Skin regeneration by terminally differentiated keratinocytes

描述（由申请人提供）：干细胞的生态位对其维持至关重要，这是公认的，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，即细胞从分化程度较高的状态向分化程度较低的状态发展，是低等脊椎动物组织再生的一个关键原理，涉及使用现有的、终末分化的细胞而不是干细胞。在哺乳动物中，去分化已经在一些器官中得到证实，如肝脏和胰腺，在这些器官中，组织再生不需要可识别的干细胞的帮助。然而，控制去分化和重编程的细胞和分子机制知之甚少。我们最近发现了一个意想不到的结果，当将经过各种方法最终分化的小鼠表皮角质形成细胞植入小鼠全层伤口床时，能够形成一个功能齐全、多系、长毛的表皮。似乎终末分化细胞是去分化为干细胞。虽然我们想提出这个模型来探索去分化和重编程的机制，但我们认为，如果没有实验证据表明这种现象发生在完整皮肤的体内，这种应用将是不成熟的。本探索性资助项目旨在为体内终末分化角质形成细胞向干细胞的逆转提供直接证据，并为研究去分化机制开发关键资源。本文提出了构建双转基因小鼠的实验，该双转基因小鼠包含Cre- LoxP系统，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，当终末分化的细胞确实经历重编程成为干细胞时，报告基因的表达将不再局限于终末分化的皮肤层，而是在基础的复制室中很明显。虽然这一应用概述了一个具体的问题，但这项工作的含义解决了广泛的问题，如——微环境在多大程度上对细胞施加了“干性”？受损的干细胞是由先前存在的干细胞补充的，还是分化程度更高的后代可以获得“干细胞性”？这些问题是干细胞的核心所在。因此，这些进展应该使我们能够探索细胞去分化和重编程的分子基础，并回答有关组织适应性反应的基本问题。项目描述：干细胞存在于许多组织中，并在生命中维持这些组织的稳态，它们被认为是损伤或移植后组织再生的主要贡献者。该项目的目标是提高我们对干细胞生物学的理解，用于修复和再生目的。

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.