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Article first published online: 27 NOV 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 12, pages 2830–2834, December 2012
How to Cite
Ramkisoensing, A. A., De Vries, A. A. F., Schalij, M. J., Atsma, D. E. and Pijnappels, D. A. (2012), Brief Report: Misinterpretation of Coculture Differentiation Experiments by Unintended Labeling of Cardiomyocytes Through Secondary Transduction: Delusions and Solutions. STEM CELLS, 30: 2830–2834. doi: 10.1002/stem.1236
Author contributions: A.A.R. and D.A.P.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; A.A.F.V.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript; M.J.S.: financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript; D.E.A.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS September 17, 2012.
- Issue published online: 27 NOV 2012
- Article first published online: 27 NOV 2012
- Accepted manuscript online: 17 SEP 2012 03:53PM EST
- Manuscript Accepted: 29 AUG 2012
- Manuscript Revised: 21 AUG 2012
- Manuscript Received: 29 JUN 2012
- Dutch Heart Foundation (NHS)
- Netherlands Organisation for Scientific Research. Grant Number: NWO, Veni Grant 91611070
- Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht
- Netherlands for providing us with VSV-neutralizing rabbit serum
- Stem cell;
- Lentiviral vector;
- Neutralizing activity
Cardiomyogenic differentiation of stem cells can be accomplished by coculture with cardiomyocytes (CMCs). To facilitate their identification, stem cells are often labeled through viral transduction with a fluorescent protein. A second marker to distinguish stem cell-derived CMCs from native CMCs is rarely used. This study aimed to investigate the occurrence of secondary transduction of unlabeled neonatal rat (nr) CMCs after coculture with human cells that had been transduced 0, 7, or 14 days earlier with a vesicular stomatitis virus (VSV) G protein-pseudotyped lentiviral vector (LV) encoding enhanced green fluorescent protein (GFP). To reduce secondary LV transfer, GFP-labeled cells were incubated with non-heat-inactivated human serum (NHI) or with VSV-neutralizing rabbit serum (αVSV). Heat-inactivated human serum and normal rabbit serum were used as controls. Immunostaining showed substantial GFP gene transfer to nrCMCs in cocultures started at the day of transduction indicated by the presence of GFP-positive/human lamin A/C-negative nrCMCs. The extent of secondary transduction was significantly reduced in cocultures initiated 7 days after GFP transduction, while it was completely abolished when human cells were added to nrCMCs 14 days post-transduction. Both NHI and αVSV significantly reduced the occurrence of secondary transduction compared to their controls. However, under all circumstances, GFP-labeled human cells had to be passaged for 14 days prior to coculture initiation to prevent any horizontal GFP gene transfer to the nrCMCs. This study emphasizes that differentiation experiments involving the use of viral vector-marked donor cells should be interpreted with caution and describes measures to reduce/prevent secondary transduction. STEM CELLS 2012;30:2830–2834