Periodic Reporting for period 4 - ORGANOMICS (Reconstructing human cortex development and malformation with single-cell transcriptomics)
Période du rapport: 2022-01-01 au 2023-06-30
Technologies to sequence and analyze single-cell transcriptomes (scRNA-seq) are revolutionizing our ability to understand cell composition and differentiation in complex tissues. In parallel, recent innovations allow the generation of three-dimensional tissues from stem cells (so-called organoids) that recapitulate human development. In this project, we shed light onto the mechanisms that underlie healthy human brain development as well as cortex malformations, both of which can be modelled by brain organoids. Our project is important for society because we will develop exciting next generation phenotyping strategies for personalized medicine using organoids grown directly from patient-derived cells. In the first objective, we developed a set of cellular barcoding tools to label individual stem and progenitor cells within developing brain organoids in order to track how cells differentiate and build cell family histories within the organoids using single-cell sequencing. In the second objective, we used high-throughput CRISPR/Cas9 perturbation screen with single-cell transcriptomic readout in mosaic brain organoids targeting genes associated with disease to understand mechanisms that regulate cell fate decisions during human brain development. Finally, we generated brain organoids from patients with cortical malformations and used single-cell multiomic and spatial transcriptomics data to reconstruct gene regulatory networks affected by the disease mutations. In summary, this project provides a new quantitative direction to study human brain development and our general strategy can be extended to various other organ systems and diseases where protocols to generate in vitro counterparts can be established.
We have completed our research goals for each of the three objectives. For objective 1 (Single-cell transcriptome coupled lineage tracing) we have developed a single-cell transcriptome coupled dual-channel lineage recorder based on DNA barcodes and CRISPR-Cas9 scarring that can be used in iPSC-derived brain organoids (He et al., Nature Methods 2021). We applied this method to brain organoid development and generated a large single cell data set, which we analyzed to infer fate-mapped whole organoid phylogenies, and reconstruct progenitor-neuron lineage trees. We extended the method to a spatial readout using spatial transcriptomics, which enabled us to spatially resolve cell lineage locations in organoids. Using this method, we uncovered the time window of organoid regionalization and identified a clonality of human brain organoid regions. For objective 2 (Gene knock-out screens in mosaic organoids) we have performed a gene knock-out screen in mosaic brain organoids with single-cell transcriptomic readout to identify regulators of early human brain development (Fleck, Jansen et al., Nature 2022). In order to identify candidate transcription factors to target in the screen, we used single-cell multiomic data to infer a gene regulatory network underlying early human brain organoid development using a novel computational tool. We then used the pooled CRISPR/Cas9 gene knock-out screen to perturb 20 of these transcription factors to assess their fuctional requirement for cell fate and state regulation in organoids. We found that certain transcription factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. For objective 3, we have applied multiple innovative single-cell technologies to characterize the effect of mutations leading to brain malformations. We have established and executed an arrayed single-cell transcriptomic CRISPR-Cas9 perturbation screen to assess the common and distinct effects of mutations of 21 genes linked to a specific type of cortex malformations (periventricular heterotopia). To complement the screening data and follow-up on some of the findings, we also obtained cells from patients with periventricular heterotopia and have grown brain organoids from them to assess mechanisms underlying the disease phenotypes using single-cell transcriptomics and spatial phenotyping.
Within this project, we have established a novel lineage tracing technology based on single-cell transcriptomics that combines cell barcoding and CRISPR/Cas9-based DNA scarring. This allows us to track cell fates within organoids with temporal resolution previously not achieved. Furthermore, we have successfully implemented genetic perturbation screens in brain organoids with single-cell resolved readouts. This allows us to test the effect of disease gene knockout in relatively high-throughput. We expect that this data will help us understand how genetic mutations impact proper fate acquisition specifically in developing human tissues. We also developed a novel computational tool to infer gene regulatory networks from single-cell multiomic data including transcriptome and accessible chromatin measurements, which has been applied by many other research groups. This tool, named Pando, allowes for the identification of genes that are very central to certain developmental processes and cell fate decisions. Finally, we believe that our innovative single cell based technologies to interrogate patient derived organoids will lead to ground breaking strategies in personalized medicine to understand mechanisms underlying neurodevelopmental diseases, and our methods can be generalized to other organ systems.