The World Health Organization reports cardiovascular diseases as the main cause of 29% of global death each year (Lozano et al., 2012). There are approximately 17.3 million cardiovascular-related deaths per year worldwide (Townsend et al., 2015). Particularly in the European region, cardiovascular diseases cause 4 million deaths; which is, 45% of death per year (Townsend et al., 2015). In all countries of Europe, the primary cause of death in women is cardiovascular diseases, estimated to be 51% of all-cause mortality in women. For men, 42% of deaths are caused by cardiovascular diseases (Townsend et al., 2015).

Due to the profound importance of cardiovascular diseases, the natural endogenous regenerative capacity of the human heart has been a topic of debate for decades. Accumulating evidence over the last decade has suggested that the human heart has the potential to undergo natural regeneration. Locally resident cardiac progenitor or stem cells might play a vital role toward the natural regeneration capacity of the heart. Myocyte proliferation happens to a low extent in the human heart, while enhanced proliferation was observed following injuries of the heart such as myocardial infarction (Beltrami et al., 2001). In addition, the muscle cells of the whole human heart are replaced every 4.5 years (Anversa et al., 2006). Cardiac progenitor cells (CPCs) can replicate in response to some pathological conditions, and they are also able to play an active role in the regeneration of injured parts of the heart (Gonzalez et al., 2008).

Of particular interest are cells with the c-Kit receptor (CD117 or SCFR-stem cell factor receptor) on the surface. Beltrami and colleagues reported the existence of CD117⁺ cells with characteristics of CPCs (Beltrami et al., 2003). In addition, other researchers described that cardiac CD117⁺ cells are potential local stem cells, which reside in the human heart (Castaldo et al., 2008; Di Meglio et al., 2010; Sandstedt et al., 2012). Bearzi and colleagues also described the typical stem cell features of the CD117⁺ cells: they are clonogenic, multipotent, and self-renewing (Bearzi et al., 2007). An increase in the number of cardiac CD117⁺ cells was observed in several cardiovascular diseases such as heart failure, cardiac hypertrophy, ischemic cardiomyopathy (ICM), acute cardiac injury, and pressure overload (Urbanek et al., 2003, 2005; Castaldo et al., 2008; Kubo et al., 2008; Altarche-Xifró et al., 2009; Itzhaki-Alfia et al., 2009; Rupp et al., 2012).

Nurzynska and colleagues conducted a comparative study of human CPCs in normal and pathological conditions (ischemic heart disease) and confirmed that the differentiation potential of CD117⁺ CPCs of the adult human pathological heart is weak in comparison to healthy cardiac tissue (Nurzynska et al., 2013). Bolli et al. conducted a phase I clinical trial for the clinical implication of CD117⁺ stem cells, and interestingly they found an increased cardiac functional capacity, reduced left ventricle scar size, and improved quality of life due to these cells (Bolli et al., 2011). They isolated CD117⁺ cells from 1 g myocardial tissue during cardiac surgery. They showed that the infusion of 1 million autologous CD117⁺ stem cells is not associated with noticeable adverse or significant positive effects. Therefore, the study of the anatomy of the human heart and the manner in which pathological states and micro-environmental conditions correlate with the availability of resident CPCs for cardiac tissue regeneration is required to further confirm the presence of local resident progenitor cells as well as for potential clinical strategies for novel forms of cardiac cell therapy by endogenous recruitment. Nevertheless, for progenitor cell detection, additional stem cell markers must be proven. Herein, we focus on the existence of CD90⁺ and CD117⁺ cells as regenerative precursor cells in the human healthy and diseased heart, that are responsible for the activation of endogenous resident progenitor cells toward tissue or cell injury. Previous investigations focused on the detection of CD90⁺ and CD117⁺ progenitor cells in the human heart along with additional information about biopsy location, diagnosis, patient age, and experimental structure are shown in Table 1. A schematic representation of the human heart provides an overview about biopsy locations used for previous identifications of CD90⁺ and CD117⁺ endogenous CPCs (Figure 1).

