Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders.

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Comparative landscape of genetic dependencies in human and chimpanzee stem cells.

Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

Comparative landscape of genetic dependencies in human and chimpanzee stem cells.

Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether changes in human cells alter requirements for essential genes. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells, providing support for the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells can reshape the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

Mapping cis- and trans-regulatory target genes of human-specific deletions.

Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution1,2. Comparative genetic studies of primates2,3 have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays4. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6,358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 19 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the liver and brain, respectively. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.

Rethinking nomenclature for interspecies cell fusions.

Cell fusions have a long history of supporting biomedical research. These experimental models, historically referred to as 'somatic cell hybrids', involve combining the plasma membranes of two cells and merging their nuclei within a single cytoplasm. Cell fusion studies involving human and chimpanzee pluripotent stem cells, rather than somatic cells, highlight the need for responsible communication and a revised nomenclature. Applying the terms 'hybrid' and 'parental' to the fused and source cell lines, respectively, evokes reproductive relationships that do not exist between humans and other species. These misnomers become more salient in the context of fused pluripotent stem cells derived from different but closely related species. Here, we propose a precise, versatile and generalizable framework to describe these fused cell lines. We recommend the term 'composite cell line', to distinguish cell lines that are experimentally created through fusions from both reproductive hybrids and natural cell fusion events without obscuring the model in overly technical terms. For scientific audiences, we further recommend technical nomenclature that describes the contributing species, ploidy and cell type.

Gene expression variability in human and chimpanzee populations share common determinants.

Inter-individual variation in gene expression has been shown to be heritable and is often associated with differences in disease susceptibility between individuals. Many studies focused on mapping associations between genetic and gene regulatory variation, yet much less attention has been paid to the evolutionary processes that shape the observed differences in gene regulation between individuals in humans or any other primate. To begin addressing this gap, we performed a comparative analysis of gene expression variability and expression quantitative trait loci (eQTLs) in humans and chimpanzees, using gene expression data from primary heart samples. We found that expression variability in both species is often determined by non-genetic sources, such as cell-type heterogeneity. However, we also provide evidence that inter-individual variation in gene regulation can be genetically controlled, and that the degree of such variability is generally conserved in humans and chimpanzees. In particular, we found a significant overlap of orthologous genes associated with eQTLs in both species. We conclude that gene expression variability in humans and chimpanzees often evolves under similar evolutionary pressures.

Getting to the heart of cardiovascular evolution in humans.

Differences in the response of cardiomyocytes to oxygen deprivation in humans and chimpanzees may explain why humans are more prone to certain heart diseases.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

A comparative study of endoderm differentiation in humans and chimpanzees.

BACKGROUND: There is substantial interest in the evolutionary forces that shaped the regulatory framework in early human development. Progress in this area has been slow because it is difficult to obtain relevant biological samples. Induced pluripotent stem cells (iPSCs) may provide the ability to establish in vitro models of early human and non-human primate developmental stages. RESULTS: Using matched iPSC panels from humans and chimpanzees, we comparatively characterize gene regulatory changes through a four-day time course differentiation of iPSCs into primary streak, endoderm progenitors, and definitive endoderm. As might be expected, we find that differentiation stage is the major driver of variation in gene expression levels, followed by species. We identify thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we consider gene-specific dynamic regulatory trajectories throughout the time course, we find that at least 75% of genes, including nearly all known endoderm developmental markers, have similar trajectories in the two species. Interestingly, we observe a marked reduction of both intra- and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of regulatory variation in endoderm progenitors. CONCLUSIONS: The reduction of variation in gene expression levels at a specific developmental stage, paired with overall high degree of conservation of temporal gene regulation, is consistent with the dynamics of a conserved developmental process.

A Comparative Assessment of Human and Chimpanzee iPSC-derived Cardiomyocytes with Primary Heart Tissues.

