Discovery of retinoic acid receptor agonists as proliferators of cardiac progenitor cells through a phenotypic screening approach

Abstract

Identification of small molecules with the potential to selectively proliferate cardiac progenitor cells (CPCs) will aid our understanding of the signaling pathways and mechanisms involved and could ultimately provide tools for regenerative therapies for the treatment of post-MI cardiac dysfunction. We have used an in vitro human induced pluripotent stem cell-derived CPC model to screen a 10,000-compound library containing molecules representing different target classes and compounds reported to modulate the phenotype of stem or primary cells. The primary readout of this phenotypic screen was proliferation as measured by nuclear count. We identified retinoic acid receptor (RAR) agonists as potent proliferators of CPCs. The CPCs retained their progenitor phenotype following proliferation and the identified RAR agonists did not proliferate human cardiac fibroblasts, the major cell type in the heart. In addition, the RAR agonists were able to proliferate an independent source of CPCs, HuES6. The RAR agonists had a time-of-differentiation-dependent effect on the HuES6-derived CPCs. At 4 days of differentiation, treatment with retinoic acid induced differentiation of the CPCs to atrial cells. However, after 5 days of differentiation treatment with RAR agonists led to an inhibition of terminal differentiation to cardiomyocytes and enhanced the proliferation of the cells. RAR agonists, at least transiently, enhance the proliferation of human CPCs, at the expense of terminal cardiac differentiation. How this mechanism translates in vivo to activate endogenous CPCs and whether enhancing proliferation of these rare progenitor cells is sufficient to enhance cardiac repair remains to be investigated.

Keywords: cell proliferation; gene expression; high throughput screening assays; induced pluripotent stem cells; nuclear receptors; small molecule libraries.

Figure 1

Phenotypic screen identifies AM580 and all‐trans retinoic acid (ATRA) as proliferators of iPS‐derived cardiac progenitor cells. A, Screening cascade for identification and validation of hits. B, ATRA and AM580 were tested at three different concentrations 0.1, 1.0, and 10 μM in the primary screen. The percentage effect is expressed relative to the control, basic FGF at 100 ng/mL. C, Percentage effect relative to max and min controls versus compounds ranked according to the highest effect at any concentration for each compound. D, AM580 concentration response effects on the proliferation of cardiac progenitor cells (CPCs) and cardiac fibroblasts (CFs) measured as nuclei count, normalized to % effect from the maximal and neutral controls for both CPCs and CFs. Data represent the mean of at least three independent experiments where each experiment consisted of technical duplicates or triplicates, with error bars = SEM

Figure 2

Cardiac progenitor cells (CPCs) retain progenitor markers after retinoic acid receptor agonist treatment. Representative images from wells where CPCs were treated for 3 days with 0.1% DMSO, 40 nM AM580, or 40 nM AM80 and then stained for cardiac progenitor markers Nkx2.5, Islet1, Gata4, and PDGFRα. Larger images are representative merged images of Hoechst nuclear stain in blue and immunofluorescence of the indicated progenitor marker in green. Insets show the progenitor marker staining in gray scale at 3.3‐fold higher magnification. Scale bars: 50 μm

Figure 3

A, Effect of a competitive retinoic acid receptor (RAR) antagonist AGN194301 in three different concentrations on RAR agonist AM580‐induced cardiac progenitor cell (CPC) proliferation, 10‐point concentration response curve. Cells were fixed and nuclei stained on day 4 postplating and 3 days postcompound addition. Proliferation was measured as nuclei count normalized to % effect from the maximal and neutral controls. pEC₅₀ for AM580 is attenuated by adding increased concentrations of AGN194301, pEC₅₀ shifts from 8.2 for AM580 to pEC₅₀ 7.0 when 0.01 μM AGN194301 is present and to 5.9 with 0.1 μM and to 5.5 with 1 μM of AGN194301. There was no effect on proliferation by AGN194301 alone. The data represent the mean of two independent experiments each conducted in quadruplicate; error bars indicate SEM. B, RARs are expressed in iPS‐derived CPCs and cardiac fibroblasts (CFs). The data for CPCs and CFs are obtained from two independent RNA preparations from each cell type

Figure 4

RNA seq analysis of AM580‐treated cardiac progenitor cells (CPCs). Expression levels are shown as logarithm base 10 of transcripts per kilobase million for DMSO and AM580‐treated CPC samples, for selected marker genes at 4 hours and 24 hours post‐treatment. Genes with expression levels significantly different (P < .05) between treatment and control are marked in red font. Significance was determined using an analysis of variance model for treatment, time, and gene followed by Tukey's correction for multiple testing

Figure 5

Retinoic acid (RA) counteracts terminal cardiac differentiation in transient progenitor cells. A, Schematic of cardiac induction protocol. Upon replating undifferentiated human pluripotent stem cells at high density, the indicated signaling pathways were simultaneously activated for 1 day, followed by WNT inhibition on days 2‐3. Spontaneously beating monolayers of immature cardiomyocytes are typically obtained after 6‐7 days. B, Characterization of cardiac induction time‐course using markers of intermediate stages (mesoderm, cardiac progenitors) and early cardiomyocytes. Data are from two independent experiments analyzed by RT‐qPCR and microarrays (merged data normalized to peak expression ± SEM). C, Cardiac progenitors replated on day 5 of differentiation and exposed for another 4 days to baseline conditions or RA (1 μM). The majority of control cells differentiates further into NKX2.5‐positive cardiomoyctes and display spontaneous beating. RA‐treated cells show an increase in cell numbers, compromised NKX2.5 induction, and sustained ISL1 expression in most cells

Figure 6

Effects of various retinoic acid receptor (RAR) agonists on proliferation and expression in cardiac progenitors derived from differentiated human pluripotent stem cells. A, Effects on proliferation and gene expression on replated day‐5 progenitors following 4 days of exposure to the indicated compounds. All‐trans retinoic acid (ATRA), EC23, TTNPB, and AM80 (all at 1.0 μM) had strong positive effects on cell proliferation (left panel, n = 3, P < .01, pairwise against untreated cells). Note the overall positive association between cell proliferation and cardiac precursor gene expression, and the negative relationship between cell numbers and cardiomyocyte marker induction (plots on the right). B, Immunocytochemistry analysis of ISL1 and cardiac troponin T (CTNT) in the same samples. Note again the increase in cell numbers, sustained ISL1 expression at the protein level, and inhibited CTNT induction with ATRA, EC23, TTNPB, and AM80