Reprogramming Non-myocytes to Cardiomyocytes in vivo

人们对产生新心肌细胞的方法非常感兴趣，不仅包括通过 心肌细胞的实验室产生，而且通过促进心肌细胞的形成治疗 通过内源性心脏发生。内源性心脏发生方法不需要递送 具有移植和存活问题的细胞，因此可能具有优势。其中一个令人兴奋的 内源性心脏发生方法是将非心脏细胞直接重编程为心肌细胞。到 在研究体内重编程时，小鼠中的诱导型cre方法是用于遗传重编程的最广泛使用的方法。 细胞的命运映射。然而，诱导型cre和其他遗传谱系作图方法可能受到以下限制： 甚至启动子的非常短暂的泄漏或在没有诱导物的情况下自发的重组酶活性 这些研究目前只能在小鼠中进行。在学习中获得信心 内源性心脏发生，补充遗传命运作图的方法可以提供令人信服的 这表明我们的领域正朝着最佳再生策略发展。我们现在已经开发了一个完全 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“代谢命运绘图”方法利用同位素富集的代谢示踪剂，特别是 肌酸，其被肌细胞摄取，磷酸化，并在细胞质磷酸肌酸中被利用。 梭肌细胞对肌酸的细胞摄取是快速的，而随后的肌酸周转是缓慢的， 使其成为肌细胞的合适代谢标记。肌酸酐阳性的细胞可以通过 多同位素成像质谱（MIMS），一种高分辨率的方法，我们已经适应了 心肌生物学我们以前已经证明了标记的胸苷在体内的使用，以证明罕见的 增殖的心肌细胞数月，我们也证明了稳定同位素成像 在人类志愿者中。我们将使用这种新的代谢命运映射方法来绘制细胞谱系图 沿着诱导型心肌细胞cre小鼠和诱导型成纤维细胞cre小鼠进行研究 在体内将非肌细胞重编程为心肌细胞。与遗传命运映射策略不同， 同位素谱系作图方法适用于任何遗传背景的任何物种，这将使 未来的大规模动物实验。最后，由于这种方法可以应用于稳定， 非放射性同位素，如13 C和15 N，已广泛用于人类，并被认为是 FDA认为是安全的，可以通过代谢命运来研究不同组织中的人体再生- 通过识别细胞类型特异性的细胞标记进行定位。

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.