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: 9563168
• 负责人: Avindra Nath
• 金额: $ 31.58万
• 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/9563168
• 关键词: Address; Adult; Affect; Age; Aging; Animals; Astrocytes; Biological Assay; Biopsy; Blood specimen; Boston; Brain; Brain Neoplasms; CD34 gene; Callithrix; Cell Differentiation process; Cell Line; Cell model; Cell physiology; Cells; Coculture Techniques; Collaborations; Cytomegalovirus; DNA Methylation; Defect; Diagnosis; Disease; Endogenous Retroviruses; Epigenetic Process; Extramural Activities; Fibroblasts; Fluorescence; Frontotemporal Dementia; GRN gene; Gene Expression; Gene-Modified; Generations; Genes; Genetic; Genetic Polymorphism; Glial Fibrillary Acidic Protein; Growth; Human; Image; In Vitro; Inflammation; Institutes; International; Medical center; Methods; Modeling; Molecular; Molecular Target; Motor; Motor Neurons; Mutate; Mutation; Nerve; Neuraxis; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Neurophysiology - biologic function; Neurosciences; Organoids; Pathogenesis; Pathway interactions; Patients; Pattern; Peripheral; Play; Pluripotent Stem Cells; Primary Lateral Sclerosis; Process; Proteins; RNA; Reporting; Research Personnel; Retroelements; Role; Sampling; Science; Signal Transduction; Skin; Small Interfering RNA; Societies; Stem Cell Research; Stem cells; System; TIMP2 gene; Testing; Therapeutic Intervention; Tissues; Training; Transcriptional Regulation; Transfection; Transgenic Organisms; Umbilical Cord Blood; United States National Institutes of Health; age effect; age related; base; cell transformation; cell type; design; env Gene Products; granulin; induced pluripotent stem cell; killings; meetings; model development; nerve stem cell; nervous system disorder; nestin protein; neural model; neurodevelopment; neurogenesis; neurotoxicity; novel; peripheral blood; preclinical study; promoter; relating to nervous system; research and development; screening; stem cell differentiation; stemness; symposium; therapeutic development; three-dimensional modeling; tool; transcriptome sequencing; ward

Specific aim 1: Use in vitro 3D brain organoids derived from human adult peripheral CD34+ cells to study neural differentiation. The in vitro neurogenesis and development model can also be used to study the mechanism of neurological disorders. In collaboration with Dr. Mary Kay Floeter, we derived iPSC cells from blood samples from patients with primary lateral sclerosis (PLS) and age-matched healthy donors. We further compared the 3D brain organoids derived from these iPSCs. We found that after 2 weeks of the culture, 3D organoids derived from PLS were significantly bigger in size compared with their age matched controls. Furthermore, the 3D organoids from PLS were more symmetric shaped, compared with the controls, which showed asymmetric growth, likely due to cell differentiation. This was confirmed by gene expression study which showed that the expression levels of genes associated with neural differentiation such as neural stem cell marker nestin, neuronal marker MAP-2, glutaminergic neuronal marker GAD and astroglial marker GFAP were all lower in PLS organoids compared with the healthy controls. However, although the absolute level was extremely low, an increase of ChAT expression was observed in the PLS 3D organoids. ChAT is a marker for peripheral motor neurons, which is spared to damage in PLS which only affect primary motor neurons. Our observation that in the brain organoids derived from PLS patients, there was a general delay of neural development in central neural cell types but not in peripheral motors, agreed with PLS pathogenesis which was specific to central nerve system. As we have shown, there was difference in DNA methylation levels between neural stem cells derived from PLS and healthy donors. It is likely that the lower DNA methylation levels in PLS neural stem cells may delay neural differentiation. While there were not enough forces to push the central neural development, as a secondary pathway, motor neuron differentiation may occur, as suggested by other researchers. We have presented the result on the annual international society of stem cell research meeting in Boston. Specific aim 2: Study the role of astroglia in the pathogenesis of neural disorders. We used the same 3D model to study the role of astroglia played in the pathogenesis of another motor neuron disorder: autosomal-dominant frontotemporal dementia (FTD), in collaboration with Dr. Michael Ward. It was recently discovered that patients with FTD may be caused by granulin (GRN) mutation. To address the mechanism, Dr. Ward has been generating FTD models with GRN gene modifications in human iPSC-derived neurons. We used the iPSCs for the 3D brain organoids culture and found that GRN mutation resulted in smaller organoids in size and significantly lower gene expression for astroglial markers. Although the number of neurons was higher in the