Unraveling the Role of Asymmetric Division in Lineage Specification of Stem Cells

 描述(由申请人提供)：由于创伤和各种肌肉骨骼疾病，迫切需要再生策略。人类多能干细胞(HPSC)因其独特的自我更新能力，为再生治疗提供了一种很有前途的工具。 几乎无限增殖，并上升为各种类型的细胞。在再生过程中，临时细胞外基质(ECM)为hPSCs提供多种信号，指导新基质的形成过程。关于hPSC如何感知和响应来自周围环境的线索的基本问题有待进一步探索。HPSC-ECM相互作用研究面临的一个主要障碍是hPSCs培养技术的局限性，这种培养技术使用的是解剖克隆中的细胞团。在这些培养方法中，在产生所需的组织特异性细胞类型之前，hPSCs不可避免地经历了自发分化到所有三个胚胎胚层的阶段。这项研究的目的是建立和表征一种单层单细胞培养模型，该模型允许研究与hPSC谱系指定相关的生物学现象。我们将评估培养在单层中的hPSCs经历上皮向间充质转化(EMT)的潜力并分化为特定的生殖层。利用单细胞单层培养的方法，我们将探索对称/不对称细胞分裂在调节hPSC多能性和谱系指定中的作用。这将通过使用微接触打印技术在单细胞水平上控制细胞极性和细胞骨架组织来实现。本项目有两个特定的目标：(1)确定培养在单层中的hPSCs进行EMT的潜力；(2)确定不对称分裂在hPSCs命运决定中的作用。本研究开发的新方法在组织工程和再生医学、疾病建模和药物测试等多个研究领域具有广泛的适用性。

 DESCRIPTION (provided by applicant): There is a profound need for regeneration strategies due to trauma and various musculoskeletal diseases. Human pluripotent stem cells (hPSCs) offer a promising tool for regenerative therapies because of their unique capability to self-renew, proliferate nearly indefinitely, and rise to various cell types. In the regeneration process, the temporary extracellular matrix (ECM) provides multiple signals to hPSCs and guides the process of new matrix formation. Fundamental questions on how hPSCs sense and respond to the cues from surrounding environment await further exploration. A main hurdle facing the studies of hPSC-ECM interactions lies in limitation of hPSCs culture techniques which use clumps of cells from dissected colonies. In these culture methods, hPSCs unavoidably pass through the stage of spontaneous differentiation to all three embryonic germ layers before the generation of the desired tissue-specific cell type. The goal of this study is to generate and characterize a monolayer single-cell culture model that allows for the investigation of biological phenomena associated with hPSC lineage specification. We will evaluate the potential of hPSCs cultured in monolayers to undergo epithelial-to-mesenchymal transition (EMT) and differentiate to specific germ layers. Using the single-cell monolayer culture method, we will explore the role of symmetric/asymmetric cell division in regulation of hPSC pluripotency and lineage specification. This will be accomplished by employing microcontact printing techniques to control cell polarity and cytoskeletal organization of at a single-cell level. This project has wo specific aims: (1) determine the potential of hPSCs cultured in a monolayer to undergo EMT, and (2) determine the role of asymmetric division in fate specification of hPSCs. The novel approach developed in this study has broad applicability to many research areas including tissue engineering and regenerative medicine, disease modeling and drug testing.