Diverse maturity-dependent and complementary anti-apoptotic brakes safeguard human iPSC-derived neurons from cell death.

In humans, most neurons are born during embryonic development and have to persist throughout the entire lifespan of an individual. Thus, human neurons have to develop elaborate survival strategies to protect against accidental cell death. We set out to decipher the developmental adaptations resulting in neuronal resilience. We demonstrate that, during the time course of maturation, human neurons install a complex and complementary anti-apoptotic signaling network. This includes i.) a downregulation of central proteins of the intrinsic apoptosis pathway including several caspases, ii.) a shift in the ratio of pro- and anti-apoptotic BCL-2 family proteins, and iii.) an elaborate regulatory network resulting in upregulation of the inhibitor of apoptosis protein (IAP) XIAP. Together, these adaptations strongly increase the threshold for apoptosis initiation when confronted with a wide range of cellular stressors. Our results highlight how human neurons are endowed with complex and redundant preemptive strategies to protect against stress and cell death.

Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids.

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.

Voltammetric Approach for Characterizing the Biophysical and Chemical Functionality of Human Induced Pluripotent Stem Cell-Derived Serotonin Neurons.

Depression is quickly becoming one of the world's most pressing public health crises, and there is an urgent need for better diagnostics and therapeutics. Behavioral models in animals and humans have not adequately addressed the diagnosis and treatment of depression, and biomarkers of mental illnesses remain ill-defined. It has been very difficult to identify biomarkers of depression because of in vivo measurement challenges. While our group has made important strides in developing in vivo tools to measure such biomarkers (e.g., serotonin) in mice using voltammetry, these tools cannot be easily applied for depression diagnosis and drug screening in humans due to the inaccessibility of the human brain. In this work, we take a chemical approach, ex vivo, to introduce a human-derived system to investigate brain serotonin. We utilize human induced pluripotent stem cells differentiated into serotonin neurons and establish a new ex vivo model of real-time serotonin neurotransmission measurements. We show that evoked serotonin release responds to stimulation intensity and tryptophan preloading, and that serotonin release and reuptake kinetics resemble those found in vivo in rodents. Finally, after selective serotonin reuptake inhibitor (SSRI) exposure, we find dose-dependent internalization of the serotonin reuptake transporters (a signature of the in vivo response to SSRI). Our new human-derived chemical model has great potential to provide an ex vivo chemical platform as a translational tool for in vivo neuropsychopharmacology.

Human cerebral organoids reveal progenitor pathology in EML1-linked cortical malformation.

Malformations of human cortical development (MCD) can cause severe disabilities. The lack of human-specific models hampers our understanding of the molecular underpinnings of the intricate processes leading to MCD. Here, we use cerebral organoids derived from patients and genome edited-induced pluripotent stem cells to address pathophysiological changes associated with a complex MCD caused by mutations in the echinoderm microtubule-associated protein-like 1 (EML1) gene. EML1-deficient organoids display ectopic neural rosettes at the basal side of the ventricular zone areas and clusters of heterotopic neurons. Single-cell RNA sequencing shows an upregulation of basal radial glial (RG) markers and human-specific extracellular matrix components in the ectopic cell population. Gene ontology and molecular analyses suggest that ectopic progenitor cells originate from perturbed apical RG cell behavior and yes-associated protein 1 (YAP1)-triggered expansion. Our data highlight a progenitor origin of EML1 mutation-induced MCD and provide new mechanistic insight into the human disease pathology.

Asymmetric Notch activity by differential inheritance of lysosomes in human neural stem cells.

Symmetric and asymmetric cell divisions are conserved strategies for stem cell expansion and the generation of more committed progeny, respectively. Here, we demonstrate that in human neural stem cells (NSCs), lysosomes are asymmetrically inherited during mitosis. We show that lysosomes contain Notch receptors and that Notch activation occurs the acidic lysosome environment. The lysosome asymmetry correlates with the expression of the Notch target gene HES1 and the activity of Notch signaling in the daughter cells. Furthermore, an asymmetry of lysosomes and Notch receptors was also observed in a human organoid model of brain development with mitotic figures showing preferential inheritance of lysosomes and Notch receptor in that daughter cell remaining attached to the apical membrane. Thus, this study suggests a previously unknown function of lysosomes as a signaling hub to establish a bias in Notch signaling activity between daughter cells after an asymmetric cell division of human NSCs.

