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Brain Chimeroids reveal individual susceptibility to neurotoxic triggers

Nature volume 631, pages 142–149 (2024)Cite this article

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Abstract

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

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Fig. 1: NSC-Chimeroids maintain donor representation across cell classes.
Fig. 2: NSC- and NPC-Chimeroids control hyperproliferative states of starting PSCs.
Fig. 3: Chimeroids are reproducible and display appropriate cell type composition.
Fig. 4: Treatment-specific alterations in the NSC-Chimeroids model.
Fig. 5: NSC-Chimeroids reveal differential donor-specific susceptibility.

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Data availability

Read-level data from scRNA-seq have been deposited at Synapse (https://www.synapse.org/; syn52132869), while count-level data and metadata have been deposited at https://singlecell.broadinstitute.org/single_cell/study/SCP2609. Any additional information required to reanalyse the data reported in this paper is available from the corresponding author on request. Data from previous publications that were used in this study were as follows: organoid reference map and fetal data20 (Synapse: syn26346373); fetal data23 (GEO: GSE162170) and fetal data24 (dbGaP: phs001836).

Code availability

Code used during data analysis is available at GitHub (https://github.com/tfaits/Arlotta_Lab_Chimeroids).

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Acknowledgements

We thank J. R. Brown for input and assistance in editing the manuscript; L. L. Lyons, N. Kozub and S. Tropp for technical assistance; all of the members of the Arlotta laboratory for discussions; B. Cohen for the Mito210 iPSC line; G. Church for the PGP1 iPSC line; M. Talkowski for the GM08330 iPSC line; the staff at the Broad Genomics Platform for sequencing; S. McCarroll for the Census-seq protocol; and B. Yeung for their support with pre-processing the Curioseeker data. Some components of the schematics were adapted from BioRender. This work was supported by grants from the Stanley Center for Psychiatric Research to P.A., R.N. and J.Z.L.; the Broad Institute of MIT and Harvard; the Blavatnik Biomedical Accelerator at Harvard University to P.A.; the National Institutes of Health (P50-MH094271 and RF1-MH123977 to P.A., R01-MH112940 to P.A. and J.Z.L., and U01-MH115727 to R.N.); and the Klarman Cell Observatory (A.R.).

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Author notes

  1. These authors contributed equally: Noelia Antón-Bolaños, Irene Faravelli

Authors and Affiliations

  1. Department of Stem Cell & Regenerative Biology, Harvard University, Cambridge, MA, USA

    Noelia Antón-Bolaños, Irene Faravelli, Tyler Faits, Sophia Andreadis, Rahel Kastli, Sebastiano Trattaro, Anqi Wei, Abhishek Sampath Kumar, Daniela J. Di Bella, Matthew Tegtmeyer, Ralda Nehme & Paola Arlotta

  2. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Noelia Antón-Bolaños, Irene Faravelli, Tyler Faits, Rahel Kastli, Sebastiano Trattaro, Xian Adiconis, Anqi Wei, Abhishek Sampath Kumar, Daniela J. Di Bella, Matthew Tegtmeyer, Ralda Nehme, Joshua Z. Levin & Paola Arlotta

  3. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Tyler Faits, Xian Adiconis & Joshua Z. Levin

