Dynamic Link between Histone H3 Acetylation and an Increase in the Functional Characteristics of Human ESC/iPSC-Derived Cardiomyocytes

<div><p>Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes <em>in vitro</em>, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA) recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.</p> </div

TSA enhances electrophysiological function in hESC-CMs.

<p>(A) Experimental scheme: 21-day-old beating colonies obtained from αMHC-EGFP transgenic hESCs were maintained for 13 to 28 days in suspension. Sensitivity to 200 nM E4031 was measured three times for each beating colony using an MEA system on Days 2, 6, and 9 after the colonies were plated on the MEA probes, as shown at the lower panel. DMSO, or 100 nM TSA, was added for 48 hrs between Days 3 and 5 and then removed for 1 day before the second MEA recording to avoid contact with ion channel inhibitors. (B) Sensitivity of EGFP-positive hESC-CMs to E4031 before TSA treatment (Day 2) and after TSA treatment (Day 6) was measured using an MEA system. Representative FPs were demonstrated in hESC-CM spheroids before and after 100 nM E4031 treatment showing phenotypic changes in the response to 100 nM E4031 from S-FPD to L-FPD in TSA-treated hESC-CM spheroids. (C) Dose-dependent QTc prolongation seen in TSA-treated hESC-CM colonies that had shown arrhythmia in response to 100 nM E4031 in the first MEA test. Each value displays the mean and SD of FPDs obtained from six contiguous FP cycles. (D) Sequential MEA analyses of every hESC-CM colony before and after TSA treatment showing that all colonies reproducibly acquired a uniform electrophysiological phenotype with 2 days of TSA treatment (n = 7).</p

Microarray data for the expression of genes involved in cardiac ion channel function.

<p>Global gene expression analyses were conducted using TSA- or DMSO-treated hESC-CMs under the Ad culture conditions (black bar) or Sus culture conditions (white bar). Genes involved in (A) sodium, (B) calcium and (C) potassium ion channels were analyzed. Each graph displays the mean and SD of two or three independent experiments.</p

Effect of suspension culture and TSA treatment on histone acetylation of hESC-CM colonies.

<p>Western blot analyses show increased histone H3 acetylation (AcH3) levels in 35-day-old hESC-CMs (A) following 14 days of suspension culture (Sus) or (B) following treatment with 100 nM TSA for 48 hrs under adhesive (Ad) culture conditions (DMSO treatment had no effect on AcH3 levels). (C) Results from western blot analyses comparing Sus to Ad cultures (n = 9) and TSA to DMSO treatments (n = 13) show increased AcH3 levels in hESC-CMs. (D) Results of qRT-PCR analysis showing that TSA increases cardiac gene expression in hESC-CMs for at least 48 hrs after treatment. The corresponding values for human adult hearts (AH) are shown for each sample. Each graph displays the mean and standard deviation (SD) of three independent experiments. *, p<0.05; **, p<0.01.</p

Heterogeneous responses to the hERG inhibitor, E4031, in MEA tests using early hESC/hiPSC-CM colonies.

<p>(A) Sensitivity to 200 nM E4031 was measured in Sus cultured hiPSC-CM colonies (d35) after an additional 2 days of Ad culture on multi-electrode array (MEA) probes. The hiPSC-CM colonies showed the following heterogeneous responses to the hERG inhibitor: lengthened field potential duration (L-FPD), shortened FPD (S-FPD), and transient FP phenotypes (T-FPs). Red arrows indicate the initiation of FPs and the black arrows show the end-point of the cardiac repolarization phase in the FP signals. The broken line shows the shift in depolarization from 0 nM through 200 nM E4031. (B, C) Dose increment analyses in Sus-cultured (d35) hESC-CM colonies. The colony showing the L-FPD response to 50 nM of E4031 showed an S-FPD response at 200 nM through to a T-FPs response at 100 nM. This indicates that some of the Sus-cultured (d35) hESC-CM colonies were more sensitive to E4031 than others. However, on the basis of the rate-corrected QT (QTc) values, the S-FPD spheroids also show QTc prolongation (red line), because of the cells’ shortened FP rates. The whole bar containing a gray part displays the mean and SD of six FP cycles, and the gray bar displays the mean and SD of FPD.</p

Responses to the ion channel blockers, E4031, Nifekalant and Sotalol, are improved in TSA-treated hESC-CMs.

