIF1α over one to nine months of culture, and positive control (a six-month-old organoid treated with cobalt (II) chloride (CoCl), an activator of the hypoxia signalling pathway). Bottom, expression of the apoptosis marker, active caspase 3 (ActCasp3) and the neuronal marker (MAP2), over one to nine months of culture. b, Expression of markers for progenitor, neuronal and glial populations over one to nine months of culture. c, One-month-old brain organoids exhibit early brain regionalization, expressing markers of forebrain (PAX6 and NKX2.1), midbrain (OTX2) and hindbrain (GBX2) progenitors. d, One-month-old brain organoids express the cortical marker EMX1, the forebrain marker FOXG1 and the retina marker VSX2, with spatial segregation between regions positive for forebrain versus retinal markers. Scale bars, 250 μm and 20 μm (insets).

Extended Data Figure 2 Decoding of the identity of cell types within the main clusters.

a, Heatmap of differentially expressed cell-type marker genes across each of the 10 main clusters. For gene lists and P values, see Supplementary Table 2, for references of cluster identification, see Extended Data Table. 1. b, Heatmap of average expression for representative marker genes and cell-type classification of the main clusters from the six-month-old organoid dataset.

Extended Data Figure 3 Quantifying variability among organoids.

tSNE plots depicting the proportion of cells each organoid contributed to each cell-type cluster. The cells are colour-coded by the organoid of origin. The large plot shows the entire cluster, and the smaller plots show the contribution of each individual organoid, labelled with the percentage of cells within the cluster contributed by that organoid.

Extended Data Figure 4 Correlation analysis for organoid cell types compared to the human fetal cortex and mouse retina.

a, Correlation between expression patterns of highly variable genes between cell populations within a previously published single-cell RNA-seq dataset of the human fetal cortex22 against the organoid astroglial cluster (c2) and the identified subclusters of the forebrain cluster (c4). IPC, intermediate progenitor cells. b, Correlation between expression patterns of highly variable genes between cell populations within a previously published single-cell RNA-seq dataset of postnatal day (P)14 mouse retina11 and the cell populations identified within the retinal cluster (c5) in our organoid dataset. Pigmented Epi, pigmented epithelium; RGC, retinal ganglion cells. Colour keys (and dot sizes) represent the range of the coefficients of determination (r2) in each analysis.

Extended Data Figure 5 Distribution and reproducibility of forebrain and retinal cell types across organoids.

a, Immunohistochemical detection in a six-month-old organoid of the forebrain marker FOXG1 with the progenitor markers Nestin and SOX2 (left) and of FOXG1 with the corticofugal projection neuron marker CTIP2 and the callosal projection neuron marker SATB2 (right), all showing extensive colocalized coexpression, which was observed in all organoids examined (6 out of 6). b, Number of cells assigned to each cortical cell type in individual organoids from flask 3, and the percentage of each cell type out of all classified cortical cells in that organoid. c, Immunohistochemical detection of retinal cell types in six-month-old organoids shows expression of known markers for amacrine cells, bipolar cells, retinal ganglion cells, Müller glia, rods and retinal pigmented epithelium, observed across all organoids in every flask, n = 11; 3 bioreactors. d, Number of cells assigned to each retinal cell type in individual organoids from flasks 1–3, and the percentage of each cell type out of all classified retinal cells in that organoid. AC, amacrine cells; BP, bipolar cells; CfuPN, corticofugal projection neurons; CPN, callosal projection neurons; IN, interneurons; IPC, intermediate progenitor cells; MG, Müller glia; PE, pigmented epithelium; photo, photoreceptors; RG, radial glial cells; RGC, retinal ganglion cells.

