Study the pathogenesis of neurological disorders using human neural cultures derived from patient peripheral blood CD34 cells

使用源自患者外周血 CD34 细胞的人类神经培养物研究神经系统疾病的发病机制

• 批准号: 9157569
• 负责人: Avindra Nath
• 金额: $ 106.13万
• 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/9157569
• 关键词: Adoption; Adult; Antibodies; Autistic Disorder; Biological Assay; Blood; CD34 gene; Cell Count; Cell Culture Techniques; Cell membrane; Cell model; Cell-Cell Adhesion; Cells; Coculture Techniques; DNA Methylation; Diagnosis; Disease; Epigenetic Process; Extramural Activities; Fluorescence; Generations; Genetic; Genomic DNA; Goals; HERVs; Human; In Vitro; Inflammation; Inflammatory; Journals; Label; Modeling; Molecular Target; Monitor; Neurodegenerative Disorders; Neurologic; Neuronal Differentiation; Neurons; Paper; Pathogenesis; Patients; Peripheral; Play; Pluripotent Stem Cells; Process; Protocols documentation; Publications; Publishing; Reaction; Research Personnel; Role; Sampling; Sclerosis; Small Interfering RNA; Staining method; Stains; Stem Cell Development; Stem cells; Symptoms; System; Systems Development; T-Lymphocyte; Therapeutic Intervention; Time; Tracer; United States National Institutes of Health; Work; base; cell type; human stem cells; induced pluripotent stem cell; inhibitor/antagonist; model development; nerve stem cell; nervous system disorder; neurogenesis; neurotoxicity; novel; peripheral blood; preclinical study; receptor; relating to nervous system; research and development; research study; screening; small molecule; therapeutic development

Objectives: 1.Establish neurogenesis models using Induced Pluripotent Stem cells (iPSC) and Induced Neural Stem Cells (iNSC) derived from patients for identifying novel genetic, epigenetic and other molecular targets for novel diagnosis and therapeutic interventions. -Screening assays for neurogenesis and neurotoxicity in human mixed T cells and neural cells co-cultures. -Identify and study the mechanisms of inflammation on neurogenesis and neurotoxicity. 2. Facilitate the utilization of our established iNSC models in basic and preclinical studies of neurological disorders by TNC, NIH and extramural investigators. Specific aim 1:Establish in vitro neurogenesis and development models using neural cultures derived from human adult peripheral CD34+ cells. We optimized our protocol and we can now derive both neural stem cells (iNS) and iPSC cells simultaneously from 10 ml of blood. We also studied the effect of certain small molecules and found that combination of retinoic receptor inhibitor G85 and small molecule UM171 will significantly inhibit CD34 differentiation and increase the cell number. Adoption of these small molecules will help us to better preserve CD34 cells in culture and provide more CD34 cells for T cells generation if needed. We also published another video protocol of culturing iNS from human CD34 cells on the Journal of Visualized Experiments (JoVE). Specific aim 2: Study the effect of inflammation on neural cells. We are using long term fluorescent cell tracer Vybrant CFDA SE to label T cells and cell membrane staining CM-Dil to label neural cells and created a dual fluorescence culture system to allow us to monitor T cell-induced neurotoxicity in a real-time manner. Using mixed T cell-neural cells cultures, we have found that T cells-derived from one undiagnosed patient blood showed neurotoxicity compared to control T cells from healthy donor. We are applying this in vitro neuroinflammatory model to the other T cell samples we collected from patients with undiagnosed neurological symptoms. This will help us to delineate whether the malfunction in T cells or abnormality in neuronal cells caused the symptoms. Specific aim 3: Study the effect of HERV K on pluripotent stem cell development. We continued the study of the effect of HERV-K Env on the pluripotent stem cells and neural differentiation. We confirmed that inhibition using siRNA or antibody against Env resulted in stem cell morphological changes and colony formation decrease. We further found that inhibition of HERV-K Env resulted in an enhanced neural induction process, likely due to its effect on the modulating of cell-cell adhesion through CD98HC. These finding suggested an important role HERV-K may have played in stem cell and human neurological system development. A paper is in prepared for publication based on these findings. Specific aim 4: Study the epigenetic mechanisms of neurodegenerative disorders. We are using induced neural stem cells generated from primary sclerosis (PLS) patients to study the possible effect of epigenetic changes in the pathogenesis of the disease. We found that general genomic DNA methylation changes during the neural stem cell induction and neuronal differentiation. The DNA methylation levels may be directly related to neuronal differentiation and maturation. Furthermore, neural stem cells derived from PLS samples showed slower neural differentiation and have lower DNA methylation level compared to normal controls, indicating epigenetic changes may play an important role in the pathogenesis of this disorder. We also collaborated with Dr. Roche to support the study on the possible role of neuroligin-4 in Autism which resulted in a recent publication in PNAS.

