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Profiling Cell Type-Specific Gene Regulatory Regions in Human Cortical Organoids

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Brain Organoid Research

Part of the book series: Neuromethods ((NM,volume 189))

Abstract

Dissecting the molecular heterogeneity of the developing human brain is a long-standing ambition of neuroscientists. This has become both a practical and ethically sustainable reality with the advent of cerebral organoids, a 3D in vitro model system of neurogenesis which recapitulates much of the cell type diversity found in the fetal brain. Detailed analysis of cell type-specific biology mandated by the heterogeneity of developing human brain tissue is thus possible without the need of primary samples. Gene regulatory regions, through differential activity, are a key factor for acquisition and maintenance of cell identity and orchestrate intricate developmental processes by directing gene transcription. Here, we describe two experimental methods to isolate distinct cell populations from cortical organoids by means of fluorescence-activated cell sorting, leveraging distinct transcription factor profiles and cell morphological aspects. We assay open chromatin in both live and cross-linked samples using ATAC-seq and validate putative enhancer sequences using luciferase assays. These methods will allow future in-depth epigenomic characterization of human neurogenesis in the context of evolution, health, and disease.

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Acknowledgments

This work was supported by the Flow Cytometry Core Facility, Light Microscopy Facility, and Deep Sequencing Facility, all core facilities of the CRTD or CMCB Technology Platforms of the Technische Universität Dresden. CRTDi004-A hiPSCs (see Human Pluripotent Stem Cell Registry) were reprogrammed from foreskin fibroblasts of a healthy, consented donor at the Stem Cell Engineering Facility, a core facility of the CMCB Technology Platform at the Technische Universität Dresden. We acknowledge technical support from Li Ding for luciferase assay setup, advice from Florian Noack on organoid dissociation and Manching Ku on bead-based ATAC-seq library size selection. The pCAG-GFP plasmid was a kind gift from Wieland B. Huttner. This work was supported by funding from the German Research Foundation (Emmy Noether, AL 2231/1-1), Schram foundation, and Center for Regenerative Therapies TU Dresden.

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Authors and Affiliations

  1. Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany

    Theresa M. Schütze, Nora Bölicke, Katrin Sameith & Mareike Albert

  2. DRESDEN-concept Genome Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany

    Katrin Sameith

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  1. Theresa M. Schütze
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  2. Nora Bölicke
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  3. Katrin Sameith
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  4. Mareike Albert
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Correspondence to Mareike Albert .

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  1. Laboratory for Centrosome and Cytoskeleton Biology, Institute for Human Genetics, Heinrich-Heine-Universität, Universitätsklinikum Düsseldorf, Düsseldorf, Germany

    Jay Gopalakrishnan

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Schütze, T.M., Bölicke, N., Sameith, K., Albert, M. (2023). Profiling Cell Type-Specific Gene Regulatory Regions in Human Cortical Organoids. In: Gopalakrishnan, J. (eds) Brain Organoid Research. Neuromethods, vol 189. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2720-4_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2720-4_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2719-8

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