Poster Presentation 6th Annual Meeting for Australasian Society for Stem Cell Research 2013

A separable upstream promoter of the human SLC8A1 (NCX1) gene efficiently marks a contractile cardiomyocyte population in human pluripotent stem cell-derived cardiomyogenic cultures (#127)

Dmitry Ovchinikov 1 , Alejandro Hidalgo 1 2 , Sean Yang 3 , Xinli Zhang 3 , James Hudson 4 , Chen Chen , Justin Cooper-White 2 5 6 , Ernst Wolvetang 1
  1. Stem Cell Engineering Group, Australian Institute for Bioengineering and nanotechnology, The University of Queensland, Brisbane, Queensland, Australia
  2. Tissue Engineering and Microfluidics Group, Australian institute for Biotechnology and Bioengineering, The University of Queensland, Queensland, Australia
  3. School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
  4. Laboratory of Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
  5. School of Chemical Engineering, The University of Queensland, Brisbane, Queensland, Australia
  6. Material Science and Engineering Division, CSIRO , Clayton, Victoria, Australia

For regenerative medicine and drug screening applications the prospective isolation of defined contractile human iPSC-derived cardiomyocytes is of great utility.  The sarcolemmal Na+/Ca2+ exchanger, encoded by the SLC8A1 (NCX1) gene, regulates cytoplasmic Ca2+ levels in cardiomyocytes from early developmental stages in vivo, and is thought to be crucial for the maintenance of cellular excitability and excitation-contraction coupling. Because the upstream-most SLC8A1(NCX1) cardiac-specific promoter (NCX1cp) is well conserved we utilized the human promoter/enhancer region to drive an NCX1cp-EGFP reporter cassette that was lentivirally delivered to human iPSCs and hESCs. Eight days into cardiac differentiation of these cells expression of the NCX1cp-EGFP was observed that transitioned to spontaneously contracting foci and individual beating cells 1-2 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was limited to contractile cells expressing high levels of cardiac troponin T (cTnT), MLC2 and α-actinin proteins, but not CD90/Thy1+ cardiac fibroblasts or CD31/PECAM+ endothelial cells. The EGFP+ flow cytometrically-sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, NKX2.5) and late (cTnT, MYH6, NPPA) cardiomyocytic markers and predominantly displayed a ventricular-like action potential during patch-clamping. We thus demonstrate the utility of a 2 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) gene that is sufficient for driving the expression of a reporter in contractile human (embryonic and induced pluripotent) stem cell-derived cardiac (predominantly ventricular-like) cells.