Explore the vast range of titles available.

Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14-84 d to complete (depending on the type of brain region-specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

A high-throughput screen of preclinical, investigational and FDA-approved drugs identifies compounds that possess antiviral and neuroprotective effects against Zika virus infection in human neural progenitor cells and astrocytes. In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.

Spatial molecular profiling has provided biomedical researchers valuable opportunities to better understand the relationship between cellular localization and tissue function. Effectively modeling multimodal spatial omics data is crucial for understanding tissue complexity and underlying biology. Furthermore, improvements in spatial resolution have led to the advent of technologies that can generate spatial molecular data with subcellular resolution, requiring the development of computationally efficient methods that can handle the resulting large-scale datasets. MISO (MultI-modal Spatial Omics) is a versatile algorithm for feature extraction and clustering, capable of integrating multiple modalities from diverse spatial omics experiments with high spatial resolution. Its effectiveness is demonstrated across various datasets, encompassing gene expression, protein expression, epigenetics, metabolomics and tissue histology modalities. MISO outperforms existing methods in identifying biologically relevant spatial domains, representing a substantial advancement in multimodal spatial omics analysis. Moreover, MISO’s computational efficiency ensures its scalability to handle large-scale datasets generated by subcellular resolution spatial omics technologies. MISO (MultI-modal Spatial Omics) integrates two or more spatial omics modalities, despite differences in data quality and spatial resolution for improved feature extraction and clustering to reveal biologically meaningful tissue organization.

Spatial molecular profiling has provided biomedical researchers valuable opportunities to better understand the relationship between cellular localization and tissue function. Effectively modeling multi-modal spatial omics data is crucial for understanding tissue complexity and underlying biology. Furthermore, improvements in spatial resolution have led to the advent of technologies that can generate spatial molecular data with sub-cellular resolution, requiring the development of computationally efficient methods that can handle the resulting large-scale datasets. MISO (MultI-modal Spatial Omics) is a versatile algorithm for feature extraction and clustering, capable of integrating multiple modalities from diverse spatial omics experiments with high spatial resolution. Its effectiveness is demonstrated across various datasets, encompassing gene expression, protein expression, epigenetics, metabolomics, and tissue histology modalities. MISO outperforms existing methods in identifying biologically relevant spatial domains, representing a significant advancement in multi-modal spatial omics analysis. Moreover, MISO’s computational efficiency ensures its scalability to handle large-scale datasets generated by sub-cellular resolution spatial omics technologies. MISO (MultI-modal Spatial Omics) integrates two or more spatial omic modalities, despite differences in data quality and spatial resolution for improved feature extraction and clustering to reveal biologically meaningful tissue organization.

The current outbreak of Zika virus-associated diseases in South America and its threat to spread to other parts of the world has emerged as a global health emergency. Insights from cell and animal models to under- stand how Zika virus causes severe birth defects may lead to treatments and prevention of these diseases.

