Great ape organoids to reconstruct uniquely human development

Información del proyecto

ANTHROPOID

Identificador del acuerdo de subvención: 803441

DOI

10.3030/803441

Proyecto cerrado

Fecha de la firma de la CE 3 Mayo 2019

Fecha de inicio 1 Junio 2019

Fecha de finalización 31 Mayo 2024

Financiado con arreglo a

• EXCELLENT SCIENCE - European Research Council (ERC)

Coste total

€ 1 500 000,00

Aportación de la UE

€ 1 500 000,00

Coordinado por

F. HOFFMANN-LA ROCHE AG
Switzerland

Periodic Reporting for period 4 - ANTHROPOID (Great ape organoids to reconstruct uniquely human development)

Período documentado: 2023-01-01 hasta 2024-05-31

Resumen del contexto y de los objetivos generales del proyecto

What Problem/Issue Are We Addressing?

Humans diverged from chimpanzees 6-10 million years ago, acquiring genetic changes that enhanced cognition, altered metabolism, resisted disease and enabled our species to colonize the planet. While genome comparisons have identified genetic differences between modern humans, Neandertals, and great apes, we have lacked experimental tools to test how these differences actually function in living tissues. We cannot study human evolutionary adaptations directly in living great apes for ethical reasons, and traditional cell culture systems are too simple to capture organ complexity.

Why Is This Important for Society?

Understanding human evolutionary adaptations has profound implications for modern health. Genetic changes that made humans successful may also create disease vulnerabilities through evolutionary trade-offs, incomplete optimization, and mutation-prone regions. For example, genomic regions that rapidly evolved are associated with neurological, autoimmune and metabolic disease. By understanding how our evolutionary history shapes biology, we can better predict disease vulnerabilities and develop targeted prevention and treatment approaches.

Overall Objectives

1: Create new model systems for studying human evolution. We generated organoids—self-organized three-dimensional tissues from human and chimpanzee stem cells—that recapitulate brain and digestive tract development. Using single-cell sequencing, we identified species-specific differences in gene expression at cellular resolution.

2: Identify genetic changes controlling human-specific traits. We mapped recently evolved transcriptional enhancers by comparing accessible chromatin regions between species, identifying genome parts that acquired new functions specifically in human cells.

3: Advance human model systems for biomedical research. We developed strategies to enhance organoid models for studying human biology and disease.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

Objective 1: Great Ape Cell Atlas

Brain organoids: We established a comprehensive atlas of cell states across cerebral organoid development using single-cell transcriptome and chromatin profiling. We analyzed differentiation from pluripotency through neuroectoderm stages to neuronal fates across forebrain, midbrain, and hindbrain regions. Comparing human, chimpanzee, and macaque organoids revealed that human neuronal development occurs at a slower pace. By pseudotemporally aligning differentiation paths, we identified human-specific gene expression in distinct cortical cell states. Chromatin accessibility analysis revealed divergence between human and chimpanzee correlating with human-specific gene expression and genetic change. Single-nucleus sequencing of adult prefrontal cortex showed developmental differences persisting into adulthood and adult-specific changes, providing a temporal cell atlas illuminating dynamic gene-regulatory features unique to humans.

Intestinal organoids: We generated a comprehensive reference atlas of developing human endodermal organs and used it to benchmark stem cell-derived intestinal organoids under multiple culture conditions. We generated chimpanzee intestinal organoids and characterized them using immunohistochemistry, single-cell transcriptomics, and chromatin sequencing. Comparisons with humans, mice, and other mammals revealed that recent primate changes associate with immune barrier function and lipid/xenobiotic metabolism, with human-specific genetic features impacting these functions.

Objective 2: Functional Genetic Switches

Using single-cell landscapes from great ape tissues, we identified cell-type-specific, human-specific regulatory regions linked to differentially expressed genes. We cataloged and ranked regions by evolutionary selection signatures, then conducted functional assays on top candidates in intestinal epithelium. We validated a MCM6 regulatory region as a functional LCT enhancer, revealing how a positively selected polymorphism associated with adult lactose tolerance disrupts a repressor-binding site.
Notably, we discovered IGFBP2 has two enhancers overlapping human accelerated regions with higher chromatin accessibility in developing human intestinal epithelial cells versus chimpanzee. In silico mutagenesis and variant-resolved enhancer assays confirmed elevated activity compared to ancestral sequences. CRISPR-Cas9 deletion of each enhancer reduced IGFBP2 expression in small intestinal organoids.
We analyzed a large iPSC repository harboring extensive Neandertal DNA, including alleles contributing to human phenotypes and diseases. We provide a database of inferred introgressed Neandertal alleles with functional variant annotations and show that organoid transcriptomic data can track Neandertal-derived RNA over development, demonstrating how iPSC resources enable experimental exploration of Neandertal DNA function.

Objective 3: Advanced Organoid Systems

We established integrated organoid atlases of brain and endoderm-derived tissues to assess model fidelity. Mapping to primary tissue references revealed in vitro-generated cell types and estimated transcriptomic similarity across protocols. These atlases serve as diverse control cohorts to annotate disease models, identifying genes and pathways underlying pathological mechanisms. We established a novel protocol generating mature intestinal epithelial cell states entirely in vitro, bypassing mouse transplantation, and incorporated tissue-resident immune cells into intestinal organoid models, providing new approaches to study human intestinal mucosal biology.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

We brought new insights into human-specific biology by comparing human development to other species and functionally assessing human-specific gene regulatory regions using organoid models (Kanton, Boyle, He et al., Nature 2019; Dannemann et al. Stem Cell Reports, 2020; Yu, Kilik et al. Cell, 2021; Pollen et al. Nature Reviews Genetics, 2023; Yu, Kilik, Secchia et al. Science, 2025).

We contributed novel organoid single-cell technologies including inducible lineage recording systems (He et al. Nature Methods 2022), pipelines for in toto light sheet microscopy of neural organoids (He et al. Nature Methods, 2022; Jain et al. Nature, 2024), multiplexed immunohistochemistry measuring subcellular localization of 60 proteins (Wahle et al. Nature Biotechnology, 2023), and multimodal integration approaches to assess organoid developmental state through reference dataset comparisons (He et al. Genome Biology 2020; Fleck et al. Cell Stem Cell, 2021; Yu, Kilik et al. Cell, 2021).
We centralized single-cell datasets of human model systems into two atlases—the Human Neural Organoid Cell Atlas (HNOCA) and Human Endoderm-derived Organoids Cell Atlas (HEOCA)—integrating RNA-sequencing data for quantitative comparison with primary tissues (He, Dony, Fleck et al. Nature 2024; Xu, Halle et al. Nature Genetics 2025).

We generated the first immunocompetent intestinal organoids demonstrating drug toxicity prediction (Recalidin et al. Nature 2024), and established engineered barrier models of human small intestine and colon with multilineage epithelium, mucus layer, microbial compartment, and autologous tissue-resident immune cells (Lopez-Sandoval et al. BioRxiv 2025). This modular system enables studying human intestinal physiology, pathologies, and evolution.

Overview