Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state

Abstract

Although both the H1 and HES2 human embryonic stem cell lines (NIH codes: WA01 and ES02, respectively) are capable of forming all three germ layers and their derivatives, various lines of evidence including the need for using different protocols to induce cardiac differentiation hint that they have distinctive preferences to become chamber-specific heart cells. However, a direct systematic comparison has not been reported. Here we electrophysiologically demonstrated that the distributions of ventricular-, atrial-and pacemaker-like derivatives were indeed different (ratios=39:61:0 and 64:33:3 for H1 and HES2, respectively). Based on these results, we hypothesized the differences in their cardiogenic potentials are imprinted in the proteomes of undifferentiated H1 and HES2. Using the multiplexing, high-resolution 2-D Differential In Gel Electrophoresis (DIGE) to minimize gel-to-gel variations that are common in conventional 2D gels, a total of 2000 individual protein spots were separated. Of which, 55 were >2-fold differentially expressed in H1 and HES2 (p<0.05) and identified by mass spectrometery. Bioinformatic analysis of these protein differences further revealed candidate pathways that contribute to the H1 and HES2 phenotypes. We conclude that H1 and HES2 have predetermined preferences to become ventricular, atrial and pacemaker cells due to discrete differences in their proteomes. These results improve our basic understanding of hESCs and may lead to mechanism-based methods for their directed cardiac differentiation into chamber-specific cardiomyocytes.

Na+/Ca2+Exchanger is a Determinant of Excitation–Contraction Coupling in Human Embryonic Stem Cell–Derived Ventricular Cardiomyocytes

“…A spectrofl uorometric method with Fura-2/ Instruments Inc., Foster City, CA) as previously described [3]. A xenon arc lamp was used to view the dsRed fl uorescence at 560/590 nm (excitation/emission).…”

“…Since cardiac differentiation of hESCs is known to generate a heterogeneous population of ventricular, atrial, and pacemaker derivatives [1,3], selection of dsRed+ hESC-CMs for experiments will enable us to study the ventricular lineage without ambiguities.…”

“…Therefore, hESC-CMs may provide an unlimited ex vivo source of genuine human CMs for transplantation and can also serve as an excellent experimental model for studying human cardiogenesis. However, current protocols for cardiac differentiation of hESCs lead to a heterogeneous population of pacemaker-, atrial-, and ventricular-like derivatives, which are conventionally functionally classifi ed based on their signature action potential (AP) profi les [1,3]. Indeed, we previously demonstrated that in vivo transplantation of a node of electrically active hESCCMs, containing a mixture of ventricular, atrial, and nodal cells, into the ventricle can collectively induce a local epicardial pacing origin [2].…”

Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

“…Neither pluripotent stem cells nor direct reprogramming methods address another critical issue, which is the need to generate chamber-specific cardiomyocytes. Pacemaker, atrial, and ventricular myocytes (VMs) have distinct functional and electrophysiological properties that may contribute to cardiac arrhythmias in the wrong environment [7,8] yield a heterogeneous population of cardiomyocytes of which only 30% -70% are VMs [9,10], and methods of purifying VMs from a population of stem cell-derived cardiomyocytes remain to be established. These issues underscore the need to better understand the molecular pathways that govern the specification of VMs from pluripotent stem cells.…”

Differentiation, Characterization and Applications of Human Embryonic Stem Cell –Derived Cardiomyocytes

“…According to the immunocytochemisty by ventricular specific marker and action potential studies, most of the differentiated cardiomyocytes were ventricular type of cells (Pekkanen-Mattila, Chapman et al 2010). Slight variation in the amount of ventricular type of cells has been observed in different cell lines (Moore, Fu et al 2008) and the amount has also been observed to vary between cardiac differentiation methods (80% and 100% of END-2 and EB-derived cardiomyocytes, respectively) (Pekkanen-Mattila, Chapman et al 2010). If compared to the human neonatal or adult atrial or ventricular cardiomyocytes, hESC-CM have relatively positive maxium diastolic potential (MDP) and slow maxium rate of rise of the AP (dV/dtmax) and therefore resemble embryonal atrial-and ventricular like cells (He, Ma et al 2003).…”