Cell diversity and network dynamics in photosensitive human brain organoids

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.

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

a. Top: Expression of the hypoxia marker HIF1-α over 1 to 9 months of culture, and positive control (a 6 month organoid treated with cobalt (II) chloride, an activator of the hypoxia signaling pathway). Bottom: Expression of the apoptosis marker active caspase 3 over 1 to 9 months of culture. b. Expression of markers for progenitor, neuronal and glial populations over 1 to 9 months of culture. c. One month old brain organoids exhibit early brain regionalization, expressing markers of forebrain, midbrain, and hindbrain 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 (low magnification), 20 μm (high magnification).

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 1. b. Heatmap of average expression for representative marker genes and cell-type classification of the main clusters from the 6 mo organoid data set. c. Table of references used for cluster identification in a.

Extended Data Figure 3. Quantifying variability among organoids

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

Extended Data Figure 4. Distribution and reproducibility of forebrain and retina cell types across organoids

a. Immunohistochemical detection in a 6 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 co-coexpression, which was observed in all organoids examined (6 of 6). b. Count 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 6 month organoids shows expression of known markers for amacrine cells, bipolar cells, retinal ganglion cells, Muller glia, rods, and retinal pigmented epithelium, observed across all organoids in every flask, n=11, 3 bioreactors. d. Count of cells assigned to each retinal cell type in individual organoids from Flasks 1, 2, and 3, and the percentage of each cell type out of all classified retinal cells in that organoid. IN: interneurons; IPC: intermediate progenitor cells; CfuPN: corticofugal projection neurons; CPN: callosal projection neurons; RG: radial glial cells; MG: Muller glia; PE: pigmented epithelium; PHOTO: photoreceptors; RGC: retinal ganglion cells; BP: bipolar cells; AC: amacrine cells.

Extended Data Figure 5. Correlation analysis for organoid cell types vs. fetal human cortex and mouse retina

a. Correlation between expression patterns of highly variable genes between cell populations within a previously published single-cell RNAseq dataset of human fetal cortex against the organoid astrogial 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 RNAseq dataset of P14 mouse retina and the cell populations identified within the retinal cluster (c5) in our organoid dataset. RGC: retinal ganglion cells; Pigmented Epi: pigmented epithelium. Scale bars represent the range of the coefficients of determination (r²) in each analysis.

Extended Data Figure 6. Electron microscopy of an 8 month old human brain organoid

a. Whole organoid before vibratome sectioning shows regional structures. Red line shows 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-haematoxylin. Scale bar, 50 μm. g. Example of a muscle cell showing a distribution of the mitochondria near the cell surface, typical for muscle cells. Scale bar, 10 μm. h. Large cell body with muscle fibers highlighted in blue. Enlarged red square region shows muscle fibers with sarcomeres. Scale bar, 10 μm (low magnification) and 1 μm (enlargement). i. Left, one of the dark cell bodies appearing as dark spots on the organoid surface. Right, cell reaching to the surface of the organoid. Scale bar, 10 μm. j. Several cell bodies with different morphologies and cytoplasm content highlighted in color. Scale bar, 10 μm.

Extended Data Figure 7. Statistical structure of spontaneous firing activity in human brain organoids

a. Electron micrograph of a 64-channel high-density probe shank (corresponding to the tip of the device shown in Fig. 4a) with two columns of 32 rows of recording sites, visible as squares along the center of the shank (top panel). The recording sites consist of 9×9 μm exposed gold pads (bottom panel) that are PEDOT electroplated to lower electrical impedances. Each recording site is connected to the outside by insulated nanofabricated wires, running along the length of the probe shank (visible as gray lines flanking the recording sites). The shank width is tapered from 35 to 60 μm wide, and is 15 μm thick. Scale bars, 100 μm (top), 20 μm (bottom). b. Example inter-spike interval (top) and auto-correlogram (bottom) plots for spontaneous activity recorded from 4 prototypical units; 1 ms bins. c. Plot of the mean spike rate in iPSC11a (o) and HuES66 (+) organoids recorded at 7–9 months. The difference between the median of the mean spike rates in the two organoid lines are non-significant (iPSC11a n= 34 cells, 12 recording sites, 7 organoids, M= 0.662, Q1= 0.186, Q3= 2.071; HuES66 n= 27 cells, 9 recording sites, 8 organoids, M=1.186, Q1= 0.463, Q3= 2.848; 2 tailed Wilcoxon rank sum test, 5% significance level, z= -1.47, p= 0.14; squared rank test suggests that the variance of mean spike rate in the two organoid lines is not significantly different, 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 that are outside the expected 99% confidence bounds are plotted in gray; those that are within the 99% confidence bounds are plotted in black. A time-series fano factor greater than 1 indicates that the unit firing is not well modeled by a stationary Poisson distribution, and points at the presence of network activity. f. Plot of the fano factor calculated across the whole recording versus the fano factor calculated during the population bursts. Shaded region highlights the 99% confidence bounds for a whole-recording fano factor of 1. Black symbols correspond to units with a fano factor close to 1, consistent with a stationary Poisson distributed system and implying a fixed firing profile during the first second of population burst. g. Spike train auto-correlogram (color) and cross-correlogram (gray) for the three units presented in Figure 4g. Color-coding as in Figure 4g. h. Left, baseline normalized spike rate (n = 25 cells from 10 organoids; o, iPSC11a, n = 9 cells 4 organoids; +, HuES66, n = 16 cells 6 organoids); color identifies the clustering of data points into responders (red) and non-responders (blue). Middle, count histogram of the data plot in the left panel overlaid by the optimal Gaussian mixture model (number of components k = 2; see Methods). The two underlying distributions are plotted as shaded regions (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. BIC is minimized at k = 2, signifying that the data is best described by a bimodal distribution (see middle panel). i. Population averaged activity for the two populations of neurons (responders in red, n=4 neurons; non-responders in blue, n=21 neurons); error envelope (shaded) is the s.e.m. In the 4 responsive organoids, light stimulation attenuated firing rate in 4 out of 5 isolated neurons.