  4. Broad Institute of MIT and Harvard, Boston, MA, USA

    Aviv Regev

  5. Genentech, San Francisco, CA, USA

    Aviv Regev

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Contributions

N.A.-B., I.F. and P.A. conceived and designed the experiments. N.A.-B., I.F. and S.A. generated, cultured and characterized all of the organoids in this study, with the help of S.T. and R.K., and P.A. supervised their work and contributed to data interpretation. N.A.-B., I.F., R.K. and S.A. performed IHC and image acquisition. R.K. performed MEA recordings and data analysis. X.A., N.A.-B. and I.F. performed scRNA-seq experiments, with help from D.J.D.B. and supervision of P.A. and J.Z.L.; N.A.-B., I.F. and S.T. performed scRNA-seq library preparation for the vast majority of the preparations included in this study. A.S.K., N.A.-B. and I.F. performed spatial transcriptomic experiments. A.S.K. and T.F. performed spatial transcriptomic computational analysis. A.W. performed the computational analysis of Extended Data Fig. 7. T.F., N.A.-B. and I.F. worked on cell type assignments and scRNA-seq data analysis, and T.F. performed all of the computational work under supervision by A.R. and P.A.; R.N. and M.T. provided the CW line, and advised on the PSC-Chimeroid experiments and on application of the Census-seq analysis pipeline, using computational tools developed by S. McCarroll’s laboratory. N.A.-B., I.F. and P.A. wrote the manuscript with contributions from all of the authors. All of the authors read and approved the final manuscript.

Corresponding author

Correspondence to Paola Arlotta.

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Competing interests

P.A. is a scientific advisory board member at Foresite Labs, CNSII, and is a co-founder and scientific advisory board member of Vesalius Therapeutics. A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until 31 August 2020, was a scientific advisory board member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov and Thermo Fisher Scientific. From 1 August 2020, A.R. has been an employee of Genentech and has equity in Roche.

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Extended data figures and tables

Extended Data Fig. 1 PSC Chimeroids display uneven donor composition but proper cell type composition.

a, Schematic of the PSC-Chimeroid protocol. Some icons were created using BioRender. b, Stacked bar plots showing donor composition, as measured via Census-seq from low-pass whole-genome sequencing, in PSC-Chimeroids from various mixes and timepoints, labelled as the number of days since the day of the seeding (DIV0). For the DIV0 and DIV1 time points, n = 4 Chimeroids have been pulled together. c, Left panel: brightfield image of 3-mo PSC-Chimeroids; scale bar: 1 mm. Right panel: immunolabelling of 3-mo PSC-Chimeroid with SATB2, TBR1 and MAP2. Scale bar: 500 μm. d, UMAP of integrated PSC-Chimeroids at 3 mo, coloured by annotated cell type (left panel) and donor line as determined with demuxlet (right panel). e, UMAPs split by donor for two different replicates. f, Barplots of cell type proportions in PSC Chimeroids (n = 2), demultiplexed by donor. g, Immunolabelling of 1-mo PSC-Chimeroids showing early progenitors (SOX2), cortical progenitors (EMX1); rosette centres are lined with ZO-1, a tight junction protein present in the endfeet of radial glia cells, and NESTIN, indicating correctly-polarized neuroepithelium. CFuPNs neurons are marked by TBR1, and the neuronal dendrites by MAP2. h, Immunolabelling of 2-mo PSC-Chimeroids showing early progenitors (SOX2), IP (TBR2) and oRG (HOPX). CTNNB1 marks the centre of the neural rosettes, indicating correctly-polarized neuroepithelium. i, Immunolabelling of 3-mo PSC-Chimeroids showing cortical markers such as SATB2 (upper layers cortical neurons), and CTIP2 (deep layers cortical neurons). White arrows point to the neural rosettes (shown at higher magnification in the lower panels). Scale bar: 100 μm.

Extended Data Fig. 2 NSC-Chimeroids created by aggregating different numbers of cells display variable development.

