Generation of Human Blood Vessel and Vascularized Cerebral Organoids.

Brain organoids have been widely used to study diseases and the development of the nervous system. Many reports have investigated the application of brain organoids, but most of these models lack vascular structures, which play essential roles in brain development and neurological diseases. The brain and blood vessels originate from two different germ layers, making it difficult to induce vascularized brain organoids in vitro. We developed this protocol to generate brain-specific blood vessel and cerebral organoids and then fused them at a specific developmental time point. The fused cerebral organoids exhibited robust vascular network-like structures, which allows simulating the in vivo developmental processes of the brain for further applications in various neurological diseases. Key Features • Culturing vascularized brain organoids using human embryonic stem cells (hESCs). • The new approach generates not only neural cells and vessel-like networks but also brain-resident microglia immune cells in a single organoid.

Generation of vascularized brain organoids to study neurovascular interactions.

Brain organoids have been used to recapitulate the processes of brain development and related diseases. However, the lack of vasculatures, which regulate neurogenesis and brain disorders, limits the utility of brain organoids. In this study, we induced vessel and brain organoids, respectively, and then fused two types of organoids together to obtain vascularized brain organoids. The fused brain organoids were engrafted with robust vascular network-like structures and exhibited increased number of neural progenitors, in line with the possibility that vessels regulate neural development. Fusion organoids also contained functional blood-brain barrier-like structures, as well as microglial cells, a specific population of immune cells in the brain. The incorporated microglia responded actively to immune stimuli to the fused brain organoids and showed ability of engulfing synapses. Thus, the fusion organoids established in this study allow modeling interactions between the neuronal and non-neuronal components in vitro, particularly the vasculature and microglia niche.

Understanding how the organs form and how their cells behave is essential to finding the causes and treatment for developmental disorders, as well as understanding certain diseases. However, studying most organs in live animals or humans is technically difficult, expensive and invasive. To address this issue, scientists have developed models called 'organoids' that recapitulate the development of organs using stem cells in the lab. These models are easier to study and manipulate than the live organs. Brain organoids have been used to recapitulate brain formation as well as developmental, degenerative and psychiatric brain conditions such as microcephaly, autism and Alzheimer's disease. However, these brain organoids lack the vasculature (the network of blood vessels) that supplies a live brain with nutrients and regulates its development, and which has important roles in brain disorders. Partly due to this lack of blood vessels, brain organoids also do not develop a blood brain barrier, the structure that prevents certain contents of the blood, including pathogens, toxins and even certain drugs from entering the brain. These characteristics limit the utility of existing brain organoids. To overcome these limitations, Sun, Ju et al. developed brain organoids and blood vessel organoids independently, and then fused them together to obtain vascularized brain organoids. These fusion organoids developed a robust network of blood vessels that was well integrated with the brain cells, and produced more neural cell precursors than brain organoids that had not been fused. This result is consistent with the idea that blood vessels can regulate brain development. Analyzing the fusion organoids revealed that they contain structures similar to the blood-brain barrier, as well as microglial cells (immune cells specific to the brain). When exposed to lipopolysaccharide ­ a component of the cell wall of certain bacteria ­ these cells responded by initiating an immune response in the fusion organoids. Notably, the microglial cells were also able to engulf connections between brain cells, a process necessary for the brain to develop the correct structures and work normally. Sun, Ju et al. have developed a new organoid system that will be of broad interest to researchers studying interactions between the brain and the circulatory system. The development of brain-blood-barrier-like structures in the fusion organoids could also facilitate the development of drugs that can cross this barrier, making it easier to treat certain conditions that affect the brain. Refining this model to allow the fusion organoids to grow for longer times in the lab, and adding blood flow to the system will be the next steps to establish this system.

The CTNNBIP1-CLSTN1 fusion transcript regulates human neocortical development.

