Abstract
Primate nonhuman and human embryonic stem (ES) cells provide a powerful model of early cardiogenesis. Furthermore, engineering of cardiac progenitors or cardiomyocytes from ES cells offers a tool for drug screening in toxicology or to search for molecules to improve and scale up the process of cardiac differentiation using high-throughput screening technology, as well as a source of cell therapy of heart failure. Spontaneous differentiation of ES cells into cardiomyocytes is, however, limited. Herein, we describe a simple protocol to commit both rhesus and human ES cells toward a cardiac lineage and to sort out early cardiac progenitors. Primate ES cells are challenged for 4 d with the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and sorted out using anti-SSEA-1 antibody-conjugated magnetic beads. Cardiac progenitor cells can be generated and isolated in 4 d using this protocol.
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Acknowledgements
The authors acknowledge the National Agency of Research (ANR) for supporting their research as well as providing salaries for J.L. and S.S. The Evry Genopole is also acknowledged for its financial support to the team. The authors thank Drs Cowan, Itskovitz and Amit, Hovatta and Mitalipov for generously providing the cell lines and for their continuous scientific support.
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Leschik, J., Stefanovic, S., Brinon, B. et al. Cardiac commitment of primate embryonic stem cells. Nat Protoc 3, 1381–1387 (2008). https://doi.org/10.1038/nprot.2008.116
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DOI: https://doi.org/10.1038/nprot.2008.116
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PUCEAT michel
In this paper we report a cell sorting protocol using an anti-CD15 antibody conjugated to magnetic beads (CD15 microbeads Miltenyi Biotec, cat. no. 130-046-601). We recently encountered problems with this antibody which lost the recognition of the BMP2-induced CD15 in human ES cells derived mesodermal cells, likely due to a derivation of the hybridoma used by Miltenyi to produce this monoclonal antibody. CD15 (or Lewis Antigen) is a complex carbohydrate antigen found on glycolipids, and in oligosaccharides of glycoproteins. It is close but not identical to SSEA-1 which contains the Lewis antigen (or CD15). CD15 can be found in cells modified by a syalyl or a sulphate group, a modification which will change the recognition of the Antigen by monoclonal antibodies. Thus, several anti-CD15 commercially available antibodies recognize not only the carbohydrate of interest but also some surrounding carbohydrate groups and possibly even amino acid of the carrier glycoprotein at the membrane of the cell. The clonal purity of the antibody used in our protocol to specifically sort out BMP2-induced mesodermal MesP1+cells is thus critical to get the right cell phenotype. We have solved the problem by screening many antibodies and in turn using another clone from another company (i.e. Exbio). We would like to alert the readers of our paper about this antibody concern to make sure that our protocol is successful in their laboratories. we apologize to some researchers who might have encountered problems in reproducing our data . Please make sure that sorted cells feature the right gene expression profile as reported in this paper or our J Clin Invest paper in 2010 Blin et al) . Please do not hesitate to contact us if you have any question. (michel.puceat@inserm.fr)