GRN-mutated 3D organoids, due to the lack of astroglia, which may play a protective role to neurons, damage was shown in the neurons as indicated by immunostaining images. This result indicates that GRN mutation may result in defect of astroglial differentiation, thus causing secondary neuronal damage. These results showed that the 3D model could be a very useful tool in modeling neurodegenerative disorders, determining the roles the different cell types may have played. Further study on the pathogenesis based on the model is ongoing. Specific aim 3: Study the role of HERV-K on human neural development. We have found that human endogenous retroviruses K (HERV-K) is expressed on human iPSC. Inhibition of HERV-K Env protein enhanced neuronal differentiation, indicating HERV-K plays a role in keep the stem cells from differentiation. We also determined that Ch22 be the likely loci for the HERV-K activation in human iPSCs. Based on the HERV-K gene sequence of Ch22, four specific siRNAs targeting HERV-K Env was designed and synthesized. The siRNAs were used to inhibit the expression of HERV-K Env in four iPSC lines. We found the siRNAs efficiently decreased HERV-K env expression in three of the four iPSC lines. In the affected three lines, Oct-4, a marker for pluripotent stem cells, was also decreased. The result further confirmed our hypothesis that HERV-K activation in iPSCs is important in maintaining the stemness of iPSCs. One of the iPSC line was not affected by siRNA indicates that in this specific human, there may be different gene patterns, thus HERV-K gene polymorphism may play a role in regulating neural differentiation. We collected the total RNA samples from the siRNA treated cells for RNA-seq analysis to study the genes that were affected by HERV-K inhibition. The result confirmed that 27 genes were significantly increased and 25 genes were significantly decreased after siRNA treatment compared to the control cells. These genes were involved in several critical molecular pathways, including DNA methylation, RNA transcriptional regulation and others specifically associated with stem cell function and differentiation. These results provide a clear mechanism by which HERV-K components used in maintaining the stemness of iPSC. By targeting the HERV-K associated pathways, we may develop novel methods to regulate neural differentiation and even treatment for neurodegenerative disorders and brain tumors where HERV-K is involved. We have submitted an abstract based on the finding to the 2017 Retropath Symposium. Specific aim 4: Study the effect of aging on central nervous system using iNSCs directly derived from CD34 cells. We found that the iNSCs we generated from cord blood CD34 cells produced much higher level of TIMP2 compared to the iNSCs generated from CD34 cells from adult donors. TIMP2 is a protein that has been reported to decrease during aging and has trophic effect on neurons. Our observation indicated that iNSCs, directly generated from CD34 cells, may maintain the epigenetic information, at least partially from the aging CD34 cells. This is different from CD34-derived iPSC, which have lost the aging information during cell transformation process. We are in collaboration with Dr. Clive Svendsen from Cedars-Sinai Medical Center to further study if the neurons derived from our iNSC from CD34 cells still maintain the epigenetic signature of the aging CD34 cells. If confirmed, our iNSC model will be very useful to study age-related neurodegenerative disorders. Specific aim 5: facilitate the research and therapeutic developments for neurological disorders using our models and methods. We are in collaboration with other investigators by providing training of the iNSC/iPSC generation or with cells. We provided iPSC generation training to Dr. Yogita K. Adlakha and Dr. Ashiwani Choudhary from Centre for Neuroscience, Indian Institute of Science. We provide consultancy to Dr. Henry Levi on developing a project studying the retroelements in neurological disorders. In collaboration with Dr. James Pickel, we tested the function of neural generation in the activation of transfected factors. Dr. James has created a marmoset with a CMV-driven transgenic fluorescent maker intend to have a ubiquitous expression on Marmoset tissues. Although fibroblasts derived from the animal skin lost fluorescent expression after passages, it was not known whether neurons express the signal or not. CMV promotor has been reported to have variable regulatory powers in different tissues. We transfected the fibroblasts with pluripotent stem cell factors to generate neural stem cells. We found after transfection, fluorescence was again observed in neuronal like cells derived from the fibroblasts. This indicated that although fibroblasts are not express the marker, once neurons are differentiated, they may still express the maker. This provide another way to answer the question without killing the animals or by doing biopsy.

