Quantitative morphology of induced phenotypes in cultured cells and tissues

在之前与Minoru Ko博士（LG-NIA）合作的工作中，我们能够建立一种仅基于细胞形态的干细胞分化试验，使用无特异性标记物的相差成像。 今年，我们与日本熊本大学的斋藤纪子合作，扩展了这项工作，以表征人类诱导多能干细胞（iPS）的质量。 作为同一合作的一部分，我们还发表了一项研究，在该研究中，我们使用荧光显微镜对培养的人类胚胎进行模式识别，以表明肌动蛋白相关蛋白ARP 6影响核仁的结构。 我们继续研究了C.线虫寿命在已发表的工作中，我们使用WND-CHARM来识别C.优雅 我们使用这种技术来分类蠕虫的基础上，他们的年龄状态在一个过渡期，其中一个老龄化的人口是均匀分布在个人之间的第一阶段和第二阶段。 这些蠕虫在遗传上是相同的，按实足年龄、生长条件和视觉外观，只能使用WND-CHARM分类为年龄状态。 对这两个亚群进行的微阵列实验揭示了数百个基因的表达谱发生了显着改变。 通过将我们的基因列表与C.在线虫中，我们能够识别出几个基因家族和功能组，这是我们研究所独有的。这项研究中唯一确定的衰老基因的一个流行主题是参与靶向蛋白水解的基因，这似乎是第一次衰老状态转变的标志。 在以前的工作中，我们已经开发了一个年龄状态分类器，使我们能够更强大地识别个别蠕虫所占据的老化状态。 最近，我们已经表明，这种状态分类器是足够强大的，以确定这些国家在其他菌株的蠕虫。 我们使用这个分类器按年龄状态对几千只蠕虫进行手动分类，以研究这些衰老状态特有的基因表达模式。 目前，我们已经完成了对C.优雅 初步结果表明，我们在四倍重复进行的生物学重复是可重复的，并且在年龄状态之间的转换中基因表达存在许多可重复的差异。我们目前正在分析这些基因表达结果。 我们已经开始表征RNAi敲除对在第一次年龄-状态转变中被鉴定为差异表达的基因的影响。 一组产生富含半胱氨酸区域的分泌蛋白的基因似乎包含可以提前或延迟第一次转换的基因。 我们还在研究一组热休克基因，它们的表达在第一次转换中发生了最显著的变化。 初步结果表明，年龄转变的时间可以通过单基因RNAi实验来操纵。 我们正在通过qPCR对所有已知的小热休克蛋白基因在C.与衰老过渡有关的线虫。 我们正在研究这样一种假设，即基因家族可能有特定于某些年龄状态的成员，这些成员在这些衰老过渡期间被交换。 在目前的工作中，我们正在将小的热休克家族基因的qPCR研究扩展到其他重要的基因家族，这些基因家族在这些老化过渡中进行转录调控。

In previous work in collaboration with Dr. Minoru Ko (LG-NIA),we were able to establish a stem-cell differentiation assay based on cell morphology alone, using phase-contrast imaging without specific markers. This year we were able to extend this work in a collaboration with Noriko Saitoh of Kumamoto University in Japan to characterize the quality of human induced pluripotent stem (iPS) cells. As part of the same collaboration, we also published a study where we used pattern recognition in fluorescence microscopy of cultured human cels to show that the actin-related protein ARP6 affects the structure of the nucleolus. We have continued work characterizing the molecular basis of morphological age-state transitions during the C. elegans life-span. In published work we used WND-CHARM to identify distinct morphological aging states in C. elegans. We used this technique to sort worms based on their age state during a transition period where an aging population is evenly divided between individuals in Stage I and Stage II. The worms were identical genetically, by chronological age, by growth conditions, and by visual appearance, and could only be sorted into age-states using WND-CHARM. Micro-array experiments performed on these two sub-populations revealed several hundred genes with significantly altered expression profiles. By comparing our gene lists with those from other aging studies in C. elegans, we were able to identify several gene families and functional groups that were unique to our study. A prevailing theme of the aging genes uniquely identified in this study were those involved in targeted proteolysis, which appears to be a hallmark of this first aging state transition. In previous work, we have developed an age-state classifier that enables us to more robustly identify the aging state occupied by individual worms. More recently we have shown that this state-classifier is robust enough to identify these states in other strains of worms. We used this classifier to manually sort several thousand worms by age-state in order to study gene expression patterns specific to these aging states. Currently, we have completed work on expression profiling of all three observed aging states and transitions in C. elegans. Preliminary results indicate that the biological replicates we performed in quadruplicate were reproducible, and that there are many reproducible differences in gene expression in the transitions between age-states. We are currently analyzing these gene expression results. We have begun to characterize the effect of RNAi knockdown on the genes identified as differentially expressed in the first age-state transition. A group of genes producing secreted proteins with cysteine-rich regions appears to contain genes that can either advance or delay the first transition. We are also looking at a set of heat-shock genes who's expression is among the most significantly altered in the first transition. Preliminary results indicate that the timing of age transitions can be manipulate with single-gene RNAi experiments. We are performing a detailed analysis by qPCR of the changes in gene expression of all of the the known small heat-shock protein genes in C. elegans associated with aging transitions. We are investigating the hypothesis that gene families may have members specific to certain age-states that get swapped out during these aging transitions. In current work, we are expanding the qPCR studies of small heat-shock family genes to other important gene families that undergo transcriptional regulation across these aging transitions.