Periodic Reporting for period 3 - ANTHROPOID (Great ape organoids to reconstruct uniquely human development)
Periodo di rendicontazione: 2021-07-01 al 2022-12-31
Humans diverged from our closest living relatives, chimpanzees and other great apes, 6-10 million years ago. Since this divergence, our ancestors acquired genetic changes that enhanced cognition, altered metabolism, and endowed our species with an adaptive capacity to colonize the entire planet and reshape the biosphere. Through genome comparisons between modern humans, Neandertals, chimpanzees and other apes we have identified genetic changes that likely contribute to innovations in human metabolic and cognitive physiology. However, it has been difficult to assess the functional effects of these genetic changes due to the lack of cell culture systems that recapitulate great ape organ complexity. Human and chimpanzee pluripotent stem cells (PSCs) can self-organize into three-dimensional (3D) tissues that recapitulate morphological, functional, and genetic aspects of organ development. Our vision is to use organoids to study the changes that set modern humans apart from our closest evolutionary relatives as well as all other organisms on the planet. This work will provide information about how the evolution of unique features in our cells might have lead to susceptibilities to certain diseases that impact modern humans.
The project has the following objectives:
1. Generate a great ape organoid cell atlas. We will grow cortex, liver, and small intestine organoids using PSCs from multiple great apes (human, chimpanzee, organgutan) and use single-cell genomics (scRNA-seq and scATAC-seq) to identify cell type-specific features of transcriptome divergence at cellular resolution.
2. Dissect enhancer function using single-cell genomic screens. We will use CRISPR/Cas9 interference screens coupled with single-cell transcriptomics in chimpanzee and human organoids to understand the function of human-specific regulatory regions.
3. Ancestralize human cells to resurrect Prehuman phenotypes. We will use gene editing to ancestralize human and modernize chimpanzee PSCs at genic and regulatory sites that emerged on the human lineage before and after the split with Neandertals, and phenotype organoids using multiple functional assays.
ANTHROPOID utilizes quantitative and state-of-the-art methods to explore exciting high-risk questions at multiple branches of the modern human lineage. This project is a ground breaking starting point to replay evolution and tackle the ancient question of what makes us uniquely human?
The project has the following objectives:
1. Generate a great ape organoid cell atlas. We will grow cortex, liver, and small intestine organoids using PSCs from multiple great apes (human, chimpanzee, organgutan) and use single-cell genomics (scRNA-seq and scATAC-seq) to identify cell type-specific features of transcriptome divergence at cellular resolution.
2. Dissect enhancer function using single-cell genomic screens. We will use CRISPR/Cas9 interference screens coupled with single-cell transcriptomics in chimpanzee and human organoids to understand the function of human-specific regulatory regions.
3. Ancestralize human cells to resurrect Prehuman phenotypes. We will use gene editing to ancestralize human and modernize chimpanzee PSCs at genic and regulatory sites that emerged on the human lineage before and after the split with Neandertals, and phenotype organoids using multiple functional assays.
ANTHROPOID utilizes quantitative and state-of-the-art methods to explore exciting high-risk questions at multiple branches of the modern human lineage. This project is a ground breaking starting point to replay evolution and tackle the ancient question of what makes us uniquely human?
With respect to objective 1, we have established a great ape atlas of cell states across cerebral and intestinal organoid development. For cerebral organoids, we have used single-cell transcriptome and accessible chromatin profiling to first analyze cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We showed that brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. We established an approach to pseudotemporally align differentiation paths, and found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans. This seminal work was published in Nature (Kanton, Boyle, He et al., Nature 2019), and has also seeded further papers on human brain development where I am a corresponding author (He et al. Nature Methods 2020; Fleck et al. Biorxiv, 2021 in revision at Nature; Fleck et al. Cell Stem Cell 2021)
For intestinal organoids, we first generated a comprehensive reference atlas of multiple human developing endodermal organs of the respiratory and gastrointestinal tract and used the atlas to provide information regarding cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. In parallel, we also used single-cell transcriptomics to analyse human intestinal organoid development, and compared the organoid data to the reference atlas. This effort was used to benchmark stem cell-derived human intestinal organoids under multiple culture conditions. A manuscript describing these results was published recently in Cell (Yu, Kilik et al. Cell, 2021) We also established a novel protocol for generated mature intestinal epithelial cell states entirely in vitro (Kilik et al. Biorxiv, 2021). Altogether, this effort established a baseline for evolutionary comparisons. Towards this aim, we have generated chimpanzee and bonobo intestinal organoids from pluripotent stem cells and have generated a single-cell transcriptome and accessible chromatin atlas from these as well as human tissues. In addition, we have generated an atlas of adult stem cell derived intestinal organoids for human, marmoset and mouse. We are bringing all of this data together to provide a comprehensive analysis of gene-regulatory mechanisms that are specific to humans. We anticipate that we will have a manuscript read for submission on this project by the end of 2022.
With regard to objective 2, we have generated single-cell transcriptome and chromatin accessibility landscapes from great ape cerebral and intestinal tissues, and have identified cell type specific and human-specific gene regulatory regions that are linked to differentially expressed genes. We have chosen the cis-regulatory regions that we would like to further characterize, and have designed the reporter constructs that we will use in assays in cerebral and intestinal organoids. We anticipate to make great progress on these experiments in the next phase of the project. We have also established a set of new vectors that can deliver two gRNAs and can be used for perturbation and lineage tracing. This system can be used for inducing gene regulatory region knock-out in mosaic organoids and has coupling to single-cell transcriptome readouts (He et al. Nature Methods, 2022).
