Reprogramming Non-myocytes to Cardiomyocytes in vivo

在体内将非心肌细胞重编程为心肌细胞

基本信息

批准号：
9493517
负责人：
RICHARD T LEE
金额：
$ 42.78万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9493517
关键词：
Animal Experiments Animals Biology Brain Stem Cardiac Myocytes Cell Lineage Cells Complement Congestive Heart Failure Creatine Effector Cell Engraftment Epidemic Extravasation Fibroblasts Future Generations Genetic Heart Heart failure Human Human Volunteers Image Injury Isotope Labeling Isotopes Label Laboratories Mammals Mass Spectrum Analysis Metabolic Methods Mus Muscle Cells Myocardial Myocardial Infarction Natural regeneration Organ Other Genetics Patients Phosphocreatine Physiologic pulse Publications Research Personnel Resolution Skeletal Muscle Stem cells Techniques Technology Testing Therapeutic Thymidine Tissues Tracer United States base cardiogenesis cell type experimental study heart cell heart function imaging approach improved in vivo interest mouse model new technology non-genetic novel strategies progenitor promoter recombinase stable isotope stem cell biology stem cell differentiation stem cell niche transdifferentiation uptake

项目摘要

There is intense interest in approaches to generating new cardiomyocytes, including not only through laboratory generation of cardiomyocytes but also by promoting cardiomyocyte formation therapeutically through endogenous cardiogenesis. The endogenous cardiogenesis approaches would not need delivery of cells with issues of engraftment and survival, and thus could have advantages. One of these exciting endogenous cardiogenesis approaches is direct reprogramming of non-cardiac cells to cardiomyocyte. To study reprogramming in vivo, the inducible cre approach in mice is the most widely used method for genetic fate-mapping of cells. However, inducible cre and other genetic lineage mapping approaches may be limited by even very transient leakage of promoters or spontaneous recombinase activity in the absence of the inducer molecule, and these studies can currently only be performed in mice. To gain confidence in the study of endogenous cardiogenesis, approaches that complement genetic fate mapping could provide compelling evidence that our field is headed toward the best regeneration strategy. We have now developed an entirely new approach to marking the identity of cells in vivo using non-radioactive isotopes in a cell-specific metabolic compound. This “Metabolic Fate-Mapping” approach utilizes an isotope-enriched metabolic tracer, specifically creatine, which is taken up by muscle cells, phosphorylated, and utilized in the cytoplasmic phosphocreatine shuttle. Cellular uptake of creatine by muscle cells is rapid, while subsequent turnover of creatine is slow, making it a suitable metabolic label for myocytes. Cells that are creatine-positive can then be identified via Multi-Isotope Imaging Mass Spectrometry (MIMS), a high resolution approach that we have adapted for myocardial biology. We have previously demonstrated usage of labeled thymidine in vivo to demonstrate rare proliferation of cardiomyocytes over months, and we have also demonstrated the stable isotope imaging approach in human volunteers. We will use this new Metabolic Fate-Mapping approach to cell lineage mapping along with an inducible cardiomyocyte cre mouse and an inducible fibroblast cre mouse to study reprogramming of non-myocytes to cardiomyocytes in vivo. Unlike genetic fate-mapping strategies, the stable isotope lineage mapping approach is amenable to any species on any genetic background, and this will enable future large animal experiments of reprogramming. Finally, because this approach can be applied with stable, non-radioactive isotopes such as 13C and 15N, which have been widely used in humans and are regarded by the FDA as safe, studies of human regeneration in diverse tissues could be enabled with Metabolic Fate- Mapping by identification of cellular labels specific to cell type.

人们对产生新的心肌细胞的方法非常感兴趣，不仅包括通过实验室产生心肌细胞，也可以通过治疗促进心肌细胞形成通过内源性心脏病。内源性心脏病方法不需要交付植入和生存问题的细胞，因此可能会有优势。这些令人兴奋的之一内源性心脏病方法是直接重编程非心肌细胞为心肌细胞。到研究重编程在体内，小鼠的诱导CRE方法是遗传最广泛使用的方法细胞的命运图。但是，可诱导的CRE和其他遗传谱图映射方法可能受到限制在没有诱导的情况下，即使是启动子或赞助重组酶活性的非常短暂的泄漏分子，这些研究目前只能在小鼠中进行。为了对研究有信心内源性心脏病发生，补充遗传脂肪图的方法可以提供引人注目证据表明我们的领域正朝着最佳的再生战略迈进。我们现在已经完全开发了使用非放射性同位素在细胞特异性代谢中使用非放射性同位素在体内标记细胞身份的新方法化合物。这种“代谢命运映射”方法利用了富含同位素的代谢示踪剂，特别是肌酸被肌肉细胞吸收，磷酸化并在细胞质磷酸蛋白穿梭。肌肉细胞创造的细胞摄取迅速，而随后创造的营业额很慢，使其成为肌细胞的合适代谢标签。然后可以通过多同位素成像质谱法（MIMS），这是一种我们已经适应的高分辨率方法心肌生物学。我们以前已经证明了在体内标记的胸苷的用法，以证明罕见心肌细胞的扩散数月，我们还展示了稳定的同位素成像人类志愿者的方法。我们将使用这种新的代谢命运映射方法进行细胞谱系映射以及可诱导的心肌细胞CRE小鼠和可诱导的成纤维细胞CRE小鼠研究在体内将非肌细胞重编程为心肌细胞。与遗传脂肪图策略不同，稳定同位素谱图映射方法适合任何遗传背景上的任何物种，这将使未来的大动物重新编程实验。最后，因为这种方法可以稳定应用，所以非放射活性同位素（例如13C和15N），这些同位素已在人类中广泛使用，并被认为 FDA是安全的，可以通过代谢命运来实现人类再生的多样性时机的研究通过识别特定于细胞类型的细胞标签来映射。