Microfluidic Platform for Genetic Guidance of Stem Cell Differentiation

胚胎干细胞和成体干细胞是再生医学的基础。他们独特的能力， 经历自我更新与产生多种分化细胞类型的能力相平衡， 彻底改变退行性疾病的治疗方法对它们的广泛翻译使用的一个核心挑战是 确定环境线索和基因调控网络，管理自我更新， 干细胞定向分化为感兴趣的组织。识别潜在的基因调控 工作中的电路是指导干细胞分化的关键步骤。此外，调节和 监测干细胞以测试预测的基因调控网络关系是评估干细胞功能的必要步骤。 合理的，可重复的和有效的方法的遗传指导分化。 在这里，我们建议调整我们现有的微技术平台，用于多路复用的动态成像。 微环境对理解和评估基因调控网络在干细胞中的作用 分化引入已知的遗传激活剂与细胞谱系报告基因的组合提供了一种新的方法。 独特的读出的遗传方法的潜在用途，以指导干细胞分化。通过病毒 外源遗传因子的表达和荧光报告基因的监测，我们将评估基因 作为鼠胚胎干细胞分化成胰腺内胚层的基础的调节网络， 牙齿和心脏瓣膜组织作为SysCODE联盟的一部分。此外，微技术的使用 工具提供了可以暴露于各种独立扰动的微环境， 高时间和空间分辨率监测的响应，允许评估许多遗传 输入沿着特定的细胞分化途径。 我们的技术平台的适度吞吐量性质与潜力的产生相结合 通过SysCODE联盟建立的基因调控网络是一个强有力的评估试验平台 网络预测和最终的潜在目标，用于开发体外方法和治疗组织 再生和器官发生。

Stem cells, embryonic and adult, represent the foundation for regenerative medicine. Their unique ability to undergo self-renewal balanced with the capacity to generate numerous differentiated cell types promises to revolutionize therapy of degenerative disorders. A central challenge to their widespread translational use is the identification of environmental cues and genetic regulatory networks that govern the self-renewal and directed differentiation of stem cells into a tissue of interest. Identification of the underlying gene regulatory circuitry at work is a key step for directing stem cell differentiation. Moreover, the ability to modulate and monitor stem cells to test predicted gene regulatory network relationships is a necessary step in evaluating plausible, reproducible and efficient methods for the genetic guidance of differentiation. Here we propose to adapt our existing microtechnology platform for dynamic imaging of multiplexed microenvironments to understanding and evaluating the role of gene regulatory networks in stem cell differentiation. The introduction of known genetic activators combined with cell lineage reporters provides a unique readout for the potential use of genetic methods to guide stem cell differentiation. Through the viral expression of exogenous genetic factors and monitoring of fluorescence reporters we will evaluate gene regulatory networks underlying the differentiation of murine embryonic stem cells into pancreatic endoderm, tooth and heart valve tissue as part of the SysCODE consortium. Furthermore, the use of microtechnology tools provide microenvironments that can be exposed to a variety of independent perturbations and the response monitored with high temporal and spatial resolution, permitting the evaluation of many genetic inputs along a specific cellular differentiation pathway. The moderate throughput nature of our technology platform in combination with the generation of potential gene regulatory networks through the SysCODE consortium represent a powerful testbed for the evaluation of network predictions and ultimately potential targets for developing in vitro methods and therapies for tissue regeneration and organogenesis.