Original article Mesoangioblasts from ventricular vessels can differentiate in vitro into cardiac myocytes with sinoatrial-like properties Andrea Barbuti a,b, ⁎, Beatriz G. Galvez c , Alessia Crespi a , Angela Scavone a , Mirko Baruscotti a,b , Chiara Brioschi a , Giulio Cossu c,d , Dario DiFrancesco a,b a Department of Biomolecular Sciences and Biotechnology, The PaceLab, University of Milano, via Celoria 26, 20133 Milan, Italy b Centro Interuniversitario di Medicina Molecolare e Biofisica Applicata (CIMMBA), University of Milan, Italy c Stem Cell Research Institute, San Raffaele Hospital, via Olgettina, 20132 Milan, Italy d University of Milano, Department of Biology, via Celoria 26, 20133 Milan, Italy abstract article info Article history: Received 17 June 2009 Received in revised form 7 September 2009 Accepted 2 October 2009 Available online 22 October 2009 Keywords: Adult stem cells Mesoangioblasts Pacemaker myocytes Funny current HCN channels Cardiac mesoangioblasts (MABs) are a class of vessel-associated clonogenic, self-renewing progenitor cells, recently identified in the post-natal murine heart and committed to cardiac differentiation. Cardiomyocytes generated during cardiogenesis from progenitor cells acquire several distinct phenotypes, corresponding to different functional properties in diverse structures of the adult heart. Given the special functional relevance to rhythm generation and rate control of sinoatrial cells, and in view of their prospective use in therapeutical applications, we sought to determine if, and to what extent, cardiac mesoangioblasts could also differentiate into myocytes with properties typical of mature pacemaker myocytes. We report here that a subpopulation of cardiac mesoangioblasts, induced to differentiate in vitro into cardiomyocytes, do acquire a phenotype with specific mature pacemaker myocytes properties. These include expression of the HCN4 isoform of pacemaker (“funny”, f-) channels and connexin 45 (Cx45), as well as reduced expression of inwardly- rectifying potassium channels. Furthermore, MAB-derived myocytes form agglomerates of pacing cells displaying stable rhythmic activity, and as in native cardiac pacemaker cells, f-channel modulation by autonomic transmitters contributes to control of spontaneous rate in differentiated mesoangioblasts. These data represent the first evidence for in vitro generation of pacemaker-like myocytes from proliferating non- embryonic stem/progenitor cells. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction During cardiac embryogenesis, common early cardiac precursors differentiate into diverse cardiovascular cell types according to specific gene programs [1,2], and some of the transcriptional networks whose activation is linked to specification of different cardiomyocytes have been elucidated [3,4]. Study of embryonic stem (ES) cell differentiation, which recapi- tulates early cardiac development, has shown generation of multiple cardiac phenotypes from common progenitors [5,6]. Adult cardiac stem cells, on the other hand, generate cardiomyocytes of the working muscle [7], but their ability to give rise to multiple cardiac phenotypes has not yet been demonstrated. MABs are vessel-associated, progenitor cells originally isolated in the embryonic aorta as clonogenic, self-renewing and multipotent cells [8]. Recently, similar cells have been identified in adult skeletal [9] and cardiac muscle [10], where they were shown to differentiate mainly to skeletal and cardiac myocytes, respectively. In this latter study, we have shown that ventricle-derived and aorta- derived MABs can differentiate spontaneously into contracting cardio- myocytes expressing cardiac-specific proteins and ion channels. During development cardiac precursors acquire different phenotypes, with specific functional features [11–13]. An important distinction exists between cells of the cardiac pacemaker/conduction tissue, whose contribution is mainly electrical (i.e. generation and propagation of the action potential) and the working myocardium, whose main function is to respond to electrical stimuli with a contraction. An obvious question arising from the finding that cardiac MABs express typical cardiac progenitor markers and have a high prolifera- tive potency, is whether their differentiation gives rise to multiple cardiac phenotypes. We specifically asked if, and to what extent, MABs could differentiate into cardiomyocytes with a mature pacemaker/conduction tissue phenotype. Our investigation was prompted by the preliminary observation that spontaneous activity was often seen to arise in a fraction of differentiating MABs. We found that in both ventricle-derived and aorta-derived MAB clones, expanded under suitable culture conditions and driven to Journal of Molecular and Cellular Cardiology 48 (2010) 415–423 ⁎ Corresponding author. Department of Biomolecular Science and Biotechnology University of Milano, Via Celoria 26, 20133 Milan, Italy. Tel.: +39 02 50314941; fax: +39 02 50314932. E-mail address: andrea.barbuti@unimi.it (A. Barbuti). 0022-2828/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2009.10.006 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc