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，非常需要再生策略。人类多能干细胞（hPSC）由于其独特的自我更新能力， 几乎无限增殖，并上升到各种细胞类型。在再生过程中，临时细胞外基质（ECM）为hPSC提供多种信号，并指导新基质形成的过程。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。研究hPSC-ECM相互作用面临的主要障碍在于使用来自解剖集落的细胞团块的hPSC培养技术的限制。在这些培养方法中，hPSC在产生所需的组织特异性细胞类型之前，可重复地通过自发分化阶段至所有三个胚胎胚层。本研究的目的是生成和表征单层单细胞培养模型，其允许研究与hPSC谱系特化相关的生物学现象。我们将评估在单层中培养的hPSC经历上皮向间充质转化（EMT）并分化为特定胚层的潜力。使用单细胞单层培养方法，我们将探索对称/不对称细胞分裂在调节hPSC多能性和谱系特化中的作用。这将通过采用微接触印刷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。该项目有两个具体目标：（1）确定单层培养的hPSC经历EMT的潜力，以及（2）确定不对称分裂在hPSC命运特化中的作用。本研究开发的新方法具有广泛的适用性，包括组织工程和再生医学，疾病建模和药物测试的许多研究领域。

 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.