Improved Atrial Differentiation of Human Pluripotent Stem Cells by Activation of Retinoic Acid Receptor Alpha (RARα)

Abstract

Human pluripotent stem cell (hPSC)-derived cardiomyocytes have proven valuable for modeling disease and as a drug screening platform. Here, we depict an optimized protocol for the directed differentiation of hPSCs toward cardiomyocytes with an atrial identity by modulating the retinoic acid signaling cascade in spin embryoid bodies. The crucial steps of the protocol, including hPSC maintenance, embryoid body (EB) differentiation, the induction of cardiac mesoderm, direction toward the atrial phenotype, as well as molecular and functional characterization of the cardiomyocytes, are described. Atrial cardiomyocytes (AMs) can be generated within 14 days. Most importantly, we show that induction of the specific retinoic acid receptor alpha (RARα) increased the efficiency of atrial differentiation to 72% compared with 45% after modulating the retinoic acid (RA) pathway with all-trans RA (atRA). In contrast, the induction of RARβ signaling only had a minor impact on the efficiency of atrial differentiation (from about 45% to 50%). Similarly, the total yield of AM per EB of 5000 hPSCs was increased from 10,350 (2.07 per hPSC) to 16,120 (3.22 per hPSC) while selectively modulating RARα signaling. For further purification of the AMs, we describe a metabolic selection procedure that enhanced the AM percentage to more than 90% without compromising the AM yield (15,542 per EB, equal to 3.11 per hPSC) or functionality of the AMs as evaluated by RNAseq, immunostaining, and optical action potential measurement. Cardiomyocytes with distinct atrial and ventricular properties can be applied for selective pharmacology, such as the development of novel atrial-specific anti-arrhythmic agents, and disease modeling, including atrial fibrillation, which is the most common heart rhythm disorder. Moreover, fully characterized and defined cardiac subtype populations are of the utmost importance for potential cell-based therapeutic approaches.

Single-cell roadmap of cardiac differentiation identifies roles for ZNF711 and retinoic acid in balanced epicardial and cardiomyocyte lineage commitment

“…S1A). We and others showed previously that RA redirects differentiation towards aCMs (Devalla et al, 2015; Schwach et al, 2017, 2022; Zhang et al, 2010), which occurs at high efficiency as evaluated by fluorescence activated cell sorting (Supplementary Fig. S1B).…”

“…Differentiation of hPSCs to ventricular or atrial EBs was performed as previously described (Schwach et al, 2022). Briefly, hPSC were seeded and aggregated in EB formation medium (Essential 8 containing 400 µg/mL Poly (vinil alcohol) (Sigma Aldrich, P8136) and 10 µM Y-27632 (Bio-Connect, S1049)) one day before starting the differentiation.…”

“…Human pluripotent stem cells (hPSC) offer an immense potential to differentiate many cell types in vitro , thus presenting a relevant alternative in mimicking the earlier steps of human tissue development (Takahashi et al, 2007; Thomson et al, 1998; Yu et al, 2007). Various culture models have been established to obtain cardiomyocytes (CMs) (Burridge et al, 2012; Devalla & Passier, 2018; Mummery et al, 2012), with variations including an early step of retinoic acid (RA) treatment to enrich for atrial CMs (aCMs) (Devalla et al, 2015; Lee et al, 2017; Schwach et al, 2022; Zhang et al, 2010) or epicardial derivatives (Guadix et al, 2017; Hofbauer et al, 2021a; Meier et al, 2023). Over time, the cardiac cultures have increased in complexity (e.g.…”

State-of-the-Art Differentiation Protocols for Patient-Derived Cardiac Pacemaker Cells

“…RArγ transcripts are specifically detected in the endocardial cushion tissue and large developing vessels [ 70 ]. Moreover, RArα activation seems to improve the atrial differentiation of hiPSCs [ 74 ]. In this way, FGF signaling, which is known to sustain ventricular development, was inhibited.…”

Towards Improved Human In Vitro Models for Cardiac Arrhythmia: Disease Mechanisms, Treatment, and Models of Atrial Fibrillation

“…Future advancements in experimental methodologies, clinical imaging modalities, computational performance, standardization, and the need for clinical evidence to support the routine use of computational models in AFib management will help move this technology forward [ 105 ]. For that, the generation of hPSC-derived atrial cardiomyocytes [ 106 , 107 , 108 ] and the development of atrial tissues [ 109 , 110 , 111 ] have indicated the potential to model AFib in vitro and open new possibilities to generate robust datasets that facilitate in silico modeling.…”