Microfluidic perfusion control of embryonic stem cell differentiation

描述（由申请人提供）：干细胞传统上作为发育和形态发生模型的宝贵资源，但它们也在旨在治疗各种退行性疾病和创伤性损伤的再生疗法的开发中发挥重要作用。多能胚胎干细胞（ESC）能够分化成一系列细胞类型，包括功能性神经元、心肌细胞和胰腺β细胞，因此代表了生物学研究和再生细胞疗法的强大细胞来源。尽管多能干细胞具有明显的潜力，但一个关键的限制是无法以可重复的、可靠的和同质的方式将ESC有效地分化为特定的细胞命运。在胚胎发育过程中，分化的时空保真度是由在适当的时间和有限的持续时间内出现的局部形态发生因子的量精确调节的。然而，通常用于检查干细胞响应于形态发生剂处理的分化的大多数体外方法缺乏以精确的时间分辨率以限定的量均匀地呈现分子的能力。由微流体培养系统产生的强制对流细胞间灌注（FCIP）可以通过以均匀的时间和剂量依赖性受控方式将分子呈递到细胞的3D培养物来克服扩散运输的限制。微流体灌注培养系统固有的灵活性和可扩展性代表了增强定向干细胞分化的创新和可转化方法。基于这一基本原理和重要的初步数据，本提案的目的是1）定义分化ESC的3D聚集体的运输限制，2）确定灌注培养对ESC分化的产量和均一性的影响，以及3）以高通量方式检查形态发生素呈递对ESC分化的剂量和时间效应。这些研究的完成将产生新的信息的能力，更可控地指导分化的胚胎干细胞的工程细胞外微环境的动态分子组成，使用微流体灌注培养。所提出的方法代表了一种从根本上新的路线，更有效地指导干细胞的分化，在体外通过同时控制剂量和时间介绍的分子因子本地，这可能是一个广泛适用的原则，在干细胞技术的发展。 公共卫生相关性：干细胞再生和诊断技术的发展目前受到不能有效控制干细胞分化的限制。该提案旨在通过微灌注培养系统控制3D干细胞微环境内形态发生因子呈递的剂量和时间来提高多能干细胞分化的效率和均一性。

DESCRIPTION (provided by applicant): Stem cells have traditionally served as a valuable resource for models of development and morphogenesis, but they also factor significantly in the development of regenerative therapies aiming to treat various degenerative diseases and traumatic injuries. Pluripotent embryonic stem cells (ESCs) are capable of differentiating into an array of cell types, including functional neurons, cardiomyocytes and pancreatic beta cells, thus representing a robust cell source for biological studies and regenerative cell therapies. Despite the clear potential of pluripotent stem cells, a critical limitation is the inability to efficiently differentiate ESCs to specific cell fates in a reproducible, reliable and homogeneous manner. During embryonic development, the spatiotemporal fidelity of differentiation is precisely regulated by the local amount of morphogenic factors presented at the appropriate time with a finite duration. However, most in vitro methods typically used to examine the differentiation of stem cells in response to morphogen treatment lack the ability to uniformly present molecules in defined amounts with precise temporal resolution. Forced convection intercellular perfusion (FCIP) created by microfluidic culture systems can overcome the limitations of diffusive transport by presenting molecules to 3D cultures of cells in a uniform temporal and dose-dependent controlled manner. The inherent flexibility and scalability of microfluidic perfusion culture systems represents an innovative and translatable approach to enhance directed stem cell differentiation. Based on this rationale and significant preliminary data, the objectives of this proposal are to 1) define the transport limitations of 3D aggregates of differentiating ESCs, 2) determine the effects of perfusion culture on the yield and homogeneity of ESC differentiation, and 3) examine the dose and temporal effects of morphogen presentation on ESC differentiation in a high-throughput manner. The completion of these studies will yield novel information about the ability to more controllably direct the differentiation of ESCs by engineering the dynamic molecular composition of the extracellular microenvironment using microfluidic perfusion culture. The proposed approach represents a fundamentally new route to more efficiently direct the differentiation of stem cells in vitro through the simultaneous control of dose and temporal presentation of molecular factors locally, which may be a broadly applicable principle in the development of stem cell technologies. PUBLIC HEALTH RELEVANCE: The development of stem cell regenerative and diagnostic technologies is currently limited by an inability to efficiently control the differentiation of the stem cells. This proposal seeks to improve the efficiency and homogeneity of pluripotent stem cell differentiation by controlling the dose and timing of morphogenic factor presentation within 3D stem cell microenvironments via microperfusion culture systems.