Neurodifferentiation/Stem Cell Unit

具体目标1：建立成人外周血CD 34+细胞体外三维脑类器官模型，用于研究神经发育和变性以及涉及人脑的感染性疾病。我们扩展了我们的能力，以获得3D神经类器官来模仿人脑。使用改进的清除技术和先进的共聚焦成像系统，我们证实，周细胞可以产生沿着内皮细胞和神经细胞在三维类器官自发地与顺序诱导培养基，使模型完美的病毒感染研究，因为周细胞已被报道为主要目标的病毒，包括SARS冠状病毒2。在另一个包含CD 34细胞的3D模型中，我们用细胞因子处理它们以促进小胶质细胞分化，并使用scRNA-Seq分析来研究所得的小胶质细胞。我们发现，虽然细胞因子的处理对于产生小胶质细胞不是必需的，但与没有细胞因子刺激的类器官相比，它确实增加了小胶质细胞的数量。当用LPS处理类器官时，其通过增加类器官中某些小胶质细胞基因的转录来诱导炎症反应，表明类器官中分化的小胶质细胞是功能性的。根据结果编写了一份手稿供出版。 具体目标2：研究HERV-K在脑发育和致瘤性中的作用。我们支持Ashish Shah博士研究HERV-K在胶质瘤发病机制中的作用。我们用含HERV-K的质粒转染星形胶质细胞，检测到更高水平的基因转录，负责干性。这一结果表明干细胞标志物被HERV-K激活，并且可能是其影响肿瘤发生的机制。我们正在使用3D模型进一步研究HERV-K在细胞增殖和肿瘤发生中的作用。 具体目标3：研究HERV-K与ALS的关系。我们从四组C9 orf 72和对照iPSC中分化出运动神经元，并进行了RNA-Seq分析。结果表明，前几个差异通路与肿瘤发生有关。由于我们已经发现在C9 orf 72运动神经元中，在HERV-K激活之后存在增加的DNA修复元件，该结果意味着运动神经元中的DNA损伤事件导致HERV-K Env激活，这可能使神经元逆转到更类似于干细胞或肿瘤细胞的状态，导致运动神经元功能障碍。 具体目标4：使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。我们将继续与其他研究人员合作，提供iNSC/iPSC生成和3D建模的材料支持和技术培训。我们正在与北京的杨仁宏博士合作，开发在微流控室中培养运动神经元的方案。我们与国立儿童医院的Kousa博士合作，研究ZIKA病毒对人脑发育的发病机制。我们从靶向和对照iPSC细胞中获得神经干细胞，发现ZIKA病毒感染因靶点故障而显著增强。我们还帮助Farinaz Safavi博士从具有突变基因的iPSC中生成3D脑类器官。我们的结果显示，与校正的iPSC相比，突变显著降低了衍生的3D类器官的生长。

Specific aim 1: To develop in vitro 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We expended our capability to derive 3D neural organoids to mimic human brain. Using improved clearance techniques and advanced confocal imaging system, we confirmed that pericytes can be produced along endothelial cells and neural cells in the 3D organoid spontaneously with sequential induction media, making the model perfect for virus infection studies, as pericytes have been reported as a main target by viruses including SARS-CoV2. In another 3D model with incorporating CD34 cells, we treated them with cytokines to promote microglial differentiation and used scRNA-Seq analysis to study the resulting microglial cells. We found that although the treatment of cytokines is not necessary for producing microglia, it did increase the numbers of microglia compared to organoids without cytokine stimulation. When the organoids were treated with LPS, which induced inflammatory reactions by increasing transcriptions of certain microglial genes in the organoids, indicating the differentiated microglia in the organoids are functional. A manuscript is prepared for publication based on the results. Specific aim 2: To study the roles of HERV-K on brain development and tumorigenicity. We supported Dr. Ashish Shah on studying the mechanism of HERV-K on the pathogenesis of glioma. We transfected astroglia with plasmid containing HERV-K and detected higher level of gene transcription responsible for stemness. This result indicates that marker for stem cells is activated by HERV-K and maybe the mechanism underlying its effect on tumorigenesis. We are further studying HERV-K effect in cell proliferation and tumorigenesis using 3D models. Specific aim 3: To study the association of HERV-K and ALS. We differentiated motor neurons from four sets of C9orf72 and control iPSCs and did RNA-Seq analysis. The results showed top differential pathways were tumorigenesis related. As we have found that in C9orf72 motor neurons, there was increased DNA repairing element followed by HERV-K activation, this result imply that DNA damaging events in motor neurons leads to HERV-K Env activation, which may reverse the neurons to a status more similar to stem or tumor cells, causing motor neuron malfunction. Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We are continuing to collaborate with other investigators by providing material support and technique trainings of the iNSC/iPSC generation and 3D modeling. We are working with Dr. In-Hong Yang in UNC to develop protocol to culture motor neurons in a microfluidic chamber. We collaborated with Dr. Kousa from National Childrens Hospital to study the pathogenesis of ZIKA virus on human brain development. We derived neural stem cells from targeting and control iPSC cells and found ZIKA virus infection was enhanced significantly by a target malfunction. We also helped Dr. Farinaz Safavi to generate 3D brain organoids from iPSCs with an mutative gene. Our result showed the mutation decreased the growth of the derived 3D organoids significantly compared to corrected iPSCs.