Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology

Abstract

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes.

Optophysiology of cardiomyocytes: characterizing cellular motion with quantitative phase imaging

“…Although traditionally hPSC-CMs are classified into the atrial-, nodal-, and ventricular-like subtypes, recent studies demonstrated that hPSC-CMs do not cleanly separate into these classes, and their properties represent a continuum, changing over time [2,38]. Our data supports this conclusion and enables observation of their development over time.…”

An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype

“…The subtype of each hiPSC‐CMs was first determined by its AP profile through the clustering analysis, an established alternative method to patch clamp for subtype classification . The AP of a ventricular‐like cell shows a more prominent plateau phase than that of an atrial‐like and pacemaker‐like cell (Figure A1‐A3).…”

“…Current hiPSC‐CMs differentiation protocols typically yield ~70% CMs with ~15% of the CMs being the pacemaking subtype . Achieving higher‐purity populations is challenging because there is no established purification method, largely due to the lack of a reliable marker that can be used to sort cells by phenotype, maturity, or subtype.…”

Induced pluripotent stem-cell-derived cardiomyocytes: cardiac applications, opportunities, and challenges

“…3) (Bett et al 2013; Burridge D r a f t Lian et al 2012;Ma et al 2011;N. Sun et al 2012;Yechikov et al 2016), although ventricular-like hiPSC-CMs usually predominate (Burridge et al 2014;Lian et al 2012;Yechikov et al 2016). Specific cardiac cells subtype diversity has also been reported to evolve throughout the differentiation process.…”

“…Specific cardiac cells subtype diversity has also been reported to evolve throughout the differentiation process. The ratio of nodal-, atrial-, and ventricular-like cells appears to be balanced during early differentiation, while ventricular-like cells predominate in older cell cultures (Burridge et al 2014;Lian et al 2012;Yechikov et al 2016). It is important to note that there is no consensus regarding the classification of hiPSC-CMs into subtypes.…”