Kinase signaling analysis of iPS cell reprogramming and differentiation

描述（由申请人提供）：与胚胎干细胞（ESCs）一样，诱导多能干细胞（iPS）能够分化成体内的每种细胞类型，但它们的衍生（称为重编程）在技术上更为直接，并且可以在不使用捐赠卵子或胚胎的情况下进行。最近发现的将人类成人细胞重编程为iPS细胞的方法引起了极大的兴奋，因为iPS细胞在患者特异性再生医学方面的潜力，以及在胚胎发育和细胞分化研究方面的新机会，以及为药物测试和疾病机制研究创造疾病特异性细胞系的能力。目前的研究工作主要集中在：1)寻找iPS细胞重编程的改进方法，因为目前的方法缓慢、效率低下，而且可能致癌；2)指导iPS细胞向体内各种细胞类型的分化；3)确定iPS细胞用于再生治疗的安全性。到目前为止，iPS细胞重编程和分化的研究主要集中在基因转录、DNA甲基化和组蛋白修饰的变化上，但令人惊讶的是，很少有人关注蛋白质磷酸化的细胞信号传导，尽管这是真核细胞中最普遍的信号传导机制。为了阐明重编程和分化过程中激酶信号传导的“黑匣子”，我们正在开发监测iPS细胞中蛋白磷酸化的方法。由于iPS重编程和分化发生在异质细胞群体中，因此使用流式细胞术获得单细胞测量对于识别感兴趣的罕见信号群体至关重要。利用Nolan实验室开发的磷酸化特异性流式细胞术，以及我们的合作者Marius Wernig的重编程专业知识，我们将表征多能和分化细胞类型之间激酶信号传导的差异，然后监测iPS细胞重编程过程中的激酶信号传导。关键信号事件的识别将指导我们努力确定提高iPS细胞重编程的速度、效率和安全性的条件。除了对重编程过程提供前所未有的见解外，我们还将为研究iPS细胞和ESCs分化为体内约200种细胞类型中的任何一种时的激酶信号传导奠定基础。研究每种细胞类型的分化超出了本提案的范围，但作为第一步，我们将确定ESC/iPS细胞分化为造血干细胞（造血干细胞）所需的激酶信号事件，这一过程对人类治疗具有很大的希望，因为它为治疗血液病和体外造血提供了患者匹配的造血干细胞的可再生来源。确定造血干细胞衍生过程中的关键信号事件将有助于找到更好的分化方案，流式细胞术单细胞分析将成为监测再生医学中iPS细胞衍生造血干细胞纯度和安全性的重要临床工具。

DESCRIPTION (provided by applicant): Like embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells are able to differentiate into every cell type in the body, but their derivation (known as reprogramming) is more straightforward technically, and can be performed without the controversial use of donated eggs or embryos. The recent discovery of methods to reprogram human adult cells into iPS cells has generated much excitement due to the potential of iPS cells for patient-specific regenerative medicine, as well as for the new opportunities made possible in the study of embryonic development and cellular differentiation, and for the ability to create disease-specific cell lines for drug testing and the study of disease mechanism. Current research efforts are focused on 1) finding improved methods for iPS cell reprogramming, because current protocols are slow, highly inefficient, and possibly oncogenic, 2) directing the differentiation of iPS cells into the various cell types of the body, and 3) determining the safety of iPS cells for regenerative therapy. Until now, the study of iPS cell reprogramming and differentiation has focused on changes in gene transcription, DNA methylation, and histone modification, but surprisingly little attention has been devoted to cellular signaling by protein phosphorylation, even though this is the most prevalent signaling mechanism in eukaryotic cells. In order to illuminate the "black box" of kinase signaling during reprogramming and differentiation, we are developing methods to monitor protein phosphorylation in iPS cells. Because iPS reprogramming and differentiation occur in heterogeneous cell populations, the use of flow cytometry to obtain single cell measurements is crucial to identify the rare signaling populations of interest. Using phospho-specific flow cytometry protocols developed in the Nolan laboratory, and the reprogramming expertise of our collaborator Marius Wernig, we will characterize the differences in kinase signaling between pluripotent and differentiated cell types, and then monitor kinase signaling during iPS cell reprogramming. The identification of key signaling events will guide our efforts to identify conditions that increase the speed, efficiency, and safety of iPS cell reprogramming. In addition to providing unprecedented insight into the reprogramming process, we will also lay the groundwork for studying kinase signaling during differentiation of iPS cells and ESCs into any of the ~200 cell types of the body. Studying the differentiation of every cell type is beyond the scope of this proposal, but as a first step we will identify the kinase signaling events required for ESC/iPS cell differentiation into Hematopoietic Stem Cells (HSCs), a process that holds great promise for human therapy because it provides a renewable source of patient-matched HSCs for treating hematologic disease and for generating blood in vitro. Identifying the key signaling events in HSC derivation will guide efforts to find improved differentiation protocols, and single cell analysis by flow cytometry will be an important clinical tool to monitor the purity and safety of iPS cell-derived HSCs for regenerative medicine. PUBLIC HEALTH RELEVANCE: Like embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells can differentiate into every cell type in the body, but their derivation (known as reprogramming) is more straightforward technically, and can be performed without the controversial use of donated eggs or embryos. iPS cells hold great promise for regenerative medicine, and for the study of disease mechanisms and human development, but the cellular signaling that controls their reprogramming and differentiation remains poorly understood. We are developing methods to measure protein phosphorylation (the most common mechanism of cellular signaling) in iPS cells, and we will use the key signaling events we identify to improve the speed and efficiency of iPS derivation, as well as the safety and utility of iPS cells for regenerative medicine.