Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3,4,5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Evolutionary honing in and mutational replacement: how long-term directed mutational responses to specific environmental pressures are possible
Theory in Biosciences Open Access 11 March 2023
Genome Biology Open Access 19 January 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Raw and processed data are publicly available through the Gene Expression Omnibus under accession GSE144825. The alignment of human and chimpanzee genomes from Ensembl is available at ftp://ftp.ensembl.org/pub/release-84/maf/ensembl-compara/pairwise_alignments/homo_sapiens.GRCh38.vs.pan_troglodytes.CHIMP2.1.4.tar. The SFARI database is available at https://www.sfari.org/resource/sfari-gene/.
All code for the described analyses of RNA-seq data and for making figures is publicly available at https://github.com/TheFraserLab/Agoglia_HumanChimpanzee2020.
Gallego Romero, I. et al. A panel of induced pluripotent stem cells from chimpanzees: a resource for comparative functional genomics. eLife 4, e07103 (2015).
Prescott, S. L. et al. Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest. Cell 163, 68–83 (2015).
Pașca, S. P. The rise of three-dimensional human brain cultures. Nature 553, 437–445 (2018).
Muchnik, S. K., Lorente-Galdos, B., Santpere, G. & Sestan, N. Modeling the evolution of human brain development using organoids. Cell 179, 1250–1253 (2019).
Qian, X., Song, H. & Ming, G. L. Brain organoids: advances, applications and challenges. Development 146, dev166074 (2019).
Pollen, A. A. et al. Establishing cerebral organoids as models of human-specific brain evolution. Cell 176, 743–756 (2019).
Kanton, S. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418–422 (2019).
Beneyto, M., Morris, H. M., Rovensky, K. C. & Lewis, D. A. Lamina- and cell-specific alterations in cortical somatostatin receptor 2 mRNA expression in schizophrenia. Neuropharmacology 62, 1598–1605 (2012).
Ádori, C. et al. Critical role of somatostatin receptor 2 in the vulnerability of the central noradrenergic system: new aspects on Alzheimer’s disease. Acta Neuropathol. 129, 541–563 (2015).
Ward, M. C. et al. Silencing of transposable elements may not be a major driver of regulatory evolution in primate iPSCs. eLife 7, e33084 (2018).
Prud’homme, B., Gompel, N. & Carroll, S. B. Emerging principles of regulatory evolution. Proc. Natl Acad. Sci. USA 104 (Suppl 1), 8605–8612 (2007).
Paşca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).
Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Camp, J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl Acad. Sci. USA 112, 15672–15677 (2015).
Sloan, S. A. et al. Human astrocyte maturation captured in 3D cerebral cortical spheroids derived from pluripotent stem cells. Neuron 95, 779–790 (2017).
Mora-Bermúdez, F. et al. Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development. eLife 5, e18683 (2016).
Otani, T., Marchetto, M. C., Gage, F. H., Simons, B. D. & Livesey, F. J. 2D and 3D stem cell models of primate cortical development identify species-specific differences in progenitor behavior contributing to brain size. Cell Stem Cell 18, 467–480 (2016).
Amps, K. et al. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat. Biotechnol. 29, 1132–1144 (2011).
Taapken, S. M. et al. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat. Biotechnol. 29, 313–314 (2011).
Müller, F.-J. et al. A bioinformatic assay for pluripotency in human cells. Nat. Methods 8, 315–317 (2011).
Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell 18, 25–38 (2016).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Stingl, J., Eaves, C. J., Zandieh, I. & Emerman, J. T. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 67, 93–109 (2001).
Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).
Greig, L. C., Woodworth, M. B., Galazo, M. J., Padmanabhan, H. & Macklis, J. D. Molecular logic of neocortical projection neuron specification, development and diversity. Nat. Rev. Neurosci. 14, 755–769 (2013).
Chen, B., Khodadoust, M. S., Liu, C. L., Newman, A. M. & Alizadeh, A. A. Profiling tumor infiltrating immune cells with CIBERSORT. Methods Mol. Biol. 1711, 243–259 (2018).
Somel, M. et al. Transcriptional neoteny in the human brain. Proc. Natl Acad. Sci. USA 106, 5743–5748 (2009).
Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).
Fraser, H. B. Genome-wide approaches to the study of adaptive gene expression evolution: systematic studies of evolutionary adaptations involving gene expression will allow many fundamental questions in evolutionary biology to be addressed. BioEssays 33, 469–477 (2011).
Oberheim, N. A., Wang, X., Goldman, S. & Nedergaard, M. Astrocytic complexity distinguishes the human brain. Trends Neurosci. 29, 547–553 (2006).
Miller, J. A., Horvath, S. & Geschwind, D. H. Divergence of human and mouse brain transcriptome highlights Alzheimer disease pathways. Proc. Natl Acad. Sci. USA 107, 12698–12703 (2010).
Bozek, K. et al. Exceptional evolutionary divergence of human muscle and brain metabolomes parallels human cognitive and physical uniqueness. PLoS Biol. 12, e1001871 (2014).
Kelley, K. W., Nakao-Inoue, H., Molofsky, A. V. & Oldham, M. C. Variation among intact tissue samples reveals the core transcriptional features of human CNS cell classes. Nat. Neurosci. 21, 1171–1184 (2018).
Basu, S. N., Kollu, R. & Banerjee-Basu, S. AutDB: a gene reference resource for autism research. Nucleic Acids Res. 37, D832–D836 (2009).
Sousa, A. M. M., Meyer, K. A., Santpere, G., Gulden, F. O. & Sestan, N. Evolution of the human nervous system function, structure, and development. Cell 170, 226–247 (2017).
Fujii, Y. et al. Somatostatin receptor subtype SSTR2 mediates the inhibition of high-voltage-activated calcium channels by somatostatin and its analogue SMS 201-995. FEBS Lett. 355, 117–120 (1994).
Liguz-Lecznar, M., Urban-Ciecko, J. & Kossut, M. Somatostatin and somatostatin-containing neurons in shaping neuronal activity and plasticity. Front. Neural Circuits 10, 48 (2016).
He, Z. et al. Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques. Nat. Neurosci. 20, 886–895 (2017).
Gokhman, D. et al. Human-chimpanzee fused cells reveal cis-regulation underlying skeletal evolution. Nat. Genet. https://doi.org/10.1038/s41588-021-00804-3 (2021).
Yoon, S. J. et al. Reliability of human cortical organoid generation. Nat. Methods 16, 75–78 (2019).
van de Geijn, B., McVicker, G., Gilad, Y. & Pritchard, J. K. WASP: allele-specific software for robust molecular quantitative trait locus discovery. Nat. Methods 12, 1061–1063 (2015).
Tehranchi, A. et al. Fine-mapping cis-regulatory variants in diverse human populations. eLife 8, e39595 (2019).
Combs, P. A. & Fraser, H. B. Spatially varying cis-regulatory divergence in Drosophila embryos elucidates cis-regulatory logic. PLoS Genet. 14, e1007631 (2018).
Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).
Zhang, Y. et al. Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron 89, 37–53 (2016).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014).
Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015).
Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48–502 (2009).
Newman, A. M. et al. Robust enumeration of cell subsets from tissue expression profiles. Nat. Methods 12, 453–457 (2015).
Paşca, S. P. et al. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat. Med. 17, 1657–1662 (2011).
We thank H. Blau and G. Markov for advice on the hybridization experiments; D. Bangs and J. Erdmann for assistance with iPS cell karyotyping; R. Jones, S. D. Conley and R. Sinha for assistance in constructing the single-cell RNA-seq libraries; and members of the Pașca and Fraser laboratories for advice and feedback on the manuscript. This work was supported by a Stanford Bio-X Interdisciplinary Initiatives Seed Grant (to S.P.P. and H.B.F.), an NIH grant T32 GM007790 (supporting R.M.A.), the Department of Defense National Defense Science and Engineering Graduate Fellowship (to R.M.A.), the Stanford Center for Computational, Evolutionary and Human Genomics (to R.M.A.), NIH grant 2R01GM097171-05A1 (supporting H.B.F.), the Stanford Medicine’s Dean’s Fellowship (to Y.M. and F.B.), the Stanford Medicine Maternal & Child Health Research Institute Postdoctoral Support Program (to Y.M. and F.B.), the American Epilepsy Society Postdoctoral Research Fellowship (to F.B.), the Stanford Wu Tsai Neurosciences Institute’s Big Idea Grants on Brain Rejuvenation and Human Brain Organogenesis (supporting S.P.P.), the Kwan Research Fund (supporting S.P.P.), the New York Stem Cell Foundation–Robertson Investigator Award (supporting S.P.P.) and the Chan Zuckerberg Ben Barres Investigator Award (to S.P.P.). This study used cell lines derived from the Yerkes National Primate Research Center, which is supported by the National Institutes of Health, Office of Research Infrastructure Programs/OD (P51OD011132).
