Microfluidic perfusion control of embryonic stem cell differentiation

描述(由申请人提供)：干细胞传统上是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的再生疗法的发展中发挥重要作用。多能胚胎干细胞(ESCs)能够分化为多种细胞类型，包括功能神经元、心肌细胞和胰岛β细胞，为生物学研究和再生细胞治疗提供了强大的细胞来源。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、可靠和同质的方式有效地将ESCs分化为特定的细胞命运。在胚胎发育过程中，分化的时空保真度受到适当时间、有限持续时间内局部形态发生因子数量的精确调节。然而，大多数通常用于检测干细胞对形态素治疗反应的分化的体外方法，缺乏以精确的时间分辨率统一呈现确定数量的分子的能力。由微流控培养系统产生的强制对流细胞间灌流(FCIP)可以以均匀的时间和剂量依赖的受控方式将分子递送到3D培养的细胞中，从而克服了扩散运输的局限性。微流控灌流培养系统固有的灵活性和可扩展性代表了一种促进干细胞定向分化的创新和可翻译的方法。基于这一理论基础和重要的初步数据，本研究的目标是：1)确定分化的胚胎干细胞3D聚集体的运输限制；2)确定灌流培养对胚胎干细胞分化的产量和均质性的影响；3)高通量地检测形态呈递对胚胎干细胞分化的剂量和时间效应。这些研究的完成将产生新的信息，即通过使用微流控灌流培养来设计细胞外微环境的动态分子组成，从而更可控地指导ESCs的分化。所提出的方法代表了一条全新的途径，通过同时控制局部分子因子的剂量和时间呈现来更有效地指导干细胞的体外分化，这可能是干细胞技术发展中广泛适用的原则。 与公共卫生相关：干细胞再生和诊断技术的发展目前受到无法有效控制干细胞分化的限制。这项建议旨在通过微灌流培养系统控制3D干细胞微环境中形态因子的呈递剂量和时机，以提高多潜能干细胞分化的效率和同质性。

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.