In the current study, we obtained myocardium biopsies from 23 patients with the following diagnoses: dilatative cardiomyopathy (DCM), ICM, myocarditis, and controls from cardiac healthy. The collected material was characterized by immunohistochemistry. Currently, paraffin-based histopathological tissue analysis represents the main conventional method for confirmation of presence or absence of histological markers, grading, or the quantification of stem cells markers in ready tissue sections. Additional quantification of these histopathological slides using an automated image analysis perspective, though providing with more sensitive and qualitative information on the presence of local CPCs in myocardium biopsies, represents a new set of challenges. In our previous studies, we compared immunohistochemical data with an automated image analysis method of digitized slides by Definiens Tissue Studio software (Abraham et al., 2005; Kaemmerer et al., 2014; Neubauer et al., 2016). In several cases, we quantified tissue morphology, staining distribution and intensity of staining, using both automated image analysis and manually performed slides (Abraham et al., 2005; Kaemmerer et al., 2014; Neubauer et al., 2016). Interestingly, in this image analysis, the digital image processing is performed by digitized histological slides, which results in numerous advantages over the conventional immunohistochemical method.

In the present study, we combined a fully automated digital image analysis with conventional histological slides to more sensitively confirm the presence of potential local endogenous CPCs and to perform a quantitative analysis of the cardiac cell signals in human myocardium biopsies from patients with various cardiac diseases.

Numerous stem cell markers were analyzed especially referring to CPCs. In the present study, the focus was mainly set on the co-expression of CD90 and CD117 in the human heart, which was already demonstrated (Gambini et al., 2011). In general, the CD90 marker is considered as a fibroblast marker but is also known as a mesenchymal stem cell (MSC) marker. Cardiac fibroblast research has been tremendously accelerated in the last decade (Gourdie et al., 2016). The novel therapeutic strategies in heart diseases with special focus on fibroblasts are reviewed elsewhere (Gourdie et al., 2016). In our previous studies, we have shown the multilineage potential of skin-derived CD90⁺ cells (Lorenz et al., 2008). Moreover, we have successfully treated acute and chronic diabetic wounds and skin ulcer of human cases by activation of local progenitor cells (Bader et al., 2011; Günter et al., 2013). In our opinion, it is important to note that the CD90 marker characterizes seemingly opposing states of cells; on the one hand fibroblasts involved in scar formation, but on the other hand, stem or progenitor cells, which could induce functional tissue regeneration. We believe that those CD90⁺ progenitor cells reside in local organs and are activated after injury. Previously, we have studied CD90 progenitor cells of the skin and shown their dependence toward localized cytokine stimulation for proliferation (Lorenz et al., 2008) and we proved the proliferation of local CD90⁺ progenitor cells after injury in the liver (unpublished data). Acute myocarditis is a process that is characterized by intense local inflammation as well. Even though the myocarditis group in the present experiments was very small (n = 3) due to the limited availability of the samples, the high expression of CD90⁺ cells corresponds to the fact that myocarditis patients more frequently undergo a complete healing pattern. The progenitor cell activation in myocarditis may explain the better functional outcome with less or no scar presence allowing the localized functional regeneration of cardiac tissue. In contrast, chronic disease states showed a depletion of CD90⁺ cells, correlating clinically to the unsuccessful healing outcomes.

Moreover, various researchers identified CD117⁺ cells in human myometrium (Ciontea et al., 2005), human fallopian tube (Popescu et al., 2005), human mammary gland stroma (Radu et al., 2005) as well as in human atrial myocardium (Hinescu et al., 2006), and ventricular myocardium (Popescu et al., 2006). We have proved here, that the number of CD117⁺ cells is upregulated in the diseased heart, especially in myocarditis. Our results validate the findings of previous studies; the number of CD117⁺ cells is increased in several cardiac diseases, for instance in advanced heart failure and aortic stenosis (Urbanek et al., 2003; Kubo et al., 2008; Itzhaki-Alfia et al., 2009). The human heart possesses a cardiac stem cell pool (Urbanek et al., 2005). The activation of the local stem cells occurs in response to ischemic injury. The stem cell pool has a crucial role in the regeneration of infarction heart (Urbanek et al., 2005). Matuszczak et al. reported that there are no differences in the number of CD117⁺ cells between various disease groups (Matuszczak et al., 2014). However, they had not used control tissue from healthy patients.

CD117⁺ local CPCs had already been used for the treatment of heart diseases in both human cases and animal models (Bolli et al., 2011). The method involves the isolation of autologous CD117⁺ cells, expansion in vitro and injection of those in high numbers (Bolli et al., 2011).

