Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Epigenetic control and manipulation of neuronal maturation timing.

During brain development, the sequence of developmental steps and the underlying transcriptional regulatory logic are largely conserved across species. However, the temporal unfolding of developmental programs varies dramatically across species and within a given species varies across brain regions and cell identities. The maturation of neurons in the human cerebral cortex is particularly slow and lasts for many years compared with only a few weeks for the corresponding mouse neurons. The mechanisms setting the 'schedule' of neuronal maturation remain unclear but appear to be linked to a cell-intrinsic 'clock'. Here, we discuss recent findings that highlight a role for epigenetic factors in the timing of neuronal maturation. Manipulations of those factors in stem cell-based models can override the intrinsic pace of neuronal maturation, including its protracted nature in human cortical neurons. We then contextualize the epigenetic regulation of maturation programs with findings from other model systems and propose potential interactions between epigenetic pathways and other drivers of developmental rates.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-D-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic ß-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

Generation of human cerebral organoids with a structured outer subventricular zone.

Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APPSWE+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APPSWE+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.

TLR3 controls constitutive IFN-ß antiviral immunity in human fibroblasts and cortical neurons.

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids.

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.

A stochastic framework of neurogenesis underlies the assembly of neocortical cytoarchitecture.

The cerebral cortex contains multiple areas with distinctive cytoarchitectonic patterns, but the cellular mechanisms underlying the emergence of this diversity remain unclear. Here, we have investigated the neuronal output of individual progenitor cells in the developing mouse neocortex using a combination of methods that together circumvent the biases and limitations of individual approaches. Our experimental results indicate that progenitor cells generate pyramidal cell lineages with a wide range of sizes and laminar configurations. Mathematical modeling indicates that these outcomes are compatible with a stochastic model of cortical neurogenesis in which progenitor cells undergo a series of probabilistic decisions that lead to the specification of very heterogeneous progenies. Our findings support a mechanism for cortical neurogenesis whose flexibility would make it capable to generate the diverse cytoarchitectures that characterize distinct neocortical areas.

Loss of SATB1 Induces p21-Dependent Cellular Senescence in Post-mitotic Dopaminergic Neurons.

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.

Extensive branching of radially-migrating neurons in the mammalian cerebral cortex.

Excitatory neurons of the cerebral cortex migrate radially from their place of birth to their final position in the cortical plate during development. Radially-migrating neurons display a single leading process that establishes the direction of movement. This leading process has been described as being unbranched, and the occurrence of branches proposed to impair radial migration. Here we have analyzed the detailed morphology of leading process in radially-migrating pyramidal neurons and its impact on radial migration. We have compared ferret and mouse to identify differences between cortices that undergo folding or not. In mouse, we find that half of radially-migrating neurons exhibit a branched leading process, this being even more frequent in ferret. Branched leading processes are less parallel to radial glia fibers than those unbranched, suggesting some independence from radial glia fibers. Two-photon videomicroscopy revealed that a vast majority of neurons branch their leading process at some point during radial migration, but this does not reduce their migration speed. We have tested the functional impact of exuberant leading process branching by expressing a dominant negative Cdk5. We confirm that loss of Cdk5 function significantly impairs radial migration, but this is independent from increased branching of the leading process. We propose that excitatory neurons may branch their leading process as an evolutionary mechanism to allow cells changing their trajectory of migration to disperse laterally, such that increased branching in gyrencephalic species favors the tangential dispersion of radially-migrating neurons, and cortical folding.

Tuning of fast-spiking interneuron properties by an activity-dependent transcriptional switch.

The function of neural circuits depends on the generation of specific classes of neurons. Neural identity is typically established near the time when neurons exit the cell cycle to become postmitotic cells, and it is generally accepted that, once the identity of a neuron has been established, its fate is maintained throughout life. Here, we show that network activity dynamically modulates the properties of fast-spiking (FS) interneurons through the postmitotic expression of the transcriptional regulator Er81. In the adult cortex, Er81 protein levels define a spectrum of FS basket cells with different properties, whose relative proportions are, however, continuously adjusted in response to neuronal activity. Our findings therefore suggest that interneuron properties are malleable in the adult cortex, at least to a certain extent.

Erbb4 deletion from fast-spiking interneurons causes schizophrenia-like phenotypes.

