Self‐organized developmental patterning and differentiation in cerebral organoids

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self‐organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso‐ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso‐ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.     

Blood vessel formation in cerebral organoids formed from human embryonic stem cells

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.     

Modelling neurodegenerative diseases with 3D brain organoids

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post‐mortem brain tissue, non‐human primate tissue, or in vitro two‐dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three‐dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.     

Knowledge graphs of ethical concerns of cerebral organoids

ObjectivesThe rapid development of cerebral organoid technology and the gradual maturity of cerebral organoids highlight the necessity of foresighted research on relevant ethical concerns. We employed knowledge graphs and conducted statistical analysis with CiteSpace for a comprehensive analysis of the status quo of the research on the ethical concerns of cerebral organoids from a bibliometric perspective.Materials and MethodsWe performed a statistical analysis of published papers on cerebral organoid ethics, keyword co‐occurrence graph, literature co‐citation and knowledge clustering graph to examine the status of the ethics research, internal relationship between technological development and ethical research, and ethical concerns of the academia. Finally, we used a keyword time zone graph and related statistics to analyze and predict the trends and popular topics of future cerebral organoids ethics research.ResultsWe demonstrated that although the ethical concerns of cerebral organoids have long been discussed, it was not until 2017 that the ethical issues began to receive more attention, when cerebral organoids were gradually mimicking the human brain more closely and increasingly being combined with chimera research. The recent key ethical concerns are primarily divided into three categories: concerns that are common in life sciences, specific to cerebral organoids, and present in cross‐fields. These increasing ethical concerns are inherently related to the continual development of technology. The analysis pointed out that future research should focus on the ethical concerns of consciousness that are unique to cerebral organoids, ethical concerns of cross‐fields, and construction and improvement of legislative and regulatory systems.ConclusionsAlthough research on cerebral organoids can benefit the biomedicine field, the relevant ethical concerns are significant and have received increasing attention, which are inherently related to the continual development of technology. Future studies in ethics regarding cerebral organoid research should focus on the ethical concerns of consciousness, and cross‐fields, as well as the improvement of regulatory systems.

We performed a statistical analysis of published papers on cerebral organoid ethics, keyword co‐occurrence graph, literature co‐citation and knowledge clustering graph to examine the status of the ethics research. We also used a keyword time zone graph and related statistics to analyse and predict the trends and popular topics of future cerebral organoids ethics research.     

Unlimited in vitro expansion of adult bi‐potent pancreas progenitors through the Lgr5/R‐spondin axis

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt‐agonistic R‐spondins (RSPOs). Intestinal, stomach and liver Lgr5⁺ stem cells grow in 3D cultures to form ever‐expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1‐based cultures, and develop into budding cyst‐like structures (organoids) that expand five‐fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi‐potentiality.     

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.     

Maternal sevoflurane exposure induces temporary defects in interkinetic nuclear migration of radial glial progenitors in the fetal cerebral cortex through the Notch signalling pathway

ObjectivesThe effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure.MethodsPregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA‐Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real‐time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)‐derived 3D cerebral organoids.ResultsMaternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA‐Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM.ConclusionsMaternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long‐term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC‐derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.     

Evolution of ASPM coding variation in apes and associations with brain structure in chimpanzees

Studying genetic mechanisms underlying primate brain morphology can provide insight into the evolution of human brain structure and cognition. In humans, loss‐of‐function mutations in the gene coding for ASPM (Abnormal Spindle Microtubule Assembly) have been associated with primary microcephaly, which is defined by a significantly reduced brain volume, intellectual disability and delayed development. However, less is known about the effects of common ASPM variation in humans and other primates. In this study, we characterized the degree of coding variation at ASPM in a large sample of chimpanzees (N = 241), and examined potential associations between genotype and various measures of brain morphology. We identified and genotyped five non‐synonymous polymorphisms in exons 3 (V588G), 18 (Q2772K, K2796E, C2811Y) and 27 (I3427V). Using T1‐weighted magnetic resonance imaging of brains, we measured total brain volume, cerebral gray and white matter volume, cerebral ventricular volume, and cortical surface area in the same chimpanzees. We found a potential association between ASPM V588G genotype and cerebral ventricular volume but not with the other measures. Additionally, we found that chimpanzee, bonobo, and human lineages each independently show a signature of accelerated ASPM protein evolution. Overall, our results suggest the potential effects of ASPM variation on cerebral cortical development, and emphasize the need for further functional studies. These results are the first evidence suggesting ASPM variation might play a role in shaping natural variation in brain structure in nonhuman primates.     

The development of an in vitro cerebral organoid model for investigating the pathomolecular mechanisms associated with the central nervous system involvement in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disorder caused by mutations in the thymidine phosphorylase gene (TYMP), leading to secondary aberrations to the mitochondrial genome. The disease is characterised by gastrointestinal dysmotility, sensorimotor peripheral neuropathy and leukoencephalopathy. The understanding of the molecular mechanisms that underlie the central nervous system (CNS) is hindered by the lack of a representative disease model; to address this we have developed an in vitro 3-D cerebral organoid of MNGIE. Induced pluripotent stem cells (iPSCs) generated from peripheral blood mononuclear cells (PBMCs) of a healthy control and a patient with MNGIE were characterised to ascertain bona fide pluripotency through the evaluation of pluripotency markers and the differentiation to the germ layers. iPSC lines were differentiated into cerebral organoids. Thymidine phosphorylase expression in PBMCs, iPSCs and Day 92 organoids was evaluated by immunoblotting and intact organoids were sampled for histological evaluation of neural markers. iPSCs demonstrated the expression of pluripotency markers SOX2 and TRA1-60 and the plasticity to differentiate into the germ layers. Cerebral organoids stained positive for the neural markers GFAP, O4, Tuj1, Nestin, SOX2 and MBP. Consistent with the disease phenotypes, MNGIE cells did not display thymidine phosphorylase expression whereas control PBMCs and Day 92 organoids did. Remarkably, control iPSCs did not stain positive for thymidine phosphorylase. We have established for the first time a MNGIE iPSC line and cerebral organoid model, which exhibited the expression of cells relevant to the study of the disease, such as neural stem cells, astrocytes and myelinating oligodendrocytes.     