a. Brightfield images of single-donor H1 NSC-Chimeroids seeded with 9,000 (upper) and 12,000 (lower) cells/well at day 3 and day 16 after reaggregation (left panels), and brightfield images of single donor PGP1 NSC-Chimeroids seeded with 9,000 (top left), 12,000 (bottom left), and 20,000 (top right) cells/well at day 3 and day 16 after reaggregation (right panels). Scale bar: 500 μm. Barplot showing growth over time of PGP1 NSC-Chimeroids compared across all different cell counts at aggregation (bottom right). Bars show median values, whiskers show upper and lower quartiles (n = 170 Chimeroids across all 4 timepoints). Significance was calculated using ANOVA followed by two-sided Tukey post-hoc pairwise tests. P-values: *** < − 0.001; **** - <0.0001. b. Immunolabelling of PGP1 single-donor NSC-Chimeroids made by aggregating the indicated number of cells, at DIV35. Upper images display whole organoids; lower images, enlargement of indicated portions. Lefthand panel, immunolabelling for SOX2 (progenitors), MAP2 (neuronal dendrites), and TBR1 (deep layer neurons). Righthand panel, immunolabelling for SOX2, ZO-1 (tight gap junctions), and EMX1 (dorsal cortical progenitors). Scale bar: 500 μm (upper) and 125 μm (lower). Arrowheads indicate non-cortical (grey) and cortical (white) regions. c. Left panel: whole-organoid brightfield images of H1 single-donor NPC-Chimeroids seeded with 100,000 cells at aggregation, at 18 and 35 days after mixing. Scale bar: 500 μm. Right panel: immunolabelling of H1 single-donor NPC-Chimeroids showing SOX2, ZO-1, and EMX1. Scale bar: 500 μm and 125 μm (zoom-in).

Extended Data Fig. 3 Multi-donor NSC Chimeroids display appropriate differentiation markers.

a, Immunolabelling of 1-mo MD-NSC Chimeroids showing early progenitors (SOX2), cortical progenitors (EMX1); rosette centres are lined with ZO-1, a tight junction protein present in the endfeet of radial glia cells, and NESTIN signal, indicating correctly-polarized neuroepithelium. CFuPNs neurons are marked by TBR1, and the neuronal dendrites by MAP2. b, Immunolabelling of 2-mo MD-NSC Chimeroids showing early progenitors (SOX2), IP (TBR2) and oRG (HOPX). CTNNB1 marks the centre of the neural rosettes, indicating correctly-polarized neuroepithelium. c, Immunolabelling of 3-mo MD-NSC Chimeroids showing cortical markers such as SATB2 (upper layers cortical neurons), and CTIP2 (deep layers cortical neurons). White arrows point to the neural rosettes. Scale bar: 100 μm. Some icons were created using BioRender.

Extended Data Fig. 4 NSC-Chimeroids develop appropriate cell-type composition across multiple donors.

a, Stacked barplots showing the donor composition, as measured via Census-seq from low-pass whole-genome sequencing, of multi-donor NSC-Chimeroids from various mixes and at various timepoints, labelled as the number of days since reaggregation (DIV; for the DIV0 and DIV1 time points, n = 4 chimeroids have been pulled together). b-e, 3-mo NSC-Chimeroids UMAPs split by donor (b, d) and stacked barplots of donor contributions for each cell type (c, e) for Mix 1 (4 donors) and Mix 2 (5 donors).

Extended Data Fig. 5 NSC-Chimeroids present uniform spatial distribution across donors.

a, Slice #1. Spatial plot of Slide-seq data from 2-mo MD-NSC Chimeroids (Mix 5), coloured by maximal donor contribution via Census-seq. b, Spatial plots showing donor prediction weights. c, Spatial plot of Slide-seq data from 2-mo NSC-Chimeroids (Mix 5), coloured by RCTD-assigned cell type. d, Spatial plots showing cell type prediction weights. e, Slice#2. Same as a. f, Slice#2. Same as b. g, Slide#2. Same as c. h, Slide#2. Same as d. i, Representative organoid placed on a Multielectrode Array (MEA). j, Representative activity map of a MD-NSC Chimeroid network burst at 4mo, where each pixel of the map represents an electrode (left) and example action potential traces from the electrodes highlighted in the activity map (right). k, Representative raster plot showing the firing activity of active electrodes (139, defined as electrodes with a mean firing rate >0.15 spikes/s inside the outline of the organoid) over a 15 min recording. Network bursts start to appear around 5 mins. l, Effect of AMPA and NMDA blockers (D-AP5, 150 µM; DNQX 60 µM) on neuronal activity/network bursts (left panel); network bursting activity of 4 recorded NSC-Chimeroids (right panel). Some icons were created using BioRender.

