Uncovering the mechanisms of small vessel disease using a CRISPR-created induced pluripotent stem cell model

血管缺陷对神经退行性变的贡献已被认为是包括阿尔茨海默病在内的多种痴呆症的关键机制之一。然而，这种情况的细胞和分子机制仍然很大程度上未知，因此，没有有效的治疗方法。在中枢神经系统（CNS）中，血管细胞和神经元细胞协同作用，实现对中枢神经系统功能的复杂调控，形成神经血管单元（NVU）。NVU内的主要细胞类型包括内皮细胞（ECs）、周细胞或血管平滑肌细胞（VSMCs）、星形胶质细胞和神经元。这些神经血管细胞在功能上整合并受到严格调控，为揭示血管性痴呆的疾病机制提供了一个有希望的靶点。同时，遗传性脑血管病为了解脑血管因素在脑认知缺陷中的作用提供了很好的模型。根据我们之前对遗传性血管性痴呆综合征CADASIL（大脑常染色体显性动脉病变伴皮层下梗死和白质脑病）的研究，我们建立了患者特异性诱导多能干细胞（iPSC）模型，并将iPSC分化为神经血管细胞类型，同时对阿尔茨海默病进行了iPSC研究。iPSC模型是有前途的人类模型，用于解剖NVU内每种细胞类型对疾病病理的功能贡献。然而，传统的二维细胞培养不太可能概括神经血管细胞之间复杂的空间和横向相互作用。通过跨学科合作，博士项目将使用CRISPR/Cas9技术创建新的遗传小血管疾病模型，并使用从疾病特异性iPSCs分化的非血管细胞的不同组合构建人类NVU，并向3D器官芯片技术迈进。3D人类NVU模型将使用分子和细胞工具进行彻底检查，以确定与痴呆相关的血管因素的关键因素或共同途径，并确定生物标志物，然后确定药物靶点，为未来的治疗提供信息。

The contribution of vascular defects to neural degeneration has been recognised to be one of the key mechanisms underlying a wide range of dementia including Alzheimer Disease. However, the cellular and molecular mechanisms underlying the condition are still largely unknown, therefore, no efficient therapy is available. In the central nervous system (CNS), vascular cells and neuronal cells act synergistically to achieve complex regulation of CNS function, forming the neurovascular unit (NVU). Major cell types within the NVU include endothelial cells (ECs), pericytes or vascular smooth muscle cells (VSMCs), astrocytes and neurones. These neurovascular cells are functionally integrated and tightly regulated, providing a promising target for uncovering disease mechanisms for vascular dementia. Meanwhile, genetic cerebral small vessel diseases provide excellent model for understanding the contribution of vascular factors to the brain cognitive defect. From our previous study on a genetic vascular dementia syndrome, CADASIL (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), we have established patient specific induced pluripotent stem cell (iPSC) model, and differentiated the iPSCs into the neurovascular cell types, along with iPSC studies on the Alzheimer Disease. The iPSC models are promising human models to be used for dissecting functional contributions of each cell type within the NVU to the disease pathologies. However, the conventional 2D cell culture is unlikely to be able to recapitulate the complicated spatial and lateral interactions between the neurovascular cells. Through interdisciplinary collaboration, the PhD project will be creating new genetic small vessel disease model using CRISPR/Cas9 technology and constructing a human NVU using different combinations of nonvascular cells differentiated from disease-specific iPSCs and move towards 3D organ-chip technology. The 3D human NVU model will be thoroughly interrogated using molecular and cellular tools, in order to identify key factors or pathways that are common to vascular factor involved dementia, and identify biomarkers, then define drug targets informing future therapy.