With respect to objective 3, we have analyzed a large iPSC repository that harbors extensive Neandertal DNA, including alleles that contribute to human phenotypes and diseases, encode hundreds of amino acid changes, and alter gene expression in specific tissues. We provide a database of the inferred introgressed Neandertal alleles for each individual iPSC line, together with the annotation of the predicted functional variants. We also show that transcriptomic data from organoids generated from iPSCs can be used to track Neandertal-derived RNA over developmental processes. We have shown that existing human iPSC resources provide an opportunity to experimentally explore Neandertal DNA function and its contribution to present-day phenotypes, and potentially study Neandertal traits. We have published a manuscript describing this work (Dannemann et al. Stem Cell Reports, 2020).
For intestinal organoids, we first generated a comprehensive reference atlas of multiple human developing endodermal organs of the respiratory and gastrointestinal tract and used the atlas to provide information regarding cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. In parallel, we also used single-cell transcriptomics to analyse human intestinal organoid development, and compared the organoid data to the reference atlas. This effort was used to benchmark stem cell-derived human intestinal organoids under multiple culture conditions. A manuscript describing these results was published recently in Cell (Yu, Kilik et al. Cell, 2021) We also established a novel protocol for generated mature intestinal epithelial cell states entirely in vitro (Kilik et al. Biorxiv, 2021). Altogether, this effort established a baseline for evolutionary comparisons. Towards this aim, we have generated chimpanzee and bonobo intestinal organoids from pluripotent stem cells and have generated a single-cell transcriptome and accessible chromatin atlas from these as well as human tissues. In addition, we have generated an atlas of adult stem cell derived intestinal organoids for human, marmoset and mouse. We are bringing all of this data together to provide a comprehensive analysis of gene-regulatory mechanisms that are specific to humans. We anticipate that we will have a manuscript read for submission on this project by the end of 2022.
With regard to objective 2, we have generated single-cell transcriptome and chromatin accessibility landscapes from great ape cerebral and intestinal tissues, and have identified cell type specific and human-specific gene regulatory regions that are linked to differentially expressed genes. We have chosen the cis-regulatory regions that we would like to further characterize, and have designed the reporter constructs that we will use in assays in cerebral and intestinal organoids. We anticipate to make great progress on these experiments in the next phase of the project. We have also established a set of new vectors that can deliver two gRNAs and can be used for perturbation and lineage tracing. This system can be used for inducing gene regulatory region knock-out in mosaic organoids and has coupling to single-cell transcriptome readouts (He et al. Nature Methods, 2022).
With respect to objective 3, we have analyzed a large iPSC repository that harbors extensive Neandertal DNA, including alleles that contribute to human phenotypes and diseases, encode hundreds of amino acid changes, and alter gene expression in specific tissues. We provide a database of the inferred introgressed Neandertal alleles for each individual iPSC line, together with the annotation of the predicted functional variants. We also show that transcriptomic data from organoids generated from iPSCs can be used to track Neandertal-derived RNA over developmental processes. We have shown that existing human iPSC resources provide an opportunity to experimentally explore Neandertal DNA function and its contribution to present-day phenotypes, and potentially study Neandertal traits. We have published a manuscript describing this work (Dannemann et al. Stem Cell Reports, 2020).
We have established several novel single-cell technologies to understand organoid development. First, we co-developed iTRACER, which is an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during organoid development. This system can be applied to any human stem cell-derived organoid system to generate fate-mapped whole organoid phylogenies over a scaring time course, and reconstructing lineage-constrained differentiation trajectories. (He et al. Nature Methods 2022). Second, we co-developed VoxHunt, which is a set of computational tools (VoxHunt) to assess brain organoid patterning, developmental state, and cell composition through comparisons to spatial and single-cell transcriptome reference datasets (Fleck et al. Cell Stem Cell, 2020). Third, we co-developed an unsupervised reference-free data representation, cluster similarity spectrum (CSS), where each cell is represented by its similarities to clusters independently identified across samples. CSS can be used to assess cellular heterogeneity and enable reconstruction of differentiation trajectories from cerebral organoid and other single-cell transcriptomic data, and to integrate data across experimental conditions and human individuals (He et al. Genome Biology 2020). Fourth, we are co-developing protocols and computational pipelines for in toto light sheet microscopy for long-term imaging of cerebral organoid development. This will be an extraordinarily powerful approach to visualize and explore human brain organoid development, and comparisons across species. (He et al. Nature Methods, 2022). Fifth, we have established an an experimental and computational toolkit to perform multiplexed immunohistochemistry to measure the subcellular localization of up to 60 proteins in the same tissue section in high-throughput (histo4I. This is a scale-crossing technique that can register protein expression from centimeter to micrometer scale in 96 tissue sections in the same experiment. We are applying this method to characterize human and primate tissues (Wahle et al. Biorxiv, 2022). Sixth, we have established a novel protocol to generate matured intestinal stem cells and differentiated epithelium from human and other primate induced pluripotent stem cells. This protocol by-passes the need for transplantation into a mouse model for functional maturation, and thereby provides an exciting new inroad into using IPSCs for studying development, diseases, and evolution of the human intestinal epithelium (Kilik et al. Biorxiv, 2021).