title: Isolation of contractile cardiomyocytes from human pluripotent stem-cell-derived cardiomyogenic cultures using a human NCX1-EGFP reporter.
abstract: The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.
authors: 1 Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St. Lucia, Australia .
authors: Dmitry A
authors: DA
authors: Ovchinnikov
authors: Alejandro
authors: A
authors: Hidalgo
authors: Seung-Kwon
authors: SK
authors: Yang
authors: Xinli
authors: X
authors: Zhang
authors: James
authors: J
authors: Hudson
authors: Stuart B
authors: SB
authors: Mazzone
authors: Chen
authors: C
authors: Chen
authors: Justin J
authors: JJ
authors: Cooper-White
authors: Ernst J
authors: EJ
authors: Wolvetang
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C485184:enhanced green fluorescent protein; C110374:sodium-calcium exchanger 1; D049452:Green Fluorescent Proteins