Human C-kit+CD45À cardiac stem cells are heterogeneous and display both cardiac and endothelial commitment by single-cell qPCR analysis Joakim Sandstedt a,⇑ , Marianne Jonsson a , Göran Dellgren b,c , Anders Lindahl a , Anders Jeppsson b,c , Julia Asp a a Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden b Department of Molecular and Clinical Medicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden c Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden article info Article history: Received 20 November 2013 Available online 2 December 2013 Keywords: FACS Cardiac stem cells Single-cell qPCR Human Heart Atrium abstract C-kit expressing cardiac stem cells have been described as multipotent. We have previously identified human cardiac C-kit+CD45À cells, but only found evidence of endothelial commitment. A small cardiac committed subpopulation within the C-kit+CD45À population might however be present. To investigate this at single-cell level, right and left atrial biopsies were dissociated and analyzed by FACS. Only right atrial biopsies contained a clearly distinguishable C-kit+CD45À population, which was single-cell sorted for qPCR. A minor portion of the sorted cells (1.1%) expressed early cardiac gene NKX2.5 while most of the cells (81%) expressed late endothelial gene VWF. VWFÀ cells were analyzed for a wider panel of genes. One group of these cells expressed endothelial genes (FLK-1, CD31) while another group expressed late cardiac genes (TNNT2, ACTC1). In conclusion, human C-kit+CD45À cells were predominantly localized to the right atrium. While most of these cells expressed endothelial genes, a minor portion expressed cardiac genes. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Traditionally, the heart has been regarded as a non-regenerative organ after the neonatal period. During the past 10 years, this no- tion has been challenged. A slow turnover of cardiomyocytes have been shown by studying 14 C content in human cardiomyocytes [1]. C-kit+ stem cells residing in the myocardium have been suggested as the source of cardiac regeneration. These cells have been described as multipotent with the capacity to differentiate into cardiac, endothelial and smooth muscle cells [2]. Most studies however have been conducted in animal models which may not reflect the human situation. We have previously identified a popu- lation of C-kit+CD45À cells in human right atrial tissue [3]. These cells showed an endothelial profile both on gene and protein levels, and could not be induced to differentiate into cardiomyocytes. This is in line with a study in mice, which showed that the capacity of cardiac C-kit+ cells to differentiate into cardiomyocytes existed in the neonatal heart but was lost in the adult [4]. On the other hand, it has been suggested that C-kit+ cells in the adult heart are heter- ogeneous in regard of differentiation potential rather than truly multipotent. When C-kit+ cells were divided based on FLK-1 expression, the FLK-1+ cells showed endothelial differential poten- tial whereas the FLK-1À cells could be induced to cardiac differen- tiation both in vitro and in vivo [5,6]. From human cardiac tissue, biopsy material is rather limited and the C-kit+ population represents only a small fraction of the cells in a biopsy. Many studies have thus adopted a primary culture step before isolation of C-kit+ cells [3,7], which may induce expansion of one committed subpopulation relative to another. Furthermore, most studies have been conducted on right atrial tissue. However, it could be hypothesized that higher pressure on the left side of the heart could affect distribution and phenotype of stem cells. In the present study, we wanted to compare distribution of C- kit+CD45À cells between right and left atrium and explore whether the C-kit+CD45À population in human adult heart show evidence of lineage commitment into the cardiac and endothelial lineages before expansion in vitro. For this purpose, we employed a novel single-cell based strategy where single C-kit+CD45À cells were sorted and subjected to quantitative PCR (qPCR) analysis of lineage and stem cell markers. 2. Materials and methods 2.1. Tissue procurement Atrial biopsies (n = 13) were obtained from patients undergoing Maze surgery at Sahlgrenska University Hospital, after informed 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.11.086 Abbreviations: qPCR, quantitative real-time polymerase chain reaction; SP, Side Population; SEM, standard error of the mean. ⇑ Corresponding author. Address: Sahlgrenska University Hospital, Department of Clinical Chemistry and Transfusion Medicine, Bruna stråket 16, SE-41345 Gothen- burg, Sweden. Fax: +46 31 828458. E-mail address: joakim.sandstedt@gu.se (J. Sandstedt). Biochemical and Biophysical Research Communications 443 (2014) 234–238 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc