In vivo two photon imaging of stem cell differentiation

干细胞分化的体内双光子成像

• 批准号: 7016557
• 负责人: PHILIP G HAYDON
• 金额: $ 21.2万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7016557
• 关键词: bioimaging /biomedical imaging; calcium flux; calcium indicator; cell differentiation; cell growth regulation; cell population study; fluorescence microscopy; fluorescence resonance energy transfer; genetic strain; growth factor; laboratory mouse; method development; molecular /cellular imaging; nervous system transplantation; optics; photolysis; sectioning; stem cell transplantation; tissue /cell preparation

DESCRIPTION (provided by applicant): Although stem cell/progenitor transplants promise unrivalled opportunities to repair damaged and aging nervous systems, success with this approach will only become possible after identification of the regulatory principles that control the functional differentiation of transplanted cells in the nervous system. Currently the technical approaches available to studying progenitor cell differentiation limit our ability to advance this field. Ideally one would repeatedly use non-invasive imaging approaches to whole animals to monitor the functional differentiation of transplanted cells. Unfortunately, available techniques such as MRI, that offer non-invasive imaging, do not provide the necessary spatial resolution. In this project we will develop a new approach in which we integrate two-photon imaging with two-photon photolysis to monitor and probe transplanted progenitor cell differentiation. To achieve this overall goal we have three objectives. First, we will refine optical stimulation and recording approaches that permit us to study the functional activity of transplanted progenitors. Using a genetically encoded Ca2+ indicator together with two-photon FRET microscopy and photolysis we will develop imaging methods that allow us to study their integration into host circuitry. Second, we will develop repeated in vivo imaging strategies that employ non-invasive two-photon imaging and photolysis to track the functional differentiation of transplanted progenitors in vivo for periods of up to one year. Finally, we will employ these imaging strategies to determine the relative merit of differentiating progenitors by exposure to growth factors either in vitro prior to or in vivo subsequent to transplantation. The development of vital imaging methods to assess progenitor cell differentiation will provide a new approach to allow the identification of the mechanisms which positively regulate differentiation and integration of transplanted progenitors. Since we will be able to assess outcomes based on cellular function that will surely be detected before behavioral consequences can be observed, the successful development of this imaging approach will accelerate the pace of research with behavioral outcomes goals.

描述（由申请人提供）：虽然干细胞/祖细胞移植有望为修复受损和老化的神经系统提供无与伦比的机会，但只有在确定控制神经系统中移植细胞功能分化的调节原则后，这种方法才有可能取得成功。目前可用于研究祖细胞分化的技术方法限制了我们推进该领域的能力。理想情况下，人们可以对整个动物重复使用非侵入性成像方法来监测移植细胞的功能分化。不幸的是，MRI 等提供非侵入性成像的现有技术无法提供必要的空间分辨率。在这个项目中，我们将开发一种新方法，将双光子成像与双光子光解相结合，以监测和探测移植的祖细胞分化。为了实现这一总体目标，我们有三个目标。首先，我们将完善光学刺激和记录方法，使我们能够研究移植祖细胞的功能活动。使用基因编码的 Ca2+ 指示剂以及双光子 FRET 显微镜和光解作用，我们将开发成像方法，使我们能够研究它们与宿主电路的整合。其次，我们将开发重复的体内成像策略，采用非侵入性双光子成像和光解作用来跟踪移植祖细胞体内长达一年的功能分化。最后，我们将采用这些成像策略来确定通过在移植前体外或移植后体内暴露于生长因子来分化祖细胞的相对优点。评估祖细胞分化的重要成像方法的发展将提供一种新方法，以识别积极调节移植祖细胞分化和整合的机制。由于我们将能够根据细胞功能来评估结果，而细胞功能肯定会在观察到行为后果之前被检测到，因此这种成像方法的成功开发将加快以行为结果为目标的研究步伐。