Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders

The use of brain organoids to investigate neural development and disease

Understanding the development and dysfunction of the human brain is a major goal of neurobiology. Much of our current understanding of human brain development has been derived from the examination of post-mortem and pathological specimens, bolstered by observations of developing non-human primates and experimental studies focused largely on mouse models. However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that enable the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of the development of the human brain and enable a detailed study of the pathogenesis of inherited and acquired brain diseases

oRGs and mitotic somal translocation - a role in development and disease.

The evolution of the human brain has been characterized by an increase in the size of the neocortex. Underlying this expansion is a significant increase in the number of neurons produced by neural stem cells during early stages of cortical development. Here we highlight recent advances in our understating of these cell populations, consisting of ventricular radial glia and outer radial glia. We highlight how gene expression studies have identified molecular signatures for radial glial cell populations and outline what has been learned about the mechanisms underlying the characteristic mode of division observed in outer radial glia cells, mitotic somal translocation. Understanding the significance of this behavior may help us explain human cortical expansion and further elucidate neurodevelopmental diseases

Expression Analysis Highlights AXL as a Candidate Zika Virus Entry Receptor in Neural Stem Cells

The recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina. We also show that AXL expression in radial glia is conserved in developing mouse and ferret cortex and in human stem cell-derived cerebral organoids, highlighting multiple experimental systems that could be applied to study mechanisms of ZIKV infectivity and effects on brain development

Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders

Zika virus alters centrosome organization to suppress the innate immune response

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response

Single-cell sequencing maps gene expression to mutational phylogenies in PDGF- and EGF-driven gliomas

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) receptors are frequently amplified and/or possess gain‐of‐function mutations in GBM. However, clinical trials of tyrosine‐kinase inhibitors have shown disappointing efficacy, in part due to intra‐tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single‐cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high‐resolution phylogenies of EGF‐driven and PDGF‐driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF‐driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor‐like cell type, expressing pro‐angiogenic factors. In contrast, phylogenetic analysis of an EGFR‐amplified tumor showed an up‐regulation of pro‐invasive genes. An in‐frame deletion in a specific dimerization domain of PDGF receptor correlates with an up‐regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In‐frame deletions in this domain are frequent in public GBM data

The Phenotypes of Proliferating Glioblastoma Cells Reside on a Single Axis of Variation.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631