Kinase signaling analysis of iPS cell reprogramming and differentiation

描述(申请人提供)：与胚胎干细胞(ESCs)一样，诱导多能干细胞(IPS)能够分化为体内的每一种细胞类型，但它们的派生(称为重新编程)在技术上更简单，而且可以在不使用有争议的捐赠卵子或胚胎的情况下进行。最近发现的将人类成体细胞重新编程为iPS细胞的方法引起了极大的兴奋，这是因为iPS细胞在患者特有的再生医学中的潜力，以及在胚胎发育和细胞分化研究中可能出现的新机会，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。目前的研究工作集中在1)寻找iPS细胞重新编程的改进方法，因为目前的方案速度慢、效率低，并且可能是致癌的；2)引导iPS细胞分化为体内各种类型的细胞；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对细胞信号的影响，尽管这是真核细胞中最普遍的信号机制。为了阐明重编程和分化过程中激酶信号的“黑匣子”，我们正在开发监测iPS细胞中蛋白质磷酸化的方法。由于iPS的重编程和分化发生在不同的细胞群体中，使用流式细胞术来获得单细胞测量结果对于识别稀有的感兴趣的信号群体至关重要。利用诺兰实验室开发的磷酸特异性流式细胞术方案，以及我们的合作者Marius Wernig的重新编程专业知识，我们将表征多潜能细胞和分化细胞类型之间的激酶信号差异，然后监控iPS细胞重新编程过程中的激酶信号。关键信号事件的识别将指导我们努力识别提高iPS细胞重新编程的速度、效率和安全性的条件。除了提供对重编程过程的前所未有的见解外，我们还将为研究iPS细胞和ESCs分化为人体~200种细胞类型中的任何一种细胞过程中的激酶信号奠定基础。研究每种细胞类型的分化超出了这一建议的范围，但作为第一步，我们将确定ESC/iPS细胞分化为造血干细胞(HSCs)所需的激酶信号事件，这一过程对人类治疗大有可为，因为它提供了用于治疗血液病和体外造血的患者匹配的HSCs的可再生来源。识别HSC来源中的关键信号事件将指导寻找改进的分化方案的努力，而用流式细胞仪进行单细胞分析将是监测再生医学中iPS细胞来源的HSC纯度和安全性的重要临床工具。 与公共卫生相关：与胚胎干细胞(ESCs)一样，诱导多能干细胞(IPS)可以分化为体内的每一种细胞类型，但它们的派生(称为重新编程)在技术上更简单，而且可以在不使用有争议的捐赠卵子或胚胎的情况下进行。IPS细胞在再生医学、疾病机制和人类发育研究方面有着巨大的希望，但控制其重新编程和分化的细胞信号仍然知之甚少。我们正在开发方法来测量iPS细胞中的蛋白质磷酸化(最常见的细胞信号机制)，我们将使用我们确定的关键信号事件来提高iPS派生的速度和效率，以及iPS细胞用于再生医学的安全性和实用性。

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.