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    • To identify the label retaining cells within

    2018-10-24

    To identify the label-retaining CX-5461 within the myocardium, BrdU was administrated and the intensity of the BrdU signal was examined after a prolonged chasing period (Urbanek et al., 2006). As expected, the number of bright BrdU-CSCs in the atria decreased rapidly after chasing while the number of dim BrdU-CSCs increased over time. The bright BrdU-CSCs detected after a chase period of 2–5 months (Fig. 2C) constitutes the subset of slow-cycling stem cell pool in the atria. Similar changes were seen in the base-mid-region and apex. The aggregate number of CSCs remained constant throughout the period of observation indicating that the growth kinetics of CSCs tends to preserve the pool of primitive cells in the young healthy heart (Urbanek et al., 2006). Thus, cardiac niches harbor a subset of BrdU-retaining cells; these CSCs may correspond to the category of undifferentiated cells that are self-renewing, clonogenic and multipotent in vitro (Beltrami et al., 2003; Bearzi et al., 2007, 2009). The BrdU pulse-chase protocol provides information on the growth kinetics of the CSC population but does not allow the recognition of the mechanism of CSC division at the single cell level. Stem cells can divide symmetrically and asymmetrically. When stem cells engage themselves in asymmetric division, one daughter-stem cell and one daughter-amplifying cell are formed. When stem cells divide symmetrically, two self-renewing daughter cells or two committed amplifying cells are generated. In the young mouse heart, the homeostasis of the cardiac niches is mediated by asymmetric and symmetric division of CSCs (Urbanek et al., 2006). Asymmetric division, however, is the predominant form of CSC replication; it accounts for ~65% of proliferating CSCs. This mechanism of cell renewal is termed “invariant” and typically occurs in organs in a steady state (Watt and Hogan, 2000). Recently, a lineage tracing study in the mouse has challenged the implications that c-kit-positive cardiac progenitors have in the modulation of myocyte turnover and regeneration in the mammalian heart (van Berlo et al., 2014), questioning the results obtained by fate mapping in rodents (Ellison et al., 2014). There are several variables that have to be considered in an effort to reconcile these contrasting findings. There is no perfect experimental strategy that can provide an undisputable answer to any scientific question. Genetic manipulations have limitations as any other methodology. The knock-in strategy employed leads inevitably to the loss of one allele of the c-kit gene which may affect significantly the physiological role of the c-kit receptor and the ability of c-kit-positive CSCs to proliferate and form a myocyte progeny (Nadal-Ginard et al., 2014). This defect in c-kit receptor function may be organ specific and the myocardium might have been more severely affected than other organs, i.e., the bone marrow and the lung. A good example consistent with this possibility can be found in the discrepancy between the number of cardiomyocytes derived from c-kit-positive CSCs in this model that is in sharp contrast with the large formation of lung cells (van Berlo et al., 2014), an organ in which c-kit-positive cells have been shown to regulate the growth and differentiation of epithelial cells (Kajstura et al., 2011). Sca1-positive cardiac progenitors are implicated in cardiomyocyte formation in the mouse (Bailey et al., 2012; Uchida et al., 2013), in which an almost 100% co-expression of c-kit and Sca1 in CSCs was detected (Urbanek et al., 2005). In contrast, Sca1-positive progenitors are not present in large mammals and humans. Thus, data in mice cannot be translated to human beings without caveats. Cardiac niches contain lineage negative cells nested together with cardiac progenitors and precursors. In close proximity to lineage negative cells, myocytes and fibroblasts are commonly visible. Within the niches, stem cells are connected to the supporting cells by gap and adherens junctions composed of connexins and cadherins, respectively. The recognition that junctional complexes are present between CSCs and myocytes or fibroblasts suggests that these cell types act as supporting cells within the cardiac niches. Conversely, endothelial cells and smooth muscle cells do not exert this role. On this basis, the cross-talk between CSCs and cardiomyocytes and fibroblasts has been carefully documented by a series of in vitro assays, which have provided strong evidence in favor of the functional coupling of these three cell populations; the transfer of dyes via gap junction channels between CSCs and cardiomyocytes or fibroblasts was shown by multiple protocols (Bearzi et al., 2009; Urbanek et al., 2006). Additionally, the translocation of microRNA-499 from cardiomyocytes to CSCs may occur physiologically resulting in lineage specification of CSCs and myocyte formation (Hosoda et al., 2011).