Original article Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells Severina Windmolders a,b , Astrid De Boeck c , Remco Koninckx a,b , Annick Daniëls a , Olivier De Wever c , Marc Bracke c , Marc Hendrikx b,d , Karen Hensen a,b , Jean-Luc Rummens a,b, ⁎ a Laboratory of Experimental Hematology, Jessa Hospital, Campus Virga Jesse, Stadsomvaart 11, 3500 Hasselt, Belgium b Faculty of Medicine and Life Sciences, Hasselt University, Martelarenlaan 42, 3500 Hasselt, Belgium c Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium d Department of Cardiothoracic Surgery, Jessa Hospital, Campus Virga Jesse, Stadsomvaart 11, 3500 Hasselt, Belgium abstract article info Article history: Received 20 July 2013 Received in revised form 12 November 2013 Accepted 28 November 2013 Available online 8 December 2013 Keywords: Myocardial infarction Mesenchymal stem cells Conditioned medium Cardiac stem cells Migration Platelet derived growth factor Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of para- crine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned me- dium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, mi- grating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet de- rived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF- AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction In the last decade, stem cell therapy has emerged as an innovative approach to restore cardiac function after myocardial infarction (MI) ei- ther directly by regeneration of functional myocardium [1] or indirectly by paracrine actions stimulating cardiac tissue healing [2]. Previously, researchers reported the existence of cardiac stem cells (CSCs) residing in the adult mammalian heart [3,4]. While phase 1 clinical studies [5,6] are completed only recently, CSC transplantations performed in the past already showed an improved cardiac function in animal models through regeneration of the damaged myocardium [7]. In the past, most experimental and clinical studies concerning ische- mic heart disease (IHD) were performed with bone marrow stem cells (BM-SCs) [8,9]. Although it was demonstrated that BM-SC implantation can reduce ventricular remodeling and improve left ventricular function after MI, the underlying mechanism is still under debate [10]. Recent data propose that among BM-SCs, mesenchymal stem cells (MSCs) are especially capable of mediating cardiac repair through the release of a broad spectrum of cytokines, growth factors and chemokines into the damaged tissue area [11]. Strong evidence comes from studies that have utilized the conditioned medium derived from MSCs (MSC-CM). Journal of Molecular and Cellular Cardiology 66 (2014) 177–188 Abbreviations: 3D, Three-dimensional; ALDH, Aldehyde dehydrogenase; AT, Annealing temperature; AxlR, Axl receptor; BM-SCs, Bone marrow stem cells; CASCs, Cardiac Atrial appendage Stem Cells; CFU-Fs, Colony-forming-unit fibroblasts; CSCs, Cardiac stem cells; cTn, Cardiac troponin; DEAB, Diethylamino-benzaldehyde; EGFR, Epidermal growth factor receptor; ELISA, Enzyme-linked immunosorbent assay; FAK, Focal adhesion kinase; FCS, Fetal calf serum; FGFR, Fibroblast growth factor receptor; FMO, Fluorescence minus one; GFP, Green fluorescent protein; IHD, Ischemic heart dis- ease; InsR, Insulin receptor; LG-DMEM, Low-glucose Dulbecco's Modified Eagle Medium; MI, Myocardial infarction; MMPs, Matrix metalloproteinases; MSCs, Mesenchymal stem cells; MSC-CM, Mesenchymal stem cell conditioned medium; NRCMs, Neonatal rat cardiomyocytes; PDGF, Platelet derived growth factor; PDGFR, Platelet derived growth fac- tor receptor; PLC-γ, Phospholipase C-γ; PI3K, Phospho-inositide-3 kinase; P/S, Penicillin/ streptomycin; RALDH2, Retinaldehyde dehydrogenase 2; RTK, Receptor tyrosine kinase; RT-PCR, Reverse transcriptase PCR; Tβ4, Thymosin β4; Tbx18, T-Box 18; TGF-β, Transforming growth factor-β; VEGF, Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor; Wt1, Wilm's tumor 1. ⁎ Corresponding author at: Laboratory of Experimental Hematology, Jessa Hospital, Campus Virga Jesse, Stadsomvaart 11, 3500 Hasselt, Belgium. Tel.: +32 11309740; fax: +32 11309750. E-mail addresses: severina.windmolders@uhasselt.be (S. Windmolders), adeboeck@ucalgary.ca (A. De Boeck), remco.koninckx@jessazh.be (R. Koninckx), annick.daniels@jessazh.be (A. Daniëls), olivier.dewever@ugent.be (O. De Wever), marc1.bracke@ugent.be (M. Bracke), marc.hendrikx@jessazh.be (M. Hendrikx), karen.hensen@jessazh.be (K. Hensen), jean-luc.rummens@jessazh.be (J.-L. Rummens). 0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.11.016 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc