Engineering neural tissue using pluripotent stem cells

大脑和脊髓由不同种类的细胞组成。复制这些复杂的结构为我们提供了一个重要的机会来发现这些组织是如何在体内形成的。这样的工程化神经组织可以用于药物筛选，而不是捐赠的人体组织。组织工程的一种流行策略是使用生物材料支架传递信号，促进干细胞分化为神经组织。我的团队致力于人类诱导多能干细胞(HiPSCs)，即成年细胞重新编程为一种状态，在这种状态下，它们可以成为生物体中发现的任何类型的细胞。这一特性使其成为组织工程的极佳细胞来源。我的团队确定了一些化学和物理线索，推动HiPSCs分化为神经组织，这是本研究计划的起点。然而，目前使用HiPSCs设计神经组织的方法需要冗长的、劳动密集型的方案。这项研究计划的首要目标是通过开发生物活性支架来从HiPSCs中设计功能神经组织，这些支架提供必要的化学和物理线索来促进快速分化，这些线索可以转化为生物打印应用。为了实现这一目标，该研究计划包括两个不同的目标，与现有方法相比，该研究计划的结果将用于快速和高通量地生成神经组织。 第一个目标是研究如何改变3D纤维蛋白支架的性质，使其能够从HiPSCs中生成功能性神经组织。我们可以使用交联剂来控制这些支架的力学性能，以增加它们的稳定性。我们还可以通过用生物活性信号来功能化它们的化学性质，比如促进HiPSCs神经分化的多肽。然后，我们将通过使用这些支架作为3D打印功能神经组织的生物墨水来确定这两种类型的线索对HiPSC分化的影响。 第二个目标将阐明从微球中控制释放新的化学信号如何快速将HiPSCs分化为神经元。这些化学信号包括转录因子Ascl1(被证明可以有效地产生神经元)，通过细胞内蛋白传递技术发挥功能的转录因子Ascl1，紫杉胺(被证明可以促进运动神经元分化)，以及谷固酮(被证明可以促进多巴胺能神经元分化)。目前，没有现有的药物传递系统能够产生这些分子的受控释放，所有这些分子都能促进HiPSCs快速分化为神经组织。我们将演示如何使用这些药物释放微球的不同组合和浓度来设计两种不同类型的神经组织，然后将其用于药物筛选应用。

Different kind of cells make up the brain and spinal cord. Replicating these complex structures provides us with a significant opportunity to discover how such tissues form in the body. Such engineered neural tissues can be used for applications in pharmacological screening instead of donated human tissues. One popular strategy for tissue engineering uses biomaterial scaffolds to deliver signals that promote stem cells to differentiate into neural tissue. My group works with human induced pluripotent stem cells (hiPSCs), adult cells reprogrammed into a state where they can become any type of cell found in an organism. This property makes them an excellent cell source for tissue engineering. My group identified a number of chemical and physical cues that drive the differentiation of hiPSCs into neural tissue, which serves as a starting point for this research program. However, current methods for engineering neural tissue using hiPSCs require lengthy, labor intensive protocols. The overarching goal of this research program is to engineer functional neural tissues from hiPSCs by developing bioactive scaffolds that present the necessary chemical and physical cues for promoting rapid differentiation that can be translated into bioprinting applications. This research program consists of two different aims for achieving this goal, and the results of this research program will be used to generate neural tissue in a rapid and high throughput manner compared to current methods. The first aim investigates how to modify the properties of 3D fibrin scaffolds so that they can generate functional neural tissue from hiPSCs. We can manipulate the mechanical properties of these scaffolds using cross-linking agents to increase their stability. We also can enhance their chemical properties by functionalizing them with bioactive cues like peptides that promote neuronal differentiation of hiPSCs. We will then determine the influence of these two types of cues on hiPSC differentiation by using these scaffolds as bioink for 3D printing of functional neural tissues. The second aim will elucidate how the controlled release of novel chemical cues from microspheres can rapidly differentiate hiPSCs into neurons. These chemical cues include the transcription factor Ascl1 (shown to efficiently produce neurons) functionalized with intracellular protein delivery technology, purmorphamine (shown to enhance motor neuron differentiation), and guggulsterone (shown to enhance dopaminergic neuron differentiation). Currently, no existing drug delivery systems can generate controlled release of these molecules all of which promote rapid differentiation of hiPSCs into neural tissue. We will demonstrate how different combinations and concentrations of these drug-releasing microspheres can be used to engineer two different types of neural tissue, which could then be used for drug screening applications.