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Stem cells and cardiac regeneration
Technology developments
for stem cell modifications
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Magnetic nanoparticles for non-viral gene transfer

Magnetically controlled gene transfer opens principally the possibility of a targeted local therapy in the frame of a systemic application. At the RTC we have succeeded to develop and establish a safe and efficient transfection method in which we apply complexes composed of non-viral vectors and low-molecular polyethylenimine (PEI) coupled to magnetic nanoparticles (MNP) such that it was possible to guide them towards their target by means of a magnetic field. With this method it was possible to achieve improved transfection efficiencies both in various cell lines as well as in primary cells while at the same time the cytotoxicity was significantly reduced.

Even without application of an external magnetic field the combination of the described complex with MNP resulted in enhanced transfection efficiency in MSCs. Using confocal microscopy we were able to show that pDNA is being released at an elevated level by the MNP in the perinuclear zone and then enters the cell nucleus. The enhanced transfection efficiency of the pDNA/PEI-complex prevailed for up to 48 hours after transfection.
In the future, we aim to genetically modify stem cells by means of this method in order to improve their therapeutic efficiency
 
 
 

miRNAs in stem cell differentiation

miRNA play a major role in stem cell differentiation as central post-transcriptional modulators for the regulation of gene expression. Investigations demonstrated a close relationship between functional changes of stem cells in cell culture under hypoxic conditions and a significant change of global gene expression profiles. In order to determine the role of miRNAs in the differentiation the activity of selected miRNAs in stem cells was effectively modified and the resultant effect was evaluated.

The angiogenesis-promoting effect of miRNA-126 is well known. By means of in-vitro angiogenesis-test systems we were able to show that an over-expression of miRNA-126 amplified the network formation which reflects an increased vessel formation. In order to utilize the large potential of anti-sense RNA in the future, we have furthermore extended our non-viral DNA-transfer techniques by the transfer of miRNA to stem cells
 

 
 

Cell seeding technologies for cardiac tissue engineering

For the improvement of reconstruction of natural tissue structures at the RTC cardiac tissue engineering methods are applied for cell seeding of cardiac valves or to develop cardiac patches. For this purpose, two different approaches for cell seeding  are  used. With a print technique by means of laser-induced forward transfer (LIFT) cells and biomaterials can be ordered in complex three-dimensional (3d) matrices in order to reproduce the biological tissue structure as precisely as possible. This method can be applied – among others - to the regeneration of infarcted tissue by means of cardiac patches.
In order to accelerate the formation of blood vessels we applied  human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC) on a cardiac patch in a precise grid structure similar to a capillary net by means of LIFT. Subsequent to the dedicatedly arranged co-culture the speed of blood vessel formation has been amplified. After application of such a cardiac patch to the infarct zone an improvement of heart function could be observed in a rat model. The second method which is currently explored at the RTC is the novel “three-channel airbrush technique”.
With help of this airbrush system we have evaluated whether it is possible to realize intraoperative heart valve tissue engineering by means of a stem cell-plus-fibrin composite. For this purpose, we sprayed a complex consisting of human CD133+-stem cells and fibrin on pulmonary valves from pig. In a bioreactor the cells were able to survive for 96 hours, and during this period the inner sides of the transplants showed the generation of adherent spindle-shaped CD31+- and VEGFR2+-cells hinting at the differentiation of CD133+-stem cells in the complex under flow conditions. So far, “tissue engineering” offers possibilities for improving the reconstruction of natural tissue structures in the cardiac field. In principle, both of the tested techniques are suited for cardiac applications. At the moment we are working on the technological optimization of both techniques to develop the best suited technique further and ultimately make it available for its application to human beings.
 

 
 

Erythropoetin as a regenerative active ingredient for the heart

Erythropoietin (EPO) has both cytoprotective and regenerative properties. In a rat model we were able to observe a reduction of infarct size, an inhibition of remodeling, a protective effect on cardiomyocytes, and an improved heart function subsequent to treatment with a single intramyocardial injection of EPO after myocardial infarction (MI). In addition, our study revealed an accelerated intracardiac cell proliferation, a significant upregulation of the stem cell homing factor SDF-1 as well as an improved recruitment of c-KIT+ and CD34+ stem cells.
An intracardiac induction of cyclin D1 and Cdc2 (Cell-division-cycle-2-Kinase) could be observed in the ischemically damaged myocardium during the early phase after MI.
It is known that both factors foster the cell cycle and play an important role in the endothelial proliferation and in the genesis of mature vessel structures. In a model of spinal cord ischemia also a neuroprotective effect with improvement of the neurological function and reduction of loss of motoneurons could be shown. These effects were due to the immigration of CD34+ stem cells and the enhanced expression of „brain-derived neurotrophic factor“ and vascular endothelial growth factor. Hence, EPO is of great interest for the regeneration processes of the heart that we are currently investigating. For this reason, we consider EPO as an important approach for improving the efficacy of regenerative therapies
 
 

SDF-1 and angiogenesis

Technological approaches on the basis of gene therapy and matrix molecules for enhancing the migration of stem cells into the myocardium have been developed. Non-viral gene therapy approaches proved to be successful and revealed an enhanced migration of bone marrow-derived CD117+ cells into the myocardium as well as an enhanced angiogenesis. Similar results were also achieved with Matrigel-application. Based on these investigations mechanism-specific applications of various different regenerative active ingredients are currently being developed.
 
 

AT2 - receptor stimulation

The cardiac stem cell-mediated therapy has a large potential for the treatment of cardiac diseases. We are working on the identification of ways to improve the therapeutic efficacy of intramyocardially applied bone marrow-derived stem cells for the myocardial regeneration. In this frame, we have also been working on Angiotensin II, the main effector of the renin-angiotensin-system, which influences the cardiac regeneration process by means of its receptor molecules (ATR1 and ATR2).
Based on the finding of a post-infarct expression of the AT2-receptor in cardiac c-kit+-progenitor cells we now intend to improve the regeneration process of the infarcted heart by means of AT2-R modulation. In addition, an improved therapeutic efficacy of bone marrow-derived progenitor cells after pre-conditioning with AT2-R stimulation has been successfully demonstrated in a rodent model. Based on these promising data we now intend to develop translational-oriented and clinically relevant approaches for the AT2-R-mediated cardiac regeneration.
 
 
Legal The RTC is supported by the BMBF and the State Mecklenburg-Western Pomerania using EU Structural Funds.