ABSTRACT
Neural organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progenies arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This high variability exceeds what can be explained by existing stochastic models of corticogenesis and indicates the existence of an additional source of stochasticity. To explain this variability, we introduce the SAN model which distinguishes Symmetrically diving, Asymmetrically dividing, and Non-proliferating cells. In the SAN model, the additional source of stochasticity is the survival time of a lineage's pool of symmetrically dividing cells. These survival times result from neutral competition within the sub-population of all symmetrically dividing cells. We demonstrate that our model explains the experimentally observed variability of lineage sizes and derive the quantitative relationship between survival time and lineage size. We also show that our model implies the existence of a regulatory mechanism which keeps the size of the symmetrically dividing cell population constant. Our results provide quantitative insight into the clonal composition of neural organoids and how it arises. This is relevant for many applications of neural organoids, and similar processes may occur in other developing tissues both in vitro and in vivo.
Subject(s)
Organoids , Organoids/cytology , Humans , Cell Lineage/physiology , Computational Biology , Neural Stem Cells/cytology , Neural Stem Cells/physiology , Stochastic Processes , Models, Biological , Neurons/physiology , Neurons/cytology , Brain/cytology , Brain/physiology , Cell Proliferation/physiology , Neurogenesis/physiologyABSTRACT
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Subject(s)
Basement Membrane/pathology , Blood Vessels/pathology , Diabetic Angiopathies/pathology , Models, Biological , Organoids/pathology , Organoids/transplantation , Adaptor Proteins, Signal Transducing , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Arteries/cytology , Arteries/drug effects , Arterioles/cytology , Arterioles/drug effects , Basement Membrane/cytology , Basement Membrane/drug effects , Blood Vessels/cytology , Blood Vessels/drug effects , Blood Vessels/growth & development , Calcium-Binding Proteins , Diabetic Angiopathies/enzymology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Hyperglycemia/complications , In Vitro Techniques , Inflammation Mediators/pharmacology , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Organoids/cytology , Organoids/drug effects , Pericytes/cytology , Pericytes/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Receptor, Notch3/metabolism , Signal Transduction , Venules/cytology , Venules/drug effectsABSTRACT
Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated. Here, we used DNA origami nanostructures to control the positions of ephrin-A5 ligands at the nanoscale and investigated EphA2 activation and transcriptional responses following ligand binding. Using RNA-seq, we determined the transcriptional profiles of human glioblastoma cells treated with DNA nanocalipers presenting a single ephrin-A5 dimer or two dimers spaced 14, 40 or 100 nm apart. These cells displayed divergent transcriptional responses to the differing ephrin-A5 nano-organization. Specifically, ephrin-A5 dimers spaced 40 or 100 nm apart showed the highest levels of differential expressed genes compared to treatment with nanocalipers that do not present ephrin-A5. These findings show that the nanoscale organization of ephrin-A5 modulates transcriptional responses to EphA2 activation.
Subject(s)
Nanostructures , Receptor, EphA2/metabolism , Transcription, Genetic , Cell Line, Tumor , DNA/chemistry , Ephrin-A5/metabolism , Humans , Ligands , Phosphorylation , RNA-SeqABSTRACT
RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , RNA Precursors/chemistry , RNA, Plant/chemistry , RNA, Transfer/chemistry , Ribonuclease P/chemistry , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Base Sequence , Cloning, Molecular , Conserved Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Evolution, Molecular , Gene Expression , Isoenzymes/chemistry , Isoenzymes/genetics , Molecular Sequence Data , Nucleic Acid Conformation , RNA Precursors/metabolism , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Plant/metabolism , RNA, Transfer/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Ribonuclease P/genetics , Substrate Specificity , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsABSTRACT
During brain development, neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells, resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids, to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids.
Subject(s)
Cell Lineage , Neural Stem Cells , Organoids , Organoids/cytology , Organoids/metabolism , Humans , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Brain/cytology , Brain/growth & development , Brain/metabolism , Cell Differentiation , Cell Proliferation , Clone Cells , Neurogenesis/genetics , DNA Barcoding, Taxonomic , AnimalsABSTRACT
Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20's nuclear localization in RBM20-DCM patients.
Subject(s)
Cardiomyopathy, Dilated , Animals , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , RNA Splicing/genetics , Alternative Splicing/genetics , Mutation , Karyopherins/geneticsABSTRACT
Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.