Skin regeneration by terminally differentiated keratinocytes

描述(申请人提供)：众所周知，干细胞的生态位对其维持至关重要，最近的研究表明，来自微环境的指导性信号能够诱导其他非干细胞的干细胞特征。去分化，即细胞从较高分化状态向较低分化状态的演变，是低等脊椎动物组织再生的关键原则，涉及到使用现有的终末分化细胞而不是干细胞。在哺乳动物中，一些器官如肝脏和胰腺的去分化已被证明，在这些器官中，组织再生发生在没有可识别的干细胞的帮助下。然而，控制去分化和重新编程的细胞和分子机制却知之甚少。我们最近得到了意想不到的结果，当将所有指标都是终末分化的小鼠表皮角质形成细胞培养后，当移植到小鼠的全厚度伤口床中时，能够重塑全功能、多谱系、有毛的表皮。终末分化的细胞似乎被去分化为干细胞。虽然我们希望提出这个模型来探索去分化和重新编程的机制，但我们认为，如果没有实验证据表明这种现象在体内、在完整的皮肤中发生，这样的应用还为时过早。这一探索性资助项目旨在为在体内将终末分化的角质形成细胞转化为干细胞提供直接证据，并为研究去分化的机制开发关键资源。提出了构建双转基因小鼠的实验，该小鼠整合了Cre-loxP系统，并仅在终末分化的角质形成细胞中表达报告基因。这种结构是这样的，如果终末分化的细胞确实经历了干细胞的重新编程，报告基因的表达将不再局限于终末分化的皮肤层，而将在基础的、复制的隔间明显。虽然这项申请概述了一个具体的问题，但这项工作的影响涉及广泛的问题，如-微环境在多大程度上强加于细胞的“干性”？受损的干细胞是由先前存在的干细胞补充的，还是更多分化的后代能够获得“干性”？这些问题是干细胞的核心。因此，这些进展应该使我们能够探索细胞去分化和重新编程的分子基础，并回答关于组织适应性反应的基本问题。项目简介：干细胞存在于许多组织中，并在一生中维持这些组织的动态平衡，它们被认为是损伤或移植后组织再生的主要贡献者。这个项目的目标是提高我们对干细胞生物学的理解，以达到修复和再生的目的。

DESCRIPTION (provided by applicant): It is well-established that the stem cell's niche is crucial for its maintenance, and recent studies suggest that instructive signals from the microenvironment are able to induce stem cell features in other non-stem cells. De-differentiation, the progression of cells from a more to a less differentiated state, is a key principle in tissue regeneration in lower vertebrates and involves the use of existing, terminally differentiated cells rather than stem cells. In mammals, dedifferentiation has been demonstrated in some organs such as liver and pancreas where tissue regeneration occurs without the aid of identifiable stem cells. However, the cellular and molecular mechanisms that control de-differentiation and reprogramming are little understood. We recently came upon unexpected results when cultures of mouse epidermal keratinocytes that were, by all measures, terminally differentiated were able to reform a fully functional, multi-lineage, hair-bearing epidermis when implanted into a full thickness wound bed in mice. It appeared that terminally differentiated cells were de-differentiated to stem cells. Although we would like to propose this model for exploring mechanisms of de-differentiation and reprogramming, we feel such a application would be premature without experimental evidence that this phenomenon occurs in vivo, in intact skin. This Exploratory Grant project is designed to provide direct evidence for reversion of terminally differentiated keratinocytes to stem cells in vivo and to develop key resources for studying the mechanism of de-differentiation. Experiments are proposed in which double transgenic mice are constructed that incorporate the Cre- LoxP system and express a reporter gene only in terminally differentiated keratinocytes. The constructs are such that if and when terminally differentiated cells do undergo reprogramming to stem cells, reporter gene expression will no longer be confined to terminally differentiated layers of the skin but will be evident in the basal, replicative compartment. Although this application outlines a specific question, implications of this work address broad issues such as - To what extent does microenvironment impose "stemness" on cells? Are damaged stem cells replenished by pre-existing stem cells or can more differentiated progeny acquire "stemness"? These issues are at the heart of what a stem cell is. Thus these advances should allow us to explore the molecular basis of cell dedifferentiation and reprogramming and to answer fundamental questions about tissue adaptive responses. PROJECT NARRATIVE: Stem cells are present in many tissues and maintain homeostasis of those tissues throughout life, and they are believed to be the major contributors to tissue regeneration following injury or transplantation. The goal of this project is to improve our understanding of stem cells biology for reparative and regenerative purposes.