Directing the Fate of Neural Stem Cells with Engineered Gradients of FGF-2

描述(由申请人提供)：人类神经干细胞(HNSC)用于治疗神经疾病或损伤取决于将干细胞分化为相关细胞类型的能力，其总体结构与体内相应组织相同。生物分子的梯度，称为形态生成素，在整个人类发育过程中普遍存在，有助于指导细胞分化以及不同类型细胞在完全形成的人类中的位置。在早期大脑皮层，成纤维细胞生长因子-2梯度被认为在建立细胞类型和组织结构方面具有重要作用，而细胞类型和组织结构是最终生物功能所必需的。利用微流控技术的最新进展，我们将把不同的FGF-2梯度应用于hNSC培养，以检测当以梯度的形式呈现时，是否可以诱导hNSC在体外概括体内观察到的各种细胞类型和组织结构。延时相和荧光显微镜将与免疫细胞化学结合使用，以检测成纤维细胞生长因子-2梯度对细胞周期、命运、大小、位置、增殖和迁移的影响。我们预测，在适当的成纤维细胞生长因子-2梯度下，高成纤维细胞生长因子-2区域将包含大部分有丝分裂的hNSCs，对称分裂形成更多有丝分裂的hNSCs。略低的成纤维细胞生长因子-2浓度的区域将被更不对称地分裂的开始产生神经元的hNSCs所填充。含有较低的成纤维细胞生长因子-2浓度的区域将只产生神经元，局部成纤维细胞生长因子-2浓度导致神经干细胞产生特定于大脑皮层某些层的神经细胞类型。我们提出的这项研究是第一次通过在微流控设备中培养人类干细胞来研究人类发育中的特定事件，该设备重建了在发育中的胎儿观察到的化学微环境的类型。我们相信，这项拟议的研究将对人类神经发育产生重大影响，并将朝着从人类干细胞开发具有功能的体外神经组织迈出重要的一步。公共卫生相关性了解大脑中给予人类独特认知能力的部分最初是如何形成的，对于了解许多大脑疾病和疾病的原因是重要的。在人脑内观察到的指导干细胞复制细胞类型和这些细胞排列的能力将极大地提高我们治疗大脑疾病、疾病和损伤的能力。

DESCRIPTION (provided by applicant): The therapeutic use of human neural stem cells (hNSC) to treat neurological disorders or injuries depends upon the ability to direct stem cell differentiation into the relevant cell types, with the same overall architecture as the corresponding in vivo tissue. Gradients of biological molecules, called morphogens, are pervasive throughout human development and help direct cell differentiation as well as the position of different cell types within the fully formed human. In the early cerebral cortex, FGF-2 gradients are thought to have an important role in establishing the cell types and tissue architecture that are essential for ultimate biological function. Capitalizing on recent advances in microfluidic technology, we will apply different FGF-2 gradients to hNSC cultures to examine if FGF-2, when presented as a gradient, can induce hNSCs to recapitulate in vitro the various cell types and tissue architecture observed in vivo. Time lapse phase and fluorescence microscopy will be used in conjunction with immunocytochemistry to examine the effects of FGF-2 gradients on cell cycle, fate, size, position, proliferation, and migration. We predict that with the appropriate FGF-2 gradient, high FGF-2 regions will contain mostly mitotic hNSCs that divide symmetrically to form more mitotic hNSCs. Regions with slightly lower FGF-2 concentrations will be populated with more asymmetrically dividing hNSCs that begin to generate neurons. Regions with yet lower FGF-2 concentrations will generate only neurons, where local FGF-2 concentration causes hNSCs to generate neuronal cell types specific to certain layers of the cortex. The study that we have proposed is the first to investigate a specific event in human development by culturing human stem cells in a microfluidic device that recreates the types of chemical microenvironments observed within the developing fetus. We believe the proposed study will yield great insight into human neural development and will make a significant step forward towards developing functional in vitro neural tissue from human stem cells. PUBLIC HEALTH RELEVANCE Understanding how the part of the brain that gives humans their unique cognitive abilities initially forms is important for understanding the cause of many disorders and diseases of the brain. The ability to direct stem cells to replicate the types of cells and the arrangement of those cells observed within the human brain would greatly advance our ability to treat brain disorders, diseases, and injuries.