Abstract 19463: Temporal Regulation of Retinoic Acid Enriches Left Ventricular Cardiomyocytes From Human Embryonic Stem Cells
Retinoic acid (RA) plays a pivotal role during the heart development; RA needs to be inactive during the early cardiac mesoderm formation, but late-stage surplus of RA is critical for the cardiac looping and formation of the ventricular myocardium. We hypothesized that mimicking the in vivo RA signaling during human embryonic stem cell (hESC) differentiation could lead to enriched left ventricular myocytes.
H9 human ESCs were differentiated to cardiac lineage by treatment with Activin A, BMP4 and bFGF (ABF, control) for the first three days (d1-4). This lead to >70% brachyury+ mesoderm formation, and >80% of the mesodermal cells became cTnT+ cardiac myocytes. RA was inhibited for 24 hrs on d1 with a pan-retinoic acid receptor antagonist, and later activated with exogenous RA (1μM) for three days from d6-9 (Ri-ABF-RA). At d14, the Ri-ABF-RA cardiac myocytes showed >4-fold higher expression of ventricular markers, IRX4 and HRT2, compared to control (n=3, p<0.05). The first heart field (FHF) populates majority of the left ventricle during development. Ri-ABF-RA cardiomyocytes expressed higher transcript and protein levels of the FHF markers, NKX2.5, TBX5and HCN4, but lower levels of the second heart field markers, ISL1, MEF2c, FGF8/10, compared to control. Use of an NKX2.5-GFP transgenic hESC line validated the gene expression data. Functionally, Ri-ABF-RA cardiomyocytes showed relatively uniform action potential durations compared to ABF myocytes, with a standard deviation of 77 vs. 268 ms, respectively, n>29) at ≥d30. This is in line with enrichment of the left ventricular myocytes in Ri+ABF+RA compared to mixed populations of nodal, atrial and ventricular myocytes in ABF. Maximum upstroke velocity was faster in Ri-ABF-RA cardiomyocytes compared to ABF (540±460 vs. 36±26 μV/sec, n>31), pointing to higher Na channel density in Ri-ABF-RA compared to control.
Taken together, the data demonstrate that early RA inhibition followed by late RA activation of hESCs leads to enrichment of the left ventricular cardiomyocytes. The de novo cardiomyocytes better represent the left ventricle, and may serve as a specific platform for cardiac regeneration and drug screen for the left ventricular myocardium.
Author Disclosures: P. Han: None. J. Li: None. H. Cho: None.
- © 2016 by American Heart Association, Inc.