Extended Data Figure 6 Electron microscopy of an eight-month-old human brain organoid.

a, Whole organoid before vibratome sectioning shows regional structures. The red line shows the approximate location of the vibratome slice. b, Example 40-nm section. c. Regular finger-like membrane stack could indicate development of the outer segment of a retinal rod cell. d, Stacked endoplasmic reticulum. e, Example synapse with crystalline-looking arrangement of vesicles. f, Putative muscle cell characterized by a large cell body with a diameter of about 30 μm highlighted in an organoid section stained with eosin and haematoxylin. g, Example of a muscle cell showing a distribution of the mitochondria near the cell surface, which is typical for muscle cells. h, Several cell bodies with different morphologies and cytoplasm content highlighted in colour. i, Large cell body with muscle fibres highlighted in blue. Inset (red square) shows muscle fibres with sarcomeres. j, One of the dark cell bodies appearing as dark spots on the organoid surface. k, Cell reaching to the surface of the organoid. Scale bars, 50 μm (f), 10 μm (gk) and 1 μm (i, inset).

Extended Data Figure 7 Statistical structure of spontaneous activity.

a, Electron micrographs of a 64-channel probe shank tip (Fig. 4a). Scale bars, 100 μm (top) and 20 μm (bottom). b, Example inter-spike interval (ISI, top) and auto-correlogram (bottom) plots for spontaneous activity recorded from 4 prototypical units; a.u., arbitrary units; freq., frequency. c, Plot of the mean spike rate in 11a cells (dots) and HuES66 (+) organoids recorded at 7–9 months. The difference between the median of the mean spike rates is not significant across lines (11a cells, n = 34 cells, 12 recording sites, 7 organoids, median = 0.662 Hz, Q1–Q3 = 0.186–2.071 Hz; HuES66 n = 27 cells, 9 recording sites, 8 organoids, median = 1.186 Hz, Q1–Q3= 0.463–2.848 Hz; two-tailed Wilcoxon rank-sum test, z = –1.47, P = 0.14; furthermore, the variance is not significantly different, squared-ranks test, z = –1.47, P = 0.141). d, Plot of the Fano factor against organoid age. e, Plot of mean spike rate against the Fano factor (n = 61 neuronal units, from 15 organoids). Fano factors outside the expected 99% confidence bounds for a Poisson distribution are plotted in grey (otherwise black). f, Plot of the Fano factors calculated across the whole recording versus the first second of population bursts. Shading, 99% confidence bounds for a whole-recording Fano factor of 1. Black symbols, Fano factor close to 1, consistent with a stationary Poisson-distributed system. g, Spike train auto-correlogram (colour-coded as in Fig. 4g) and cross-correlogram (grey) for the three units presented in Fig. 4g. h, Left, baseline normalized spike rate (n = 25 cells, 10 organoids; 11a cells, n = 9 cells, 4 organoids; HuES66, n = 16 cells, 6 organoids); the clustering of data of responders (red) and non-responders (blue). Middle, count histogram of the left data plot overlaid by the optimal Gaussian mixture model (number of components k = 2, see methods; μk = kth mean, σk = kth s.d.). The underlying distributions are shaded (μ1 = 0.16, σ1 = 0.11, mixing proportion = 0.16; μ2 = 0.99, σ2 = 0.18, mixing proportion = 0.84). Right, plot of the Bayes information criterion (BIC) generated for the Gaussian mixture models with k components. i, Population averaged activity for light responsive (red, n = 4), and non-responsive (blue, n = 21), neurons; error envelope (shaded) is the s.e.m. In the four responsive organoids, light stimulation attenuated firing rate in 4 out of 5 isolated neurons.

Extended Data Table 1 References used for cluster identification in Extended Data Fig. 2

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion and Supplementary Table 1, a list of primary antibodies employed in this study. (PDF 244 kb)

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). (XLSX 752 kb)

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

Organoid surface towards the top of the images. (MP4 27916 kb)

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

Organoid surface towards the top. (MP4 17598 kb)

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. (MP4 3795 kb)

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Quadrato, G., Nguyen, T., Macosko, E. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017).

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