Fusion transcripts or RNAs have been found in both disordered and healthy human tissues and cells; however, their physiological functions in the brain development remain unknown. In the analysis of deposited RNA-sequence libraries covering early to middle embryonic stages, we identify 1,055 fusion transcripts present in the developing neocortex. Interestingly, 98 fusion transcripts exhibit distinct expression patterns in various neural progenitors (NPs) or neurons. We focus on CTNNBIP1-CLSTN1 (CTCL), which is enriched in outer radial glial cells that contribute to cortex expansion during human evolution. Intriguingly, downregulation of CTCL in cultured human cerebral organoids causes marked reduction in NPs and precocious neuronal differentiation, leading to impairment of organoid growth. Furthermore, the expression of CTCL fine-tunes Wnt/ß-catenin signaling that controls cortex patterning. Together, this work provides evidence indicating important roles of fusion transcript in human brain development and evolution.

TBC1D3 promotes neural progenitor proliferation by suppressing the histone methyltransferase G9a.

Genomic changes during human linage evolution contribute to the expansion of the cerebral cortex to allow more advanced thought processes. The hominoid-specific gene TBC1D3 displays robust capacity of promoting the generation and proliferation of neural progenitors (NPs), which are thought to contribute to cortical expansion. However, the underlying mechanisms remain unclear. Here, we found that TBC1D3 interacts with G9a, a euchromatic histone lysine N-methyltransferase, which mediates dimethylation of histone 3 in lysine 9 (H3K9me2), a suppressive mark for gene expression. TBC1D3 displayed an inhibitory role in G9a's histone methyltransferase activity. Treatment with G9a inhibitor markedly increased NP proliferation and promoted human cerebral organoid expansion, mimicking the effects caused by TBC1D3 up-regulation. By contrast, blockade of TBC1D3/G9a interaction to disinhibit G9a caused up-regulation of H3K9me2, suppressed NP proliferation, and impaired organoid development. Together, this study has demonstrated a mechanism underlying the role of a hominoid-specific gene in promoting cortical expansion.

Heterogeneous nuclear ribonucleoprotein A3 controls mitotic progression of neural progenitors via interaction with cohesin.

Cortex development is controlled by temporal patterning of neural progenitor (NP) competence with sequential generation of deep and superficial layer neurons, but underlying mechanisms remain elusive. Here, we report a role for heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) in regulating the division of early cortical NPs that mainly give rise to deep-layer neurons via direct neurogenesis. HNRNPA3 is expressed at high levels in NPs of mouse and human cortex at early stages, with a unique peri-chromosome pattern. Intriguingly, downregulation of HNRNPA3 caused chromosome disarrangement, which hindered normal separation of chromosomes during NP division, leading to mitotic delay. Furthermore, HNRNPA3 is associated with the cohesin-core subunit SMC1A and controls its association with chromosomes, implicating a mechanism for the role of HNRNPA3 in regulating chromosome segregation in dividing NPs. Hnrnpa3-deficient mice exhibited reduced cortical thickness, especially of deep layers. Moreover, downregulation of HNRNPA3 in cultured human cerebral organoids led to marked reduction in NPs and deep-layer neurons. Thus, this study has identified a crucial role for HNRNPA3 in NP division and highlighted the relationship between mitosis progression and early neurogenesis.

Single-cell transcriptomes reveal molecular specializations of neuronal cell types in the developing cerebellum.

The cerebellum is critical for controlling motor and non-motor functions via cerebellar circuit that is composed of defined cell types, which approximately account for more than half of neurons in mammals. The molecular mechanisms controlling developmental progression and maturation processes of various cerebellar cell types need systematic investigation. Here, we analyzed transcriptome profiles of 21119 single cells of the postnatal mouse cerebellum and identified eight main cell clusters. Functional annotation of differentially expressed genes revealed trajectory hierarchies of granule cells (GCs) at various states and implied roles of mitochondrion and ATPases in the maturation of Purkinje cells (PCs), the sole output cells of the cerebellar cortex. Furthermore, we analyzed gene expression patterns and co-expression networks of 28 ataxia risk genes, and found that most of them are related with biological process of mitochondrion and around half of them are enriched in PCs. Our results also suggested core transcription factors that are correlated with interneuron differentiation and characteristics for the expression of secretory proteins in glia cells, which may participate in neuronal modulation. Thus, this study presents a systematic landscape of cerebellar gene expression in defined cell types and a general gene expression framework for cerebellar development and dysfunction.

