Characterization of Human Epidermal Stem Cells

人表皮干细胞的表征

• 批准号: 7931700
• 负责人: RUBY GHADIALLY
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931700
• 关键词: Aging; Amphiregulin; Antigens; Basal cell carcinoma; Benign; Biological Assay; Burn injury; Carcinogens; Cell Cycle; Cells; Chronic; Cutaneous; Dermatitis; Disease; Epidermal Growth Factor Receptor; Epidermis; Erinaceidae; Frequencies; Goals; Growth; Healthcare; Hematopoiesis; Hematopoietic; Homeostasis; Human; In Vitro; Individual; Injection of therapeutic agent; Interleukin-1 alpha; Longitudinal Studies; MCAM gene; Malignant - descriptor; Mus; Neoplasms; Oncogenic; Phenotype; Population; Production; Psoriasis; Recombinant Proteins; Recombinants; Regenerative Medicine; Regulation; Relative (related person); Sorting - Cell Movement; Stem cells; Stimulus; System; TFRC gene; Testing; Therapeutic; Time; Transgenic Mice; Transplantation; Veterans; Work; Wound Healing; base; carcinogenesis; cell behavior; experience; gene therapy; genetic manipulation; in vivo; inhibitor/antagonist; insight; keratinocyte; neoplastic; novel; overexpression; pifithrin; progenitor; research and development; self-renewal; stem; stem cell population; treatment strategy

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.

描述（由申请人提供）： 该项目的短期目标是获得人类表皮干细胞（EpiSC）在一个几乎纯的人口，以帮助他们的表征，提供证据支持或反对的表皮祖细胞的层次结构，并研究在EpiSC的动态平衡发生的变化，由于良性和肿瘤性过度增殖。长期目标是更好地了解表皮分化和致癌作用，从而实现干细胞靶向基因治疗，扩大EpiSC群体用于伤口愈合应用，并为良性和致癌性皮肤过度增殖提供新疗法。 在目标1A中，为了量化选择策略分离EpiSC的能力的持续目标，并且基于20年的造血经验，我们设计了一种策略以获得几乎纯的EpiSC群体，并根据其标记EpiSC与短期再增殖（转运扩增细胞，TAC）的能力来评估推定的标记物。在目的1A中，细胞内干细胞抗原的表达将用于选择FACS相容的推定EpiSC标志物，用于体内移植测定中的定量测试。在目标1A ii中，将优化谱系阴性标志物的混合物，类似于造血中使用的混合物，以消除表达与增殖或分化状态相关的抗原的细胞。在Aim 1A iii中，将使用来自Aim 1A i的最有效的选择策略沿着来自Aim 1A ii的谱系阴性混合物对EpiSC进行FACS分选，并在移植测定中定量富集程度。我们不仅将研究长期再增殖能力，还将研究我们获得的最富集的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推定的TAC的富集，并且这项工作将为单个表皮祖细胞或祖细胞层级提供证据。在目标2中，我们将检验致癌剂（Bmi-1、Shh、p53）促进EpiSC对称自我更新分裂从而增加干细胞群体的假设，而促进良性皮肤过度增殖的剂（IL-1 α、双调蛋白）将增加不对称EpiSC分裂，从而维持EpiSC群体并仅产生TAC的增加。 正在不断地做出巨大努力，以开发基于各种无法比较的测定的EpiSC标志物组。我们的计划是在干细胞行为的定量和可重复的功能测定中使用可用的标记物，以确定现有推定标记物分离EpiSC的相对能力，并且最重要的是策略性地联合收割机组合它们的使用，以获得几乎纯的EpiSC群体。我们的研究还将有助于理解EpiSC分化，并提供关于是否有一个祖细胞层次或单个表皮祖细胞负责表皮的产生的见解。研究良性和致癌性表皮过度增殖中EpiSC稳态的改变不仅可以为良性和恶性过度增殖性皮肤疾病的可能治疗策略提供信息，还可以增加我们对正常和失调的EpiSC稳态的理解。 公共卫生关系： 在这个建议中，我们将获得一个几乎纯的表皮干细胞群体，并提供证据的祖先或一个单一的表皮祖细胞的层次。分离几乎纯的表皮干细胞群体对于再生医学应用（例如创伤、烧伤）、靶向干细胞用于遗传操作以及对于表皮干细胞调节/分化的更基础的研究将是重要的。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改变，以更好地了解良性过度增殖和肿瘤性皮肤病中失调的干细胞稳态，并提供可能的治疗策略。用于伤口愈合的再生医学与VA医疗保健特别相关，良性过度增殖性疾病（银屑病，慢性皮炎）和皮肤肿瘤也是如此，所有这些都在老年退伍军人中增加。