Stem cells for cardiac regeneration

Recent studies have demonstrated a turnover of cardiomyocytes throughout the adult life. Whether this regenerative potential is due to proliferation and differentiation of resident stem cells or to the dedifferentiation of adult cardiomyocytes remains unclear. Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury; however the mechanisms by which injury responses modulate differentiation and proliferation of cardiomyocyte progenitors is not clear. In Study I, we show that sublethal apoptotic stimuli modulate the differentiation of mouse embryonic stem cells (ESC) into cardiomyocytes through a caspasedependent mechanism. This indicates a direct link between caspase activation and initiation of the cardiogenic programs. Further unfolding of these mechanisms may have significant implications for the understanding of the dynamics of cardiac regeneration after myocardial injury involving both exogenous and endogenous cell sources. The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. In Study II our aim was to establish a culture protocol for large-scale expansion of human fetal cardiac MSCs with characteristics of cardiovascular progenitor cells. By culturing the cardiac MSCs on defined laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway we could generate multipotent cells which could differentiate into endothelial, smooth muscle cells, and spontaneously beating cardiomyocytes. The expanded cardiac MSCs stained positive for the progenitor markers: Pdgfr-α, Isl1, and Nkx2.5, and subpopulations also expressed: Tbx18, Kdr, c-Kit, and Ssea-1. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo. Subsequently, in Study III we performed bioenergetic and metabolic profiling of human fetal cardiac MSCs as a tool for investigating metabolic changes in embryonic hearts during development. The critical role of metabolism in the active control of cell renewal and lineage specification has recently come into focus, and Wnt has been proposed as one of the signaling pathways which links energy metabolism to cell fate decision. In the MSCs derived from hearts of different gestational age, we observed an increase in oxidative metabolism during first trimester of development. Finally, stimulation of the canonical Wnt/β-catenin signaling pathway in hypoxia appeared to enhance long-term MSC expansion, and resulted in a cell population with higher cardiogenic differentiation potential. Our findings suggest that manipulation of metabolic signals may facilitate endogenous stem cell activation, tissue renewal and induction of cytoprotective properties. LIST OF PUBLICATIONS I. Sublethal Caspase Activation Promotes Generation of Cardiomyocytes from Embryonic Stem Cells Ivana Bulatovic*, Cristian Ibarra*, Cecilia Österholm, Heng Wang, Antonio Beltrán-Rodríguez, Manuel Varas-Godoy, Agneta MånssonBroberg, Per Uhlén, András Simon, Karl-Henrik Grinnemo PLoS One. 2015; 10(3):e0120176 II. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells Agneta Mansson-Broberg, Sergey Rodin, Ivana Bulatovic, Cristian Ibarra, Marie Lofling, Rami Genead, Eva Wardell, Ulrika Felldin, Carl Granath, Evren Alici, Katarina Le Blanc, C.I. Edvard Smith, Alena Salasova, Magnus Westgren, Erik Sundstrom, Per Uhlen, Ernest Arenas, Christer Sylven, Karl Tryggvason, Matthias Corbascio, Oscar E. Simonson, Cecilia Osterholm and Karl-Henrik Grinnemo Stem Cell Reports. 2016; 6(4):607-17 III. Metabolomic Profiling of Human Fetal Cardiac Mesenchymal Stromal Cells Ivana Bulatovic, Clifford Folmes, Ivan Vuckovic, Song Zhang, Ryounghoon Jeon, Ulrika Felldin, Marie Lofling, Cecilia Osterholm, Andre Terzic, Karl-Henrik Grinnemo Manuscript *These authors contributed equally PUBLICATIONS NOT INCLUDED IN THIS THESIS Human fetal cardiac progenitors: The role of stem cells and progenitors in the fetal and adult heart. Bulatovic I, Månsson-Broberg A, Sylvén C, Grinnemo KH. Best Pract Res Clin Obstet Gynaecol. 2016; 31:58-68. Local control of nuclear calcium signaling in cardiac myocytes by perinuclear microdomains of sarcolemmal insulin-like growth factor 1 receptors. Ibarra C, Vicencio JM, Estrada M, Lin Y, Rocco P, Rebellato P, Munoz JP, Garcia-Prieto J, Quest AF, Chiong M, Davidson SM, Bulatovic I, Grinnemo KH, Larsson O, Szabadkai G, Uhlén P, Jaimovich E, Lavandero S. Circ Res. 2013; 112(2):236-45. Human Embryonic Non-haematopoietic SSEA-1+ Cells are Cardiac Progenitors Expressing Markers of Both the First and Second Heart Field. Elsheikh E, Genead R, Mansson-Broberg A, Bulatovic I, Ljung K, Wärdell E, Felldin U, Ibarra C, Alici E, Sylvén C and Grinnemo KH J Cytol Histol 2013; 4. 192 Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells. Genead R, Fischer H, Hussain A, Jaksch M, Andersson AB, Ljung K, Bulatovic I, Cereceda AF, Elsheikh E, Corbascio M, Smith Edvard C.I. , Sylvén C, Grinnemo KH PLoS ONE. 2012;7(5):e36804. IgM phosphorylcholine antibodies inhibit cell death and constitute a strong protection marker for atherosclerosis development, particularly in combination with other auto-antibodies against modified LDL. Fiskesund R, Su J, Bulatovic I, Vikström M, de Faire U, Frostegård Results in Immunology. 2012 8;2:13-8.