Cerebral organoids to unravel the mechanisms underlying malformations of human cortical development.

Genetic studies identified multiple mutations associated with malformations of cortical development (MCD) in humans. When analyzing the underlying mechanisms in non-human experimental models it became increasingly evident, that these mutations accumulate in genes, which functions evolutionary progressed from rodents to humans resulting in an incomplete reflection of the molecular and cellular alterations in these models. Human brain organoids derived from human pluripotent stem cells resemble early aspects of human brain development to a remarkable extent making them an attractive model to investigate MCD. Here we review how human brain organoids enable the generation of fundamental new insight about the underlying pathomechanisms of MCD. We show how phenotypic features of these diseases are reflected in human brain organoids and discuss challenges and future considerations but also limitations for the use of human brain organoids to model human brain development and associated disorders.

Corrigendum: Genes and Mechanisms Involved in the Generation and Amplification of Basal Radial Glial Cells.

[This corrects the article DOI: 10.3389/fncel.2019.00381.].

Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing.

Expansions of short tandem repeats are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with current polymerase-based methods1-4. Here we introduce a CRISPR-Cas-based enrichment strategy for nanopore sequencing combined with an algorithm for raw signal analysis. Our method, termed STRique for short tandem repeat identification, quantification and evaluation, integrates conventional sequence mapping of nanopore reads with raw signal alignment for the localization of repeat boundaries and a hidden Markov model-based repeat counting mechanism. We demonstrate the precise quantification of repeat numbers in conjunction with the determination of CpG methylation states in the repeat expansion and in adjacent regions at the single-molecule level without amplification. Our method enables the study of previously inaccessible genomic regions and their epigenetic marks.

Drug discovery in psychopharmacology: from 2D models to cerebral organoids
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Psychiatric disorders are a heterogeneous group of mental illnesses associated with a high social and economic burden on patients and society. The complex symptomatology of these disorders, coupled with our limited understanding of the structural and functional abnormalities affecting the brains of neuropsychiatric patients, has made it difficult to develop effective medical treatment strategies. With the advent of reprogramming technologies and recent developments in induced pluripotent stem (iPS) cell-based protocols for differentiation into defined neuronal cultures and 3-dimensional cerebral organoids, a new era of preclinical disease modeling has begun which could revolutionize drug discovery in psychiatry. This review provides an overview of iPS cell-based disease models in psychiatry and how these models contribute to our understanding of pharmacological drug action. We also propose a refined iPSC-based drug discovery pipeline, ranging from cell-based stratification of patients through improved screening and validation steps to more precise psychopharmacology.
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Mettre la traduction ES.

Mettre la traduction FR.

Genes and Mechanisms Involved in the Generation and Amplification of Basal Radial Glial Cells.

The development of the cerebral cortex relies on different types of progenitor cell. Among them, the recently described basal radial glial cell (bRG) is suggested to be of critical importance for the development of the brain in gyrencephalic species. These cells are highly numerous in primate and ferret brains, compared to lissencephalic species such as the mouse in which they are few in number. Their somata are located in basal subventricular zones in gyrencephalic brains and they generally possess a basal process extending to the pial surface. They sometimes also have an apical process directed toward the ventricular surface, similar to apical radial glial cells (aRGs) from which they are derived, and whose somata are found more apically in the ventricular zone. bRGs share similarities with aRGs in terms of gene expression (SOX2, PAX6, and NESTIN), whilst also expressing a range of more specific genes (such as HOPX). In primate brains, bRGs can divide multiple times, self-renewing and/or generating intermediate progenitors and neurons. They display a highly specific cytokinesis behavior termed mitotic somal translocation. We focus here on recently identified molecular mechanisms associated with the generation and amplification of bRGs, including bRG-like cells in the rodent. These include signaling pathways such as the FGF-MAPK cascade, SHH, PTEN/AKT, PDGF pathways, and proteins such as INSM, GPSM2, ASPM, TRNP1, ARHGAP11B, PAX6, and HIF1α. A number of these proteins were identified through transcriptome comparisons in human aRGs vs. bRGs, and validated by modifying their activities or expression levels in the mouse. This latter experiment often revealed enhanced bRG-like cell production, even in some cases generating folds (gyri) on the surface of the mouse cortex. We compare the features of the identified cells and methods used to characterize them in each model. These important data converge to indicate pathways essential for the production and expansion of bRGs, which may help us understand cortical development in health and disease.