<p>Relative FPD values were obtained with the MEA system on Day 2 to Day 6 for each colony in the presence of ion channel inhibitor (200 nM E4031; 20 µM Nifekalant; 200 µM Sotalol). Each graph displays changes in the relative FPD values in each hESC-CM colony after DMSO or TSA treatment and the mean and SD (E4031, n = 5 for DMSO and n = 6 for TSA; Nifekalant, n = 3 for DMSO and n = 4 for TSA; Sotalol, n = 3 for DMSO and n = 3 for TSA). *, p<0.05; †, tendency to differ (p<0.1).</p

TSA enhances electrophysiological function in hiPSC-CMs accompanied by histone H3 acetylation.

<p>(A) Western blot analyses show increased histone H3 acetylation levels in hiPSC-CMs treated with 100 nM TSA for 48 hrs. (B) Results from western blot analyses comparing Sus to Ad cultures (n = 7) and TSA to DMSO treatments (n = 9) show increased AcH3 levels in hiPSC-CMs. (C) Sequential MEA analyses in all hiPSC-CM colonies on Day 2 and Day 6 showed that TSA altered the electrophysiological phenotypes evaluated on the basis of their sensitivity to 200 nM of E4031 (n = 8). (D) Sequential MEA analyses in all hiPSC-CM spheroid on Days 2, 6 and 9 after plating showed electrophysiological phenotypes improved reproducibly following 2 days of TSA treatment in the presence of 200 nM E4031 (n = 7 for DMSO, n = 8 for TSA). Changes in relative FPD values (Day 2 = 1) from Day 2 to Days 6 and 9 and the mean±SD are shown. †, tendency to differ (p<0.1) only on Day 6.</p

The TSA-mediated positive effect on MEA testing is transient.

<p>(A) Results of qRT-PCR analyses of cardiac gene expression in hESC-CMs showed that the effect of TSA was transient. Day 5 (d5): immediately after 2 days of TSA treatment; Day 10 (d10): 5 days of culture after TSA washout. Values relative to the DMSO-treated hESC-CMs (DMSO = 100%) are shown (n = 3). Values represent the mean ± SD for each set of measurements. (B) Sequential MEA analyses of all hESC-CM colonies were performed on Days 2, 6, and 9 in the presence of 200 nM E4031. Changes in the relative FPD values on Day 6 or Day 9 from Day 2 for each colony are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045010#pone-0045010-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045010#pone-0045010-g006" target="_blank">6B</a>, respectively. Values represent the mean ± SD (n = 5 for DMSO, n = 6 for TSA). Significant differences between DMSO and TSA treatment detected on Day 6 were not detected on Day 9.</p

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

<div><p>The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.</p></div

The Small Molecule Method Functions via RARβ Receptors.

<p>(A) Results of the flow cytometric analyses showing induction of OSR1⁺ cells by the small molecule method, when AM580 or TTNPB was replaced by all-trans retinoic acid (ATRA), adapalene, and CD1530. (B) Effects of adding RAR antagonists, BMS493, LE135, and MM11253, on the induction of OSR1⁺ cells by the TTNPB method. (C) The knockdown efficiency of siRNAs targeting <i>RARB</i>, and (D and E) effects on expression levels of <i>OSR1</i> and the induction of OSR1⁺ cells by the TTNPB method. (F) Effects of adding a pan-RXR agonist (SR11237) or a pan-RXR antagonist (UVI3003) to the induction efficiency of OSR1⁺ cells by the TTNPB method. (G) The induction of OSR1⁺ cells by the small molecule method, when SR11237 replaced AM580 or TTNPB. (H) Results of qRT-PCR analyses showing <i>OSR1</i> expression activated by the AM580 and TTNPB methods, and when ATRA, adapalene, or CD1530 were used instead of AM580 or TTNPB. OSR1-GFP knock-in hiPSCs prior to treatments were used to normalize the data. The data in (A–H) are presented as the mean±SD on culture day 6 of three independent experiments (n = 3).</p