Stanford University holds a patent covering the generation of brain region-specific organoids (US patent serial no. 62/163,870;8) (S.P.P.).
Peer review information Nature thanks Megan Munsie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, FACS of fused hybrid cells (representative plots for fusion of H20961 and C3649). Top, co-cultured cells with no PEG; bottom, co-cultured cells with PEG; from left, initial size selection, gating out doublets, gating out dead cells, sorting for red (human) and green (chimpanzee) double-positive population; FSC, forward scatter; SSC, side scatter; A, area; H, height. Pacific Blue measures DAPI, FITC measures green CMFDA (chimpanzee) and APC measures deep red (human). b, Representative karyotype for female (XX/XX) hybrid iPS cell lines. c–e, Immunostaining for the pluripotency markers NANOG and TRA-1-81 (c), OCT4 and SSEA4 (d), and SOX2 and TRA-1-60 (e). f, Results from PluriTest analysis of RNA-seq data from this study and from Ward et al.10 (see Methods); benchmarked thresholds are 20 or higher for pluripotency, 1.6 or lower for novelty (dotted lines). Scale bars, 200 μm (c–e).
a, b, Plots showing aneuploidies on chromosome 20 indicating a gain of a chimpanzee chromosome (a) or a combined loss of the human short arm and gain of the human long arm (b). Top, scatter plot of ASE (log2(human/chimpanzee)) versus genomic location; middle, median ASE in a sliding window of 20 genes; bottom, P-values from a two-sided Wilcoxon rank-sum test comparing a sliding window of 20 genes to the background of the entire genome. c, Total (top) and allelic (bottom; human allele pink, chimpanzee allele blue) expression of XIST in RNA-seq samples; symbols indicate the sex of each iPS cell line; n = 2 technical replicates per cell line. d, Plots of ASE across the X chromosome (as in a, b). e, Total and allelic expression of RNR1 (chrMT), as in c; n = 2 technical replicates per cell line.
a, Heat map of correlations (Pearson’s) between RNA-seq samples from human (H1, H2 and H3), chimpanzee (C1, C2 and C3) and hybrid (Hy1-25, Hy1-29, Hy1-30, Hy2-9 and Hy2-16) iPS cells. b, Top, pipeline for analysis of RNA-seq data and separation of species-specific sequencing reads; bottom, pile-up of phased allelic reads from human, chimpanzee and hybrid RNA-seq samples for a representative gene. c, Representative scatter plot (from line Hy1-30) showing total gene expression when samples are mapped to the human genome (GRCh38, x-axis) versus the chimpanzee genome (PanTro5, y-axis); n = 1, out of 10 total hyiPS samples sequenced with similar results. d, Scatter plot of ASE in all hybrid samples when mapped to the human versus the chimpanzee genome; genes represented by the points in red are considered to have mapping bias and are eliminated from subsequent analyses; data merged from n = 10 samples from 5 hyiPS cell lines (2 replicates each).
a, b, Principal components plots for iPS and cortical spheroid (pilot study) RNA-seq samples based on total (a) or allelic (b) gene expression. c, Rates of success of three protocols used to derive hyCS (success is defined as at least one cortical spheroid from a given cell line surviving to 100 days of differentiation). n refers to the number of independent attempts to differentiate any of 3 hyiPS cell lines. d, Bright-field imaging of hCS and hyCS at day 7 or 8 of differentiation. The experiment was repeated across 3 independent differentiation experiments with 3 hyiPS and 1 hiPS cell lines with similar results. e, Bright-field images of Matrigel-embedded hCS and hyCS, as well as non-embedded hCS, at days 16 and 35 of differentiation. f, Heat map of correlations (Pearson’s) between bulk RNA-seq samples for hyCS. g, Principal components plot for iPS and hyCS (full dataset) RNA-seq samples based on allelic gene expression h, Heat map coloured by the percentage of human reads in each single cell, stratified by chromosome; rows are ordered by hybrid cell line; top bar shows read depth of each chromosome across all cells; bottom left, colour key and histogram for heat map values; bottom middle, scatter plot of total read depth versus variance per chromosome, wherein fewer reads results in higher variance; bottom right, histogram showing the percentage of human reads in each cell, genome-wide. i, j, Histogram of the percentage of human reads in each cell for aneuploid chromosomes 18 (i) and 20 (j), stratified by cell line. Scale bars, 1 mm (d, e).