具体目标 1：使用源自人类成人外周 CD34+ 细胞的体外 3D 脑类器官来研究神经分化。体外神经发生和发育模型还可用于研究神经系统疾病的机制。我们与 Mary Kay Floeter 博士合作，从原发性侧索硬化症 (PLS) 患者和年龄匹配的健康捐赠者的血液样本中提取了 iPSC 细胞。我们进一步比较了源自这些 iPSC 的 3D 大脑类器官。我们发现，培养 2 周后，与年龄匹配的对照相比，源自 PLS 的 3D 类器官尺寸明显更大。此外，与对照组相比，来自 PLS 的 3D 类器官形状更加对称，而对照组表现出不对称生长，可能是由于细胞分化所致。基因表达研究证实了这一点，该研究表明，与健康对照相比，PLS 类器官中与神经分化相关的基因（如神经干细胞标记物巢蛋白、神经元标记物 MAP-2、谷氨酰胺能神经元标记物 GAD 和星形胶质细胞标记物 GFAP）的表达水平均较低。然而，尽管绝对水平极低，但在 PLS 3D 类器官中观察到 ChAT 表达增加。 ChAT 是外周运动神经元的标记物，在仅影响初级运动神经元的 PLS 中不会受到损伤。我们观察到，在来自 PLS 患者的脑类器官中，中枢神经细胞类型的神经发育普遍延迟，但外周运动细胞没有，这与中枢神经系统特有的 PLS 发病机制一致。正如我们所表明的，来自 PLS 的神经干细胞和健康供体的神经干细胞之间的 DNA 甲基化水平存在差异。 PLS 神经干细胞中较低的 DNA 甲基化水平可能会延迟神经分化。正如其他研究人员所建议的，虽然没有足够的力量来推动中枢神经发育，但作为次要途径，运动神经元分化可能会发生。我们在波士顿举行的国际干细胞研究学会年度会议上公布了这一结果。 具体目标 2：研究星形胶质细胞在神经疾病发病机制中的作用。我们与 Michael Ward 博士合作，使用相同的 3D 模型来研究星形胶质细胞在另一种运动神经元疾病：常染色体显性额颞叶痴呆 (FTD) 发病机制中的作用。 最近发现，患者FTD可能是由颗粒蛋白（GRN）突变引起的。为了解决这一机制，Ward 博士一直在人类 iPSC 衍生神经元中构建带有 GRN 基因修饰的 FTD 模型。我们使用 iPSC 进行 3D 脑类器官培养，发现 GRN 突变导致类器官尺寸更小，并且星形胶质细胞标记的基因表达显着降低。尽管GRN突变的3D类器官中的神经元数量较多，但由于缺乏可能对神经元起保护作用的星形胶质细胞，但免疫染色图像显示神经元出现损伤。这一结果表明GRN突变可能导致星形胶质细胞分化缺陷，从而引起继发性神经元损伤。这些结果表明，3D 模型可能是模拟神经退行性疾病、确定不同细胞类型可能发挥的作用的非常有用的工具。基于该模型的发病机制的进一步研究正在进行中。 具体目标3：研究HERV-K对人类神经发育的作用。我们发现人内源性逆转录病毒 K (HERV-K) 在人 iPSC 上表达。抑制 HERV-K Env 蛋白可增强神经元分化，表明 HERV-K 在阻止干细胞分化中发挥作用。 我们还确定 Ch22 可能是人类 iPSC 中 HERV-K 激活的位点。基于Ch22的HERV-K基因序列，设计并合成了4种针对HERV-K Env的特异性siRNA。 siRNA 用于抑制 4 个 iPSC 系中 HERV-K Env 的表达。