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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ORGANOMICS (Reconstructing human cortex development and malformation with single-cell transcriptomics)

Teaser

Summary

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 want to shed light onto the mechanisms that underlie healthy human cortex development as well as cortex malformations, both of which can be modelled by brain organoids. Currently, the molecular mechanisms leading to human brain malformations are largely unexplored in human tissues, especially not in tissues derived from patients. 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 develop a set of cellular barcoding tools to label individual stem and progenitor cells within the 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 aim, we will use high-throughput CRISPR/Cas9 perturbation screens targeting genes associated with disease to understand mechanisms that regulate cell lineage decisions during cortex development. Finally, we will generate cerebral organoids from patients with cortical malformations and reconstruct networks and infer differentiation hierarchies using single-cell genomics and spatial transcriptomics. This project provides a new quantitative direction to study human cortex development. Our general strategy can be extended to various other organ systems and diseases where protocols to generate in vitro counterparts can be established.

Work performed

We have already made significant progress towards our research goals for each of the three Aims. For Aim 1 (Single-cell transcriptome coupled lineage tracing) we have developed the lineage-tracing method in iPSC-derived cerebral organoids (Step 1) and have generated lineage-coupled single-cell transcriptome data from organoids at multiple time points (Step 2). We will continue to generate data from additional time points and conditions, and in the next phase of the project we will deeply analyse the data to infer lineages and cell state transition probabilities (Step 3). For Aim 2 (Gene knock-out screens in mosaic organoids) we have developed the assay to edit iPSCs, grow mosaic organoids composed of cells with different genotypes, and sequence transcriptomes or DNA scars (Step 1). We are currently generating and analysing data from several gene panels (Step 2). In addition, we have also already started to follow-up on interesting candidates by generating isogenic knockout or wild type organoids and dissecting the organoids using scRNA-seq (Step 3). In the next phase of the project we will further generated data using single-cell based readouts for these high-throughput gene knockout screens. For Aim 3 (High-throughput reconstructions of cortex malformations), we have obtained ethical permissions to collect patient-derived fibroblasts and have started to generate iPSCs for selected disorders (Step 1). The next phase of this work will be to generate high-throughput and lineage coupled single-cell transcriptomics data on mutant and control organoids (step 2), analyse the data and spatially map dysregulated cell states using spatial transcriptomics (step 3). Along the lines of this Aim, we have published our analysis of organoids generated from iPSC lines from patients with periventricular nodular heterotopia in a high impact journal (Kraus, Kanton et al. Nature Medicine, 2019).

Final results

Within this project we have now 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. We expect to use this method to build a comprehensive map of brain organoid development in healthy and perturbed/malformed organoids. Furthermore, we have successfully implemented genetic perturbation screens in brain organoids with single-cell resolved readouts. This allows us to now 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. Finally, though our analysis of patient-derived organoids, we expect to develop exciting new computational methods to integrate, analyze, and interpret single-cell transcriptome, accessible chromatin, and spatial cell state data from cerebral organoids from different human iPSC lines, time points, conditions, and genotypes. This we believe 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.