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）用于治疗神经系统疾病或损伤的治疗性使用取决于引导干细胞分化为相关细胞类型的能力，其整体结构与相应的体内组织相同。生物分子的梯度，被称为形态原，在整个人类发育过程中普遍存在，并帮助指导细胞分化以及在完全形成的人类中不同细胞类型的位置。在早期大脑皮层中，FGF-2梯度被认为在最终生物学功能所必需的细胞类型和组织结构的建立中起着重要作用。利用微流体技术的最新进展，我们将在hNSC培养中应用不同的FGF-2梯度，以检查当FGF-2以梯度呈现时，是否可以诱导hNSC在体外重现体内观察到的各种细胞类型和组织结构。时间推移相和荧光显微镜将与免疫细胞化学结合使用，以检查FGF-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FGF-2梯度下，高FGF-2区域将包含大部分有丝分裂的hNSCs，这些hNSCs对称分裂形成更多有丝分裂的hNSCs。FGF-2浓度稍低的区域将被更多不对称分裂的hNSCs填充，这些hNSCs开始产生神经元。FGF-2浓度较低的区域只会产生神经元，而局部FGF-2浓度会导致hNSCs产生特定于某些皮层层的神经元细胞类型。我们提出的研究是第一个通过在微流体装置中培养人类干细胞来研究人类发育中的特定事件，该装置重现了在发育中的胎儿中观察到的化学微环境类型。我们相信这项研究将对人类神经发育产生深刻的见解，并将朝着从人类干细胞中开发出具有功能的体外神经组织迈出重要的一步。了解赋予人类独特认知能力的大脑部分最初是如何形成的，对于了解许多大脑紊乱和疾病的原因非常重要。指导干细胞复制在人脑中观察到的细胞类型和细胞排列的能力将大大提高我们治疗大脑紊乱、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.