Trends and challenges in modeling glioma using 3D human brain organoids

The human brain organoids derived from pluripotent cells are a new class of three-dimensional tissue systems that recapitulates several neural epithelial aspects. Brain organoids have already helped efficient modeling of crucial elements of brain development and disorders. Brain organoids’ suitability in modeling glioma has started to emerge, offering another usefulness of brain organoids in disease modeling. Although the current state-of-the organoids mostly reflect the immature state of the brain, with their vast cell diversity, human brain-like cytoarchitecture, feasibility in culturing, handling, imaging, and tractability can offer enormous potential in reflecting the glioma invasion, integration, and interaction with different neuronal cell types. Here, we summarize the current trend of employing brain organoids in glioma modeling and discuss the immediate challenges. Solving them might lay a foundation for using brain organoids as a pre-clinical 3D substrate to dissect the glioma invasion mechanisms in detail.Subject terms: Cancer stem cells, CNS cancer     

R‐spondin 1 and noggin facilitate expansion of resident stem cells from non‐damaged gallbladders

Pioneering studies within the last few years have allowed the in vitro expansion of tissue‐specific adult stem cells from a variety of endoderm‐derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R‐spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5‐positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non‐regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R‐spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder‐derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride‐treated mice. Human gallbladders were also a source of organoid‐forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.     

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

Human pluripotent stem cells (hPSCs) have shown the ability to self-organize into different types of neural organoids (e.g., whole brain organoids, cortical spheroids, midbrain organoids etc.) recently. The extrinsic and intrinsic signaling elicited by Wnt pathway, Hippo/Yes-associated protein (YAP) pathway, and extracellular microenvironment plays a critical role in brain tissue morphogenesis. This article highlights recent advances in neural tissue patterning from hPSCs, in particular the role of Wnt pathway and YAP activity in this process. Understanding the Wnt-YAP interactions should provide us the guidance to predict and modulate brain-like tissue structure through the regulation of extracellular microenvironment of hPSCs.     

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

AbstractUnderstanding 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.

Subject terms: Neuroscience, Neurological disorders

     

Dispersion of the neurons expressing layer specific markers in the reeler brain

Neurons with similar functions including neuronal connectivity and gene expression form discrete condensed structures within the vertebrate brain. This is exemplified within the circuitry formed by the cortical layers and the neuronal nuclei. It is well known that the Reelin protein is required for development of these neuronal structures in rodents and human, but the function of Reelin remains controversial. In this report, we used “layer‐specific markers” of the cerebral cortex to carry out detailed observations of spatial distribution of the neuronal subpopulations in the brain of the Reelin deficient mouse, reeler. We observed a spatially dispersed expression of the markers in the reeler cerebral cortex. These markers are expressed also in other laminated and non‐laminated structures of brain, in which we observed similar abnormal gene expression. Our observations suggest that neurons within the brain structures (such as the layers and the nuclei), which normally exhibit condensed distribution of marker expressions, loosen their segregation or scatter by a lack of Reelin.     

Inhibitory Effect of Silkworm-Extract(SE) on Monoamine Oxidase Activity in Vitro and in Vivo

In vitro.MAO‐A activity was inhibited 16‐25%, and MAO‐B activity was inhibited 20‐50% by SE treatment (12.5, 25 and 50 μg), In vivo.male C57BL/6 mice Received intraperitoneal injection of SE (20 mg/kg/day) for 14 days. The results showed that MAO‐A activity of pre‐SE‐treatment mice brain was inhibited in whole brain, cerebral cortex, substantia nigra. MAO‐B activity of pre‐SE‐treatment mice brain was inhibited in substantia nigra and cerebellum than saline‐treated control group. These results suggest that SE inhibits MAO activity in vivo.which would be expected to results in anti‐depressive and neuroprotective effects.     

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.     

Effect of larval brain extracts derived from three swallowtail butterflies,Papilio helenus L., P. machaon L. and P. memnon L., on seasonal morph development of P. xuthus L. (Lepidoptera: Papilionidae)

Adults of the three papilionid butterflies, Papilio helenus L., Papilio machaon L. and Papilio memnon L., exhibit seasonal diphenism comprising spring and summer morphs. To elucidate the physiological mechanism underlying seasonal morph development in papilionid butterflies, we investigated whether a cerebral factor showing summer‐morph‐producing hormone (SMPH) activity is present in the brain of three Papilio species using an assay system with chilled male short‐day pupae of P. xuthus L. When 2% NaCl extracts derived from 20 larval brains of the three species were injected into abdomens of chilled male short‐day pupae of P. xuthus, all recipients destined to develop into spring‐morph adults developed into summer‐ and intermediate‐morph adults. On the other hand, all recipients injected with distilled water as a control developed into spring‐morph adults. These results indicate that a cerebral factor showing SMPH activity is present in the larval brain of the three Papilio species. Additionally, all recipients injected with 2% NaCl extracts derived from 20 adult brains of Bombyx mori L. also developed into summer‐ and intermediate‐morph adults. The results revealed that SMPH or a cerebral factor showing SMPH activity is widely distributed among lepidopteran insects.