Inovative biotechnologies for generation of functional cardiac tissue

Introduction: Cardiac tissue bioengineering represents an important research direction for the patient-specific myocardial reconstruction therapies. Tridimensional cardiac tissues generation requires a structural matrix, specialized cardiac cells and an optimal medium in which cells can multiply and form a mature tissue, capable of sustaining the complex cardiac functions.
Objective: To obtain functional cardiac tissue on a rat animal model, starting with a decellularized heart used as a matrix for grafting the human cells- adult mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) obtained through peripheral blood lymphocytes T transduction. The research also studied the differentiation capacity into cardiomyocytes.
Methods: The heart of Sprague-Dowley rats was can-nulated at aortic level, attached to decellularization devices – Langendorff-Radnoti and simplified Lan-gendorff in alternative electric field, and perfused with SDS/EDTA solutions in different concentrations. The decellularization efficiency was evaluated by optic and electron microscopy, and measurement of DNA and protein level. For cardiac matrix recellularization, adult mesenchymal stem cells (MSC) were used, differen-tiated in 2D and 3D models towards cardiomyocytes using 5-azacytidine, as well as induced pluripotent stem cells (iPSC), obtained through reprogramming of T cells; iPsc were stained for specific marker expressi-on. Recellularization was performed on decellularized cardiac tissue fragments and in 3 D models, throu-gh intraventricular injection or aortic perfusion with myocardial cells.
Results: Simplified Langendorff system in alternative electric field was more effective in decellularization of rat heart (5.5 hours), compared to classic system (12 hours), and the process was progressive, from the RV, to atria and LV. DNA and proteins concentrations in decellularization solution had a quasi-linear increase, and the heart was completely decellularized when a plateau was reached (600 ng/μl for DNA and 0.9 ng/ μl for proteins). Optic microscopy and electron micro-scopy revealed maintenance of collagen fibers architec-ture, the absence of cells, and vascular permeability. T cells were expanded in specific media (Dynabeads© T-activator CD3/CD28). T cells reprogramming towards iPSC used ReproTeSR™ media, and iPSC were obtained after 21 days in cuture. Immunofluorescence revealed positive marker expression on 80% of iPSC – Oct-4, Nanog, Sox-2, SSEA-4 and Tra-1-81. Human adult me-senchymal stem cells induced towards cardiomyocytes with 5-azacytidine in vitro 2D had a differentiation rate of 50%, while 3D differentiation, in continuous perfu-sion of 5-azacytidine for 24 hours, increased the rate to 80%. Matrix recellularization reached 50-60% after 40 days of maintenance in incubator.
Conclusions: Our study showed that decellularization of the rat heart using the simplified Langendorff in alternative electric field is more efficient, because it used smaller amount of potentially noxious substances for the cardiac matrix. Adult human mesenchymal stem cells can recellularize the matrix with reduce efficiency, while the iPSC could have a better grafting and cardiac differentiation rate. Simultaneous Intraventricular in-jection and coronary perfusion with recellularization cells is more efficient compared with only perfusion. iPSC obtained from peripheral blood T lymphocytes could be used for personalized therapy of the patients with myocardial degenerative diseases.

ISSN – online: 2734 – 6382
ISSN-L 1220-658X
ISSN – print: 1220-658X
The Romanian Journal of Cardiology is indexed by:
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CODE: 379
CME Credits: 10 (Romanian College of Physicians)
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