Organoids: An intermediate modeling platform in precision oncology.

Cancer harbors variable heterogeneity and plasticity. Thus far, our comprehension is greatly based on cell lines, organoids, and patient-derived tumor xenografts (PDTXs). Organoids are a three-dimensional in vitro culture platform constructed from self-organizing stem cells. They can almost accurately recapitulate tumor heterogeneity and microenvironment "in a dish," which surpass established cell lines and are not as expensive and time-consuming as PDTXs. As an intermediate model, tumor organoids are also used to study the fundamental issues of tumorigenesis and metastasis. They are specifically applied for drug testing and stored as "living biobanks." In this review, we highlight the translational applications of organoid technologies in tumor research and precision medicine, discuss the advantages and limitations compared with other mentioned methods, and provide our outlook on its future.

[Molecular and cellular mechanisms of cortical expansion and folding in brain development and evolution].

During the evolution from primates to humans, the size of cerebral cortex is increased by forming more gyri and sulci, which is believed to be highly associated with cognitive abilities and the basis of higher brain functions in humans. Accumulating lines of evidence have shown that the cortical size is regulated both by protein-coding genes and non-coding RNAs. In particular, the recently identified outer radial glial cells (oRGs) distributed in the outer subventricular zone (oSVZ) of gyrencephalic brains, have been considered to be important for cortical expansion and folding. This review summarizes recent progresses in the understanding of cortex expansion and discusses the potential molecular and cellular mechanisms of cortical folding.

The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice.

Cortical expansion and folding are often linked to the evolution of higher intelligence, but molecular and cellular mechanisms underlying cortical folding remain poorly understood. The hominoid-specific gene TBC1D3 undergoes segmental duplications during hominoid evolution, but its role in brain development has not been explored. Here, we found that expression of TBC1D3 in ventricular cortical progenitors of mice via in utero electroporation caused delamination of ventricular radial glia cells (vRGs) and promoted generation of self-renewing basal progenitors with typical morphology of outer radial glia (oRG), which are most abundant in primates. Furthermore, down-regulation of TBC1D3 in cultured human brain slices decreased generation of oRGs. Interestingly, localized oRG proliferation resulting from either in utero electroporation or transgenic expression of TBC1D3, was often found to underlie cortical regions exhibiting folding. Thus, we have identified a hominoid gene that is required for oRG generation in regulating the cortical expansion and folding.

JIP1 mediates anterograde transport of Rab10 cargos during neuronal polarization.

Axon development and elongation require strictly controlled new membrane addition. Previously, we have shown the involvement of Rab10 in directional membrane insertion of plasmalemmal precursor vesicles (PPVs) during neuronal polarization and axonal growth. However, the mechanism responsible for PPV transportation remains unclear. Here we show that c-Jun N-terminal kinase-interacting protein 1 (JIP1) interacts with GTP-locked active form of Rab10 and directly connects Rab10 to kinesin-1 light chain (KLC). The kinesin-1/JIP1/Rab10 complex is required for anterograde transport of PPVs during axonal growth. Downregulation of JIP1 or KLC or disrupting the formation of this complex reduces anterograde transport of PPVs in developing axons and causes neuronal polarity defect. Furthermore, this complex plays an important role in neocortical neuronal polarization of rats in vivo. Thus, this study has demonstrated a mechanism underlying directional membrane trafficking involved in axon development.

Laminin/ß1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization.

Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through ß1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.