Figure 1. Large-scale, single-cell sequencing demonstrates development of a broad spectrum of cell types in human brain organoids

a. Schematic of long-term culture of brain organoids. Dissociated human iPSCs are seeded at day 0 into round-bottom plates to allow EB formation (day 2–5). After a two-step neural induction (day 6–10), EBs are embedded in Matrigel (day 10) and transferred to spinning bioreactors (day 15) for long-term culture. BDNF is added starting at 1 month. Immunohistochemistry (IHC), single cell RNA-sequencing (Drop-seq), electrophysiology (E-phys) and electron microscopy (EM) were performed at different timepoints. b. t-SNE plot of single-cell mRNA sequencing data from 6 mo organoids. A total of 66,889 cells were clustered into 10 distinct groups. c. Same as in b, with cells color-coded by organoid of origin.

Figure 2. Human brain organoids contain subclasses of forebrain and retina cells

a. Subclustering of the forebrain cluster (c4): five major cell subtypes were identified as radial glia (7 subclusters), interneurons (1 subcluster), intermediate progenitors (1 subcluster), corticofugal neurons (2 subclusters), or callosal neurons (8 subclusters). b. All cells in c4, color-coded by organoid of origin. c. Subclustering of the retina cluster (c5): six major cell subtypes were identified as Muller glia (3 subclusters), photoreceptors (4 subclusters), retinal ganglion cells (2 subclusters), bipolar cells (3 subclusters), amacrine cells (4 subclusters), or pigmented epithelium (2 subclusters). d. All cells in c5, color-coded by organoid of origin. e. Expression of genes identified as retinal cell subtype markers across the retinal subclusters (see Extended Data Figure 2c for sources).

Figure 3. Extended culture permits development of mature cell types including differentiated photoreceptors

a. Average expression of selected marker genes in organoids, by cell type and organoid age (3 vs. 6 month). ASTRO: astrocytes; MESO: mesodermal cells; NE: neuroepithelial cells; IN: interneurons; CPN: callosal projection neurons; CfuPN: corticofugal projection neurons; IPC: intermediate progenitor cells; RG: radial glial cells; BP: bipolar cells; MG: Muller glia; AC: amacrine cells; RGC: retinal ganglion cells. b. Reclustering of CRX⁺ cells in the combined 3 mo and 6 mo data sets identifies 17 clusters. c. Among CRX⁺ cells, sub-cluster sb-c7 is present only at 6 mo. d. Genes related to rod phototransduction (brown bar) are highly differentially expressed in the photoreceptor cluster (sb-c7). e–k. Synapse and dendritic spine development in organoids. e. Expression of the synaptic marker SYN1 is absent at 1 mo and appears with organoid maturation by 3 mo. f. Immunohistochemical detection of the synaptic proteins VGAT and VGLUT1 in a 6 mo organoid. g. EM slice from an 8 mo organoid (outer surface at left). Red frame, reconstructed area used in h–k. Scale bar, 10 μm. h. Example EM images of synaptic structures in the reconstructed volume. Scale bars, 1 μm. i. 3D renderings of all traced axons (blue) and dendrites (red) that establish synapses in the volume. Scale bar, 1 μm. j. Example dendrite with two spines (orange) making synapses with two axons (blue and green). Synaptic vesicles, yellow. k. Synaptic contact sites (yellow) on the 29 spines identified in the volume. Asterisks, synapses from j. Scale bar, 1 μm.

Figure 4. Brain organoids develop spontaneous networks and photosensitive neurons that can be modulated by sensory stimulation with light

a. Schematic and photograph of extracellular recordings from intact organoids. (arrow, probe; double arrow, organoid). b. Human brain organoids display spontaneous activity. Top, example raw traces and spike raster plots from a single unit (scale bar 0.5 s). Bottom, individual (gray) and average (blue) spike waveforms (scale bars, 0.5 ms, 50 μV). c. Organoids display spontaneous activity at 8 months (6 of 7) but not 4 months (0 of 4; Fisher’s exact test, p=0.015). d. Example spike-train cross-correlogram. The positive peak with a short time lag indicates mono-synaptic connections. Red line, estimated mean spike rate; Blue line, statistical threshold for identification of connected pairs (see Methods). e. NBQX attenuates activity. Left, example raster plots and population-averaged spiking rate (2 s bins; arrows, break to apply NBQX). Right, summary plot (n=12; pre-NBQX, M=0.380 Hz, Q1–3=0.115–1.145; post-NBQX, M=0.073 Hz, Q1–3=0.007–0.163; exact sign test, p<0.0005). f. Example population rate histograms (1 s bins), for organoids displaying bursts of activity (bottom) and without obvious bursting (top). Inset, raster plots of isolated units. g. Neuronal activity during population bursts shows temporal structure. Left, neuron mean firing rate aligned to burst onset and raster plots for 3 neurons (color-coded) in 3 example bursts. Right, mean spike waveforms recorded by probes (color, peak response; vertical order reflects probe geometry). Scale bars, 2 ms, 50 μV. h. Example rate histograms (10 s bins) for 2 cells showing light-attenuated activity (see Extended Data Figure 7 h–i). i. Light stimulation increases c-fos expression (red); rod-like cells are indicated by rhodopsin staining (green). Scale bars, main 250 μm, inset 20 μm. All: * denotes significance p < 0.05.