Extended Data Fig. 6 NPC-Chimeroids display appropriate differentiation markers.

a, Immunolabelling of 1-mo NPC-Chimeroids showing early progenitors (SOX2), cortical progenitors (EMX1); rosette centres are lined with ZO-1, a tight junction protein present in the endfeet of radial glia cells, and NESTIN signal, indicating correctly-polarized neuroepithelium. CFuPN neurons are marked by TBR1, and the neuronal dendrites by MAP2. b, Immunolabelling of 2-mo NPC-Chimeroids showing early progenitors (SOX2), IP (TBR2) and oRG (HOPX). CTNNB1 marks the centre of the neural rosettes, indicating correctly-polarized neuroepithelium. c, Immunolabelling of 3-mo NPC-Chimeroids showing cortical markers such as SATB2 (upper layers cortical neurons), and CTIP2 (deep layers cortical neurons). White arrows point to the neural rosettes. Scale bar: 100 μm. d, Brightfield images of 3-mo Chimeroids across all protocols (scale bar: 1 mm). Some icons were created using BioRender.

Extended Data Fig. 7 Nascent Chimeroid input to aggregation steps is composed of expected cell-type populations.

a, UMAP of integrated dataset containing DIV23 organoids from our reference map of Velasco-protocol organoid development (“Ref. Map”), and MD-NSC and MD-NPC-Chimeroids 1 day after aggregation (INPUT), colour-coded by annotated cell type. b. UMAPs split by protocol. c, UMAP of the INPUT for MD-NSC Chimeroids (mix 2), colour-coded by annotated cell type. d, UMAPs split by donors. e, UMAP of the INPUT for MD-NPC Chimeroids (Mix 6), colour-coded by annotated cell type. f, UMAPs split by donors. g, Donor demultiplexed after sc-RNA-seq. h, Bar plots of cell-type proportions in INPUT MD-NSC and MD-NPC Chimeroids for each donor. Some icons were created using BioRender.

Extended Data Fig. 8 Single-donor Chimeroids display appropriate differentiation markers.

a, Immunolabelling of 1-mo 11a SD-NSC Chimeroids showing early progenitors (SOX2), cortical progenitors (EMX1); rosette centres are lined with ZO-1, a tight junction protein present in the endfeet of radial glia cells, NESTIN and CTNNB1 signal, indicating correctly-polarized neuroepithelium surrounded by early new born CFuPN neurons. b, Same as panel a, for PGP1 SD-NSC Chimeroids. c, Immunolabelling of 3-mo 11a SD-NSC Chimeroids showing cortical markers such as SATB2 (upper layers cortical neurons), and CTIP2 (deep layers cortical neurons). d, Same as panel c, for PGP1 SD-NSC Chimeroids. White arrows point to the neural rosettes. Scale bar: 100 μm. Some icons were created using BioRender.

Extended Data Fig. 9 Chimeroids comparison across protocols and donors.