a, UMAP clustering of all cells (n = 706); clusters are identified by colour and labelled by letter (A, astroglia; P, cycling progenitors; N1, glutamatergic neurons; N2, GABAergic neurons; M1, mesenchyme cluster 1; M2, mesenchyme cluster 2; E, epithelial cells). b, Proportion of cells from each hybrid cell line in each single cell cluster (from a). c, Dot plot for expression of marker genes for each cluster in a; size corresponds to the percentage of cells in each cluster that express each gene. d, UMAP coloured by expression of mesenchymal and epithelial marker genes. e, Scatter plot of normalized gene expression between embedded (y-axis) and non-embedded (x-axis) hybrid (line Hy1-29) cortical spheroids at day 50 of differentiation; points in red and green indicate genes whose expression is induced by the addition of Matrigel (see Methods). f, t-SNE of all single cells from this study aggregated with cells from non-embedded spheroids in Sloan et al.15, coloured by study. g, t-SNE from f, coloured by expression of cell-type marker genes. h, UMAP from a, coloured according to which cells were defined as neural and used for further analysis in Fig. 2. i, Histograms of per-gene ASE, where ASE is defined as the ratio of all human reads across cells of a given cell type to all chimpanzee reads in those cells.
a, b, Representative bright-field images of three cortical spheroids per line for three human (a) and three chimpanzee (b) cell lines at day 166. c, Immunostaining of hCS and cCS for SOX9, PAX6 and CTIP2. At each time point, a maximum of 2 spheroids were fixed for immunostaining across 3 hiPS and 3 ciPS cell lines with 4 independent differentiation experiments per cell line. d, e, Heat map of Pearson’s correlations between bulk RNA-seq samples for hCS (d) and cCS (e). Scale bars, 1 mm (a, b) and 50 μm (c).
a, b, Principal components plots for RNA-seq samples based on total gene expression of parent and hybrid samples. c, e, g, Per-sample estimated cell-type proportions in hyCS (c), hCS (e) and cCS (g) (see Methods). d, f, h, Normalized expression across time of cell-type-specific marker genes in hyCS (d), hCS (f) and cCS (h).
a, Dendrogram of all genes used in WGCNA; genes in the same colour block belong to the same co-expressed module. b, Eigengene values for genes in the blue, brown and red modules over time in hCS and cCS; chimpanzee blue, human red; in order of time points, n = 6, 6, 6, 6, 6, 6 and 5 hCS and n = 6, 6, 6, 6, 5, 5 and 5 cCS samples from 3 human and 3 chimpanzee iPS cell lines (1–2 replicates per cell line). c, Expression of module genes (eigengene, see Methods) in single-cell data; cell clusters are defined in Extended Data Fig. 5a. d, Allelic eigengene values for genes in these modules over time in hyCS (see Methods); chimpanzee blue, human red; in order of time points, n = 7, 9, 7 and 2 hyCS from 3 hyiPS cell lines (2–3 replicates per cell line). e, Single-cell gene expression of PMP2. f, Expression of PMP2 in parental bulk time course; chimpanzee blue, human red; n as in b. g, Allelic expression of PMP2 in hybrid bulk time course; chimpanzee allele blue, human allele red; n as in d. Box plots in b, d, f, g: the centre line shows median, box limits represent upper and lower quartiles and whiskers extend to 1.5× the interquartile range.
