Creation of adult epithelial stem cells from differentiated epithelial

描述（申请人提供）：近年来，利用胚胎干细胞（ES）及其分化后代再生各种成人组织的前景备受关注。然而，我们相信，在可预见的未来，至少在上皮组织的情况下，一个更可行和可实现的策略将从实验方案中衍生出来，该实验方案旨在诱导分化的成人细胞重编程为承诺的祖细胞和干细胞，然后它们可以作为载体，用于因衰老或各种病理过程而耗尽的成人组织的再生。为此，我们打算开发一种可应用于分化上皮细胞的信号剂混合物，以便将其重新编程为具有谱系承诺的上皮干细胞。我们的策略是重新激活一个潜在的发育程序，称为上皮-间质转化（EMT）。我们之前已经证明，诱导进入EMT赋予分化的乳腺上皮细胞许多与上皮干细胞相关的特征。重要的是，我们相信我们的理论适用于广泛的分化上皮细胞，包括分层鳞状上皮，如口腔黏膜。EMTs影响胚胎发育各个阶段的关键形态发生步骤。总的来说，研究集中在emt将细胞命运从上皮细胞转变为间充质细胞的能力上，从而引起细胞形态和功能的深刻变化。然而，我们最近的研究结果表明，emt在干细胞自我更新和组织稳态中具有新的生理作用。人类和小鼠乳腺干细胞已被证明具有间充质特性，进一步将EMT与干细胞生物学联系起来，这一发现强化了EMT产物与干细胞状态之间的联系。重要的是，EMT和干细胞状态之间的密切相似之处似乎并不是乳腺上皮的特质：新出现的证据表明，EMT诱导的转录因子（TF）蜗牛和蛞蝓在皮肤形态发生中发挥了重要作用。在发育过程中，上皮间质转化（epithelial-mesenchymal transition, emt）是由相邻细胞层的单个细胞接收到的环境信号诱导和协调的。因此，几个旁分泌信号在胚胎上皮细胞上的聚合诱导上皮细胞经历EMT。基于这些观察结果，我们假设细胞内EMT程序的初始触发取决于一组确定的旁分泌信号对该细胞的伴随刺激，并且由此产生的间充质/干细胞状态的维持取决于随后自分泌信号回路的激活，这些信号回路涉及先前触发进入EMT的相同因素。事实上，我们的初步数据与该模型一致，强调了细胞外信号环境对调节emt的重要性，并加强了我们开发一种实验方案的基本原理，该实验方案使用信号剂的混合物作为一种手段，将分化的上皮细胞去分化和重编程为干细胞样状态。从长远来看，我们打算开发一种方案，以诱导多种不同的成人上皮细胞类型受控地去分化为相应的干细胞。这种方案具有产生大量承诺上皮干细胞的潜力，有望再生各种耗尽的成人上皮组织。

DESCRIPTION (provided by applicant): Much attention has focused in recent years on the promise of using embryonic stem (ES) cells and their differentiated progeny to regenerate various adult tissues. We believe, however, that for the foreseeable future, a far more viable and attainable strategy will derive, at least in the case of epithelial tissues, from experimental protocols designed to induce the reprogramming of differentiated adult cells into committed progenitors and stem cells, which can then serve as vehicles for the regeneration of adult tissues depleted by aging or by various pathological processes. For this purpose, we intend to develop a cocktail of signaling agents that can be applied to differentiated epithelial cells in order to reprogram them to lineage-committed epithelial stem cells. Our strategy is to re-activate a latent developmental program, termed the Epithelial-Mesenchymal Transition (EMT). We have previously shown that induction into EMT confers on differentiated mammary epithelial cells many of the traits associated with epithelial stem cells. Importantly, we believe that our rationale is applicable to a wide range of differentiated epithelial cells, including stratified squamous epithelia, such as the mucosa of the mouth. EMTs effect key morphogenetic steps during various steps of embryonic development. In general, research has focused on the ability of EMTs to switch cellular fate from epithelial to mesenchymal, thereby causing profound changes in cell morphology and function. However, our recent findings indicate a novel physiological role for EMTs in stem-cell self-renewal and thus in tissue homeostasis. Reinforcing this association between the products of EMT and the stem-cell state is the finding that putative human and mouse mammary stem cells have been shown to possess mesenchymal traits, further linking EMT to stem cell biology. Importantly, the close parallels between EMT and the stem-cell state do not appear to be an idiosyncrasy of the mammary epithelium: emerging evidence suggests an essential role for the EMT-inducing Transcription Factors (TF) Snail and Slug in skin morphogenesis. During the course of development, epithelial-mesenchymal transitions (EMTs) are induced and coordinated by contextual signals received by individual cells from adjacent cell layers. Thus, the convergence of several paracrine signals on an embryonic epithelial cell induces that epithelial cell to undergo an EMT. Based on these observations, we have hypothesized that the initial triggering of an EMT program within a cell depends on concomitant stimulation of that cell by a defined set of such paracrine signals, and that maintenance of the resulting mesenchymal/stem-cell state depends subsequently on the activation of autocrine signaling loops involving the same factors that previously triggered entrance into the EMT. Indeed, our preliminary data are in concordance with this model, underscoring the importance of the extracellular signaling environment for regulating EMTs, and reinforcing our rationale to develop an experimental protocol with a cocktail of signaling agents as a means to dedifferentiate and reprogram differentiated epithelial cells to a stem cell-like state. In the longer term, we intend to develop a protocol for inducing the controlled dedifferentiation of a number of distinct adult epithelial cell types into their corresponding stem cells. Such protocols have the potential of generating large numbers of committed epithelial stem cells that hold promise for the regeneration of a variety of depleted adult epithelial tissues. PUBLIC HEALTH RELEVANCE: Much attention has focused in recent years on the promise of using embryonic stem (ES) cells to regenerate various adult tissues. We believe, however, that for the foreseeable future, a far more viable and attainable strategy will derive from experimental protocols designed to induce the reprogramming of differentiated adult cells into committed progenitors and stem cells. These reprogrammed cells can then serve as vehicles for the regeneration of adult tissues depleted by aging or by various pathological processes. For this purpose, we intend to develop a cocktail of signaling agents that can be applied to in vitro cultures of differentiated epithelial cells in order to reprogram them to committed epithelial stem cells via activation of a latent developmental program. We believe that this strategy can be applied to many epithelial tissues, such as the skin or mouth mucosa.