As bona fide p53 transcriptional targets, miR-34 microRNAs (miRNAs) exhibit frequent alterations in many human tumor types and elicit multiple p53 downstream effects upon overexpression. Unexpectedly, miR-34 deletion alone fails to impair multiple p53-mediated tumor suppressor effects in mice, possibly due to the considerable redundancy in the p53 pathway. Here, we demonstrate that miR-34a represses HDM4, a potent negative regulator of p53, creating a positive feedback loop acting on p53. In a Kras-induced mouse lung cancer model, miR-34a deficiency alone does not exhibit a strong oncogenic effect. However, miR-34a deficiency strongly promotes tumorigenesis when p53 is haploinsufficient, suggesting that the defective p53-miR-34 feedback loop can enhance oncogenesis in a specific context. The importance of the p53/miR-34/HDM4 feedback loop is further confirmed by an inverse correlation between miR-34 and full-length HDM4 in human lung adenocarcinomas. In addition, human lung adenocarcinomas generate an elevated level of a short HDM4 isoform through alternative polyadenylation. This short HDM4 isoform lacks miR-34-binding sites in the 39 untranslated region (UTR), thereby evading miR-34 regulation to disable the p53-miR-34 positive feedback. Taken together, our results elucidated the intricate cross-talk between p53 and miR-34 miRNAs and revealed an important tumor suppressor effect generated by this positive feedback loop.

SummaryMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

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The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

Abstract

Supplemental Digital Content is available in the text.

SummaryHuman pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration.

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