Comparative genomic studies in primates have the potential to reveal the genetic and mechanistic basis for human specific traits. These studies may also help us better understand inter-species phenotypic differences that are clinically relevant. Unfortunately, the obvious limitation on sample collection and experimentation in humans and non-human apes severely restrict our ability to perform dynamic comparative studies in primates. Induced pluripotent stem cells (iPSCs), and their corresponding differentiated cells, may provide a suitable alternative system for dynamic comparative studies. Yet, to effectively use iPSCs and differentiated cells for comparative studies, one must characterize the extent to which these systems faithfully represent biological processes in primary tissues. To do so, we compared gene expression data from primary adult heart tissue and iPSC-derived cardiomyocytes from multiple human and chimpanzee individuals. We determined that gene expression in cultured cardiomyocytes from both human and chimpanzee is most similar to that of adult hearts compared to other adult tissues. Using a comparative framework, we found that 50% of gene regulatory differences between human and chimpanzee hearts are also observed between species in cultured cardiomyocytes; conversely, inter-species regulatory differences seen in cardiomyocytes are found significantly more often in hearts than in other primary tissues. Our work provides a detailed description of the utility and limitation of differentiated cardiomyocytes as a system for comparative functional genomic studies in primates.

Silencing of transposable elements may not be a major driver of regulatory evolution in primate iPSCs.

Transposable elements (TEs) comprise almost half of primate genomes and their aberrant regulation can result in deleterious effects. In pluripotent stem cells, rapidly evolving KRAB-ZNF genes target TEs for silencing by H3K9me3. To investigate the evolution of TE silencing, we performed H3K9me3 ChIP-seq experiments in induced pluripotent stem cells from 10 human and 7 chimpanzee individuals. We identified four million orthologous TEs and found the SVA and ERV families to be marked most frequently by H3K9me3. We found little evidence of inter-species differences in TE silencing, with as many as 82% of putatively silenced TEs marked at similar levels in humans and chimpanzees. TEs that are preferentially silenced in one species are a similar age to those silenced in both species and are not more likely to be associated with expression divergence of nearby orthologous genes. Our data suggest limited species-specificity of TE silencing across 6 million years of primate evolution.

Impact of regulatory variation across human iPSCs and differentiated cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for studying cellular differentiation and cell types that are otherwise difficult to access. We investigated the use of iPSCs and iPSC-derived cells to study the impact of genetic variation on gene regulation across different cell types and as models for studies of complex disease. To do so, we established a panel of iPSCs from 58 well-studied Yoruba lymphoblastoid cell lines (LCLs); 14 of these lines were further differentiated into cardiomyocytes. We characterized regulatory variation across individuals and cell types by measuring gene expression levels, chromatin accessibility, and DNA methylation. Our analysis focused on a comparison of inter-individual regulatory variation across cell types. While most cell-type-specific regulatory quantitative trait loci (QTLs) lie in chromatin that is open only in the affected cell types, we found that 20% of cell-type-specific regulatory QTLs are in shared open chromatin. This observation motivated us to develop a deep neural network to predict open chromatin regions from DNA sequence alone. Using this approach, we were able to use the sequences of segregating haplotypes to predict the effects of common SNPs on cell-type-specific chromatin accessibility.

Genetic Variation, Not Cell Type of Origin, Underlies the Majority of Identifiable Regulatory Differences in iPSCs.

The advent of induced pluripotent stem cells (iPSCs) revolutionized human genetics by allowing us to generate pluripotent cells from easily accessible somatic tissues. This technology can have immense implications for regenerative medicine, but iPSCs also represent a paradigm shift in the study of complex human phenotypes, including gene regulation and disease. Yet, an unresolved caveat of the iPSC model system is the extent to which reprogrammed iPSCs retain residual phenotypes from their precursor somatic cells. To directly address this issue, we used an effective study design to compare regulatory phenotypes between iPSCs derived from two types of commonly used somatic precursor cells. We find a remarkably small number of differences in DNA methylation and gene expression levels between iPSCs derived from different somatic precursors. Instead, we demonstrate genetic variation is associated with the majority of identifiable variation in DNA methylation and gene expression levels. We show that the cell type of origin only minimally affects gene expression levels and DNA methylation in iPSCs, and that genetic variation is the main driver of regulatory differences between iPSCs of different donors. Our findings suggest that studies using iPSCs should focus on additional individuals rather than clones from the same individual.

A panel of induced pluripotent stem cells from chimpanzees: a resource for comparative functional genomics.

Comparative genomics studies in primates are restricted due to our limited access to samples. In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates, we must have access to faithful model systems for a wide range of cell types. To facilitate this, we generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell (iPSC) lines derived from healthy donors. To demonstrate the utility of comparative iPSC panels, we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines, as well as from 7 human iPSCs and their source lines, which encompass multiple populations and cell types. We observe much less within-species variation in iPSCs than in somatic cells, indicating the reprogramming process erases many inter-individual differences. The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude.

Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature.

Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs), have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC) system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10-15). Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.