我们发现 siRNA 有效降低了 4 个 iPSC 系中的 3 个系中的 HERV-K env 表达。在受影响的三个细胞系中，多能干细胞标记物 Oct-4 也有所下降。结果进一步证实了我们的假设，即 iPSC 中 HERV-K 的激活对于维持 iPSC 的干性非常重要。其中一个iPSC系没有受到siRNA的影响，这表明在这个特定的人类中，可能存在不同的基因模式，因此HERV-K基因多态性可能在调节神经分化中发挥作用。 我们从 siRNA 处理的细胞中收集总 RNA 样本进行 RNA-seq 分析，以研究受 HERV-K 抑制影响的基因。结果证实，与对照细胞相比，经过siRNA处理后，27个基因显着增加，25个基因显着减少。这些基因参与了几个关键的分子途径，包括 DNA 甲基化、RNA 转录调控以及其他与干细胞功能和分化特别相关的分子途径。这些结果提供了 HERV-K 成分用于维持 iPSC 干性的明确机制。通过针对 HERV-K 相关通路，我们可以开发新的方法来调节神经分化，甚至治疗涉及 HERV-K 的神经退行性疾病和脑肿瘤。 我们已向 2017 年 Retropath 研讨会提交了一份基于该发现的摘要。 具体目标4：利用直接源自CD34+细胞的iNSCs研究衰老对中枢神经系统的影响。 我们发现，与从成人捐赠者的 CD34 细胞生成的 iNSC 相比，从脐带血 CD34 细胞生成的 iNSC 产生的 TIMP2 水平要高得多。 TIMP2 是一种蛋白质，据报道会随着衰老而减少，并对神经元具有营养作用。 我们的观察表明，直接由 CD34+ 细胞产生的 iNSC 可以维持表观遗传信息，至少部分来自老化的 CD34+ 细胞。这与CD34衍生的iPSC不同，后者在细胞转化过程中丢失了老化信息。 我们与 Cedars-Sinai 医学中心的 Clive Svendsen 博士合作，进一步研究来自 CD34 细胞的 iNSC 的神经元是否仍保持衰老 CD34 细胞的表观遗传特征。如果得到证实，我们的 iNSC 模型对于研究与年龄相关的神经退行性疾病将非常有用。 具体目标 5：利用我们的模型和方法促进神经系统疾病的研究和治疗发展。我们与其他研究人员合作，提供 iNSC/iPSC 一代或细胞的培训。我们为印度科学研究所神经科学中心的 Yogita K. Adlakha 博士和 Ashiwani Choudhary 博士提供了 iPSC 生成培训。我们为 Henry Levi 博士提供咨询，以开发研究神经系统疾病中的逆转录因子的项目。 我们与 James Pickel 博士合作，测试了神经生成在转染因子激活中的功能。 James 博士创造了一只带有 CMV 驱动的转基因荧光标记的狨猴，旨在在狨猴组织中普遍表达。尽管来自动物皮肤的成纤维细胞在传代后失去了荧光表达，但尚不清楚神经元是否表达信号。据报道，CMV 启动子在不同组织中具有不同的调节能力。我们用多能干细胞因子转染成纤维细胞以产生神经干细胞。我们发现转染后，在源自成纤维细胞的神经元样细胞中再次观察到荧光。这表明，尽管成纤维细胞不表达该标记物，但一旦神经元分化，它们仍可能表达该标记物。这提供了另一种回答问题的方法，无需杀死动物或进行活检。