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.

该项目的目标和目标是识别和表征鼠毛囊中静态黑素细胞群，以前的研究表明，这些细胞毛囊中含有黑素细胞干细胞群体。成年黑素细胞干细胞是在每个连续的头发周期中产生黑色素发色单元的自我更新细胞。由于各种不同的原因，了解该细胞种群很重要。 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会提供有用的信息，可用于阐明干细胞行为的重要原理。由于鼠毛囊的凸起面积不仅藏有黑素细胞干细胞，还包括角质形成细胞和神经rest干细胞，定义了黑素细胞干细胞所需的信号，这将使我们能够理解不同的成年干细胞类型如何在定义的干细胞小细胞元素中相互作用并传达。最后，由于在成年，组织特异性干细胞和相应的癌症干细胞类型之间已注意到细胞表面蛋白表达的保存，很可能表征了黑素细胞干细胞的分子表型，尤其是细胞表面标志物表达，将提供有用的信息，可提供用于确认或排除该细胞瘤干细胞和分析的靶向分析和实验性分析的信息。为了鉴定黑色素细胞干细胞，我们使用的是假设毛囊较低的永久性部分（凸起区域）具有干细胞特性的假设。该假设是基于先前的报道，即该区域中表达某些黑素细胞标记物的细胞表现出静止或保留标签的特性，并且可以在抗体介导的分化黑色素细胞种群消融后更新卵泡色素单元。为了鉴定这些先前报告中的静态细胞，向小鼠注射了核苷衍生物5-bromo-2'-脱氧尿苷（BRDU），以标记新合成的DNA，随着时间的推移，其持久性表明这些细胞的慢速循环性质。为了鉴定这些先前研究中的静态细胞，需要对皮肤标本的固定，从而使细胞不可生存且不适合获得基因表达分析的RNA。我们正在大规模表征黑素细胞干细胞的基因表达谱，以定义一个描述这些细胞并将其与其他黑素细胞区分开的基因特征。我们还希望发现这种细胞类型的特定标记。因此，对于我们的研究，我们开发了一种系统，该系统利用可耐二霉素诱导的转基因小鼠以及稳定的绿色荧光蛋白（GFP）报告基因来标记静态细胞。这种方法类似于先前报道的研究，在该研究中，使用荧光激活的细胞分选（FACS）来分离角质形成细胞特异性启动子与鼠滤泡性膨胀分离鼠卵泡凸起（FACS）进行基因表达分析。在我们的情况下，我们已经产生了表达四环素调节的反式激活剂TTA（'tet-off'）的转基因小鼠，该小鼠是从黑素细胞干细胞中表达的黑素细胞特异性启动子（一种黑素细胞特异性启动子）的。我们已经确定了一条创始人线，在总共17个筛选中，该产品驱动了组蛋白2b（H2b）-GFP融合蛋白报道蛋白蛋白的成功表达，以强力霉素调节的方式。当小鼠不给予强力霉素时，在成年鼠毛囊的漏斗，凸起和下部根鞘段中检测到GFP的表达。但是，遵循强力霉素给药，表达GFP的细胞的数量大大减少了。在这些条件下，表达卵泡GFP的细胞通常仅限于毛囊的较低永久性部分，并且位于标记凸起的卵泡的CD34表达区域。我们已经使用FACS将表达GFP的细胞与成年皮肤细胞悬浮液分离。从组成型表达的GFP细胞和保留标记的GFP细胞的多种生物学重复获得的RNA已被放大并用于微阵列分析。我们与Glenn Merlino博士在NCI的Glenn Merlino小组中与Aleksandra Michalowska的合作导致了200多个基因的鉴定，这些基因的表达在静态黑素细胞固定的细胞及其非质量对应物之间存在显着差异。我们使用来自不同细胞池的RNA样品选择了大约35个最显着差异和生物学上有趣的基因来通过定量实时PCR分析进行验证，而不是用于微阵列研究的细胞。为此验证分析生成了生物学独立的样品。在静态细胞子群中，发现大约15个基因的表达大于2倍。当前，我们将这些基因作为蛋白质水平的验证作为静态黑素细胞的特定标记。我们正在使用流式细胞术，荧光激活的细胞分选和原位免疫荧光研究来验证这些发现。尽管这些研究仍在进行中，但我们仍然有一些有趣的初步发现。经过验证的两个基因在其他细胞系统中祖细胞行为的调节中具有重要作用，这表明祖细胞状态在卵泡黑色素细胞中的治理与其他细胞的治理之间存在潜在的联系。这些基因的鉴定还立即提出了将基因表达专门指向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的实验，我们还开发了测定法，允许我们评估黑素细胞干细胞行为。这些测定包括在体外测定中，以评估单个细胞形成菌落和体内测定的潜力，使用缺乏色素的黑素细胞缺陷小鼠的皮肤，以评估静态黑素细胞亚群的潜力，以持续的方式将色素沉着赋予毛囊。这些实验的结果是有希望的，并提供了对黑素细胞干细胞不同亚类别的活性的更多见解。