Genetic variation in neuregulin and its ErbB4 receptor has been linked to schizophrenia, although little is known about how they contribute to the disease process. Here, we have examined conditional Erbb4 mouse mutants to study how disruption of specific inhibitory circuits in the cerebral cortex may cause large-scale functional deficits. We found that deletion of ErbB4 from the two main classes of fast-spiking interneurons, chandelier and basket cells, causes relatively subtle but consistent synaptic defects. Surprisingly, these relatively small wiring abnormalities boost cortical excitability, increase oscillatory activity, and disrupt synchrony across cortical regions. These functional deficits are associated with increased locomotor activity, abnormal emotional responses, and impaired social behavior and cognitive function. Our results reinforce the view that dysfunction of cortical fast-spiking interneurons might be central to the pathophysiology of schizophrenia.

Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults.

In the forebrain, cortical structures consist of networks of excitatory and inhibitory neurons born in distant locations. Understanding how these two major classes of neurons integrate into unique functional cell assemblies may shed light on the organization of cortical circuits. In this review, we provide an overview of the mechanisms used by GABAergic interneurons to reach their final position, with an emphasis on the final steps of this process. To this end, we analyze similarities and differences between the integration of GABAergic interneurons in the developing cerebral cortex and in the postnatal brain, using the neocortex and the olfactory bulb as model systems.

Lineage-specific laminar organization of cortical GABAergic interneurons.

In the cerebral cortex, pyramidal cells and interneurons are generated in distant germinal zones, and so the mechanisms that control their precise assembly into specific microcircuits remain an enigma. Here we report that cortical interneurons labeled at the clonal level do not distribute randomly but rather have a strong tendency to cluster in the mouse neocortex. This behavior is common to different classes of interneurons, independently of their origin. Interneuron clusters are typically contained within one or two adjacent cortical layers, are largely formed by isochronically generated neurons and populate specific layers, as revealed by unbiased hierarchical clustering methods. Our results suggest that different progenitor cells give rise to interneurons populating infra- and supragranular cortical layers, which challenges current views of cortical neurogenesis. Thus, specific lineages of cortical interneurons seem to be produced to primarily mirror the laminar structure of the cerebral cortex, rather than its columnar organization.

Slit/Robo signaling modulates the proliferation of central nervous system progenitors.

Neurogenesis relies on a delicate balance between progenitor maintenance and neuronal production. Progenitors divide symmetrically to increase the pool of dividing cells. Subsequently, they divide asymmetrically to self-renew and produce new neurons or, in some brain regions, intermediate progenitor cells (IPCs). Here we report that central nervous system progenitors express Robo1 and Robo2, receptors for Slit proteins that regulate axon guidance, and that absence of these receptors or their ligands leads to loss of ventricular mitoses. Conversely, production of IPCs is enhanced in Robo1/2 and Slit1/2 mutants, suggesting that Slit/Robo signaling modulates the transition between primary and intermediate progenitors. Unexpectedly, these defects do not lead to transient overproduction of neurons, probably because supernumerary IPCs fail to detach from the ventricular lining and cycle very slowly. At the molecular level, the role of Slit/Robo in progenitor cells involves transcriptional activation of the Notch effector Hes1. These findings demonstrate that Robo signaling modulates progenitor cell dynamics in the developing brain.

Focal adhesion kinase modulates radial glia-dependent neuronal migration through connexin-26.

Focal adhesion kinase (FAK) is an intracellular kinase and scaffold protein that regulates migration in many different cellular contexts but whose function in neuronal migration remains controversial. Here, we have analyzed the function of FAK in two populations of neurons with very distinct migratory behaviors: cortical interneurons, which migrate tangentially and independently of radial glia; and pyramidal cells, which undergo glial-dependent migration. We found that FAK is dispensable for glial-independent migration but is cell-autonomously required for the normal interaction of pyramidal cells with radial glial fibers. Loss of FAK function disrupts the normal morphology of migrating pyramidal cells, delays migration, and increases the tangential dispersion of neurons arising from the same radial unit. FAK mediates this process by regulating the assembly of Connexin-26 contact points in the membrane of migrating pyramidal cells. These results indicate that FAK plays a fundamental role in the dynamic regulation of Gap-mediated adhesions during glial-guided neuronal migration in the mouse.