Error bars represent SEM; n=5 cells; **p <0. 01 versus HCASMCs, analyzed by the Student'st-test. (E)Kinetic parameters of calcium transients after phenylephrine addition. both human epicardial-like cells and SMCs. Keywords:: human pluripotent stem cell, epicardial cell, WNT, RA, smooth muscle cell == Introduction == Epicardial cells arerequired for embryonic heart formation. The proepicardium, a transient embryonic structure composed of epicardial progenitor cells, develops at the inflow of the heart tube and localizes posterior to the sinus venosus (SV) and atria [1]. Although the precise origin of the proepicardium is unclear, several genetic lineage-tracing analyses have suggested that the proepicardium and myocardium develop from a common cardiogenic precursor pool [2]. At the looping stage of heart development, proepicardial cells migrate onto the heart tube and cover the heart surface, thereby forming the epicardium [3]. Rabbit polyclonal to SRP06013 A subset of epicardial cells undergo epithelial-to-mesenchymal transition (EMT) and invade the subepicardium and myocardial layer as epicardium-derived cells (EPDCs) to generate cardiac fibroblasts (CFs), smooth muscle cells (SMCs), and coronary endothelial cells that contribute to coronary vessel formation [47]. In addition , embryonic epicardial cells promote cardiomyocyte proliferation and maturation by producing paracrine factors, including retinoic acid (RA), fibroblast growth factor (FGF)-9, and insulin-like growth factor-2 [810]. In the adult mammalian heart, the proliferative ability of cardiomyocytes is not retained and the vast majority of epicardial cells have lost expression of the embryonic epicardial marker genes TBX18 and WT1. Myocardial infarction (MI) and thymosin 4 injection can reactivate expression of these two genes in adult epicardium and subepicardium, leading to neovascularization in infarcted ventricular tissue [1114]. Furthermore, transplantation of adult human EPDCs into a mouse infarction model has been shown to improve heart function and vascularization [15, 16]. Thus, epicardial cells and EPDCs, particularly TBX18- and WT1-expressing epicardial cells, are potential cell sources for repair of injured heart tissue using cell-based transplantation. Recently, two groups reported WT1+epicardial cell differentiation methods that used the cytokine basic FGF (bFGF), Z-Ile-Leu-aldehyde bone morphogenetic protein (BMP) 4, activin A, vascular endothelial growth factor, and WNT3A in an pet product-containing medium [17, 18]. Our differentiation method is based on an Z-Ile-Leu-aldehyde understanding of signals involved in epicardial development. Early studies in chicken embryo showed expression of retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme for RA synthesis, in the posterior part of the lateral mesoderm, which includes proepicardial precursors, the atria, and SV progenitors [19, 20]. At this developmental stage, WNT-8cand the Z-Ile-Leu-aldehyde competitive WNT inhibitor, Crescent, are distributed at opposite ends of the embryo. The posterior part of the heart field, which develops into the atria, is located within theCrescent-expressing region, while proepicardial precursors are located within the anterior edge of theWNT-expressing zone [21]. Therefore , we hypothesized that RA is required for both atrium and proepicardium development, with WNT signals inducing the fate separation of atrial myocytes and epicardial cells. In this study, we showed that WNT activated WT1 expression in human pluripotent stem cell (hPSC)-derived cardiac progenitor cells (CPCs), and RA promoted TBX18 expression in the WT1+cell population. Manipulating the WNT and RA signaling pathways with small molecular compounds in a chemically defined albumin-free medium led to 80% of hPSCs differentiating into TBX18+/WT1+epicardial-like cells. These cells possessed epicardial cell characteristics, exhibited a cobblestone-like morphology following passaging, and differentiated into functional SMCs or CF-like cells Z-Ile-Leu-aldehyde when induced with transforming growth factor 1 (TGF1) and bFGF. This method could Z-Ile-Leu-aldehyde potentially be used to efficiently and stably generate minimally pathogenic epicardial cells and coronary SMCs for clinical cardiovascular tissue engineering and heart regeneration in the future. == Materials and Methods == == Animals and tissue sections == Institute for Cancer Research mice were obtained from the Center for Animal Research, Institute of Biophysics, Chinese Academy of Sciences. All experiments with mice were performed according to protocols approved by the Institute of Biophysics. Pregnant females were sacrificed and embryonic hearts (E10. 5, E12. 5,.