Dissertation committee:
Ρουμπελάκη Μαρία, Αναπληρώτρια Καθηγήτρια, Τμήμα Ιατρικής, ΕΚΠΑ
Καπετανάκη Γιασεμή, Ερευνήτρια Α, Κέντρο Βασικής Έρευνας, ΙΙΒΕΑΑ
Μπέης Δημήτρης, Ερευνητής Β, Κέντρο Πειραματικής Χειρουργικής και Μεταφραστικής Έρευνας, ΙΙΒΕΑΑ
Ηλιόπουλος Αριστείδης, Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Λάζου Αντιγόνη, Καθηγήτρια, Τμήμα Βιολογίας, ΑΠΘ
Σανούδου Δέσποινα, Αναπληρώτρια Καθηγήτρια, Τμήμα Ιατρικής, ΕΚΠΑ
Μαυροειδής Μανώλης, Ερευνητής Γ, Κέντρο Βασικής Έρευνας, ΙΙΒΕΑΑ
Summary:
Cardiomyopathies are the major cause of death worldwide, leading to heart failure in most cases. Unfortunately, the implementation of a treatment is not always possible, especially in cases of heart failure where a heart transplant is required. The discovery that the adult heart has a low turnover capacity, as well as finding progenitor cells in the adult myocardium, was a great achievement for the development of pioneering therapies for the treatment of heart diseases.
Numerous studies have been conducted in this direction in the last two decades, where the basic properties and differentiating capacity of adult cardiac progenitor cells have been studied. Although experimental results are often quite optimistic about their regenerative capacity, the success rate is very low, which makes it difficult to use them clinically. In fact, this observation has triggered a long scientific debate over the endogenous differentiating ability of cardiac progenitor cells into myocardial cells. The aim of this PhD thesis was to isolate and characterize the adult progenitor cells of the heart that express the phenotype of "Cardiac Side Population" (CSP), as well as the cells that express the stem cell surface marker Sca1 (Sca1+) from the mouse model with desmin deficiency.
Desmin, the major muscle specific protein of Intermediate Filaments (IF) and the earliest myogenic marker, has been shown to play an important role during the differentiation of embryonic stem cells (ESCs), while it is expressed in the CSP cells of adult mouse. Taking into account the importance of desmin in ensuring the proper development and function of myocardial cells (in its absence are observed mitochondrial damage, severe cell death and development of Dilated Cardiomyopathy) both during development and in the adult stages, it is hypothesised that it may also be involved in the appearance of the characteristics of CSP and Sca1+ progenitor cells with a direct impact on their regenerative capacity.
In order to answer the above questions, cardiac CSP and Sca1+ cells were isolated from desmin deficient (des-/-) and wild type (wt) adult mice and were transcriptionally characterized in terms of the expression of cardiac gene markers. At the same time, as CSP cells constitute a heterogeneous population, they were also phenotypically characterized in terms of the expression of cell surface markers in order to determine the role of desmin in the various CSP subpopulations. From the experiments performed, it was found that unlike Sca1+ cells, the absence of desmin affected the percentage of total CSP cells, which was significantly increased in des-/- mice compared to wt, as well as the various subpopulations of CSP cells. This increase was found to be unrelated to a direct effect of desmin on CSP cell proliferation, as differences in the expression of cell cycle gene markers observed immediately after CSP cell isolation, a condition that reflects the in vivo condition, were eliminated upon culture, while at the same time the proliferation rate of CSP cells in vitro did not differ between the two genotypes. In contrast, desmin appears to play an indirect role in the proliferation of CSP cells through the microenvironment and the conditions prevailing in des-/- myocardium, such as fibrosis, inflammation and cardiomyocyte death, as in 15 days old des-/- mice, which have not developed yet the pathological phenotype, and also in des-/- mice in which the phenotype was rescued after overexpression of the small heat-shock protein αB-crystallin, the percentage of CSP cells did not differ compared to the corresponding wt mice.
During the transcriptional characterization, CSP and Sca1+ cells were observed to express gene markers of cardiac progenitor cells, but not of differentiated cardiomyocytes. In addition, it was found that the lack of desmin is involved in the proper expression of cardiac transcription markers mef2c and Nkx2.5, the levels of which were significantly reduced immediately after isolation or after culture, respectively, compared to wt, which advocates for the role of desmin in the cardiac commitment of CSP and Sca1+ cells.
In order to furtherly investigate the role of desmin in the differentiation of CSP and Sca1+ towards cardiomyocytes, their differentiation was induced using the methyltransferase inhibitor 5-azacitidine. It was observed that des-/- CSP cells in relation to wt showed a reduced expression of the Nkx2.5 gene, while des-/- Sca1+ cells showed a reduced expression of the Myh6 gene, facts which on the one hand indicate that desmin is differentially involved in the differentiation of the two cell types and, on the other hand, its role relies mainly on their early commitment to cardiomyocytes. However, with this methodology, no satisfactory cardiomyogenic differentiation of the cells was observed, as no expression of any of the markers of mature cardiomyocytes was detected in protein level. In order to overcome the above obstacle and to fully clarify the role of desmin in cardiomyocyte differentiation, an alternative experimental approach was followed in which induction of Sca1+ cell differentiation was performed after overexpression of the cardiac transcription factors Gata4, Mef2c and Tbx5 (GMT), which can induce the reprogramming of cardiac fibroblasts directly into cardiomyocytes. The results showed that desmin affected the expression of Myh6 gene only at mRNA level which was significantly reduced in des-/- Sca1+ cells compared to wt, while at protein level no differences were observed in the expression of proteins of the mature cardiomyocytes between the two genotypes.
As can be seen from the above, the role of desmin in the characteristics of the cardiac progenitor cells seems to be limited in vivo, mainly through the events that follow its absence. As many studies suggest that conditions similar to those prevalent in des-/- myocardium are involved in the aging of stem cells, it was studied whether the absence of desmin affects the aging of CSP and Sca1+ cells, which would have a negative impact on their ability to differentiate and, consequently, to their further use in clinical trials to repair myocardial damage. The results indicted that desmin’s absence leads to the overexpression of the aging marker p16INK4α only in Sca1+ cells, however without the correlation of the telomere length and the induction of Sca1+ aging in vitro.
It is concluded that the role of desmin in the CSP and Sca1+ progenitor populations of the adult mouse heart, is mainly limited to their early cardiomyocyte commitment rather to their eventual differentiation, which is laso confirmed by differentiation studies in ESCs. The above finding is important for the development of new improved methodologies in order to more effectively and targeted differentiate progenitor cells into cardiomyocytes, as their limited differentiation towards cardiomyocytes is the major problem in their use in clinical trials which has provoked much controversy in the scientific community.
