Neurodifferentiation/Stem Cell Unit

神经分化/干细胞单位

• 批准号: 10708659
• 负责人: David Wang
• 金额: $ 89.24万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708659
• 关键词: 2019-nCoV; 3-Dimensional; Adult; Astrocytes; Biological Assay; Brain; C9ORF72; CD34 gene; Cell Proliferation; Cells; Coculture Techniques; Communicable Diseases; DNA Damage; DNA Repair; Development; Diagnosis; Disease; Elements; Endogenous Retroviruses; Endothelial Cells; Epigenetic Process; Event; Extramural Activities; Gene Mutation; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Glioma; Goals; Growth; Human; In Vitro; Inflammation; Inflammatory; Manuscripts; Methods; Microfluidics; Microglia; Modeling; Molecular Target; Motor Neurons; Mutation; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Neurodevelopmental Disorder; Neurons; Organoids; Pathogenesis; Pathway interactions; Patients; Pediatric Hospitals; Pericytes; Peripheral; Plasmids; Protocols documentation; Publications; Reaction; Reporting; Research Personnel; Role; Sampling; Techniques; Therapeutic Intervention; Training Technics; Tumorigenicity; United States National Institutes of Health; Virus; Virus Diseases; Yang; ZIKV infection; Zika Virus; base; cell type; confocal imaging; cytokine; imaging system; improved; induced pluripotent stem cell; neoplastic cell; nerve stem cell; nervous system disorder; neural; neural model; neurodevelopment; neurogenesis; neuroimmunology; neurotoxicity; novel; preclinical study; research and development; screening; single-cell RNA sequencing; stem cell biomarkers; stem cell model; stem cells; stemness; therapeutic development; three-dimensional modeling; transcriptome sequencing; translational neuroscience; tumorigenesis

Specific aim 1: To develop in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We expended our capability to derive 3D neural organoids to mimic human brain. Using improved clearance techniques and advanced confocal imaging system, we confirmed that pericytes can be produced along endothelial cells and neural cells in the 3D organoid spontaneously with sequential induction media, making the model perfect for virus infection studies, as pericytes have been reported as a main target by viruses including SARS-CoV2. In another 3D model with incorporating CD34 cells, we treated them with cytokines to promote microglial differentiation and used scRNA-Seq analysis to study the resulting microglial cells. We found that although the treatment of cytokines is not necessary for producing microglia, it did increase the numbers of microglia compared to organoids without cytokine stimulation. When the organoids were treated with LPS, which induced inflammatory reactions by increasing transcriptions of certain microglial genes in the organoids, indicating the differentiated microglia in the organoids are functional. A manuscript is prepared for publication based on the results. Specific aim 2: To study the roles of HERV-K on brain development and tumorigenicity. We supported Dr. Ashish Shah on studying the mechanism of HERV-K on the pathogenesis of glioma. We transfected astroglia with plasmid containing HERV-K and detected higher level of gene transcription responsible for stemness. This result indicates that marker for stem cells is activated by HERV-K and maybe the mechanism underlying its effect on tumorigenesis. We are further studying HERV-K effect in cell proliferation and tumorigenesis using 3D models. Specific aim 3: To study the association of HERV-K and ALS. We differentiated motor neurons from four sets of C9orf72 and control iPSCs and did RNA-Seq analysis. The results showed top differential pathways were tumorigenesis related. As we have found that in C9orf72 motor neurons, there was increased DNA repairing element followed by HERV-K activation, this result imply that DNA damaging events in motor neurons leads to HERV-K Env activation, which may reverse the neurons to a status more similar to stem or tumor cells, causing motor neuron malfunction. Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We are continuing to collaborate with other investigators by providing material support and technique trainings of the iNSC/iPSC generation and 3D modeling. We are working with Dr. In-Hong Yang in UNC to develop protocol to culture motor neurons in a microfluidic chamber. We collaborated with Dr. Kousa from National Childrens Hospital to study the pathogenesis of ZIKA virus on human brain development. We derived neural stem cells from targeting and control iPSC cells and found ZIKA virus infection was enhanced significantly by a target malfunction. We also helped Dr. Farinaz Safavi to generate 3D brain organoids from iPSCs with an mutative gene. Our result showed the mutation decreased the growth of the derived 3D organoids significantly compared to corrected iPSCs.

具体目的1：建立成人外周血CD34+细胞来源的三维脑器官模型，用于研究神经发育、变性及脑内感染性疾病。我们扩大了我们的能力，以获得3D神经器官来模仿人类的大脑。利用改进的清除技术和先进的共聚焦成像系统，我们证实了在序贯诱导介质中，周细胞可以自发地沿着3D器官中的内皮细胞和神经细胞产生，这使得该模型非常适合于病毒感染的研究，因为周细胞已经被报道为包括SARS-CoV2在内的病毒的主要靶点。在另一个包含CD34细胞的3D模型中，我们用细胞因子处理它们以促进小胶质细胞分化，并使用scRNA-Seq分析来研究由此产生的小胶质细胞。我们发现，尽管细胞因子的治疗不是产生小胶质细胞所必需的，但与没有细胞因子刺激的器官相比，它确实增加了小胶质细胞的数量。当内毒素处理器官时，通过增加器官中某些小胶质基因的转录而引起炎症反应，表明器官中分化的小胶质细胞是有功能的。根据结果准备了一份手稿以供出版。 特定目的2：研究Herv-K在脑发育和肿瘤发生中的作用。我们支持Ashish Shah博士研究Herv-K在胶质瘤发病机制中的作用。我们用含有HERV-K基因的载体转染星形胶质细胞，检测到与茎的形成有关的基因转录水平较高。这一结果表明，HERV-K激活了干细胞标志物，这可能是其影响肿瘤发生的机制。我们正在利用3D模型进一步研究Herv-K在细胞增殖和肿瘤发生中的作用。 具体目的3：研究HERV-K与ALS的相关性。我们从四组C9orf72和对照IPSCs中分化出运动神经元，并进行RNA-Seq分析。结果表明，顶端分化途径与肿瘤的发生有关。我们发现，在C9orf72运动神经元中，DNA修复元件的增加伴随着Herv-K的激活，这一结果表明运动神经元中的DNA损伤事件导致了Herv-K Env的激活，这可能使神经元逆转到更类似于干细胞或肿瘤细胞的状态，导致运动神经元功能障碍。 具体目标4：使用我们的模型和方法促进神经疾病的研究和治疗发展。我们继续与其他研究人员合作，为iNSC/IPSC的生成和3D建模提供物质支持和技术培训。我们正在与北卡罗来纳大学的杨英宏博士合作，制定在微流体室中培养运动神经元的方案。我们与国家儿童医院的库萨博士合作，研究寨卡病毒对人脑发育的致病机制。我们从靶向和对照IPSC细胞中提取神经干细胞，发现目标故障显著增强了寨卡病毒的感染。我们还帮助Farinaz Safavi博士从带有突变基因的iPSCs中生成了3D脑器官。我们的结果表明，与校正后的IPSCs相比，该突变显著降低了衍生的3D有机物的生长。