Differentiation of tissue- and devlopment of tumor stem cells

组成组织和器官的细胞处于不断更新的状态。衰老的、分化的细胞通过细胞脱落或凋亡被清除，并被来自组织特异性干细胞或祖细胞的细胞所取代。同样，肿瘤也由终末分化细胞和干细胞样细胞组成。后一种亚群被认为是驱动肿瘤生长和对治疗产生潜在抵抗的因素。虽然作为一个概念已经确立，但肿瘤干细胞是出了名的难以识别。到目前为止，还没有描述出癌症干细胞的独特标记。此外，肿瘤干细胞的起源细胞仍不清楚。干细胞被定义为能够进行自我更新的对称和分化的非对称有丝分裂的细胞。它们可以分化成几种特殊的细胞类型。虽然没有稳定的标记物，但组织干细胞可以通过表面标记物或转录因子（如Oct4、nanog或Sox2）的表达来识别。干细胞生活在一个高度特化的微环境中，即干细胞生态位，它严格调节着干细胞的维持、增殖和分化。在干细胞行为调控中描述的信号分子包括Wnt、Notch、FGF-2和tgf - β。tgf - β是一种多效细胞因子，参与发育过程和组织维持和修复。tgf - β通过Smad2/3通路和MAPK通路等多种细胞内信号级联传递信号，并与PI3K/mTOR和BMP信号级联广泛相互作用，参与调控干细胞和分化细胞的分化、存活、增殖和凋亡的信号网络。此外，tgf - β通过改变细胞外基质的组成影响细胞生物学；一般来说，tgf - β通过增加基质胶原蛋白的产生来增加基质的刚性。进一步研究表明，干细胞分化受基质硬度本身的强烈影响。除了这些因素外，核限制蛋白/脑（nuclear restricted protein/brain, NRP/B）已被证明在各种肿瘤的恶性转化中起关键的调节作用，并在分化的胚胎干细胞中过表达。为了稳定培养肿瘤干细胞，我们需要一种工具来分析给定细胞群在缺乏强大标记的情况下的干细胞特征。在我们的研究中，具有CD15、CD24、CD44、CD133和醛脱氢酶活性的facs分选细胞系和原代细胞不具有干细胞特性。我们的下一步将是使用荧光蛋白表达报告质粒，用于组织干细胞（如Oct4和nanog）中表达的转录因子，以识别上皮细胞、基质细胞和肿瘤细胞中的干细胞亚群。我们最近建立了人乳腺上皮细胞和基质细胞在无血清条件下的分离和培养。目前，我们通过免疫荧光和FACS分析鉴定这些培养中的亚群，并优化培养条件以延长培养时间，使细胞保持稳定的形态表型。为了测试维生素、氨基酸或脂类等物质对细胞增殖和形态的影响，我们采用高通量成像（Opera系统），使我们能够研究来自同一细胞群的细胞数量、细胞活力/死亡、细胞形态和蛋白质表达。利用人乳腺癌细胞系，我们观察到有丝分裂肿瘤细胞亚群中tgf - β的不对称活化。这些数据暗示在干细胞有丝分裂过程中tgf - β可能决定子细胞的命运。然而，由于肿瘤干细胞的稳定标记物尚未确定，我们目前与Ron McKay合作，利用皮质神经元干细胞（NSC）研究tgf - β对干细胞有丝分裂和分化的影响。我们能够通过免疫荧光证明NSCs内源性表达活跃的tgf - β， tgf - β以浓度依赖的方式降低NSC死亡，而ALK5抑制剂SB431542增加细胞死亡。接下来，我们将使用McKay实验室已经建立并可用的高通量成像技术（Opera系统）和延时成像技术（荧光/相位对比/ DIC）来研究tgf - β信号网络在NSC分化中的作用。我们将使用外源性tgf - β和小分子抑制剂来影响信号级联反应。细胞增殖、分化和信号级联的激活将通过荧光活性染料、荧光蛋白偶联报告试验和免疫细胞化学/免疫荧光来监测。使用这些方法，我们的目标不仅是确定tgf - β是否影响干细胞分化，而且还确定在哪个时间框架内信号被激活。所有的方法都可以很容易地应用于其他类型的干细胞，特别是一旦确定了乳腺组织/肿瘤干细胞。由于基质弹性一方面影响干细胞分化，另一方面参与乳腺癌的肿瘤发生和肿瘤进展，我们在这里的目的是确定基质弹性是否通过改变肿瘤干细胞池的大小来影响肿瘤进展。我们建立了生成丙烯酰胺和硅树脂基质的协议，这些基质具有一定的弹性，可以用纤维连接蛋白等基质成分功能化。细胞系的初步实验表明，随着基质密度的增加，乳腺上皮细胞确实表现出肿瘤细胞中常见的蛋白质表达改变。我们现在将研究干细胞、瞬时扩增细胞和祖细胞群的基质相关变化的生物学相关性。特别是，我们将使用已经建立的MCF10A模型询问基质密度是否会增加上皮细胞的恶性肿瘤，以及这是否由于干细胞样细胞池的扩大。我们将进一步研究，如果组织干细胞样细胞生长在与组织密度不合适的基质上（例如，如果神经干细胞生长在骨骼硬度的基质上），是否会产生肿瘤细胞群。我们还开始建立以下项目：使用Smad null小鼠模型，我们将确定正常和乳腺癌干细胞之间异染色质结构的特定改变或修饰。我们进一步计划利用微阵列和Affymetrix分析Smad野生型和敲除小鼠获得的乳腺上皮细胞和造血干细胞，以确定参与干细胞增殖和分化的维持。将确定目标分子的分子/生化机制，并计划进行体内研究，以验证已确定的分子在维持干细胞特性方面的重要性。最后，我们将研究NRP/B对胚胎干细胞分化作用的分子机制。NRP/B诱导敲除小鼠将用于研究NRP/B在胚胎发育和干细胞分化中的生物学功能。

Cells comprising tissues and organs are in a constant state of turnover. Senescent, differentiated cells are removed by cell shedding or apoptosis, and are replaced by cells derived from tissue specific stem or progenitor cells. Similarly, tumors are composed of terminally differentiated as well as stem cell like cells. This latter subpopulation is believed to drive tumor growth and to underly resistance to therapy. Although well established as a concept, tumor stem cells are notoriously difficult to identify. So far no unique marker for cancer stem cells has been described. Furthermore, the cell of origin of tumor stem cells remains unclear. Stem cells are defined as cells that can undergo self renewing symmetric and differentiating asymmetric mitosis. They can differentiate into several specialized cell types. Though stable markers are unavailable, tissue stem cells can be identified by surface markers or by expression of transcription factors such