Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro

Abstract

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

Retinoic acid signaling in heart development: Application in the differentiation of cardiovascular lineages from human pluripotent stem cells

“…In contrast to in vivo findings, in vitro studies show that treatment with RA steers proepicardial differentiation of hPSCs (Iyer et al, 2015;Guadix et al, 2017;Zhao et al, 2017;Tan et al, 2021). Manipulation of BMP, WNT, RA signaling pathways, or a combination of these allowed the generation of epicardial-like cells from hPSCs (Figure 5) (Witty et al, 2014;Iyer et al, 2015;Bao et al, 2016;Guadix et al, 2017;Zhao et al, 2017).…”

“…This is in accordance with in vivo findings that revealed the contribution of NKX2–5 + /ISL1 + cardiac progenitors to the proepicardium ( Zhou et al., 2008 ). In other protocols, an earlier population at the cardiac mesoderm stage marked by PDGFRα was steered toward the epicardial lineage ( Witty et al., 2014 ; Guadix et al., 2017 ; Tan et al., 2021 ), implying that there are multiple differentiation routes toward epicardial cells. Whether these correspond to the different progenitor populations as shown in vivo is yet to be evaluated ( Mommersteeg et al., 2010 ; Tyser et al., 2021 ; Zhang et al., 2021 ).…”

“…Co-culture of hPSCderived epicardial cells with cardiomyocytes in 2D cultures as well as 3D constructs enhanced contractility and calcium handling properties of the cardiomyocytes. It was shown that the proliferative effect of proepicardial cells on co-cultured ventricular cardiomyocytes was mediated at least in part by RA-dependent IGF2 signaling (Tan et al, 2021). IGF2 has also been utilized for the maturation of compact ventricular cells derived from hPSCs (Funakoshi et al, 2021).…”

“…Manipulation of BMP, WNT, RA signaling pathways, or a combination of these allowed the generation of epicardial-like cells from hPSCs ( Figure 5 ) ( Witty et al., 2014 ; Iyer et al., 2015 ; Bao et al., 2016 ; Guadix et al., 2017 ; Zhao et al., 2017 ). In a more recent protocol, BMP4 + RA + vascular endothelial growth factor (VEGF) was used to generate proepicardial cells; however, the added advantage of including VEGF as opposed to using BMP4 + RA alone was not investigated ( Tan et al., 2021 ). Like RA, WNT signaling is also only implicated in later stages of epicardial development, epicardial EMT, and formation of coronary vessels in vivo ( Zamora et al., 2007 ; von Gise et al., 2011 ).…”

“…Besides its role in the pSHF, Tbx5 is also implicated in proepicardial development and epicardial migration ( Liu and Stainier, 2010 ; Diman et al., 2014 ). Proepicardial cells generated from hPSCs in vitro using BMP4 + RA also express Tbx5 ( Tan et al., 2021 ). Moreover, microarray analysis of Tbx5-induced genes identified Wt1 as a possible target ( Plageman and Yutzey, 2006 ).…”