a, UMAP showing overlapping neighbourhoods of cells, as calculated using Milo. Red and blue colours indicate neighbourhoods with significant enrichment for cells from single-donor or multi-donor Chimeroids, respectively. Point size indicates the number of cells in a neighbourhood, and edge thickness indicates the number of cells shared between pairs of neighbourhoods. b, Beeswarm plot showing shifts in the composition of neighbourhoods of cells, grouped by the cell-type identity of those neighbourhoods. Each point represents a neighbourhood of 50-200 cells with similar gene expression profiles. The vertical axis indicates the enrichment of single-donor cells within a neighbourhood, with positive log fold change values indicating more than expected single-donor cells, and negative values indicating fewer than expected single-donor cells. Neighbourhoods are coloured based on statistical significance of that enrichment: grey, not significantly different from random; red, significant over-enrichment; blue, significant under-enrichment. c-d, Volcano plots showing DEGs, overall and for each donor (c), and for each major cell type (d), between the MD- and SD-NSC Chimeroid protocols. Positive log2 fold change indicates higher expression in the SD protocol. e, Correlation plots comparing the DEGs between each pair of donors in MD-NSC Chimeroids to those between the same pair of donors in the SD-NSC Chimeroids. The x- and y-axies shows log2 fold change in MD and SD, respectively. Point size and colour indicate statistical significance in MD and SD, respectively. f, Heatmap showing the mean absolute value of the log2 fold change between each donor in the MD-NSC Chimeroid protocol (columns) and each donor in the SD-NSC Chimeroid protocol (rows). Lower values imply more similarity; samples from each donor were transcriptionally most similar to samples from the same donor in the other protocol. g, Aitchison distance measuring the dissimilarity in cell type composition between replicates within each protocol, split into comparisons within batches and between batches, and limited to cells derived from the PGP1 and Mito210 donors (as only these two donors are present in all organoid/Chimeroid protocols assayed here). (n = 23 PGP1, 23 Mito210, and 10 fetal samples; Boxes show upper and lower quartiles and median, whiskers show highest/lowest values within 1.5 interquartile range (IQR) of the nearest hinge). The dissimilarity in cell type composition between samples within each of three fetal cortical datasets is also shown, indicating natural variability between individuals. Dotted lines represent mean inter-sample distances within each fetal dataset20,23,24. Some icons were created using BioRender.

Extended Data Fig. 10 Chimeroids display similar cell type complexity as single-donor organoids and fetal tissue.

Rank-Rank Hypergeometric Overlap (RR-HO) plots comparing the expression signatures of cell types in multi-donor Chimeroids to all cell types in single-donor Chimeroids, cortical organoids from our reference map of Velasco-protocol organoid development, or endogenous human fetal tissue20. The horizontal axes represent lists of marker genes for multi-donor NSC-Chimeroid cell types compared to all other Chimeroid cells, ranked from most upregulated to most downregulated; the vertical axes represent similarly-ranked lists of marker genes for single-donor NSC-Chimeroids, reference map organoids, or human fetal cells. Colour at a given position represents the significance (negative log p-value) of the overlap of the gene lists up to that point, as calculated by Fisher’s exact tests. High significance (i.e., red colour) in the lower left and upper right quadrants indicates strong concordance between the expression profiles which define the compared cell types. a, b, c, d and e represent different cell types analysed. f, Explanatory schematic for the RR-HO plots. Some icons were created using BioRender.

Extended Data Fig. 11 Chimeroids display similar developmental trajectories and metabolic hallmarks across donors and across protocols.

a, Density plot showing the distribution of scaled pseudotimes assigned to each donor within each organoid/Chimeroid protocol. Pseudotime was calculated independently for each protocol using Monocle 3, with aRGs in each dataset set as root cells. b, Glycolysis module scores calculated in each cell type/donor/Chimeroid with linear MEMs (using lme4’s lmer) on the PGP1/Mito210 cells using donor as a random effect (n = 40 replicates across 4 protocols; boxes show upper and lower quartiles and median, whiskers show highest/lowest values within 1.5 interquartile range (IQR) of the nearest hinge.). P-values were generated via one-sided F-test comparing models with and without “protocol” as a covariate. c-d, Expression of the glycolysis geneset is similar across donors (12,887 cells from n = 45 donor/Chimeroids) and protocols (20,425 cells from n = 40 donor/Chimeroids and organoids). Violin plots showing the module scores for the MSigDB Hallmark Glycolysis gene set across cell types, donors, and protocols; boxes show upper and lower quartiles and median, whiskers show highest/lowest values within 1.5 interquartile range (IQR) of the nearest hinge. Module scores were calculated with Seurat’s addModuleScores function.

