Robustness of the Intestinal Stem Cell Niche

 描述(申请人提供)：肠上皮是体内再生最快的组织之一。再生是由肠隐窝底部的一组干细胞维持的，称为隐窝基柱状细胞(CBCs)。在过去的5年里，对CBCs的研究帮助改变了干细胞生物学的教条。CBCs不是静止的，经历着不对称的分裂，遵循单向分化的层次，它高度增殖，能够对称分裂，并可被更多分化的细胞类型所取代。从那时起，在许多其他组织中也发现了类似的干细胞群体和调控原则。然而，所有这些发现都集中在一个中心问题上：干细胞利基如何控制可塑性，以保持恒定数量的CBCs？由于增殖性CBCs与结肠癌有关，而且干细胞移植正被用于临床治疗各种疾病，了解其潜在的控制机制是很重要的。细胞动力学创新的多光子成像和激光消融技术将被用来研究干细胞利基内细胞如何分裂、移动和恢复损失，重点放在四个基本问题上：(1)细胞壁细胞是否能够进行对称和不对称分裂，(2)同一细胞基座中的单个细胞是否具有相同的增殖潜力，(3)细胞壁龛如何从细胞损失中恢复，以及(4)壁龛外的细胞如何去分化和重新进入壁龛？信号动力学利用3D肠道器官分析，将进行系统的研究，以寻找主要信号通路之间的反馈和串扰机制。将进行动态系统分析，以了解它们对利基控制电路的影响。整合和验证为了整合实验结果和计算洞察力，将建立一个随机的、多尺度的模型，捕捉干细胞利基中的细胞分裂、运动和信号事件。这个模型将被用来检验关于利基控制电路的假设。一种新的通过将基因工程干细胞注射到胚泡中的植入试验已经被开发出来用于体内验证。通过使用可打开的腹部窗口、3D打印的肠道支持、标记的血管路线图和跟踪算法，创新的活体GI成像技术已经被开发出来。通过窗户，一种特殊的激光可以在不损害周围组织的情况下，消融壁龛内的单个细胞。一个计算框架将整合动态系统分析和多尺度建模，以研究控制细胞和信号动力学的调节电路。预测将在新型嵌合小鼠身上得到验证，这些嵌合小鼠的肠道隐窝来自胚泡注射细胞。初步发现，在失去一个CBC后，干细胞生态位经历了广泛的重组，证明了其动态性质。已经确定了各种反馈和串扰机制来提高稳健性。

 DESCRIPTION (provided by applicant): The intestinal epithelium is one of the fastest regenerative tissues in the body. Regeneration is sustained by a group of stem cells, termed crypt base columnar cells (CBCs), at the bottom of the intestinal crypt. In the past 5 years, studies of CBCs have helped transform the dogma of stem cell biology. Rather than being largely quiescent, undergoing asymmetric division, and following the unidirectional differentiation hierarchy, CBCs are highly proliferative, capable of symmetric division, and replaceable by more differentiated cell types. Since then, similar stem cell populations and regulatory principles have been identified in many other tissues. However, all these discoveries converge on a central question: how does the stem cell niche control plasticity in order to keep a constant number of CBCs? Since proliferative CBCs have been linked to colon cancer, and stem cell transplants are being used clinically for treating a variety of diseases, it is importantto understand the underlying control. Cellular dynamics Innovative multiphoton imaging and laser ablation technologies will be used to investigate how cells divide, move and recover loss inside the stem cell niche, focusing on four fundamental questions: (1) are CBCs capable of both symmetric and asymmetric division, (2) do individual CBCs in the same niche have equal proliferative potential, (3) how does the niche recover from cell loss, and (4) how do cells outside the niche dedifferentiate and reenter the niche? Signaling dynamics Using the 3D intestinal organoid assay, a systematic study will be carried out to search for feedback and crosstalk mechanisms among major signaling pathways. Dynamical systems analysis will be performed to understand their impact on the niche control circuitry. Integration and Validation To integrate experimental findings and computational insights, a stochastic, multiscale model will be built that captures cellular division, movement and signaling events in the stem cell niche. This model will be used to test hypotheses on the niche control circuitry. A novel engraftment assay through injection of genetically engineered stem cells into blastocysts has been developed for in vivo validation. Innovation Novel in vivo GI imaging techniques have been developed by using openable abdominal window, 3-D printed intestinal support, labeled vasculature roadmap, and tracking algorithms. Through the window, a special laser can ablate a single cell inside the niche without damaging the surrounding tissue. A computational framework will integrate dynamical systems analysis and multiscale modeling to study the regulatory circuitry that controls cellular and signaling dynamics inside the niche. Predictions will be validated in novel chimeric mice with intestinal crypts derived from blastocyst-injected cells. Preliminary findings The stem cell niche undergoes extensive reorganization after loss of one CBC, attesting to its dynamic nature. Various feedback and crosstalk mechanisms have been identified to improve robustness.