Isolation, Characterization, and Behavior of Melanocyte Stem Cells

黑素细胞干细胞的分离、表征和行为

• 批准号: 7965565
• 负责人: thomas j hornyak
• 金额: $ 47.13万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965565
• 关键词: Ablation; Adult; Antibodies; Area; Behavior; Biological; Biological Assay; Bromodeoxyuridine; Cell Surface Proteins; Cell surface; Cells; Chimeric Proteins; Collaborations; DNA; Dermal; Development; Dopachrome isomerase; Doxycycline; Exhibits; Flow Cytometry; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Proteins; Genes; Goals; Green Fluorescent Proteins; Hair; Hair follicle structure; Hair shaft structure; Histones; Immunofluorescence Immunologic; In Situ; Individual; Label; Malignant Neoplasms; Mediating; Microarray Analysis; Mus; Nature; Neural Crest; Nucleosides; Oncogenes; Pigmentation physiologic function; Pigments; Plant Roots; Population; Property; Proteins; RNA; Regulation; Reporter; Reporting; Role; Sampling; Signal Transduction; Skin; Specimen; Stem cells; Subcategory; Suspension substance; Suspensions; System; Tetanus Helper Peptide; Tetracyclines; Time; Tissues; Trans-Activators; Transgenic Mice; Validation; adult stem cell; base; cancer stem cell; candidate marker; cell behavior; cell type; experimental analysis; in vitro Assay; in vivo; insight; interest; keratinocyte; melanocyte; melanoma; molecular phenotype; neoplastic cell; precursor cell; progenitor; promoter; protein expression; research study; sample fixation; self renewing cell; self-renewal; stem cell niche; stem cell population; therapeutic target; tumor

The goals and objectives of this project are to identify and characterize the quiescent melanocyte cell population in the murine hair follicle that previous studies have shown contains the melanocyte stem cell population. The adult melanocyte stem cell is the self-renewing cell that generates the melanogenic pigmentary unit during each successive hair cycle. Understanding this cell population is important for a variety of different reasons. Insight into the determinants of quiescence and cellular self-renewal of these cells may provide information useful for elucidating important principles of stem cell behavior. Since the bulge area of the murine hair follicle harbors not only melanocyte stem cells but also keratinocyte and neural crest stem cells, defining the signals required by melanocyte stem cells will allow us to understand how different adult stem cell types interact and communicate within a defined stem cell niche. Finally, because of the conservation of cell surface protein expression that has been noted between adult, tissue-specific stem cells and the corresponding cancer stem cell type, it is likely that characterizing the molecular phenotype of melanocyte stem cells, especially cell surface marker expression, will provide information useful for confirming or refuting the existence of melanoma stem cells and dissecting this cellular subset from tumors for experimental analysis and therapeutic targeting. To identify melanocyte stem cells, we are using the assumption that quiescent melanocytes in the lower permanent portion of the hair follicle, the bulge region, possess stem cell properties. This assumption is based upon prior reports that cells in this region expressing certain melanocyte markers exhibit quiescent, or label-retaining, properties, and can renew the follicular pigmentary unit following antibody-mediated ablation of the differentiated melanocyte population. To identify quiescent cells in these prior reports, mice were injected with the nucleoside derivative 5-bromo-2'-deoxyuridine (BrdU) to mark newly synthesized DNA, whose persistence over time indicated the slow-cycling nature of these cells. To identify the quiescent cells in these previous studies, fixation of skin specimens was required, thus rendering the cells non-viable and unsuitable to obtain RNA for gene expression profiling. We are characterizing on a large scale the gene expression profile of