A high throughput platform for the selective generation of neurons from stem cells

人类诱导多能干细胞（iPSCs）可以在培养物中无限生长，并且可以被指定为人体中发现的几乎任何细胞类型。因此，它们代表了组织工程和细胞替代疗法的一个有吸引力的途径。然而，将iPSCs转化为特定的细胞类型仍然很困难，这阻碍了它们的应用。该项目使用一种独特的工程设备，同时检查许多不同的化学线索对iPSC规格的影响。要研究的模型系统涉及一种神经元亚型的产生，这种神经元亚型的丧失导致帕金森病（PD）。因此，该项目的直接结果将对PD患者产生积极影响，并对使用iPSCs替代其他病变组织具有潜在的长期意义。该项目还将通过与纳什维尔公立学校系统的现有外展计划，为本科生和高中生提供研究经验。人类诱导多能干细胞（iPSCs）作为疾病建模、药物筛选和组织工程中患者来源的材料的无限来源，具有革新个性化医疗的潜力。仍然存在的一个关键挑战是单个iPSC系可重复分化为所需细胞类型的均匀群体。大多数iPSC分化过程产生异质细胞混合物，并且为一个iPSC系确定的分化条件通常不能转化为其他系。鉴于体内发育依赖于不同的时空线索，其中生长因子浓度的细微差异可以改变谱系轨迹，标准的iPSC分化程序利用静态培养，培养基变化不频繁，缺乏对可溶性蛋白和小分子浓度的连续控制。因此，该方案的目标是利用一个独特的微配方平台，该平台允许将独立的介质输送到标准的96孔板的每口井中，以实现iPSC分化。该提案的具体目标将集中在产生A9中脑多巴胺能（DA）神经元，这是帕金森病（PD）中丢失的细胞类型。DA神经元以前已经从iPSCs分化出来，但具有异质性和行间变异性。因此，微制剂的实用性和重要性将通过以下方式得到证明：1)从单个iPSC细胞系中产生高纯度中脑DA祖细胞的分化条件的反复筛选；2)反复筛选高纯度A9中脑DA神经元优于其他DA亚型的分化条件；3)多能干细胞分化条件快速优化示范。

Human induced pluripotent stem cells (iPSCs) can grow indefinitely in culture and be specified to almost any cell type found in the human body. As such, they represent an attractive avenue for tissue engineering and cell replacement therapy. However, it remains difficult to convert iPSCs into sspecific cell types, which hinders their applications. This project uses a unique engineered device to simultaneously examine the effects of many different chemical cues on iPSC specification. The model system to be studied involves the generation of a subtype of neurons whose loss causes Parkinson's Disease (PD). Thus, the immediate outcomes of this project will have positive impacts on PD patients, and potential long-term implications for using iPSCs to replace other diseased tissues. The project will also support research experiences for undergraduates and for high school students through an existing outreach program with the Nashville public school system.Human induced pluripotent stem cells (iPSCs) have the potential to revolutionize personalized medicine by serving as an unlimited source of patient-derived material for disease modeling, drug screening, and tissue engineering. A key challenge remaining is the reproducible differentiation of individual iPSC lines to homogeneous populations of the desired cell type. A majority of iPSC differentiation procedures yield heterogeneous cell mixtures, and differentiation conditions identified for one iPSC line often do not translate to other lines. Whereas in vivo development relies on diverse spatiotemporal cues, where subtle differences in growth factor concentrations can alter lineage trajectories, standard iPSC differentiation procedures utilize static cultures with infrequent media changes that lack continuous control over soluble protein and small molecule concentrations. As such, the objective of this proposal is utilize a unique microformulator platform, which permits independent media delivery to each well of a standard 96-well plate, for iPSC differentiations. The proposal's specific aims will be focused on producing A9 midbrain dopaminergic (DA) neurons, which are the cell type lost in Parkinson's Disease (PD). DA neurons have been previously differentiated from iPSCs but with heterogeneous identity and line-to-line variability. Thus, the utility and significance of the microformulator will be demonstrated by: 1) iterative screening of differentiation conditions that yield highly pure midbrain DA progenitors from a single iPSC line; 2) iterative screening of differentiation conditions that favor high purity A9 midbrain DA neurons over other DA subtypes; and 3) demonstration of rapid optimization of differentiation conditions to multiple iPSC lines.