Early Human Brain Development Research Articles

Organoids Modeling Stroke in a Petri Dish

Stroke is a common neurological disorder, the second leading cause of death, and the third leading cause of disability. Unfortunately, the only approved drug for it is tissue plasminogen, but the therapeutic window is limited. In this context, preclinical studies are relevant to better dissect the underlying mechanisms of stroke and for the drug screening of potential therapies. Brain organoids could be relevant in this setting. They are derived from pluripotent stem cells or isolated organ progenitors that differentiate to form an organ-like tissue, exhibiting multiple cell types that self-organize to form a structure not unlike the organ in vivo. Brain organoids mimic many key features of early human brain development at molecular, cellular, structural, and functional levels and have emerged as novel model systems that can be used to investigate human brain diseases including stroke. Brain organoids are a promising and powerful tool for ischemic stroke studies; however, there are a few concerns that need to be addressed, including the lack of vascularization and the many cell types that are typically present in the human brain. The aim of this review is to discuss the potential of brain organoids as a novel model system for studying ischemic stroke, highlighting both the advantages and disadvantages in the use of this technology.

Establishment of human cerebral organoid systems to model early neural development and assess the central neurotoxicity of environmental toxins

Abstract Human brain development is a complex process, and animal models often have significant limitations. To address this, researchers have developed pluripotent stem cell-derived three-dimensional structures, known as brain-like organoids, to more accurately model early human brain development and disease. To enable more consistent and intuitive reproduction of early brain development, in this study, we incorporated forebrain organoid culture technology into the traditional unguided method of brain organoid culture. This involved embedding organoids in matrix glue for only 7 days during the rapid expansion phase of the neural epithelium and then removing them from the matrix glue for further cultivation, resulting in a new type of human brain organoid system. This cerebral organoid system replicated the temporospatial characteristics of early human brain development, including neuroepithelium derivation, neural progenitor cell production and maintenance, neuron differentiation and migration, and cortical layer patterning and formation, providing more consistent and reproducible organoids for developmental modeling and toxicology testing. As a proof of concept, we applied the heavy metal cadmium to this newly improved organoid system to test whether it could be used to evaluate the neurotoxicity of environmental toxins. Brain organoids exposed to cadmium for 7 or 14 days manifested severe damage and abnormalities in their neurodevelopmental patterns, including bursts of cortical cell death and premature differentiation. Cadmium exposure caused progressive depletion of neural progenitor cells and loss of organoid integrity, accompanied by compensatory cell proliferation at ectopic locations. The convenience, flexibility, and controllability of this newly developed organoid platform make it a powerful and affordable alternative to animal models for use in neurodevelopmental, neurological, and neurotoxicological studies.

Scaling patterns of cortical folding and thickness in early human brain development in comparison with primates.

Across mammalia, brain morphology follows specific scaling patterns. Bigger bodies have bigger brains, with surface area outpacing volume growth, resulting in increased foldedness. We have recently studied scaling rules of cortical thickness, both local and global, finding that the cortical thickness difference between thick gyri and thin sulci also increases with brain size and foldedness. Here, we investigate early brain development in humans, using subjects from the Developing Human Connectome Project, scanned shortly after pre-term or full-term birth, yielding magnetic resonance images of the brain from 29 to 43 postmenstrual weeks. While the global cortical thickness does not change significantly during this development period, its distribution does, with sulci thinning, while gyri thickening. By comparing our results with our recent work on humans and 11 non-human primate species, we also compare the trajectories of primate evolution with human development, noticing that the 2 trends are distinct for volume, surface area, cortical thickness, and gyrification index. Finally, we introduce the global shape index as a proxy for gyrification index; while correlating very strongly with gyrification index, it offers the advantage of being calculated only from local quantities without generating a convex hull or alpha surface.