a–d, Overlap in genes with significant ASE between hyCS at days 50 versus 100 (a), hyCS at days 100 versus 150 (b), hyiPS cells versus hyCS at day 150 (c), and differential expression between hCS and cCS at day 150 versus ASE in hyCS at day 150 (d). e, Scatter plot showing differences in gene expression between parental lines (y-axis) versus between alleles in the hybrid (x-axis) at day 150; data are from bulk RNA-seq of 6 human, 5 chimpanzee and 7 hybrid cortical spheroid samples, collected across 3 human, 3 chimpanzee and 3 hybrid iPS cell lines. f, Overlap between ASE genes and SFARI genes. g, ASE in SFARI genes from the overlapping genes in f. h, i, Allelic expression over time in GRIN2A (h) and SCN1A (i); human allele pink, chimpanzee allele blue; in order of time points n = 7, 9, 7 and 2 hyCS samples (1–2 spheroids per sample) from 3 independent differentiations of 3 hyiPS cell lines. j, Filtering pipeline for prioritizing candidate genes. k, Scatter plot of hybrid ASE (x-axis) and parental differential expression (y-axis) for top candidate genes at day 150; n = 7 hyCS, 6 hCS and 5 cCS samples (1–3 spheroids per sample) derived from 3 iPS cell lines per species and 2 independent differentiations per hiPS and ciPS cell line, n = 3 independent differentiations per hyiPS cell line. Box plots in h, i: the centre line shows median, box limits represent upper and lower quartiles and whiskers extend to 1.5× the interquartile range.
a, Expression of SSTR2 in parental bulk time course; chimpanzee blue, human red; in order of time points, n = 6, 6, 6, 6, 6, 6 and 5 hCS and n = 6, 6, 6, 6, 5, 5 and 5 cCS samples from 3 human and 3 chimpanzee iPS cell lines (1–2 replicates per cell line). b, Expression of SSTR2 across cortical sections in adult primate brain tissue (data from He et al.38); dotted lines indicate approximate boundaries of cortical layers; WM, white matter. c, Immunostaining for MAP2 (neuronal) and SSTR2 protein in dissociated hCS (H20682) and cCS (C3649) at day 225–250; right panels show SSTR2 only; 10 images were taken per sample and quantified. d, Quantification of fluorescence intensity (arbitrary units) of MAP2 for the images in c. n = 13 cells for hCS, 14 cells for cCS; ****P < 0.0001, two-tailed Mann–Whitney test. e, Quantification of fluorescence intensity (arbitrary units) of SSTR2 relative to MAP2 for the images in c; n = 13 cells for hCS, 14 cells for cCS; ****P < 0.0001, two-tailed Mann–Whitney test. f, Additional immunostaining for TUBB3 (neuronal) and SSTR2 in dissociated hCS (H20682) and cCS (C3649) at day 225–250; 10 images were taken per sample and quantified. g, Immunostaining for MAP2 and SSTR2 in whole hCS (H20961) and cCS (C3651) at day 160; imaging was reproduced across 3 human and 2 chimpanzee cell lines from 1 differentiation experiment with n = 3, 2, 3, 2 and 3 images for lines H21792, H20682, H20961, C3649 and C3651, respectively. h, Representative still frame images of hCS (H20682)- and cCS (C3649)-derived neurons infected with AAV-DJ-hSyn1-eYFP; images are taken from one of the samples in Fig. 4g–i; the experiment was reproduced across 2 human and 1 chimpanzee cell lines. i, Representative still frame images of hCS (H20682) infected with the viral vector co-encoding stable red fluorophore mRuby2 and genetically encoded calcium indicator GCaMP6s; images are taken from one of the samples in Fig. 4j–l; the experiment was reproduced across 3 human and 3 chimpanzee cell lines. Box plots in a, d, e: the centre line shows median, box limits represent upper and lower quartiles and whiskers extend to 1.5× the interquartile range.; dotted lines connect average values (a). Scale bars, 50 μm (f), 10 μm (c, g), 60 μm (h), 30 μm (i).
About this article
Cite this article
Agoglia, R.M., Sun, D., Birey, F. et al. Primate cell fusion disentangles gene regulatory divergence in neurodevelopment. Nature 592, 421–427 (2021). https://doi.org/10.1038/s41586-021-03343-3
This article is cited by
Genome Biology (2023)
Nature Reviews Bioengineering (2023)
Nature Reviews Neuroscience (2023)
Nature Reviews Genetics (2023)
Nature Reviews Genetics (2023)