as Oct4, nanog, or Sox2. Stem cells reside in a highly specialized microenvironment, the stem cell niche, which tightly regulates stem cell maintenance, proliferation, and differentiation. Signaling molecules described in the regulation of stem cell behavior include Wnt, Notch, FGF-2, and TGF-beta. TGF-beta is a pleiotropic cytokine that is involved in developmental processes and in tissue maintenance and repair. TGF-beta signals through several intracellular signaling cascades such as the Smad2/3 pathway, and MAPK pathways, and extensively interacts with other signaling cascades such as PI3K/mTOR and BMP signaling, and as such participates in a signaling network that regulates differentiation, survival, proliferation and apoptosis of stem cells and differentiated cells. Additionally, TGF-beta influences cell biology by altering the composition of the extracellular matrix; generally, TGF-beta increases matrix rigidity via increased stromal collagen production. It has further been shown that stem cell differentiation is strongly influenced by the matrix rigidity itself. In addition to these factors, nuclear restricted protein/brain (NRP/B) has been shown to play a critical regulatory role in malignant transformation of various tumors and to be overexpressed in differentiated embryonic stem cells. In order to work with stable culture of tumor stem cells, we need a tool to analyze stem cell characteristics of a given cell population in the absence of a robust marker. In our hands, FACS-sorting cell lines and primary cells for CD15, CD24, CD44, CD133, and aldehyde dehydrogenase activity did not cells with stem cells properties. Our next step will be to use fluorescence protein expressing reporter plasmids for transcription factors that are expressed in tissue stem cells such as Oct4 and nanog to identify stem cell like subpopulation in epithelial cells, stromal cells, and tumor cells. We recently established the isolation and culture of human breast epithelial and stromal cells under serum free conditions. Currently, we identify subpopulations in these cultures by immunofluorescence and FACS analysis, and optimize the culture conditions to extend the culture time during which cells remain in a stable morphological phenotype. To test the influence of substances such as vitamins, amino acids, or lipids on cell proliferation and morphology we employ high throughput imaging (Opera system) that enables us to investigate cell number, cell vitality / death, cell morphology, and expression of proteins from the same cell population. Using a human breast cancer cell line we observed asymmetric activation of TGF-beta in a subset of mitotic tumor cells. These data imply that TGF-beta may determine the fate of daughter cells during stem cell mitosis. However, since since stable markers for tumor stem cells are not yet identified, we currently employ cortical neuronal stem cells (NSC) to investigate the influence of TGF-beta on stem cell mitosis and differentiation in collaboration with Ron McKay. We were able to demonstrate by immunofluorescence that NSCs endogenously express active TGF-beta, and that TGF-beta decreases NSC