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of amyloid‐beta and hyperphosphorylated tau (P‐tau) proteins in the brain. P‐tau accumulates in neurons and is strongly associated with AD severity and affected brain regions. However, only a subset of neurons in AD exhibit tau pathology. The molecular mechanisms behind heterogeneous tau pathology and how it contributes to AD are not well understood. Method We developed a fluorescence‐activated nuclear sorting (FANS) method to separate P‐tau+ neuronal nuclei from P‐tau‐ neuronal nuclei from the same brain tissue. To validate the specificity of P‐tau+ neurons, we mixed non‐AD control tissue with AD tissue and examined the origin of neuronal nuclei based on their genotype. We also subjected nuclei with P‐tau signal from FANS to immunofluorescence microscopy to examine the morphology and localization of P‐tau aggregates. Results We observed disease‐specific P‐tau signal for nuclei in advanced AD (Braak stage V‐VI) cases. These P‐tau+ nuclei are highly distinguished from the P‐tau‐free nuclei. Confocal immunofluorescence microscopy showed P‐tau adherent to the outside of nuclei that exhibited P‐tau signal by FANS. We demonstrated that the nuclei sorting method based on P‐tau levels can highly enrich for P‐tau+ nuclei (>380 fold) and has a high accuracy of 98% based on the mixing experiments. In addition, we were able to obtain high‐quality single‐cell genome amplification for P‐tau‐sorted nuclei using primary template‐directed amplification (PTA), permitting single‐nucleus genome interrogation. Conclusion We developed a highly efficient method using fluorescence‐activated nuclear sorting (FANS) to separate the population of P‐tau+ neuronal nuclei in AD brains. This method allows interrogation of human neuronal nuclei based on single‐cell tau pathology and enables single‐cell genomic studies of heterogeneous tau pathology in AD, toward insight into disease pathogenesis and therapeutic targets.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of amyloid-beta and hyperphosphorylated tau (P-tau) proteins in the brain. P-tau accumulates in neurons and is strongly associated with AD severity and affected brain regions. However, only a subset of neurons in AD exhibit tau pathology. The molecular mechanisms behind heterogeneous tau pathology and how it contributes to AD are not well understood. We developed a fluorescence-activated nuclear sorting (FANS) method to separate P-tau+ neuronal nuclei from P-tau- neuronal nuclei from the same brain tissue. To validate the specificity of P-tau+ neurons, we mixed non-AD control tissue with AD tissue and examined the origin of neuronal nuclei based on their genotype. We also subjected nuclei with P-tau signal from FANS to immunofluorescence microscopy to examine the morphology and localization of P-tau aggregates. We observed disease-specific P-tau signal for nuclei in advanced AD (Braak stage V-VI) cases. These P-tau+ nuclei are highly distinguished from the P-tau-free nuclei. Confocal immunofluorescence microscopy showed P-tau adherent to the outside of nuclei that exhibited P-tau signal by FANS. We demonstrated that the nuclei sorting method based on P-tau levels can highly enrich for P-tau+ nuclei (>380 fold) and has a high accuracy of 98% based on the mixing experiments. In addition, we were able to obtain high-quality single-cell genome amplification for P-tau-sorted nuclei using primary template-directed amplification (PTA), permitting single-nucleus genome interrogation. We developed a highly efficient method using fluorescence-activated nuclear sorting (FANS) to separate the population of P-tau+ neuronal nuclei in AD brains. This method allows interrogation of human neuronal nuclei based on single-cell tau pathology and enables single-cell genomic studies of heterogeneous tau pathology in AD, toward insight into disease pathogenesis and therapeutic targets.

Brain organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate brain development. High cost, variability and heterogeneity currently limit the broad application of existing brain organoid technologies. My thesis work focuses on developing methods to generate forebrain organoids from human induced pluripotent stem cells (hiPSCs) using a custom-designed spinning bioreactor. Forebrain organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a well-defined outer radial glial cell (RGCs) layer resembling the human outer subventricular zone. An immediate application for brain organoids is to address the global health emergency of Zika virus (ZIKV)-induced microcephaly. Exposure of ZIKV to forebrain organoids revealed preferential, productive infection of RGCs. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell layer volume and reduced overall organoid size, resembling microcephaly. Our forebrain organoids and SpinΩ bioreactor provide an accessible and versatile platform for modeling human brain development and disease, and for compound testing, including potential ZIKV antiviral drugs. Results from our ZIKV experiments reveal cellular mechanisms of ZIKV-induced microcephaly and have implications for therapeutic development. Another limitation of current organoid methods is interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture representative of late developmental stages. In order to generate more mature organoids, we developed the sliced neocortical organoid (SNO) method, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustains neurogenesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third-trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/β-Catenin signaling in regulating cortical neuron fate specification, which is disrupted by a psychiatric disorder-associated genetic mutation in patient iPSC-derived SNOs. These results demonstrate the utility of SNOs as a model for investigating previously inaccessible human-specific late-stage cortical development and disease-relevant mechanisms. Together, my work has tremendously advanced brain organoid technologies, and provided a novel platform to elucidate the underlying mechanisms and potential therapeutic strategies for neurodevelopmental diseases.