Fabrice F Darche1, Rasmus Rivinius1, Eva Köllensperger2, Uwe Leimer2, Günter Germann2, Anja Seckinger3, Dirk Hose3, Julian Schröter1, Claus Bruehl4, Andreas Draguhn4, Richard Gabriel5, Manfred Schmidt5, Michael Koenen6, Dierk Thomas7, Hugo A Katus7, Patrick A Schweizer8. 1. Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120 Heidelberg, Germany. 2. ETHIANUM Klinik Heidelberg, Voßstraße 6, D-69115 Heidelberg, Germany. 3. Department of Hematology, Oncology and Rheumatology, Medical University Hospital Heidelberg, INF 410, D-69120 Heidelberg, Germany. 4. Institute for Physiology and Pathophysiology, University of Heidelberg, INF 326, D-69120 Heidelberg, Germany. 5. Molecular and Gene Therapy, National Center for Tumor Diseases (NCT) Heidelberg, INF 460, D-69120 Heidelberg, Germany. 6. Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120 Heidelberg, Germany; Department of Molecular Neurobiology, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany. 7. Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120 Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, University of Heidelberg, INF 410, D-69120 Heidelberg, Germany. 8. Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120 Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, University of Heidelberg, INF 410, D-69120 Heidelberg, Germany. Electronic address: patrick.schweizer@med.uni-heidelberg.de.
Abstract
AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.
AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.
Authors: Fabrice F Darche; Rasmus Rivinius; Ann-Kathrin Rahm; Eva Köllensperger; Uwe Leimer; Günter Germann; Miriam Reiss; Michael Koenen; Hugo A Katus; Dierk Thomas; Patrick A Schweizer Journal: World J Stem Cells Date: 2020-10-26 Impact factor: 5.326
Authors: Rizwan Malik; Fabrice A Darche; Rasmus Rivinius; Anja Seckinger; Ulf Krause; Michael Koenen; Dierk Thomas; Hugo A Katus; Patrick A Schweizer Journal: Cell Transplant Date: 2020 Jan-Dec Impact factor: 4.139