Abstract
Mesp1 directs multipotential cardiovascular cell fates, even though it’s transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1’s transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1Cre/+; Rosa26EYFP/+ ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.
Highlights
To track the Mesp1-marked progenitor cell lineage, we previously crossed the murine Mesp1Cre/+ line with the Rosa26EYFP/EYFP line to generate a Mesp1Cre/+; Rosa26EYFP/+ ESC reporter line as we reported in Solbam et al.[19]
To characterize further specification of these Mesp1-EYFP+cells, we investigated their contribution to multiple lineages in ES cell differentiation and heart disease (Fig. 1A)
Though Mesp[1] transcripts were present in the EYFP- fraction, this likely reflects the delay between activation of the Mesp[1] locus (Cre) and subsequent Cre-mediated activation of the Rosa locus (EYFP) in EYFP- cells, which would later turn EYFP+.At day 8, Nkx2.5, αMHC, and Ryr[2] transcripts were almost exclusively present in EYFP+cells, supporting that cardiomyocytes arise from Mesp1+progenitors (Fig. 1C)
Summary
Generation of embryonic stem cell lines and cell culture. The Mesp1-Cre mouse strain was developed by Yumiko Saga[13]. The Rosa26-EYFP mouse strain was developed by Frank Costantini[18] We had crossed these two strains to create the Mesp1Cre/+; Rosa26EYFP/+ ESC reporter line, as we reported in Soibam et al.[19]. The cells were grown as 20 ul hanging droplets (2 × 104 cells/ml) for 2 days in serum-free differentiation media (SFDM, a 3:1 mixture of IMDM/F12 containing 0.05% BSA, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, 2 mM L-glutamate, 1x chemically defined lipids, 450 uM 1-thioglycerol, 0.5x B27, 1X N2, 0.4mg/ml PVA, 10 ug/ml Insulin, 1 uM Y-27632 and 20 ng/ml BMP4). Beating clusters were observed 6 days after the initiation of hanging droplets
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