EURON and THEME joint PhD meeting

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64 EURON and THEME joint meeting 2011 Local gene targeting and cell positioning using magnetic nanoparticles for the generation of biological cardiac pacemakers Carsten Kilgus, Tobias Bruegmann, Bernd K. Fleischmann and Philipp Sasse Institute of Physiology I, Life&Brain Center, University Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Implantation of electronic implantable pacemakers is the primary therapy for patients with sinus node dysfunction or high degree of atrio-ventricular block. Although complications are rare, this treatment has some limitations which is why the development of biological pacemakers is studied. A biological pacemaker should be confined to an area within the heart and we have therefore developed new technologies for localized cell therapy and gene transfer to cardiomyocytes using magnetic nanoparticles (MNP) and magnetic fields. First initial proof of principle studies, a monolayer cardiomyocytes from the HL-1 line transfected with a lentivirus that expressed the green fluorescent protein (GFP). Localization was achieved by generation of complexes with MNP and lentiviruses (200 fg iron per virus particle) and transduction of cells in a confined magnetic field that was generated by neodymium-iron-boron permanent magnets with specially designed soft iron tips. This approach allowed reliably and stable transduction of cells within an area of < 1mm diameter indicated by localized GFP expression. To provide a functional pacemaker we plated HL-1 cells onto multi-electrode arrays (MEA, 60 electrodes, 200 µm spacing) to read out the local field potentials and calculated the site from where pacemaking was initiated. We performed localized transduction with MNP-lentivirus complexes that express the light-gated ion channel channelrhodopsin2 (ChR2). This lead to localized ChR2 expression and light flashes paced the whole monolayer. Importantly latency analysis of the MEA recordings proved that pacing was always initiated from the transduced sites despite global illumination. To be able to generate an autonomeous pacemaker, we have successfully generated lentiviruses to express the classical pacemaker channels HCN2 and HCN4 as well as a dominant-negative potassium channel (Kir2.1-AAA). Preliminary long-term MEA recordings and localized transduction with the Kir2.1-AAA virus indicated that pacemaking is initiated from the transduced site 2-3 days after localized gene transfer. Besides gene transfer we have also tested localized cell positioning for pacemaker generation. Therefore we have differentiated and purified ChR2-expressing
65 EURON and THEME joint meeting 2011 cardiomyocytes from embryonic stem cells, which could be paced by light. These were loaded with MNP, guided onto HL-1 monolayers on MEA by localized magnetic fields. By this approach only 5000 cells were needed to generate a confined cell cluster (~ 1mm diameter). Importantly, global light stimulation could be used to pace the monolayer of HL-1 cells and pacing started from the guided ChR2-cell cluster. In summary, magnetic nanoparticles and localized magnetic field are very effective tools for localized gene transfer and cell positioning to generate cardiac pacemakers in vitro. The application of this technology for light-induced or autonomic cardiac pacing in vivo is currently under investigation.
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EURON and THEME joint PhD meeting U
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The local organizing committee: Pro
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Information about THEME 3 EURON and
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EURON a short introduction 7 EURON
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Page 15 and 16: Programme 13 EURON and THEME joint
Page 17 and 18: Thursday September 22 nd 9:00 - 10:
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Page 25 and 26: EURON Lecture Frank Kirchhoff ADDRE
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Page 29 and 30: THEME Lecture Eckhard Mandelkow POS
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Page 33 and 34: Abstracts 31 EURON and THEME joint
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Page 105 and 106: List of Participants 103 EURON and
Page 107 and 108: Nuersailike Abuduwali University of
Page 109 and 110: Marianne Eisenhardt University of B
Page 111 and 112: Sabine Klein University of Bonn PhD
Page 113 and 114: Viktoriya Peeva University of Bonn
Page 115: Sylvie van der Kruijs Maastricht Un
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EURON and THEME joint PhD meeting

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