ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum.

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

Cerebral organoids display dynamic clonal growth and tunable tissue replenishment.

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.

Neutral competition explains the clonal composition of neural organoids.

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.

In vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain-striatum-cortex assembloids.

Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson's disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson's disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain-striatum-cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids.

During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the posttranscriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5'TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach proposes novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.

The HUSH complex controls brain architecture and protocadherin fidelity.

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

A nomenclature consensus for nervous system organoids and assembloids.

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.

Amplification of human interneuron progenitors promotes brain tumors and neurological defects.

Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.

ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

ISSCR guidelines for the transfer of human pluripotent stem cells and their direct derivatives into animal hosts.

The newly revised 2021 ISSCR Guidelines for Stem Cell Research and Clinical Translation includes scientific and ethical guidance for the transfer of human pluripotent stem cells and their direct derivatives into animal models. In this white paper, the ISSCR subcommittee that drafted these guidelines for research involving the use of nonhuman embryos and postnatal animals explains and summarizes their recommendations.

Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly.

Viral infection in early pregnancy is a major cause of microcephaly. However, how distinct viruses impair human brain development remains poorly understood. Here we use human brain organoids to study the mechanisms underlying microcephaly caused by Zika virus (ZIKV) and herpes simplex virus (HSV-1). We find that both viruses efficiently replicate in brain organoids and attenuate their growth by causing cell death. However, transcriptional profiling reveals that ZIKV and HSV-1 elicit distinct cellular responses and that HSV-1 uniquely impairs neuroepithelial identity. Furthermore, we demonstrate that, although both viruses fail to potently induce the type I interferon system, the organoid defects caused by their infection can be rescued by distinct type I interferons. These phenotypes are not seen in 2D cultures, highlighting the superiority of brain organoids in modeling viral infections. These results uncover virus-specific mechanisms and complex cellular immune defenses associated with virus-induced microcephaly.

Brain organoids: an ensemble of bioassays to investigate human neurodevelopment and disease.

Understanding etiology of human neurological and psychiatric diseases is challenging. Genomic changes, protracted development, and histological features unique to human brain development limit the disease aspects that can be investigated using model organisms. Hence, in order to study phenotypes associated with human brain development, function, and disease, it is necessary to use alternative experimental systems that are accessible, ethically justified, and replicate human context. Human pluripotent stem cell (hPSC)-derived brain organoids offer such a system, which recapitulates features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation of neural progenitors into neurons and glial cells and the complex interactions among the diverse, emergent cell types of the developing brain in three-dimensions (3-D). In recent years, numerous brain organoid protocols and related techniques have been developed to recapitulate aspects of embryonic and fetal brain development in a reproducible and predictable manner. Altogether, these different organoid technologies provide distinct bioassays to unravel novel, disease-associated phenotypes and mechanisms. In this review, we summarize how the diverse brain organoid methods can be utilized to enhance our understanding of brain disorders. FACTS: Brain organoids offer an in vitro approach to study aspects of human brain development and disease. Diverse brain organoid techniques offer bioassays to investigate new phenotypes associated with human brain disorders that are difficult to study in monolayer cultures. Brain organoids have been particularly useful to study phenomena and diseases associated with neural progenitor morphology, survival, proliferation, and differentiation. OPEN QUESTION: Future brain organoid research needs to aim at later stages of neurodevelopment, linked with neuronal activity and connections, to unravel further disease-associated phenotypes. Continued improvement of existing organoid protocols is required to generate standardized methods that recapitulate in vivo-like spatial diversity and complexity.

A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.

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.

LifeTime and improving European healthcare through cell-based interceptive medicine.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Reflections on the past two decades of neuroscience.

The first issue of Nature Reviews Neuroscience was published 20 years ago, in 2000. To mark this anniversary, in this Viewpoint article we asked a selection of researchers from across the field who have authored pieces published in the journal in recent years for their thoughts on notable and interesting developments in neuroscience, and particularly in their areas of the field, over the past two decades. They also provide some thoughts on current lines of research and questions that excite them.

DigiTAG-a RNA Sequencing Approach to Analyze Transcriptomes of Rare Cell Populations in Drosophila melanogaster.

Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.

Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors.

During central nervous system development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. Here, we perform ChIP-seq on distinct subtypes of Drosophila FACS-purified NSCs and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, we show that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. Our analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts (NBs), one of the two main Drosophila NSC identities. Finally, we demonstrate that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group-mediated repression.

The transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop.

Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. Here, we developed a method to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. Our analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. We propose that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities.