Neurodifferentiation/Stem Cell Unit

神经分化/干细胞单位

基本信息

批准号：
10916077
负责人：
David Wang
金额：
$ 89.58万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
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/10916077
关键词：
2019-nCoV 3-Dimensional Adopted Adult Agreement Animal Model Astrocytes Binding Biological Assay Blood - brain barrier anatomy Brain CD34 gene COVID-19 test Cell Line Cells Clinical Coculture Techniques Collaborations Communicable Diseases Congresses Development Diagnosis Disease Endogenous Retroviruses Endothelial Cells Endothelium Epigenetic Process Equipment Extramural Activities Fibroblasts Future Generations Genes Genetic Genetic Transcription Genetic study Glioblastoma Goals Growth Home Hospitals Human In Vitro India Induced pluripotent stem cell derived neurons Infection Inflammation Inflammatory Macaca mulatta Methods Microfluidic Microchips Microglia Mitochondria Modeling Molecular Target Motor Neurons National Institute of Neurological Disorders and Stroke Neoplasm Metastasis Nerve Degeneration Neurodevelopmental Disorder Neurons Neurosciences Organoids Pathogenesis Patients Peripheral Peripheral Blood Mononuclear Cell Plasmids Pluripotent Stem Cells Predisposition Protocols documentation Publications Reporting Research Personnel Rhesus Role SARS-CoV-2 infection SARS-CoV-2 spike protein Sampling Shapes Skin Spain Stains Study models Techniques Therapeutic Intervention Training Transfection Tumorigenicity United States National Institutes of Health Variant Virion Virus Activation cell type density design dopaminergic neuron induced pluripotent stem cell motor neuron degeneration nerve stem cell nervous system disorder neural neural model neurodevelopment neurogenesis neuroimmunology neurotoxicity novel preclinical study reconstitution research and development screening stem cells stemness therapeutic development three-dimensional modeling translational neuroscience tumorigenesis

项目摘要

Specific aim 1: To develop in vitro 2D neuronal and 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We set up a new brain-on-the-chip microfluidic culture equipment into our unit, we now can reconstitute 3D brain cultures in a precise way by combining four types of essential cells in the brain: neurons, microglia, astroglia and endothelial cells. This approach, in additional to our existing 3D brain organoid models, is very useful to delineate the role of specific cell types in the neural development or neurodegeneration. In the preliminary study, we have used iPSC-derived neurons and commercially obtained primary astroglia, endothelial cells and a microglial cell line to make the mixed brain-on-the-chip successfully. We also optimized protocols to successfully generate microglia, astrocyte and endothelial cells from iPSCs and will use these methods to generate disease-specific brain-on-the-chip in the future study. In addition, we used the home developed endothelial incorporated 3D brain model to test if SARS-CoV2 can directly infect it. Using a pseudo-virus with SARS-CoV2 spike protein, we found that the SARS-CoV2 pseudo-virus can hardly infect the brain organoids, with only scarce weak positive cells along endothelial linings, not in the inner neuronal mass. This result is agreed to the clinical observation that SARS-CoV2 is seldom detected from neurons in patient brains. Although this is different with other reports which used traditional brain organoids to show high levels of SARS-CoV2 infections. These conflicts may be due to the existence of endothelial cells and blood-brain-barrier. We have been invited to present the model and findings in the upcoming 11th IBRO World Congress of Neuroscience, Granada, Spain. Specific aim 2: To study the roles of HERV-K on brain development and tumorigenicity. We continued the collaboration with Dr. Ashish Shah on studying the mechanism of HERV-K on the pathogenesis of glioblastoma. We used the human CD34-derived neural stem cells to generate a 3D model for astroglial growth. In transfected astroglia with plasmid containing HERV-K we detected higher level of gene transcription responsible for stemness and tumorigenesis and the organoids grow faster than controls with an irregular shape, indicating a potential for easier metastasis. These results contributed to a new publication. Specific aim 3: To study the association of HERV-K and motor neuron degeneration like ALS. We continued our study of retro virus activation and motor neuron degeneration. For functional study, we collaborated with Dr. InHong Yang from UNC to use mitotrackers to stain the mitochondria of motor neurons. Use this method, we counted mitochondrial density from both ALS neurons and controls. While our preliminary result indicated a difference of mitochondrial staining between the two groups, more studied using rigor differentiation conditions are needed to confirm the result. We also used specifically designed ASO targeting HERV-K Env to study the effect of 3D brain development and motor neuron degeneration, we observed that ASO resulted in slower growth of 3D organoids as expected. We also used an in vitro neural culture derived from rhesus skin fibroblasts to study its susceptibility of HERV-K infection, in collaboration with Dr. Dr. Catherine Demarino from SINS, NINDS. We found that NSC derived from Rhesus skin fibroblasts express HERV-K Env binding target and high titer of HERV-K virions infects the NSC, indicating Rhesus monkey could be used as the animal model for studying HERV-K infection and neurodegeneration. Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We Generated a new iPSC line from fibroblast of a patient with a specific FRMD4 gene variation, in collaborated with Dr. Maria Isis Atallah Gonzalez, CHUV. We trained Dr. Sanjay Yadav, a collaborator from AIIMS Raebareli, India on generating dopaminergic neurons from patient PBMCs and since generated 4 lines of iPSCs from PD patients. We continued our collaborate with Dr. Youssef Kousa by providing material support and technique advice of the iNSC/iPSC generation and 3D modeling that he has adopted in his newly established lab in National Childrens Hospital.

具体目标1：开发源自成人外周 CD34+ 细胞的体外 2D 神经元和 3D 脑类器官模型，以研究涉及人脑的神经发育和变性以及传染病。我们在我们的单位建立了一种新的片上脑微流控培养设备，现在我们可以通过组合大脑中四种必需细胞：神经元、小胶质细胞、星形胶质细胞和内皮细胞，以精确的方式重建3D大脑培养物。除了我们现有的 3D 大脑类器官模型之外，这种方法对于描述特定细胞类型在神经发育或神经变性中的作用非常有用。在初步研究中，我们使用iPSC衍生的神经元和商业获得的原代星形胶质细胞、内皮细胞和小胶质细胞系成功制作了混合芯片脑。我们还优化了方案，以成功地从 iPSC 生成小胶质细胞、星形胶质细胞和内皮细胞，并将在未来的研究中使用这些方法生成疾病特异性的芯片大脑。此外，我们还利用自主研发的内皮整合3D大脑模型来测试SARS-CoV2是否可以直接感染它。使用带有 SARS-CoV2 刺突蛋白的假病毒，我们发现 SARS-CoV2 假病毒几乎不能感染大脑类器官，仅在内皮层上有稀缺的弱阳性细胞，而不是在内部神经元团中。这一结果与临床观察一致，即患者大脑神经元中很少检测到 SARS-CoV2。尽管这与其他使用传统脑类器官显示高水平 SARS-CoV2 感染的报告不同。这些冲突可能是由于内皮细胞和血脑屏障的存在所致。我们受邀在即将于西班牙格拉纳达举行的第 11 届 IBRO 世界神经科学大会上展示该模型和研究结果。具体目标2：研究HERV-K对大脑发育和致瘤性的作用。我们继续与Ashish Shah博士合作研究HERV-K在胶质母细胞瘤发病机制中的作用。我们使用人类 CD34 衍生的神经干细胞生成星形胶质细胞生长的 3D 模型。在用含有 HERV-K 的质粒转染的星形胶质细胞中，我们检测到负责干细胞性和肿瘤发生的基因转录水平更高，并且类器官比形状不规则的对照生长得更快，表明有可能更容易转移。这些结果促成了新的出版物。具体目标 3：研究 HERV-K 与 ALS 等运动神经元变性的关联。我们继续研究逆转录病毒激活和运动神经元变性。对于功能研究，我们与北卡罗来纳大学的 Inhong Yang 博士合作，使用线粒体追踪器对运动神经元的线粒体进行染色。使用这种方法，我们计算了 ALS 神经元和对照组的线粒体密度。虽然我们的初步结果表明两组之间线粒体染色存在差异，但需要使用严格的分化条件进行更多研究来证实结果。我们还使用专门设计的针对 HERV-K Env 的 ASO 来研究 3D 大脑发育和运动神经元变性的影响，我们观察到 ASO 导致 3D 类器官的生长减慢，正如预期的那样。我们还与来自 SINS、NINDS 的 Catherine Demarino 博士合作，使用源自恒河猴皮肤成纤维细胞的体外神经培养物来研究其对 HERV-K 感染的敏感性。我们发现源自恒河猴皮肤成纤维细胞的NSC表达HERV-K Env结合靶标，并且高滴度的HERV-K病毒粒子感染NSC，表明恒河猴可以作为研究HERV-K感染和神经变性的动物模型。具体目标 4：利用我们的模型和方法促进神经系统疾病的研究和治疗开发。我们与 CHUV 的 Maria Isis Atallah Gonzalez 博士合作，从具有特定 FRMD4 基因变异的患者的成纤维细胞中生成了新的 iPSC 系。我们对来自印度 AIIMS Raebareli 的合作者 Sanjay Yadav 博士进行了从患者 PBMC 中生成多巴胺能神经元的培训，此后从 PD 患者中生成了 4 系 iPSC。我们继续与 Youssef Kousa 博士合作，为他在国家儿童医院新建立的实验室中采用的 iNSC/iPSC 生成和 3D 建模提供材料支持和技术建议。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Retrotransposon insertions associated with risk of neurologic and psychiatric diseases.

逆转录转座子插入与神经系统和精神疾病的风险相关。

DOI：
发表时间：
2023-01-09
期刊：
影响因子：
7.7
作者：
Ahn, Hyo Won;Worman, Zelia F;Lechsinska, Arianna;Payer, Lindsay M;Wang, Tongguang;Malik, Nasir;Li, Wenxue;Burns, Kathleen H;Nath, Avindra;Levin, Henry L
通讯作者：
Levin, Henry L