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The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region–specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell–cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3–4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.A protocol is described for generating human brain assembloids and performing viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics with calcium imaging and electrophysiological recordings to model neural circuits.

Cortico-striatal projections are critical components of forebrain circuitry that regulate motivated behaviors. To enable the study of the human cortico-striatal pathway and how its dysfunction leads to neuropsychiatric disease, we developed a method to convert human pluripotent stem cells into region-specific brain organoids that resemble the developing human striatum and include electrically active medium spiny neurons. We then assembled these organoids with cerebral cortical organoids in three-dimensional cultures to form cortico-striatal assembloids. Using viral tracing and functional assays in intact or sliced assembloids, we show that cortical neurons send axonal projections into striatal organoids and form synaptic connections. Medium spiny neurons mature electrophysiologically following assembly and display calcium activity after optogenetic stimulation of cortical neurons. Moreover, we derive cortico-striatal assembloids from patients with a neurodevelopmental disorder caused by a deletion on chromosome 22q13.3 and capture disease-associated defects in calcium activity, showing that this approach will allow investigation of the development and functional assembly of cortico-striatal connectivity using patient-derived cells. Organoids representing the human striatum are generated and used to build circuits in cortico-striatal assembloids.

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease 1 – 5 . However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered. Human stem cell-derived cortical organoids transplanted into rats mature and integrate into sensory and motivation circuits to influence behaviour.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease. A human stem cell–derived model helps to uncover neuronal phenotypes associated with genetic forms of neuropsychiatric disease.

3D object retrieval is a hot research field in computer vision and multimedia analysis domain. Since the appearance feature and points of view of 3D objects are very different, thus, the distribution of the training set and test set are variant which is very suitable for transfer learning or cross-domain learning. In the transfer learning or cross-domain learning, the feature extraction is very important which should have good robust for different domains. Thus, in this work, we pay attention to the feature extraction of 3D objects. So far, different feature representations and object retrieval approaches have been proposed. Among them, view-based deep learning retrieval methods achieve state-of-the-art performance, but the existing deep learning retrieval methods only simply use a deep neural network to extract features from each view and directly obtain the view-level shape descriptors without utilizing the spatial relationship between the views. In order to mine the spatial relationship among different views and obtain more discriminative 3D shape descriptors, in this work, 3D object retrieval based on non-local graph neural networks (NGNN) is proposed. In detail, the residual network is firstly utilized as the infrastructure, and then the non-local structure is embedded in the resnet to learn the intrinsic relationship between the views. Finally, the view pooling layer is employed to further fuse the information from different views, and obtain the discriminate feature for the 3D object. Experimental results on two public MVRED and NTU 3D datasets show that the non-local graph network is very efficient for exploring the latent relationship among different views, and the performance of NGNN significantly outperforms state-of-the-art approaches whose improvement can reaches 12.4%-22.7% on ANMRR.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7 , we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Antisense oligonucleotides effectively decrease the inclusion of exon  8A of CACNA1C in human cells both in vitro and in rodents transplanted with human brain organoids, and a single intrathecal administration rescued both calcium changes and in vivo dendrite morphology of patient neurons.

Studying the early function of essential genes is an Here, we established a method for rapidly inducing important and challenging problem in developmental biology. CRISPR-Cas9-mediated mutations in one blastomere of two- cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection （2CC）, to study the in vivo function of essential （or unknown） genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of in- terest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different develop- mental stages, offering internal control for phenotypic analyses of mutant cells. Using this method, we identified nov- el functions of the essential gene Tet3 in regulating excitatory and inhibitory synaptic transmission in the developing mouse cerebral cortex. By generating chimeric mutant mice, the 2CC method allows for the rapid screening of gene function in multiple tissues and cell types in founder chimeric mice, significantly expanding the current armamentar- ium of genetic tools.

