Challenging dogma: an alternative non-hierarchical, epigenetically regulated model of the urothelium.

上皮组织出现在身体内外表面的交界处，在那里它们进化出高度专业化的形式和功能。例如，皮肤进化用于物理保护和防止脱水，而肠道则专门用于消化食物和吸收营养。这两种上皮组织都表现出不断的更替，专门化细胞被来自基础祖细胞或干细胞池的增殖和迁移产生的其他细胞所取代。细胞的分层模型被用来解释组织如何在细胞丢失和获得之间保持平衡(动态平衡)，甚至解释为什么癌症干细胞应该成为癌症治疗的靶点。这个项目基于对另一种上皮组织--尿路的观察，挑战了这种通用的上皮调节和分化模型。尿路上皮是一种特殊的上皮，排列在膀胱和输尿管之间，起着紧密的尿液屏障的作用。与肠道或皮肤不同，尿路上皮细胞没有持续的更新周期：尿路上皮细胞寿命长，处于有丝分裂静止状态，但通过快速重新进入有丝分裂周期，保持有助于有效修复和再生尿路屏障的能力。在人的尿路上皮中还没有发现特定的干细胞，在这三层中的任何一层都可以观察到分裂细胞。在表型和位置上，尿路上皮细胞采用四种不同的细胞类型中的一种(我们可以分离每种细胞)。目前的分子知识是基于匀浆的尿路上皮制备，因此缺乏足够的亚型特异性细节。我们的假设是，尿路上皮细胞不是线性分化程序的一部分，而是对外源信号或其在组织中的“生态位”做出反应，包括适应变化(如损伤)而表现出适当的表型。我们的假设预测了表观遗传和信号转导机制之间的密切关系，以影响细胞表型的变化。我们将使用输尿管和膀胱的尿路上皮进行比较，以检验我们的假设，因为它们来自不同的胚胎学来源。从每个来源中，我们将分离出四种不同的尿路上皮细胞表型，并对每个亚型的转录组和表观基因组进行深入的表征，以确定不同和相似之处。我们还将分析在适应非许可细胞培养系统后分离的细胞类型，在该系统中，我们预测所有细胞将采用基线或默认参考鳞状细胞表型，而不考虑来源。我们将使用细胞培养系统来研究每个“生态位”的信号，并测试改变不同的调控途径如何改变细胞表型，我们预测表观遗传机制将发挥迄今未被认识到的关键作用，可以操纵和利用。该项目的成果将是一个数据丰富的、空间分辨率的尿路上皮图，其中包括定义不同尿路上皮亚型的调控网络和机制。这项研究的结果将是对尿路上皮组织动态平衡的重要新理解，这将挑战组织生物学的长期模型，并为影响膀胱的慢性衰老疾病带来新的视角，包括未来组织工程和再生医学的治疗机会。

Epithelial tissues occur at the interface of internal and external surfaces of the body, where they have evolved highly specialised forms and functions. For example, the skin has evolved for physical protection and to protect against dehydration, whereas the gut has specialised to digest food and absorb nutrients. Both these epithelial tissues show constant turnover, with specialised cells being replaced by others generated by proliferation and migration from a basal progenitor or stem cell pool. The hierarchical model of cells programmed to pursue a one-way journey from a stem cell to the specialised, differentiated cell is used to explain how tissues maintain a balance (homeostasis) between cell loss and gain, and even why cancer stem cells should be targeted in cancer therapy. This project challenges this generic model of epithelial regulation and differentiation, based on observations from a different epithelial tissue, the urothelium.Urothelium is the specialised epithelium which lines the bladder and ureters and functions as a tight urinary barrier. Unlike gut or skin, there is no constant cycle of renewal: cells of the urothelium are long-lived and mitotically-quiescent, but retain the capacity to contribute to efficient urinary barrier repair and regeneration by rapid re-entry into the mitotic cycle. No specific stem cell has ever been identified in human urothelium and dividing cells can be observed in any of the three layers. Phenotypically and positionally, urothelial cells adopt one of four distinct cell types (each of which we can isolate). Current molecular knowledge is based on homogenised urothelial preparations and therefore lacks sufficient subtype-specific detail. Our hypothesis is that rather than being part of a linear differentiation programme, urothelial cells display the appropriate phenotype in response to exogenous cues or their "niche" within the tissue, including adaptation to change (eg damage). Our hypothesis predicts an intimate relationship between epigenetic and signal transduction machineries to effect changes in cell phenotype. We will test our hypothesis using urothelium from the ureter and the bladder for comparison, as these are of different embryological derivations. From each source, we will isolate, to high purity, each of the four distinct urothelial cell phenotypes and perform an in depth characterisation of the transcriptome and epigenome of each subtype to determine differences and similarities. We will also analyse the separated cell types after adaptation to a non-permissive cell culture system where we predict that all cells will adopt a baseline or default reference squamous phenotype irrespective of derivation. We will use the cell culture system to investigate the signals of each "niche" and test how altering different regulatory pathways can modify cell phenotype, where we predict that the epigenetic machinery will play a hitherto unrecognised key role that can be manipulated and exploited. A deliverable from this project will be a data-rich, spatially-resolved urothelial map of the regulatory networks and machinery that defines the different urothelial subtypes. The outcome of this study will be important new understanding of urothelial tissue homeostasis that will challenge longstanding models of tissue biology and bring new perspectives to chronic diseases of ageing that affect the bladder, including future therapeutic opportunities in tissue engineering and regenerative medicine.