melanocyte stem cells in order to define a gene signature that describes these cells and differentiates them from other melanocytic cells. We also hope to discover specific markers of this cell type. Hence, for our studies, we have developed a system that utilizes doxycycline-inducible transgenic mice in conjunction with a stable green fluorescent protein (GFP) reporter to label quiescent cells. This approach is similar to a previously reported study in which a keratinocyte-specific promoter was utilized to isolate keratinocyte stem cells from the murine follicular bulge using fluorescence-activated cell sorting (FACS) for gene expression profiling. In our case, we have generated transgenic mice expressing the tetracycline-regulated transactivator tTA ('Tet-Off') from the dopachrome tautomerase (Dct) promoter, a melanocyte-specific promoter expressed in melanocyte stem cells. We have identified one founder line, out of a total of 17 screened, which drives successfully expression of a histone 2B (H2B)-GFP fusion protein reporter protein in a doxycycline-regulated manner. When mice are not administered doxycycline, expression of GFP is detected in melanocytic cells in the infundibular, bulge, and lower outer root sheath segment of the adult murine hair follicle. However, following doxycycline administration, the number of GFP-expressing cells is greatly reduced. Follicular GFP-expressing cells under these conditions are generally restricted to the lower permanent portion of the hair follicle, and are located in the CD34-expressing region of the follicle that marks the bulge. We have used FACS to isolate GFP-expressing cells from adult dermal cellular suspensions. RNA obtained from multiple biological replicates of constitutively-expressing GFP cells and label-retaining GFP cells has been amplified and used for microarray analysis. Our collaboration with Aleksandra Michalowska in Dr. Glenn Merlino's group at NCI has led to the identification of over 200 genes whose expression differs significantly between quiescent melanocyte label-retaining cells and their non-quiescent counterparts. We chose approximately 35 of the most significantly different and biologically interesting genes for validation by quantitative real-time PCR analysis, using RNA samples derived from different pools of cells than those used for the microarray studies. Biologically independent samples were generated for this validation analysis. Approximately 15 of these genes were found to have greater than 2-fold expression in the quiescent cell subset. Currently, we are focusing on the validation of these genes on the protein level as specific markers of quiescent melanocytes. We are using flow cytometry, fluorescence-activated cell sorting, and in situ immunofluorescence studies to validate these findings. Although these studies are still ongoing, we nonetheless have some intriguing preliminary findings. Two of the validated genes have significant roles in the regulation of progenitor cell behavior in other cellular systems, suggesting a potential connection between governance of the progenitor state in follicular melanocytes and that of other cells. Identification of these genes also immediately suggests strategies to direct gene expression specifically to the melanocyte stem cell compartment. In conjunction with experiments validating markers of cellular quiescence that may represent melanocyte stem cell markers, we are also developing assays permitting us to evaluate melanocyte stem cell behavior. These assays include in vitro assays to evaluate the potential of individual cells to form colonies as well as in vivo assays, using the skin of melanocyte-deficient mice that lack pigment, to evaluate the potential of subsets of quiescent melanocytes to confer pigmentation to hair follicles in a sustained manner. Results from these experiments are promising and provide additional insight into the activities of distinct subcategories of melanocyte stem cells.