Mutations in the Heterotopia Gene Eml1/EML1 Severely Disrupt the Formation of Primary Cilia.

Apical radial glia (aRGs) are predominant progenitors during corticogenesis. Perturbing their function leads to cortical malformations, including subcortical heterotopia (SH), characterized by the presence of neurons below the cortex. EML1/Eml1 mutations lead to SH in patients, as well as to heterotopic cortex (HeCo) mutant mice. In HeCo mice, some aRGs are abnormally positioned away from the ventricular zone (VZ). Thus, unraveling EML1/Eml1 function will clarify mechanisms maintaining aRGs in the VZ. We pinpoint an unknown EML1/Eml1 function in primary cilium formation. In HeCo aRGs, cilia are shorter, less numerous, and often found aberrantly oriented within vesicles. Patient fibroblasts and human cortical progenitors show similar defects. EML1 interacts with RPGRIP1L, a ciliary protein, and RPGRIP1L mutations were revealed in a heterotopia patient. We also identify Golgi apparatus abnormalities in EML1/Eml1 mutant cells, potentially upstream of the cilia phenotype. We thus reveal primary cilia mechanisms impacting aRG dynamics in physiological and pathological conditions.

In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.

During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440.

Cortical organoids: why all this hype?

The development of organoids derived from human pluripotent stem cells heralded a new area in studying human organ development and pathology outside of the human body. Triggered by the seminal work of pioneers in the field such as Yoshiki Sasai or Hans Clevers, organoid research has become one of the most rapidly developing fields in cell biology. The potential applications are manifold reaching from developmental studies to tissue regeneration and drug screening. In this review, we will concentrate on brain organoids of cortical identity. We will describe the 'state of the art' in generating cortical organoids and discuss potential applications. Finally, we will provide future perspectives including suggestions how further innovations can broaden the application of brain organoids.

Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity.

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477-486.

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

A Little Bit of Guidance: Mini Brains on Their Route to Adolescence.

Cerebral organoids represent a new model in which to study human brain development outside the human body. Recently in Nature Biotechnology, Lancaster et al. (2017) tackled the lack of reproducibility, tissue homogeneity, and complexity of this system by bioengineering organoids to establish the next generation of human mini brains.

An Organoid-Based Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker Syndrome.

Miller-Dieker syndrome (MDS) is caused by a heterozygous deletion of chromosome 17p13.3 involving the genes LIS1 and YWHAE (coding for 14.3.3ε) and leads to malformations during cortical development. Here, we used patient-specific forebrain-type organoids to investigate pathological changes associated with MDS. Patient-derived organoids are significantly reduced in size, a change accompanied by a switch from symmetric to asymmetric cell division of ventricular zone radial glia cells (vRGCs). Alterations in microtubule network organization in vRGCs and a disruption of cortical niche architecture, including altered expression of cell adhesion molecules, are also observed. These phenotypic changes lead to a non-cell-autonomous disturbance of the N-cadherin/ß-catenin signaling axis. Reinstalling active ß-catenin signaling rescues division modes and ameliorates growth defects. Our data define the role of LIS1 and 14.3.3ε in maintaining the cortical niche and highlight the utility of organoid-based systems for modeling complex cell-cell interactions in vitro.

Arylsulfatase A Overexpressing Human iPSC-derived Neural Cells Reduce CNS Sulfatide Storage in a Mouse Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Auto-attraction of neural precursors and their neuronal progeny impairs neuronal migration.

Limited neuronal migration into host brain tissue is a key challenge in neural transplantation. We found that one important mechanism underlying this phenomenon is an intrinsic chemotactic interaction between the grafted neural precursor cells (NPCs) and their neuronal progeny. NPCs secrete the receptor tyrosine kinase ligands FGF2 and VEGF, which act as chemoattractants for neurons. Interference with these signaling pathways resulted in enhanced migration of human neurons from neural clusters.