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Light and Matter of the Interdisciplinary Faculty at Rostock University

6 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Christian Rimmbach¹, Julia J. Jung¹, Robert David¹

¹Reference and Translation Center for Cardiac Stem Cell Therapy, University of Rostock

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These “Induced-sinoatrial-bodies” (“iSABs”) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Medicine

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Natalia Voronina¹, Heiko Lemcke¹, Frank Wiekhorst², Jens-Peter Kühn³, Markus Frank⁴, Gustav Steinhoff¹, Robert David¹

¹Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, University of Rostock, ²Physikalisch-Technische Bundesanstalt, ³Department of Radiology and Neuroradiology, Ernst-Moritz-Arndt-University Greifswald, ⁴Electron Microscopy Center, University of Rostock

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Biology

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Heiko Lemcke^1,2,3, Natalia Voronina^1,2,3, Gustav Steinhoff^1,2,3, Robert David^1,2,3

¹Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), ²Department of Cardiac Surgery, University of Rostock, ³Department of Life, Light and Matter of the Interdisciplinary Faculty, University of Rostock

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Bioengineering

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Frauke Hausburg^*1,2, Paula Müller^*1,2, Natalia Voronina^*1, Gustav Steinhoff^1,2, Robert David^1,2

¹Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, Rostock University Medical Center, ²Department Life, Light and Matter of the Interdisciplinary Faculty, Rostock University

This protocol illustrates a safe and efficient procedure to modify CD133⁺ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Genetics

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

Paula Müller^*1,2, Katharina Ekat^*3, Anne Brosemann³, Anne Köntges³, Robert David^1,2, Hermann Lang³

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

Biology

Analyzing the α-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

Lisa Johann^1,2, Oleksandra Chabanovska^1,2, Cajetan Immanuel Lang³, Robert David^1,2, Heiko Lemcke^1,2

¹Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Rostock University Medical Center, ²Faculty of Interdisciplinary Research, Department Life, Light & Matter, University Rostock, ³Department of Cardiology, Rostock University Medical Center

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.