Carbon birth dating is an approach devised by the Frisen

Carbon-14 birth dating is an approach devised by the Frisen group to monitor cellular turnover in tissue (Bergmann et al., 2009). This strategy makes use of the transient increase of carbon-14 (14C) in the purchase S63845 due to a period of above ground nuclear testing that ended in the 1960s. The carbon-14 was taken up by humans through the diet and incorporated into the genomic DNA, which can be used as a time-stamp to calculate the mean birth date of a stable cell population (Bergmann et al., 2009). The carbon-14 content in cardiomyocyte nuclear DNA is quantified using accelerator mass spectrometry (AMS, which has a lower detection limit of 10 moles). The mean age of cardiomyocytes is then compared with the age of the source individual. This indicated generation of new cardiomyocytes at a rate of ~1% after 25years of age. The fidelity of this approach depends on the quality of cardiomyocyte nuclei isolated from post-mortem myocardium. AMS provides a tissue-average, which poses a challenge since in cardiomyocytes, S-phase can be connected with cell division and differentiation. Hence, the analysis relied on additional assumptions that had to be made for mathematical modeling of the data (Bergmann et al., 2009), including assumptions about potential changes of the number of cardiomyocytes after birth, rates of cardiomyocyte cell death, and the extent of non-proliferative cell cycles (multinucleation and ploidy) with age (Elser and Margulies, 2012). Fate mapping with genetic tags has been used to label distinct cell populations to determine the source of new cells with time or intervention. Multiple groups have used the α-MHC promoter driven Cre model crossed with reporter lines to assay the role of progenitor cells in the generation of new cardiomyocytes after injury (Senyo et al., 2013; Malliaras et al., 2013; Ellison et al., 2013). After tamoxifen induction, a majority of differentiated cardiomyocytes is labeled with a reporter such as green fluorescent protein (GFP) or LacZ (Senyo et al., 2013; Porrello et al., 2012). Using this technique, we have demonstrated that during normal aging in mice, the percent of preexisting cardiomyocytes, indicated by the percentage of GFP-positive cardiomyocytes, remains unchanged (Hsieh et al., 2007). Influx of cardiomyocytes generated from undifferentiated progenitor cells should result in a decrease of the percentage of GFP-labeled (preexisting) cardiomyocytes. This “dilution” was the case after experimental myocardial infarction, leading to the conclusion that myocardial injury induced an influx of progenitor cell-derived cardiomyocytes (Hsieh et al., 2007). However, multiple other studies showed very little or no contribution of progenitor cells to generating cardiomyocytes in adult mammals (Senyo et al., 2013; Malliaras et al., 2013; Ellison et al., 2013; Porrello et al., 2012; Loffredo et al., 2011). Considering one specific progenitor type, Ellison et al. reported a 0.15% generation rate for c-kit progenitor-derived cardiomyocytes in a 4-week period of normal aging (Ellison et al., 2013). This contrasts with results from using a knock-in of an inducible Cre into the c-kit gene locus (van Berlo et al., 2014). This more direct approach demonstrated that c-kit positive cells do not generate cardiomyocytes in mice after birth to a significant degree (van Berlo et al., 2014). In this context, it is important to note that c-kit positive cells isolated from neonatal mice can generate cardiomyocytes (Zaruba et al., 2010). Systematic molecular characterization of myocardial c-kit cells and comparison with the cardiomyocyte lineage should help resolve this controversy. Potential explanations of our discrepant results with the genetic α-MHC-MerCreMer/ZEG fate mapping techniques include cardiomyocyte toxicity due to high expression levels of Cre (Lexow et al., 2013; Bersell et al., 2013). However, we did not observe these effects in the 14-day induction protocol with 5-hydroxy-tamoxifen (Loffredo et al., 2011; Lexow et al., 2013; Bersell et al., 2013; Koitabashi et al., 2009). A concern of genetic tags expressed under control of cell-specific promoters is the dependence on the fidelity of transcriptional activity. In addition, it may be possible that the labeled cell population is phenotypically heterogenous. This possibility must be considered when the genetic labeling technique does not label the entire cell population, as is the case with the α-MHCMerCreMer mice (Sohal et al., 2001). Other genetic approaches such as retrospective clonal assays utilize random labeling of precursor cells and have been used to address lineage in early cardiac development (Ali et al., 2014; Buckingham and Meilhac, 2011). This system selectively labels daughter cells of mononucleated cells in divergent fluorescent tags so that one cell is labeled with red and the other with green. Using two transgenic models for a cardiomyocyte specific source and a non-discriminate source, respectively, Ali et al. demonstrated that very few new mononucleated cardiomyocytes are generated primarily from pre-existing cardiomyocytes (Ali et al., 2014). Genetic labeling remains one of the most powerful approaches to monitor cell population dynamics, if applied appropriately.