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：利用来源于成人外周CD34+细胞的体外三维脑类器官研究神经分化。体外神经发生和发育模型也可用于研究神经系统疾病的机制。与Mary Kay Floeter博士合作，我们从原发性侧索硬化症（PLS）患者和年龄匹配的健康供者的血液样本中提取了iPSC细胞。我们进一步比较了这些iPSCs衍生的3D脑类器官。我们发现，在培养2周后，来自PLS的3D类器官的大小明显大于与其年龄匹配的对照组。此外，与对照组相比，来自PLS的3D类器官形状更对称，可能是由于细胞分化导致的不对称生长。基因表达研究证实，神经干细胞标记物nestin、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等神经分化相关基因在PLS类器官中的表达水平均低于健康对照组。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PLS中仅影响初级运动神经元的损伤中不受其影响。我们观察到，在PLS患者衍生的脑类器官中，中枢神经细胞类型普遍存在神经发育延迟，而外周运动细胞类型则没有，这与PLS特异性的中枢神经系统发病机制一致。正如我们所显示的，来自PLS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。虽然没有足够的力量来推动中枢神经的发育，但正如其他研究人员所提出的那样，作为次要途径，运动神经元的分化可能会发生。我们已经在波士顿举行的年度国际干细胞研究学会会议上提出了这一结果。