In comparison with conventional stem cell therapy, the activation of local endogenous progenitor cells is a holistic approach to both preventing and regulating the heart. We are aware that the characterization of progenitor cells requires the identification of various stem cell markers and it is of high importance to exclude unspecific signals. Here, we analyzed the co-expression of CD117, CD90, and partially CD105, also. Previous studies underline our analyses: Gambini and colleagues demonstrated a co-expression of CD117 and CD90 or rather CD105 in human heart auricle primary cultured cells (Gambini et al., 2011). Several other studies also identified CD117⁺ cells which were positive for CD105 and CD90, too (Li et al., 2012; Matuszczak et al., 2014). CD90 and CD105 were detected on cardiosphere-derived cells (CDCs) (Smith et al., 2007; Davis et al., 2009; Mishra et al., 2011; Chan et al., 2012). A series of other markers was proven on CD117⁺ cells, including CD29, CD44, CD31, CD34, and Sca1 (Gambini et al., 2011; Fang et al., 2012; Matuszczak et al., 2014). Furthermore, it was already displayed that CD117+ cardiac stem cells are negative for CD45 (Matuszczak et al., 2014) and that not all CD117⁺ cells are mast cells (Kubo et al., 2008; Zhou et al., 2010).

Vicinanza et al. also showed that cardiac CD117⁺/CD45⁻ cells are clonogenic and multipotent, but they determined that >90% of cardiac CD117⁺ cells contain endothelial cells and their precursors (Vicinanza et al., 2017). They demonstrated the myogenic and regenerative potential of CD117⁺/CD45⁻ cells in the damaged myocardium after injection of CD117⁺/CD45⁻ cells into damaged myocardium. Therefore, they conclude that CD117 is still an essential marker for CPCs (Vicinanza et al., 2017).

Nevertheless, future experiments with additional stem cell markers are necessary to prove the progenitor cell identity of CD117⁺ cardiac cells and to exclude unspecific labeling. We identified many CD117⁺ stained cardiomyocytes, which do not belong to the local progenitor cells. Other researchers studied CD117⁺ hematopoietic bone marrow cells and their ability to act as cardiac progenitors and to transdifferentiate into cardiomyocytes (Orlic et al., 2001; Rota et al., 2007). Rota et al. reported that CD117⁺ bone marrow cells lose their hematopoietic CD45 phenotype and obtain a cardiomyocyte phenotype (Rota et al., 2007). Probably these cells were also enriched in cases of inflammation or other pathological conditions. Further studies would be necessary to analyze this kind of cells.

In the present study, it was not our purpose to perform a series analysis of cardiac stem cell markers. Rather than that, the present study mainly focuses on investigating the combination of a traditional histological method with a novel digital image analysis technology. This technology enables a quantitative evaluation of two CPC markers in comparison of paraffin–embedded tissue sections of healthy and diseases heart samples. It would be of high interest to complement these experiments with other techniques such as immunofluorescence and high magnification confocal microscopy in the future.

In the present experiment, we identified CD90⁺ and CD117⁺ cells in all patient groups: Myocarditis, ICM, DCM, and healthy cardiac patients. With the novel digital image analysis technology, a comparison of differently sized paraffin-embedded tissue sections is available. Taking into consideration that the sample size in the present experiment was limited, we proved an increase of CD90⁺/CD117⁺ cells in acute myocarditis. This finding supports our theory that endogenous cardiac stem or progenitor cell activation is part of the repairing mechanism after acute myocardial damage, as in cases of acute myocarditis. A similar inflammation process is described for acute myocardial infarction. Nevertheless, in most cases, acute myocardial infarction leads to the development of a scar. Further studies may show, how an amplification of the local myocardial stem or progenitor cell activation may contribute to the myocardial regeneration and healing process.

This study aimed at the identification and the quantitative analysis of CD90⁺ and CD117⁺ cardiac cells from human myocardium biopsies of 23 patients. Besides the conventional histological image analysis, the digital image analysis enabled a computer-based immunohistochemical quantification of some stem cell markers, using whole-slide images created by the virtual slide scanning microscopy. In our experiments, the number of CD90 and CD117 signals in patients with myocarditis was higher than in all other groups. Taking into consideration the regenerative healing potential prospects of myocarditis, it is likely that there is a relation between CD90 and CD117 expression and clinical outcome. Future studies with larger sample size are necessary to confirm that theory. The proof on the existence of endogenous resident progenitor cells not only in the healthy but also in the diseased human heart opens up the promising concept of regeneration instead of repair, which has an impressive scope in treating or preventing cardiovascular diseases.

MG, JS, SE, SG, and AB: conceived and designed the experiments; NS, WM, and JS: organized the human tissue samples; MG and SE: performed the experiments; MG, SE, BB-S, and MA: analyzed the data; MG, JS, SE, SG, AB, and MA: wrote the paper; SG and AB: provided guidance on the whole study.