Apically localized PANX1 impacts neuroepithelial expansion in human cerebral organoids

Dysfunctional paracrine signaling through Pannexin 1 (PANX1) channels is linked to several adult neurological pathologies and emerging evidence suggests that PANX1 plays an important role in human brain development. It remains unclear how early PANX1 influences brain development, or how loss of PANX1 alters the developing human brain. Using a cerebral organoid model of early human brain development, we find that PANX1 is expressed at all stages of organoid development from neural induction through to neuroepithelial expansion and maturation. Interestingly, PANX1 cellular distribution and subcellular localization changes dramatically throughout cerebral organoid development. During neural induction, PANX1 becomes concentrated at the apical membrane domain of neural rosettes where it co-localizes with several apical membrane adhesion molecules. During neuroepithelial expansion, PANX1−/− organoids are significantly smaller than control and exhibit significant gene expression changes related to cell adhesion, WNT signaling and non-coding RNAs. As cerebral organoids mature, PANX1 expression is significantly upregulated and is primarily localized to neuronal populations outside of the ventricular-like zones. Ultimately, PANX1 protein can be detected in all layers of a 21–22 post conception week human fetal cerebral cortex. Together, these results show that PANX1 is dynamically expressed by numerous cell types throughout embryonic and early fetal stages of human corticogenesis and loss of PANX1 compromises neuroepithelial expansion due to dysregulation of cell-cell and cell-matrix adhesion, perturbed intracellular signaling, and changes to gene regulation.

MRI-based structural covariance network in early human brain development.

MRI-based structural covariance network in early human brain development.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial–temporal regulation of histone arginine methylation in neurodevelopment

BackgroundOxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency.ResultsWe report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial–temporal regulation of histone arginine methylation in specific brain regions.ConclusionsThis study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

A review on current brain organoid technologies from a biomedical engineering perspective

Brain organoids are 3D cytoarchitectures resembling the embryonic human brain. This review focuses on current advancements in biomedical engineering methods to develop organoids such as pluripotent stem cells assemblies, quickly aggregated floating culture, hydrogel suspension, microfluidic systems (both photolithography and 3D printing), and brain organoids-on-a-chip. These methods have the potential to create a large impact on neurological disorder studies by creating a model of the human brain investigating pathogenesis and drug screening for individual patients. 3D brain organoid cultures mimic not only features of patients' unknown drug reactions, but also early human brain development at cellular, structural, and functional levels. The challenge of current brain organoids lies in the formation of distinct cortical neuron layers, gyrification, and the establishment of complex neuronal circuitry, as they are critically specialized, developmental aspects. Furthermore, recent advances such as vascularization and genome engineering are in development to overcome the barrier of neuronal complexity. Future technology of brain organoids is needed to improve tissue cross-communication, body axis simulation, cell patterning signals, and spatial-temporal control of differentiation, as engineering methods discussed in this review are rapidly evolving.

The single-cell and spatial transcriptional landscape of human gastrulation and early brain development

The emergence of the three germ layers and the lineage-specific precursor cells orchestrating organogenesis represent fundamental milestones during early embryonic development. We analyzed the transcriptional profiles of over 400,000 cells from 14 human samples collected from post-conceptional weeks (PCW) 3 to 12 to delineate the dynamic molecular and cellular landscape of early gastrulation and nervous system development. We described the diversification of cell types, the spatial patterning of neural tube cells, and the signaling pathways likely involved in transforming epiblast cells into neuroepithelial cells and then into radial glia. We resolved 24 clusters of radial glial cells along the neural tube and outlined differentiation trajectories for the main classes of neurons. Lastly, we identified conserved and distinctive features across species by comparing early embryonic single-cell transcriptomic profiles between humans and mice. This comprehensive atlas sheds light on the molecular mechanisms underlying gastrulation and early human brain development.

