University Rostock

8 ARTICLES PUBLISHED IN JoVE

Biology

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Peter Donndorf¹, Marion Ludwig¹, Fabian Wildschütz¹, Dritan Useini¹, Alexander Kaminski¹, Brigitte Vollmar², Gustav Steinhoff¹

¹Reference and Translation Centre for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, University Rostock, ²Institute for Experimental Surgery, University of Rostock

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

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.

Medicine

Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

Christian Klopsch¹, Alexander Kaminski¹, Friedrich Prall², Pascal Dohmen^1,3

¹Department of Cardiac Surgery, Heart Center Rostock, Rostock University Medical Center, University of Rostock, ²Institute of Pathology, Rostock University Medical Center, University of Rostock, ³Department of Cardiothoracic Surgery, Faculty of Health Science, University of the Free State

Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

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.