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Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro.

ABSTRACT: Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

SUBMITTER: Tan JJ

PROVIDER: S-EPMC8370973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4? and ?-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

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Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro.

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