Mechanics of morphogenesis in neural development: In vivo, in vitro, and in silico

Morphogenesis in the central nervous system has received intensive attention as elucidating fundamental mechanisms of morphogenesis will shed light on the physiology and pathophysiology of the developing central nervous system. Morphogenesis of the central nervous system is of a vast topic that includes important morphogenetic events such as neurulation and cortical folding. Here we review three types of methods used to improve our understanding of morphogenesis of the central nervous system: in vivo experiments, organoids (in vivo), and computational models (in silico). The in vivo experiments are used to explore cellular- and tissue-level mechanics and interpret them on the roles of neurulation morphogenesis. Recent advances in human brain organoids have provided new opportunities to study morphogenesis and neurogenesis to compensate for the limitations of in vivo experiments, as organoid models are able to recapitulate some critical neural morphogenetic processes during early human brain development. Due to the complexity and costs of in vivo and in vitro studies, a variety of computational models have been developed and used to explain the formation and morphogenesis of brain structures. We review and discuss the advantages and disadvantages of these methods and their usage in the studies on morphogenesis of the central nervous system. Notably, none of these methods alone is sufficient to unveil the biophysical mechanisms of morphogenesis, thus calling for the interdisciplinary approaches using a combination of these methods in order to test hypotheses and generate new insights on both normal and abnormal development of the central nervous system.Statement of Significance: The understanding of the central nervous system is essential to provide supports to treat and prevent neurological conditions. Mechanisms of morphogenesis therein can be elucidated from multiple unique perspectives via multidisciplinary approaches. The in vivo experiments, organoid models, and computational modeling are three most effective ways to study brain morphogenesis. In vivo experiments on live animals provide important evidence for studying the roles of mechanical forces in morphogenetic events. The human brain organoid models can greatly assist to study early human brain development and closely simulate the in-vivo counterpart. Moreover, computational models based on physical principles can test hypotheses in conjunctions with experiments to facilitate understanding of the spatial and temporal evolution of these complex structures. The combination of these approaches can complement each other to unveil fundamental mechanisms of the neural morphogenesis and shed light on the development, prevention, and treatment of neurological disorders.

A multi-omics approach to visualize early neuronal differentiation from hESCs in 4D

SummaryNeuronal differentiation of pluripotent stem cells is an established method to study physiology, disease, and medication safety. However, the sequence of events in human neuronal differentiation and the ability of in vitro models to recapitulate early brain development are poorly understood. We developed a protocol optimized for the study of early human brain development and neuropharmacological applications. We comprehensively characterized gene expression and epigenetic profiles at four timepoints, because the cells differentiate from embryonic stem cells towards a heterogeneous population of progenitors, immature and mature neurons bearing telencephalic signatures. A multi-omics roadmap of neuronal differentiation, combined with searchable interactive gene analysis tools, allows for extensive exploration of early neuronal development and the effect of medications.

Neuronal hyperexcitability and ion channel dysfunction in CDKL5-deficiency patient iPSC-derived cortical organoids

Early epilepsy is a prominent feature in patients with CDKL5-deficiency disorder (CDD). The underlying mechanism for excessive excitability in CDD is largely unknown. The brain organoid model has been recently developed to resemble many critical features of early human brain development. Here, we used a brain organoid model to investigate the cellular electrophysiological basis for hyper-excitability in CDD patients. Our study employed cortical organoids derived from two CDD patients harboring the same CDKL5 mutation (R59X) and two controls from their healthy parents. Whole-cell patch-clamp recordings revealed higher action potential (AP) firing rate and lower rheobase in both CDD organoids, indicating increased intrinsic neuronal excitability. We further found dysfunction of voltage-gated ion channels in CDD neurons that leads to hyperexcitability, including higher Na+ and K+ current densities and a negative shift in Na+ channel activation. In contrast to neuronal properties, we found that glutamatergic neurotransmission and the electrophysiological properties of glial cells were not altered in CDD organoids. In support of our CDD findings, we further discovered similar electrophysiologic properties in cortical organoids derived from a Rett syndrome (RTT) patient, including alterations in AP firings and Na+ and K+ channel function suggesting a convergent mechanism. Together, our study suggests a critical role of intrinsic neuronal hyperexcitability and ion channel dysfunction, seen in early brain development in both CDD and RTT disorders. This investigation provides potential novel drug targets for developing treatments of early epilepsy in such disorders.

Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. Results: Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A –/– ) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A –/– hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. Conclusions: We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.

Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome.

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.

Revealing the Impact of Mitochondrial Fitness During Early Neural Development Using Human Brain Organoids.

Mitochondrial homeostasis -including function, morphology, and inter-organelle communication- provides guidance to the intrinsic developmental programs of corticogenesis, while also being responsive to environmental and intercellular signals. Two- and three-dimensional platforms have become useful tools to interrogate the capacity of cells to generate neuronal and glia progeny in a background of metabolic dysregulation, but the mechanistic underpinnings underlying the role of mitochondria during human neurogenesis remain unexplored. Here we provide a concise overview of cortical development and the use of pluripotent stem cell models that have contributed to our understanding of mitochondrial and metabolic regulation of early human brain development. We finally discuss the effects of mitochondrial fitness dysregulation seen under stress conditions such as metabolic dysregulation, absence of developmental apoptosis, and hypoxia; and the avenues of research that can be explored with the use of brain organoids.

Understanding the Role of the Gut Microbiome in Brain Development and Its Association With Neurodevelopmental Psychiatric Disorders.

The gut microbiome has a tremendous influence on human physiology, including the nervous system. During fetal development, the initial colonization of the microbiome coincides with the development of the nervous system in a timely, coordinated manner. Emerging studies suggest an active involvement of the microbiome and its metabolic by-products in regulating early brain development. However, any disruption during this early developmental process can negatively impact brain functionality, leading to a range of neurodevelopment and neuropsychiatric disorders (NPD). In this review, we summarize recent evidence as to how the gut microbiome can influence the process of early human brain development and its association with major neurodevelopmental psychiatric disorders such as autism spectrum disorders, attention-deficit hyperactivity disorder, and schizophrenia. Further, we discuss how gut microbiome alterations can also play a role in inducing drug resistance in the affected individuals. We propose a model that establishes a direct link of microbiome dysbiosis with the exacerbated inflammatory state, leading to functional brain deficits associated with NPD. Based on the existing research, we discuss a framework whereby early diet intervention can boost mental wellness in the affected subjects and call for further research for a better understanding of mechanisms that govern the gut-brain axis may lead to novel approaches to the study of the pathophysiology and treatment of neuropsychiatric disorders.

Modeling Somatic Mutations Associated With Neurodevelopmental Disorders in Human Brain Organoids.

Neurodevelopmental disorders (NDDs) are a collection of diseases with early life onset that often present with developmental delay, cognitive deficits, and behavioral conditions. In some cases, severe outcomes such as brain malformations and intractable epilepsy can occur. The mutations underlying NDDs may be inherited or de novo, can be gain- or loss-of-function, and can affect one or more genes. Recent evidence indicates that brain somatic mutations contribute to several NDDs, in particular malformations of cortical development. While advances in sequencing technologies have enabled the detection of these somatic mutations, the mechanisms by which they alter brain development and function are not well understood due to limited model systems that recapitulate these events. Human brain organoids have emerged as powerful models to study the early developmental events of the human brain. Brain organoids capture the developmental progression of the human brain and contain human-enriched progenitor cell types. Advances in human stem cell and genome engineering provide an opportunity to model NDD-associated somatic mutations in brain organoids. These organoids can be tracked throughout development to understand the impact of somatic mutations on early human brain development and function. In this review, we discuss recent evidence that somatic mutations occur in the developing human brain, that they can lead to NDDs, and discuss how they could be modeled using human brain organoids.

A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development.

The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.

Cell-Type-Specific Impact of Glucocorticoid Receptor Activation on the Developing Brain: A Cerebral Organoid Study.