该项目的目标和目的是鉴定和表征小鼠毛囊中的静止黑素细胞群，这些细胞群先前的研究表明含有黑素细胞干细胞群。成体黑素细胞干细胞是一种自我更新的细胞，在每个连续的毛发周期中产生黑色素色素单位。由于各种不同的原因，了解这种细胞群很重要。深入了解这些细胞的静止和细胞自我更新的决定因素可能为阐明干细胞行为的重要原则提供有用的信息。由于小鼠毛囊的凸起区域不仅包含黑素细胞干细胞，还包含角化细胞和神经嵴干细胞，因此确定黑素细胞干细胞所需的信号将使我们能够了解不同类型的成体干细胞如何在确定的干细胞生态位内相互作用和交流。最后，由于在成人、组织特异性干细胞和相应的癌症干细胞类型之间已经注意到细胞表面蛋白表达的保守性，很可能表征黑素细胞干细胞的分子表型，特别是细胞表面标志物的表达，将为证实或反驳黑色素瘤干细胞的存在提供有用的信息，并从肿瘤中解剖这一细胞亚群，以进行实验分析和治疗靶向。为了鉴定黑素细胞干细胞，我们假设毛囊较低的永久性部分，即凸起区域的静止黑素细胞具有干细胞特性。这一假设是基于先前的报道，即该区域表达某些黑素细胞标记的细胞表现出静止或标签保留的特性，并且可以在抗体介导的黑素细胞群消融后更新滤泡色素单位。为了鉴定这些先前报道中的静止细胞，给小鼠注射了核苷衍生物5-溴-2'-脱氧尿苷（BrdU）来标记新合成的DNA，其随时间的持续存在表明这些细胞的缓慢循环性质。在这些先前的研究中，为了鉴定静止细胞，需要将皮肤标本固定，从而使细胞无法存活，不适合获得RNA进行基因表达谱分析。我们正在大规模地表征黑素细胞干细胞的基因表达谱，以便定义描述这些细胞并将它们与其他黑素细胞区分开来的基因特征。我们也希望发现这种细胞类型的特定标记。因此，在我们的研究中，我们开发了一种系统，利用强力霉素诱导的转基因小鼠与稳定的绿色荧光蛋白（GFP）报告基因结合来标记静止细胞。该方法类似于先前报道的一项研究，该研究利用角化细胞特异性启动子从小鼠滤泡凸起中分离角化细胞干细胞，使用荧光激活细胞分选（FACS）进行基因表达谱分析。在我们的研究中，我们从多巴胺自变酶（Dct）启动子中产生了表达四环素调节的反激活子tTA（“Tet-Off”）的转基因小鼠，Dct启动子是黑素细胞干细胞中表达的一种黑素细胞特异性启动子。我们已经从17个筛选的菌株中鉴定出一个方正菌株，它以强力霉素调节的方式成功地驱动了组蛋白2B (H2B)-GFP融合蛋白报告蛋白的表达。当小鼠未被给予强力霉素时，在成年小鼠毛囊的基底、凸起和下外根鞘段的黑色素细胞中检测到GFP的表达。然而，给予强力霉素后，表达gfp的细胞数量大大减少。在这种情况下，毛囊中表达gfp的细胞通常局限于毛囊的较低的永久性部分，并且位于毛囊中cd34表达区域，标志着凸起。我们使用FACS从成人真皮细胞悬浮液中分离出表达gfp的细胞。从组成表达的GFP细胞和保留标记的GFP细胞的多个生物复制中获得的RNA已被扩增并用于微阵列分析。我们与NCI Glenn Merlino博士小组的Aleksandra Michalowska合作，鉴定了200多个基因，这些基因的表达在静止的黑素细胞标记保留细胞和非静止的对应细胞之间存在显著差异。我们选择了大约35个最显著的差异和生物学上有趣的基因，通过定量实时PCR分析进行验证，使用来自不同细胞池的RNA样本，而不是用于微阵列研究的RNA样本。为验证分析生成了生物独立的样品。这些基因中大约有15个在静止细胞亚群中有大于2倍的表达。目前，我们正专注于在蛋白质水平上验证这些基因作为静止黑素细胞的特定标记。我们正在使用流式细胞术、荧光激活细胞分选和原位免疫荧光研究来验证这些发现。虽然这些研究仍在进行中，但我们已经有了一些有趣的初步发现。其中两个被证实的基因在其他细胞系统中对祖细胞行为的调节中起着重要作用，这表明滤泡黑素细胞中祖细胞状态的调控与其他细胞的调控之间存在潜在的联系。这些基因的鉴定也立即提出了直接基因表达到黑素细胞干细胞室的策略。结合实验验证可能代表黑素细胞干细胞标记的细胞静止标记，我们也在开发允许我们评估黑素细胞干细胞行为的分析。这些试验包括体外试验，评估单个细胞形成集落的潜力；体内试验，利用缺乏色素的黑色素细胞缺陷小鼠的皮肤，评估静止黑色素细胞亚群以持续的方式赋予毛囊色素沉着的潜力。这些实验的结果是有希望的，并为黑素细胞干细胞不同亚类的活性提供了额外的见解。