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Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

ABSTRACT: OBJECTIVES:Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. MATERIALS AND METHODS:Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. RESULTS:Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although ?-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on ?-AR signalling than ?-AR signalling. In addition, selective activation of ?1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of ?2 - or ?-AR signalling induced no or less differentiation, respectively. EPI- and ?1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. CONCLUSIONS:These results demonstrate that activation of ARs, particularly of ?1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling.

SUBMITTER: Li XL

PROVIDER: S-EPMC6529101 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

Li Xiao-Li XL Zeng Di D Chen Yan Y Ding Lu L Li Wen-Ju WJ Wei Ting T Ou Dong-Bo DB Yan Song S Wang Bin B Zheng Qiang-Sun QS

Cell proliferation 20161027 1

<h4>Objectives</h4>Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and deve ...[more]

PMID: 27790820

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Project description:Pluripotent stem cell-derived endothelial cells (ECs) have great potential to be used in vascular therapy or tissue engineering. It is also much desired to obtain arterial or venous ECs for specific applications. Factors that are critical for the proper arterial or venous differentiation from pluripotent stem cells still need to be understood. Here, we aim at investigating this problem deeper by examining neuropilin-1 (Nrp1), an early arterial marker that may be critical for arterial cell fate commitment. Using murine embryonic stem cells as the model system, this study investigates the neuropilin-1 (Nrp1) expression during the differentiation of pluripotent stem cells toward a vascular progenitor population. We hypothesize that Nrp1, an early arterial marker present in a developing embryo, may be more responsive when further induced in vitro toward an arterial fate. We developed a two-step differentiation approach that yielded a large percentage of Nrp1+ vascular progenitor cells (VPCs) and investigated their potential to become arterial ECs. We have defined the culture parameters that contribute greatly to the emergence of Nrp1+ VPCs: certain soluble factors, especially Wnt and BMP4, early cell-cell contact, and hypoxia. Subsequent isolation of this population demonstrated a highly proliferative and network-forming behavior. The Nrp1+ VPCs exhibited increased gene expression of several Notch pathway-related arterial markers compared with Nrp1- VPCs. Most importantly, Nrp1+ VPCs demonstrated a dramatically greater response to hemodynamic stimuli by upregulating many arterial markers whereas Nrp1- VPCs have very little response. Surprisingly, these differences between Nrp1+ and Nrp1- VPCs are not evident with vascular endothelial growth factor (VEGF) treatment. Our data suggest that Nrp1+ VPCs may serve as the arterial progenitor by enhanced response to hemodynamic flow but not to VEGF, whereas Nrp1- VPCs lack the plasticity to become arterial ECs. The findings of this research indicate that Nrp1+ VPCs in the murine model act as an important step in the arterial differentiation process.

Project description:Induced pluripotent stem cells (iPSCs) provide a model of cardiomyocyte (CM) maturation. Nitric oxide signaling promotes CM differentiation and maturation, although the mechanisms remain controversial. The study tested the hypothesis that in the absence of S-nitrosoglutathione reductase (GSNOR), a denitrosylase regulating protein S-nitrosylation, the resultant increased S-nitrosylation accelerates the differentiation and maturation of iPSC-derived cardiomyocytes (CMs). iPSCs derived from mice lacking GSNOR (iPSCGSNOR-/-) matured faster than wildtype iPSCs (iPSCWT) and demonstrated transient increases in expression of murine Snail Family Transcriptional Repressor 1 gene (Snail), murine Snail Family Transcriptional Repressor 2 gene (Slug) and murine Twist Family BHLH Transcription Factor 1 gene (Twist), transcription factors that promote epithelial-to-mesenchymal transition (EMT) and that are regulated by Glycogen Synthase Kinase 3 Beta (GSK3β). Murine Glycogen Synthase Kinase 3 Beta (Gsk3β) gene exhibited much greater S-nitrosylation, but lower expression in iPSCGSNOR-/-. S-nitrosoglutathione (GSNO)-treated iPSCWT and human (h)iPSCs also demonstrated reduced expression of GSK3β. Nkx2.5 expression, a CM marker, was increased in iPSCGSNOR-/- upon directed differentiation toward CMs on Day 4, whereas murine Brachyury (t), Isl1, and GATA Binding Protein (Gata4) mRNA were decreased, compared to iPSCWT, suggesting that GSNOR deficiency promotes CM differentiation beginning immediately following cell adherence to the culture dish-transitioning from mesoderm to cardiac progenitor. Together these findings suggest that increased S-nitrosylation of Gsk3β promotes CM differentiation and maturation from iPSCs. Manipulating the post-translational modification of GSK3β may provide an important translational target and offers new insight into understanding of CM differentiation from pluripotent stem cells. Deficiency of GSNOR or addition of GSNO accelerates early differentiation and maturation of iPSC-cardiomyocytes.

Dataset Information

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

Publications

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

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