death in a concentration dependent fashion, while the ALK5 inhibitor SB431542 increased cell death. We will next investigate the role of the TGF-beta signaling network on NSC differentiation using high throughput imaging techniques (Opera system) and time lapse imaging (fluorescence / phase contrast / DIC) which are established and available in the McKay lab. We will use exogenous TGF-beta and small molecule inhibitors to influence signaling cascades. Cell proliferation, differentiation, and activation of signaling cascades will be monitored by using fluorescent vital dyes, fluorescent protein coupled reporter assays, and immunocytohemistry / immunofluorescence. Using these methods we aim to identify not only if TGF-beta affects stem cells differentiation, but also in which time frame signaling is activated. All methods can be readily applied to other types of stem cells, in particular breast tissue/tumor stem cells as soon as those are identified. Since matrix elasticity influences stem cells differentiation on on side, and is involved in tumorigenesis and tumor progression in breast cancer on the other side, we here aim to identify whether matrix elasticity influences the tumor progression by altering the size of the tumor stem cell pool. We established protocols for generating acrylamide and silicone matrices of defined elasticity that can be functionalized with matrix components such as fibronectin. Preliminary experiments using cell lines indicate that with increasing matrix density breast epithelial cells indeed show alterations of protein expression that are typically found in tumor cells. We will now investigate the biological relevance of matrix-related changes of the stem cell, transiently amplifying cell, and progenitor cell populations. Particularly, we will ask whether matrix density increases malignancy in epithelial cells using the already established MCF10A model, and if this is due to the expansion of a stem cell like cell pool. We will furthermore investigate if tissue stem cell like cells can give rise to tumor cell populations if grown on a matrix of inappropriate density for the tissue (e.g. if neuronal stem cell are grown on matrix of the rigidity of bone). We have also begun to establish the following projects: Using the Smad null mouse model, we will identify the specific alteration(s) or modification(s) of heterochromatin structure between normal and breast cancer stem cells. We further plan to identify the involved in the maintenance of stem cell proliferation and differentation by utilizing microarray and Affymetrix analysis of mammary epithelial and hematopoetic stem cells obtained from Smad wild type and knockout mice. Molecular/biochemical mechanisms of the target molecule(s) will be determined and in vivo studies are planned to verify the significance of the identified molecules on the maintenance of stem cell properties. Finally, we will study the molecular mechanism of NRP/B's action on the differentiation of embryonic stem cell. NRP/B inducible knockout mice will be generated and used for the biological function of NRP/B on the embryonic development and stem cell differentiation.