Neurodifferentiation/Stem Cell Unit

具体目标1： 目的：建立成人外周血CD34+细胞来源的2D神经元和3D脑器官模型，以研究神经发育、变性和脑内感染性疾病。我们在我们的单位中安装了一种新的脑片上微流控培养设备，现在我们可以通过结合大脑中四种类型的基本细胞：神经元、小胶质细胞、星形胶质细胞和内皮细胞，以一种精确的方式重建3D脑培养。除了我们现有的3D脑器官模型之外，这种方法对于描述特定细胞类型在神经发育或神经退化中的作用非常有用。在初步研究中，我们利用IPSC来源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地制作了混合脑芯片。我们还优化了方案，成功地从IPSCs中产生了小胶质细胞、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来产生疾病特异性的芯片上脑。此外，我们还利用自行研制的内皮细胞结合的3D脑模型来验证SARS-CoV2是否可以直接感染它。使用带有SARS-CoV2刺突蛋白的伪病毒，我们发现SARS-CoV2伪病毒几乎不能感染脑器官，只有内皮衬里的弱阳性细胞，而不是内神经元团。这一结果与临床观察到的SARS-CoV2很少从患者大脑中的神经元中检测到的结果是一致的。尽管这与其他报告不同，其他报告使用传统的脑器官来显示SARS-CoV2感染的高水平。这些冲突可能与血管内皮细胞和血脑屏障的存在有关。我们被邀请在即将在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发现。 特定目的2：研究Herv-K在脑发育和肿瘤发生中的作用。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上的作用。我们使用人类CD34来源的神经干细胞来生成星形胶质细胞生长的3D模型。在含有HERV-K基因的星形胶质细胞中，我们检测到与干性和肿瘤形成有关的基因转录水平较高，并且细胞体生长得更快，形状不规则，这表明更容易发生转移。这些结果促成了一份新的出版物。 具体目的3：研究Herv-K与肌萎缩侧索硬化症等运动神经元变性的关系。我们继续我们对逆转录病毒激活和运动神经元变性的研究。在功能研究方面，我们与北卡罗来纳大学的杨英红博士合作，使用有丝分裂追踪器对运动神经元的线粒体进行染色。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的线粒体染色有所不同，但还需要更多的严格分化条件的研究来证实这一结果。我们还使用了专门设计的针对Herv-K Env的ASO来研究其对3D脑发育和运动神经元变性的影响，我们观察到ASO如预期的那样导致3D有机物的生长缓慢。我们还与NINDS SINS的Catherine DeMarino博士合作，使用了来自恒河猴皮肤成纤维细胞的体外神经培养来研究其对HERV-K感染的敏感性。我们发现恒河猴皮肤成纤维细胞来源的神经干细胞表达HERV-K Env结合靶点，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：使用我们的模型和方法促进神经疾病的研究和治疗发展。我们与CHUV的Maria Isis Atallah Gonzalez博士合作，从一名具有特定FRMD4基因变异的患者的成纤维细胞中产生了一种新的IPSC系。我们培训了印度AIIMS Raebareli的合作者Sanjay Yadav博士从患者PBMCs中培养多巴胺能神经元，并从PD患者中培养了4株IPSCs。我们继续与优素福·库萨博士合作，为他在国立儿童医院新成立的实验室中采用的iNSC/IPSC一代和3D建模提供物质支持和技术建议。

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.