Extended Data Fig. 12 VPA and EtOH treatment differently affect cell-type composition in NSC-Chimeroids.

a, Stacked barplot showing cell type and donor composition for control Chimeroids; the width of each bar corresponds to the proportion of the indicated donor in Mix 1 (4 donors, left panel) and Mix 2 (5 donors, right panel). b, Brightfield images of whole cortical NSC-Chimeroids at 3 mo, in the EtOH treatment condition. Scale bar: 1 mm. c, Stacked barplot showing cell type and donor composition for EtOH treated Chimeroids; the width of each bar corresponds to the proportion of the indicated donor in Mix 1 (4 donors, left panel) and Mix 2 (5 donors, right panel). d, UMAPs of EtOH treated NSC-Chimeroids, split by mixes and replicates. e, Brightfield images of whole cortical MD-NSC Chimeroids at 3 mo, in the VPA treatment condition. Scale bar: 1 mm. f, Stacked barplot showing cell type and donor composition for EtOH treated Chimeroids; the width of each bar corresponds to the proportion of the indicated donor in Mix 1 (4 donors, upper panel) and Mix 2 (5 donors, lower panel). g, UMAPs of VPA treated NSC-Chimeroids, split by mixes and replicates. Some icons were created using BioRender.

Extended Data Fig. 13 VPA treatment cause protein and transcriptome alterations in single-donor Chimeroids.

a, Immunolabelling of multi donor NSC-Chimeroids (a, Mix 1, upper images and b, Mix 2, lower images). Left panel, MAP2 (neuronal dendrites), EMX1 (cortical progenitors), and DLX2 (GABAergic cells). Right panel, DCX (migrating neurons), SATB2 (upper layers cortical neurons), and CTIP2 (deep layers cortical neurons). Scale bar: 100 μm. c, Stacked barplot showing cell type composition for control and treated single-donor NSC-Chimeroids. d, Left panel: cell-type specific changes in single-donor Chimeroids treated with VPA (yellow) vs control (grey). Right panel: UMAP showing overlapping neighbourhoods of cells, as calculated using Milo. Red and blue colours indicate neighbourhoods with significant enrichment for VPA-treated cells or control cells, respectively. Point size indicates the number of cells in a neighbourhood, and edge thickness indicates the number of cells shared between pairs of neighbourhoods. e, Beeswarm plot showing shifts in the composition of neighbourhoods of cells in response to VPA treatment in single-donor Chimeroids, grouped by the cell-type identity of those neighbourhoods. Each point represents a neighbourhood of 50-200 cells with similar gene expression profiles. The vertical axis indicates the enrichment of VPA-treated cells within a neighbourhood, with positive log fold change values indicating more than expected VPA-treated cells, and negative values indicating fewer than expected VPA-treated cells. Neighbourhoods are coloured based on statistical significance of that enrichment: grey, not significantly different from random; red, significant over-enrichment; blue, significant under-enrichment. If most neighbourhoods within a cell type collectively shift up or down, it implies an overall gain or loss, respectively, of that cell type in VPA-treated Chimeroids. Cell types with neighbourhoods that form long tails of both over-enrichment and under-enrichment are likely to have treatment-induced changes in expression profile, without necessarily changing in abundance. f, GSEA of donor specific genes from MD-NSC Chimeroids ranked on the corresponding single-donor datasets. g, GSEA of donor specific genes from SD-NSC Chimeroids ranked on the corresponding multi-donor datasets (lower panels). P-values calculated via two-sided Kolmogrov-Smirnov test. Some icons were created using BioRender.

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Antón-Bolaños, N., Faravelli, I., Faits, T. et al. Brain Chimeroids reveal individual susceptibility to neurotoxic triggers. Nature 631, 142–149 (2024). https://doi.org/10.1038/s41586-024-07578-8

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