Generation of Pure Cardiomyocytes from Murine Induced Pluripotent Stem Cells by Puromycin Purification of Cardiomyocytes
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- Category: Vol. 85, March 2017
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Generation of Pure Cardiomyocytes from Murine Induced Pluripotent Stem Cells by Puromycin Purification of Cardiomyocytes, MARIANNE E. YASSA, IMAN A. MANSOUR, NADIA I. SEWELAM and TAGHRID GAAFAR
Abstract
Background: Embryonic and induced Pluripotent Stem cells (ES/iPS cells) are promising cell sources for cardiac regenerative medicine but their clinical use is associated with tumor formation as generated cardiac cells will always have few escaping pluripotent cells that proliferate after transplan-tation and make problems. To realize ES/iPS cell-based cardiac cell therapy, efficient induction, purification, and transplan-tation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose.
Aim of Work: In this study, we attempted to purify murine iPS cell-derived cardiomyocytes. The desired result is to eliminate all pluripotent cells and to deliver 100% pure cardiomyocytes.
Methods and Results: With the use of a standard Embryoid Body (EB)-based differentiation protocol for ES cells, murine iPS cells were differentiated into cardiomyocytes. The differ-entiation resulted in an average of 70% of spontaneously contracting EBs at day 9 of differentiation. As murine iPS expressed the neomycin-resistance gene under the control of the cardiac a-Myosin Heavy Chain (a-MHC) promoter, addition of puromycin to the beating EBs resulted in 100% beating EBs. Further puromycin purification of plated single cells derived from dissociated EBs results in pure beating cardiomyocyte production. It constitutes 92% of cultured cells by flow cytometry.
Conclusion: We succeeded in efficiently inducing cardi-omyocytes from murine iPS cells and selecting the murine iPS cell-derived cardiomyocytes for robust, efficient and scalable purification. These findings would offer a valuable technological basis for iPS cell-based cell therapy.