目的： 1.使用来自患者的诱导多能干细胞（iPSC）和诱导神经干细胞（iNSC）建立神经发生模型，用于识别新的遗传、表观遗传和其他分子靶标，用于新的诊断和治疗干预。 - 人类混合T细胞和神经细胞共培养物中神经发生和神经毒性的筛选测定。 - 识别和研究炎症对神经发生和神经毒性的机制。 2.促进TNC、NIH和校外研究者在神经系统疾病的基础和临床前研究中利用我们建立的iNSC模型。 具体目标1：使用人成人外周血CD 34+细胞来源的神经培养物建立体外神经发生和发育模型。我们优化了我们的方案，现在我们可以从10 ml血液中同时获得神经干细胞（iNS）和iPSC细胞。我们还研究了某些小分子的作用，发现维甲酸受体抑制剂G85和小分子UM 171的组合可以显著抑制CD 34的分化并增加细胞数量。 采用这些小分子将有助于我们更好地保存培养中的CD 34细胞，并在需要时为T细胞生成提供更多的CD 34细胞。 我们还在Journal of Visualized Experiments（JoVE）上发表了另一个从人CD 34细胞培养iNS的视频方案。 具体目标2：研究炎症对神经细胞的影响。我们正在使用长期荧光细胞示踪剂Vybrant CFDA SE标记T细胞和细胞膜染色CM-Dil标记神经细胞，并创建了双荧光培养系统，使我们能够实时监测T细胞诱导的神经毒性。使用混合的T细胞-神经细胞培养物，我们发现与来自健康供体的对照T细胞相比，来自一个未确诊患者血液的T细胞显示出神经毒性。我们正在将这种体外神经炎症模型应用于我们从未确诊的神经系统症状患者中收集的其他T细胞样本。 这将有助于我们描述是否T细胞的功能障碍或神经元细胞的异常引起的症状。 具体目标3：研究HERV K对多能干细胞发育的影响。我们继续研究HERV-K Env对多能干细胞和神经分化的影响。 我们证实，使用针对Env的siRNA或抗体的抑制导致干细胞形态学变化和集落形成减少。我们进一步发现，HERV-K Env的抑制导致增强的神经诱导过程，这可能是由于其对通过CD 98 HC调节细胞-细胞粘附的作用。这些发现表明HERV-K可能在干细胞和人类神经系统发育中发挥了重要作用。正在根据这些调查结果编写一份文件供出版。 具体目标4：研究神经退行性疾病的表观遗传机制。我们正在使用从原发性硬化症（PLS）患者产生的诱导神经干细胞来研究表观遗传变化在疾病发病机制中的可能作用。我们发现在神经干细胞诱导和神经元分化的过程中，基因组DNA的甲基化发生了变化。DNA甲基化水平可能与神经元的分化和成熟直接相关。此外，与正常对照相比，来自PLS样品的神经干细胞显示出较慢的神经分化和较低的DNA甲基化水平，表明表观遗传变化可能在这种疾病的发病机制中起重要作用。 我们还与Roche博士合作，支持关于neuroligin-4在自闭症中可能作用的研究，该研究最近发表在PNAS上。