A fine-tuned balance of glucocorticoid receptor (GR) activation is essential for organ formation, with disturbances influencing many health outcomes. In utero, glucocorticoids have been linked to brain-related negative outcomes, with unclear underlying mechanisms, especially regarding cell-type-specific effects. An in vitro model of fetal human brain development, induced human pluripotent stem cell (hiPSC)-derived cerebral organoids, was used to test whether cerebral organoids are suitable for studying the impact of prenatal glucocorticoid exposure on the developing brain. The GR was activated with the synthetic glucocorticoid dexamethasone, and the effects were mapped using single-cell transcriptomics across development. The GR was expressed in all cell types, with increasing expression levels through development. Not only did its activation elicit translocation to the nucleus and the expected effects on known GR-regulated pathways, but also neurons and progenitor cells showed targeted regulation of differentiation- and maturation-related transcripts. Uniquely in neurons, differentially expressed transcripts were significantly enriched for genes associated with behavior-related phenotypes and disorders. This human neuronal glucocorticoid response profile was validated across organoids from three independent hiPSC lines reprogrammed from different source tissues from both male and female donors. These findings suggest that excessive glucocorticoid exposure could interfere with neuronal maturation in utero, leading to increased disease susceptibility through neurodevelopmental processes at the interface of genetic susceptibility and environmental exposure. Cerebral organoids are a valuable translational resource for exploring the effects of glucocorticoids on early human brain development.

Maternal Diet, Infection, and Risk of Cord Blood Inflammation in the Bangladesh Projahnmo Pregnancy Cohort.

Inflammation may adversely affect early human brain development. We aimed to assess the role of maternal nutrition and infections on cord blood inflammation. In a pregnancy cohort in Sylhet, Bangladesh, we enrolled 251 consecutive pregnancies resulting in a term livebirth from July 2016–March 2017. Stillbirths, preterm births, and cases of neonatal encephalopathy were excluded. We prospectively collected data on maternal diet (food frequency questionnaire) and morbidity, and analyzed umbilical cord blood for interleukin (IL)-1α, IL-1β, IL-6, IL-8 and C-reactive protein. We determined associations between nutrition and infection exposures and cord cytokine elevation (≥75% vs. <75%) using logistic regression, adjusting for confounders. One-third of mothers were underweight (BMI < 18.5 kg/m2) at enrollment. Antenatal and intrapartum infections were observed among 4.8% and 15.9% of the sample, respectively. Low pregnancy intakes of B vitamins (B1, B2, B3, B6, B9 (folate)), fat-soluble vitamins (D, E), iron, zinc, and linoleic acid (lowest vs. middle tertile) were associated with higher risk of inflammation, particularly IL-8. There was a non-significant trend of increased risk of IL-8 and IL-6 elevation with history of ante-and intrapartum infections, respectively. In Bangladesh, improving micronutrient intake and preventing pregnancy infections are targets to reduce fetal systemic inflammation and associated adverse neurodevelopmental outcomes.

5-hydroxymethylcytosine is dynamically regulated during forebrain organoid development and aberrantly altered in Alzheimer's disease.

SUMMARY5-hydroxymethylcytosine (5hmC) undergoes dynamic changes during mammalian brain development, and its dysregulation is associated with Alzheimer’s disease (AD). The dynamics of 5hmC during early human brain development and how they contribute to AD pathologies remain largely unexplored. We generate 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients. Stage-specific differentially hydroxymethylated regions demonstrate an acquisition or depletion of 5hmC modifications across developmental stages. Additionally, genes concomitantly increasing or decreasing in 5hmC and gene expression are enriched in neurobiological or early developmental processes, respectively. Importantly, our AD organoids corroborate cellular and molecular phenotypes previously observed in human AD brains. 5hmC is significantly altered in developmentally programmed 5hmC intragenic regions in defined fetal histone marks and enhancers in AD organoids. These data suggest a highly coordinated molecular system that may be dysregulated in these early developing AD organoids.