Abnormalities in crosstalk between the thalamus and the cerebral cortex are thought to lead to severe neuropsychiatric disorders, such as epilepsy and psychotic disorders. Pathogenic variants in the CACNA1G gene, which encodes the α1G subunit of the thalamus-enriched T-type voltage-gated calcium channel CaV3.1, are associated with absence seizures, intellectual disability, and schizophrenia, but the cellular and circuit level consequences of these genetic variants in humans remain unknown. Here, we developed an in vitro human assembloid model of the thalamocortical pathway to systematically dissect the contribution of genetic variants in T-type calcium channels. We discovered that a CACNA1G variant (M1531V) associated with seizures led to hypersynchronous activity in the thalamus and in cortical neurons in thalamo-cortical assembloids. In contrast, CACNA1G loss, which has been associated with risk of schizophrenia, resulted in abnormal thalamocortical connectivity that was related to both increased spontaneous thalamic activity and aberrant thalamic axonal projections. Taken together, these results illustrate the utility of organoid and assembloid systems for interrogating human genetic disease risk variants at both cellular and circuit level.Competing Interest StatementStanford University has filed a provisional patent application covering the generation of multi-region assembloids. M.H.P. is on the Board of Directors and holds equity in Graphite Bio. M.H.P. serves on the SAB of Allogene Tx and is an advisor to Versant Ventures.

Galangin, a natural flavonoid product derived from the root of galangal, is emerging as a promising anticancer agent against multiple cancers. Yet, whether it also has antitumor effects on cholangiocarcinoma (CCA) and the underlying mechanism is still unknown. Herein, we demonstrate that galangin exhibits multiple antitumor effects on CCA cells including decreases cell viability; inhibits proliferation, migration, and invasion; and induces apoptosis. Moreover, those phenotypic changes are associated with downregulated microRNA-21 (miR-21) expression. To support, overexpression of miR-21 blocks galangin-mediated antisurvival and metastasis effects on CCA cells. Mechanically, galangin increases the expression of phosphatase and tensin homolog (PTEN), a direct target of miR-21, resulting in decreased phosphorylation of AKT, a protein kinase which plays a critical role in controlling survival and apoptosis. In contrast, overexpression of miR-21 abrogates galangin-regulated PTEN expression and AKT phosphorylation. Taken together, these findings indicate that galangin inhibits CCA cell proliferation and metastasis and induces cell apoptosis through a miR-21-dependent manner, and galangin may provide a novel potential therapeutic adjuvant to treat CCA.

Aim & Hypothesis: The primary objective of this prospective clinical study was to compare the dimensional changes of peri-implant buccal bone between grafted and native bone during the submerged healing period. We hypothesized the dimensional changes of peri-implant buccal bone were greater in implants placed in grafted bone than native bone. Secondarily, the influence of initial buccal bone thickness on the stability of peri-implant buccal bone was also assessed.Materials & Methods: Patients receiving dental implants were enrolled based on three different healing conditions: 1) augmented alveolar ridges, 2) grafted sockets, and 3) pristine bone. The thickness of peri-implant buccal bone was measured during implant surgery at implant shoulder and 2, 4 and 6 mm apical to the shoulder using a manufactured measuring device. After 3–4 months of submerged healing, the second stage implant surgery was performed, and the above measurements were repeated. Mann-Whitney U test was used to compare the differences in vertical and horizontal bone changes. Spearman rank correlation was applied to investigate the correlation between initial buccal bone thickness and peri-implant buccal bone change.Results: A total of eighteen patients–two in GBR group, eight in RP group and eight in Control group–were enrolled in this study. For the horizontal change of buccal bone, RP group showed statistically significant higher horizontal bone resorption than control group at 2mm apical to the implant shoulder level (p = 0.03), and there were no statistically significant differences at shoulder and 4, 6 mm apical to the shoulder. No differences were detected in the vertical changes of peri-implant buccal bone. The initial buccal bone thickness had a moderate negative linear correlation with the peri-implant buccal bone horizontal change at the implant shoulder level. (Rs: −0.65, p = 0.02)Conclusion: To our knowledge, this is the first prospective clinical study to compare peri-implant buccal bone dimensional changes in three different healing conditions. Our current sample size has a limited power to detect more statistically significant differences. However, the preliminary results showed the trend that implants placed in preserved ridges have more horizontal bone resorption than native bone at 2mm apical to the implant shoulder level during the submerged healing period. Additionally, the amount of peri-implant buccal bone horizontal resorption was inversely associated with initial buccal thickness at the implant shoulder level.