Individual evaluation of cardiac marker expression and self‐beating during cardiac differentiation of P19CL6 cells on different culture substrates

Abstract

Cell-based therapies using self-beating cardiomyocytes have been attracting great attention for use in cardiac regeneration, although an effective procedure to improve cardiac differentiation and self-beating induction is required. The purpose of this study is to clarify the effect of the culture substrate on cardiac maturation by separately evaluating the cardiac differentiation step and the beating induction step in vitro. To this end, the well-studied cardiomyocyte-like progenitor cell line P19CL6 and neonatal cardiomyocytes (NCMs) were selected and cultured on substrates coated with collagen type I (Col-I), gelatin (Gel), fibronectin (FN), or poly-l-lysine (PLL). It was found that the cardiac differentiation step, which was assessed using cardiac marker gene expression (GATA-binding protein 4 (GATA4), myocyte-specific enhancer factor 2D (MEF2D), and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)) in the P19CL6 embryonal carcinoma cells, was greatly enhanced on Col-I, Gel, and PLL. In contrast, the spontaneous beating step, which was directly assessed by counting the beating colonies and measuring contractile protein gene expression (α-myosin heavy chain (α-MHC), troponin C type 1 (TnC1), and troponin T type 2 (TnT2)) in the rat NCMs, was enhanced on the FN and PLL surfaces. In the present study, for the first time, it was found that PLL enhances both the cardiac differentiation and the beating induction steps of cardiac maturation, which can aid in preparing beating cardiomyocytes for regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1166-1174, 2017.

Cardiac specific transcription factor Csx/Nkx2.5 regulates transient-outward K+ channel expression in pluripotent P19 cell-derived cardiomyocytes

“…Such regulatory coordination also depends on the developmental changes in the cardiac substrates that are responsible for actions of transcriptional/translational feedback modifications. In recent years, in vitro models using iPSC-derived cell types or pluripotent cell lines have rapidly emerged as a powerful genetic system to study cardiac development and function [25,26].…”