Cell fate and tissue turnover in the aged studied with multi-isotope imaging mass

描述(由申请人提供)：有必要在老龄化的背景下量化细胞周转。我们将建立一种新方法的基础，以提供关键信息来满足这一需求。这种方法将允许跟踪稳定同位素标记的细胞，并使用稳定同位素测量细胞分裂的数量。我们将使用多同位素成像质谱仪(MIMS)，这是我们开发的一种新方法，MIMS被称为“一场成像革命”，它“开始产生曾经被认为无法获取的信息”。我们将使用MIMS成像和测量细胞核内稳定同位素标记的DNA。这将使我们能够监测细胞分裂，时间范围可能从几分钟到几年，因为稳定的同位素不会衰退，不会改变新陈代谢，也没有毒性。MIMS通过定量了解衰老过程中的细胞内稳态，有可能给细胞周转和更新的研究带来革命性的变化。一种新的理论认为，与衰老相关的表型可能至少部分归因于组织干细胞数量或功能的下降。组织再生能力随着年龄的增长而下降，在肌肉、血液、肝脏和脑等组织中，这种下降被归因于组织特异性干细胞和祖细胞的数量或功能减少。驻留的自我更新细胞在许多器官的动态平衡维持中起着重要作用。大量研究表明，衰老会改变成人干细胞的功能。干细胞潜能的这种损害可能在一定程度上导致随着年龄的增长而观察到的进行性组织恶化。因此，了解衰老过程中体内细胞的更新是了解衰老过程的一个重要目标。虽然量化细胞分裂速率的能力是干细胞生物学中的一个关键因素，但在体内测量细胞刷新一直是极具挑战性的。有许多追踪干细胞进入分化组织的技术，但这些技术有重要的局限性。我们将开发的新方法使用稳定的非放射性同位素来获得高分辨率的高精度定量数据，从而消除了对放射性脉冲或基因标记的需要。用稳定的同位素标记DNA实际上消除了毒性和长期剂量的问题，因为没有放射性，而且这些稳定的同位素在体内安全的证据是压倒性的。MIMS具有比放射性碳标记高几个数量级的灵敏度，可以同时分析同一细胞内的多个同位素，允许在很长一段时间内从不同的时间脉冲中定量结合的同位素，从而描述单个细胞的分裂历史，这将是一个福音。 公共卫生相关性：我们将开发一种新的方法，使用多同位素成像质谱仪(MIMS)来研究衰老过程中的细胞稳态。我们的工具将允许我们跟踪稳定同位素标记的细胞并测量细胞分裂的数量。这种方法有可能给细胞周转和再生的研究带来革命性的变化，并为量化和可视化衰老过程中的细胞动态平衡开辟了一条强大的新途径。

DESCRIPTION (provided by applicant): There is a need to quantify cellular turnover in the context of aging. We will establish the foundations of a new method to contribute crucial information to fulfill this need. This method will permit to track stable-isotope labeled cells and to measure the number of cell divisions using stable-isotopes. We will use Multi-Isotope Imaging Mass Spectrometry (MIMS), a novel methodology that we have developed, MIMS has been called "an imaging revolution" that is "beginning to yield information once considered inaccessible". We will use MIMS to image and measure stable isotope labeled DNA within nuclei of cells. This will allow us to monitor cell division with time scales potentially ranging from minutes to years, since stable isotopes do not decay, do not modify metabolism and are not toxic. MIMS has the potential to revolutionize the study of cell turnover and refreshment by permitting quantitative understanding of cellular homeostasis during aging. An emerging theory is that aging-related phenotypes might be due, at least in part, to a decline in the number or function of tissue stem cells. Tissue regenerative capacity declines with age, and in tissues such as muscle, blood, liver and brain, this decline has been attributed to a diminished number or function of tissue-specific stem and progenitor cells. Resident self-renewing cells have a significant role in the homeostatic maintenance of many organs. Numerous studies have shown that aging alters adult stem cell function. This impairment of stem cell potential could, in part, cause the progressive tissue deterioration that is observed with aging. Thus, understanding in vivo cell refreshment during aging is an important goal for understanding the aging process. Although the ability to quantify the rate of cell divisions is a crucial factor in stem cell biology, it has been extremely challenging to measure cellular refreshment in vivo. Numerous techniques exist for tracking stem cells incorporation into differentiated tissues but these techniques have important limitations. The new method that we will develop uses stable, nonradioactive isotopes to obtain highly precise quantitative data at high resolution, eliminating the need for radioactive pulses or genetic labeling. Labeling DNA with stable isotopes practically eliminates issues of toxicity and long-term dosing since there is no radioactivity and the evidence for in vivo safety of these stable isotopes is overwhelming. MIMS has sensitivities several orders of magnitude above radioactive carbon labeling and can analyze multiple isotopes simultaneously inside the same cell, permitting the quantification of incorporated isotopes from different time pulses over very long period of time and thus to describe the history of division of a single cell, which will be a boon. PUBLIC HEALTH RELEVANCE: We will develop a new methodology employing Multi-Isotope Imaging Mass Spectrometry (MIMS) to study cellular homeostasis during aging. Our tools will allow us to track stable-isotope labeled cells and measure the number of cell divisions. This method has the potential to revolutionize the study of cell turnover and regeneration and opens a powerful new way to quantify and visualize cellular homeostasis during aging.