Characterization of Human Epidermal Stem Cells

描述(由申请人提供)： 该项目的短期目标是在几乎纯净的人群中获得人表皮干细胞(EpiSC)，以帮助鉴定它们，为支持或反对表皮前体细胞的分级提供证据，并研究由于良性和肿瘤性过度增殖而发生的EpiSC动态平衡的变化。长期目标是更好地了解表皮分化和癌变，实现干细胞靶向基因治疗，扩大EpiSCs用于伤口修复的种群，并为良性和致癌性皮肤过度增殖提供新的治疗方法。在目标1A中，为了量化分离EpiSCs的选择策略的能力，并基于20年的造血学经验，我们设计了一种策略，以获得几乎纯的EpiSCs群体，并根据它们标记EpiSCs的能力与短期再填充(运输放大细胞，TAC)的能力来评估假定的标记。在目标1AI中，细胞内干细胞抗原的表达将被用来选择与FACS相容的假定的EpiSC标记，用于体内移植试验的定量测试。在目标1AII中，将优化谱系阴性标记物的鸡尾酒，类似于用于造血的标记物，以消除表达与增殖或分化状态相关的抗原的细胞。在目标1AIII中，将使用目标1AI中最有效的选择策略和目标1AII中的谱系阴性鸡尾酒，对EpiSCs进行FACS分选，并在移植试验中量化浓缩程度。我们不仅将研究EpiSCs的长期再繁殖能力，还将研究我们获得的最丰富的EpiSCs群体的多能性、自我更新和体外生长潜力。在目标1B中，将确定在选定的角质形成细胞群体中假定的TAC的丰度，这项工作将为单一的表皮祖细胞或祖细胞的分级提供证据。在目标2中，我们将检验这一假说，即致癌药物(Bmi-1、Shh、P53)促进EpiSC对称性自我更新分裂，从而增加干细胞数量，而促进良性皮肤过度增殖的药物(IL-1α、两性调节素)将增加EpiSC的不对称分裂，从而维持EpiSC数量并仅产生TAC的增加。基于各种无法比较的分析方法，人们一直在努力开发EpiSC标记物。我们的计划是在干细胞行为的定量和可重复性的功能分析中使用可用的标记，确定现有假定标记分离EpiSCs的相对能力，最重要的是战略性地结合它们的使用，以获得几乎纯净的EpiSCs群体。我们的研究还将有助于理解EpiSC的分化，并为了解是一系列祖细胞还是单个表皮祖细胞负责表皮的产生提供了洞察力。研究良性和致癌性表皮过度增殖中EpiSC稳态的变化，不仅有助于研究良性和恶性过度增生性皮肤病的可能治疗策略，而且有助于加深我们对正常和失调的EpiSC稳态的理解。 公共卫生相关性： 在这项建议中，我们将获得几乎纯净的表皮干细胞群体，并为祖细胞或单个表皮干细胞的分级提供证据。分离出几乎纯净的表皮干细胞群对于再生医学应用(如创伤、烧伤)、针对干细胞进行基因操作以及更基础的表皮干细胞调控/分化研究具有重要意义。我们还将研究与良性和肿瘤性过度增殖刺激相关的干细胞稳态的变化，以更好地了解良性过度增生性和肿瘤性皮肤病中失调的干细胞稳态，并为可能的治疗策略提供见解。创伤愈合的再生医学与退伍军人医疗保健特别相关，良性增生性疾病(牛皮癣、慢性皮炎)和皮肤肿瘤也是如此，所有这些在老年退伍军人中都会增加。

DESCRIPTION (provided by applicant): The short-term objective of this project is to obtain human epidermal stem cells (EpiSCs) in a nearly pure population in order to aid their characterization, to provide evidence for or against a hierarchy of epidermal progenitors, and to study alterations in EpiSC homeostasis that occur due to benign and neoplastic hyperproliferation. The long-term objective is to better understand epidermal differentiation and carcinogenesis enable stem cell-targeted gene therapy, expand populations of EpiSCs for wound-healing applications, and to provide novel therapies for benign and oncogenic cutaneous hyperproliferation. In Aim 1A, for the ongoing goal of quantifying the ability of selection strategies to isolate EpiSCs, and based on 20 years of hematopoietic experience, we have devised a strategy to obtain a nearly pure population of EpiSCs and to assess putative markers in terms of their ability to mark EpiSCs versus short term repopulating (transit amplifying cells, TACs). In Aim 1A i the expression of intracellular stem cell antigens will be used to choose FACS-compatible putative EpiSC markers for quantitative testing in an in vivo transplantation assay. In Aim 1A ii a cocktail of lineage-negative markers will be optimized, akin to that used in hematopoesis, to eliminate cells expressing antigens associated with a proliferative or differentiated state. In Aim 1A iii EpiSCs will be FACS-sorted, using the most effective selection strategy from Aim 1A i along with the lineage-negative cocktail from Aim 1A ii, and the degree of enrichment quantified in the transplantation assay. We will not only study the long-term repopulation ability, but the multipotency, self-renewal, and in vitro growth potential of the most enriched population of EpiSCs that we obtain. In Aim 1B the enrichment of putative TACs in selected keratinocyte populations will be determined and this work will provide evidence for a single epidermal progenitor or a hierarchy of progenitors. In Aim 2 we will examine the hypothesis that oncogenic agents (Bmi-1, Shh, p53) promote EpiSC symmetric self-renewal divisions thereby increasing the stem cell population, while agents that promote benign cutaneous hyperproliferation (IL-1alpha, amphiregulin) will increase asymmetric EpiSC divisions, thus maintaining the EpiSC population and producing an increase in TACs only. Great effort is continuously being exerted to develop sets of EpiSC markers based on a variety of assays that cannot be compared. Our plan is to use available markers in a quantitative and reproducible functional assay of stem cell behavior, to determine the relative ability of existing putative markers to isolate EpiSCs, and most importantly to combine their use strategically, to obtain a nearly pure population of EpiSCs. Our studies will also help in the understanding of EpiSC differentiation and provide insight as to whether a hierarchy of progenitors or a single epidermal progenitor is responsible for production of the epidermis. Studying alterations in EpiSC homeostasis in benign and oncogenic epidermal hyperproliferation will not only inform regarding possible treatment strategies for benign and malignant hyperproliferative cutaneous diseases but also increase our understanding of normal and dysregulated EpiSC homeostasis. PUBLIC HEALTH RELEVANCE: In this proposal we will obtain a nearly pure population of epidermal stem cells and provide evidence for a hierarchy of progenitors or a single epidermal progenitor. Isolation of a nearly pure epidermal stem cell population will be important for regenerative medicine applications (e.g. wounds, burns), targeting stem cells for genetic manipulation, and for more basic studies of epidermal stem cell regulation/differentiation. We will also examine the alterations in stem cell homeostasis associated with benign and neoplastic hyperproliferative stimuli, to better understand dysregulated stem cell homeostasis in benign hyperproliferative and neoplastic cutaneous disease, as well as providing insight into possible therapeutic strategies. Regenerative medicine for wound healing is especially relevant for VA healthcare, as are benign hyperproliferative disorders (psoriasis, chronic dermatitis) and cutaneous neoplasia, all of which are increased in aging veterans.