Correspondence to: L. M. POPESCU, M.D., Ph.D., Department of Cellular and Molecular Medicine, ‘Carol Davila’ University of Medicine and Pharmacy, P.O. Box 35–29, Bucharest 35, Romania. E-mail: LMP@jcmm.org
A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6–10 μm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.
A highly heterogeneous population of stem and progenitor cells [1–11] has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells (CSC) remains unknown. All these resident stem cells seem to have regenerative potential and to be the most appropriate source for cardiac cellular therapy . However, it is still not clear that the different putative adult cardiac stem and progenitor cells represent different developmental and/or physiological stages of a unique type of resident stem cell [3, 11].
In this paper, we try to identify by electron microscopy (EM) CSC in a cardiac stem cell niche (CSCN) which contains cardiomyocyte progenitors (CMP) already described in the peri-coronary epicardial space . The identity of putative CSC is still indefinite and their ultrastructural phenotype(s) should be of particular interest for their precise location in the myocardial complex architecture in order to understand the regenerative physiology of the heart. We show here the ability of putative CSC to continuously self-renew and produce differentiated CMP.
Materials and methods
Small fragments from mouse atrioventricular myocardium with epicardium were processed for transmission electron microscopy (EM) according to routine procedures, as we previously described . Hearts from six 4-, 6- and 12-month-old C57BL/6 mice have been examined after the institutional ethical committee approval.
We have previously reported the existence of CMP, cells with immature features, in the adult mouse heart , exclusively located in the subepicardium surrounding coronary artery (Fig. 1). EM showed that in CSCN the connective tissue is loose and particularly rich in capillaries and interstitial cells: mast cells, adipocytes, macrophages, fibroblasts, nerves, telocytes , CMP and mononuclear cells (Fig. 2).
Using EM and exclusion criteria we found that some mononuclear cells (Fig. 2A) present ultrastructural features of stem cells (Fig. 3). These putative CSC have been found in the loose connective tissue surrounding the epicardial coronary artery (Fig. 1). CSC are small, round-oval cells (6–10 μm), with large, irregular nuclei, scattered chromatin and large nucleolus, few mitochondria, few long endoplasmic reticulum cisternae and a large amount of free ribosomes (Fig. 3A and B).
Another type of mononuclear cell in CSCN is a cell which is slightly larger (10–15 μm) than CSC. These cells have large and indented nuclei with less scattered chromatin and prominent nucleoli, more numerous endoplasmic reticulum cisternae, few mitochondria, clusters of small and clear vacuoles generating a multilocular appearance, few dense granules and lysosomes and well-developed Golgi apparatus (Fig. 3C). These cells could be considered an early committed cell, an intermediate stage between CSC and CMP, because some of their ultrastructural features (e.g. clusters of cytoplasmic vesicles, well-developed Golgi apparatus) are still visible in the early differentiated CMP (Fig. 4).
Unlike CSC, the CMP  are recognizable without difficulty (Figs 2 and 4). These cells display typical ultrastructural features of immature cardiomyocytes including: high nucleo-cytoplasmic ratio, unorganized bundles of filaments, lipid droplets, intracytoplasmatic dense bodies (similar to primordial Z-lines), intracytoplasmatic desmosome-like structures (primordial intercalated discs) and cortical leptofibrils. Moreover, these cells have large mitochondria, numerous caveolae and a continuous basal lamina. A central element of the niche is represented by telocytes, stromal supporting cells. EM showed that telocytes (Figs 2B, 3 and 4A) are real candidates for this role, offering physical and informational support for CMP .
The mononuclear cells presented in this paper as putative stem cells have been found to undergo mitosis (Fig. 5A and B) and apoptosis (Fig. 5C and D) in the CSCN. Because we have found mitosis and apoptosis in cells with ‘stemness’ features with EM, sometimes in the same section, we appreciate that this behaviour is quite common.
The integration of newly formed cardiomyocytes into the cardiac muscle is of particular interest. In Fig. 6, we illustrate a possible integration mechanism: committed cells connect with adult cardiomyocytes in the presence of telocytes (Fig. 6A), gradually differentiate into cardiomyocytes and develop their particular junctional complexes at the same time (Fig. 6B and C).
This EM study supports the idea of a unique type of cardiac stem cell in different stages of differentiation and maturation  and demonstrates the ability of putative CSC to continuously self-renew and produce differentiated cardiomyocyte progenitors. The mitotic and apoptotic CSC (Fig. 5), the presence of CMP in different stages of development (Figs 4 and 6) and the integration of newly formed cardiomyocytes suggest that there is a dynamic pool of stem cells in a continuous process of commitment and differentiation. In our opinion, stem cells, progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart .
The next step would be to identify surface molecules (markers) which accurately differentiate stem cells from progenitors. Another important aspect to be clarified is telocyte involvement (Figs 1–6) [12–24] in stem cell homeostasis and cardiac renewal.