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Inhibition of ?-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

ABSTRACT: During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/?-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/?-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations. Cardiogenic mesoderm was generated under conditions specifying anterior-like mesoderm, whereas blood-forming endothelium was generated from posterior-like mesoderm, and vessel-forming CD31(+) endothelial cells were generated from all mesoderm origins. Surprisingly, inhibition of ?-catenin signaling led to the highly efficient respecification of anterior-like endothelium into beating cardiomyocytes. Cardiac respecification was not observed in posterior-derived endothelial cells. Thus, activin/BMP gradients specify distinct mesodermal subpopulations that generate cell derivatives with unique angiogenic, hemogenic and cardiogenic properties that should be useful for understanding embryogenesis and developing therapeutics.

SUBMITTER: Palpant NJ

PROVIDER: S-EPMC4582173 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Inhibition of β-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

Palpant Nathan J NJ Pabon Lil L Roberts Meredith M Hadland Brandon B Jones Daniel D Jones Christina C Moon Randall T RT Ruzzo Walter L WL Bernstein Irwin I Zheng Ying Y Murry Charles E CE

Development (Cambridge, England) 20150707 18

During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/β-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior ax ...[more]

PMID: 26153229

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Project description:BackgroundThe Wnt/β-catenin signaling pathway plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of the right ventricle, outflow tract (OFT), and a portion of the inflow tract (IFT). During early cardiogenesis, these AHF CPCs reside within the pharyngeal mesoderm (PM) that provides a microenvironment for them to receive signals that direct their cell fates. Here, N-cadherin, which is weakly expressed by CPCs, plays a significant role by promoting the adhesion of CPCs within the AHF, regulating β-catenin levels in the cytoplasm to maintain high Wnt signaling and cardioproliferation while also preventing the premature differentiation of CPCs. On the contrary, strong expression of N-cadherin observed throughout matured myocardium is associated with downregulation of Wnt signaling due to β-catenin sequestration at the cell membrane, inhibiting cardioproliferation. As such, upregulation of Wnt signaling pathway to enhance cardiac tissue proliferation in mature cardiomyocytes can be explored as an interesting avenue for regenerative treatment to patients who have suffered from myocardial infarction.MethodsTo investigate if Wnt signaling is able to enhance cellular proliferation of matured cardiomyocytes, we treated cardiomyocytes isolated from adult mouse heart and both murine and human ES cell-derived matured cardiomyocytes with N-cadherin antibody or CHIR99021 GSK inhibitor in an attempt to increase levels of cytoplasmic β-catenin. Immunostaining, western blot, and quantitative PCR for cell proliferation markers, cell cycling markers, and Wnt signaling pathway markers were used to quantitate re-activation of cardioproliferation and Wnt signaling.ResultsN-cadherin antibody treatment releases sequestered β-catenin at N-cadherin-based adherens junction, resulting in an increased pool of cytoplasmic β-catenin, similar in effect to CHIR99021 GSK inhibitor treatment. Both treatments therefore upregulate Wnt signaling successfully and result in significant increases in matured cardiomyocyte proliferation.ConclusionAlthough both N-cadherin antibody and CHIR99021 treatment resulted in increased Wnt signaling and cardioproliferation, CHIR99021 was found to be the more effective treatment method for human ES cell-derived cardiomyocytes. Therefore, we propose that CHIR99021 could be a potential therapeutic option for myocardial infarction patients in need of regeneration of cardiac tissue.

Project description:Endothelial wingless-related integration site (Wnt)-/β-catenin signaling is a key regulator of the tightly sealed blood-brain barrier. In the hepatic vascular niche angiokine-mediated Wnt signaling was recently identified as an important regulator of hepatocyte function, including the determination of final adult liver size, liver regeneration, and metabolic liver zonation. Within the hepatic vasculature, the liver sinusoidal endothelial cells (LSECs) are morphologically unique and functionally specialized microvascular endothelial cells (ECs). Pathological changes of LSECs are involved in chronic liver diseases, hepatocarcinogenesis, and liver metastasis. To comprehensively analyze the effects of endothelial Wnt-/β-catenin signaling in the liver, we used endothelial subtype-specific Clec4g-iCre mice to generate hepatic ECs with overexpression of Ctnnb1. In the resultant Clec4g-iCre tg/wt ;Ctnnb1(Ex3) fl/wt (Ctnnb1 OE-EC ) mice, activation of endothelial Wnt-/β-catenin signaling resulted in sinusoidal transdifferentiation with disturbed endothelial zonation, that is, loss of midzonal LSEC marker lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve1) and enrichment of continuous EC genes, such as cluster of differentiation (CD)34 and Apln. Notably, gene set enrichment analysis revealed overrepresentation of brain endothelial transcripts. Activation of endothelial Wnt-/β-catenin signaling did not induce liver fibrosis or alter metabolic liver zonation, but Ctnnb1 OE-EC mice exhibited significantly increased plasma triglyceride concentrations, while liver lipid content was slightly reduced. Ctnnb1 overexpression in arterial ECs of the heart has been reported previously to cause cardiomyopathy. As Clec4g-iCre is active in a subset of cardiac ECs, it was not unexpected that Ctnnb1 OE-EC mice showed reduced overall survival and cardiac dysfunction. Altogether, balanced endothelial Wnt-/β-catenin signaling in the liver is required for normal LSEC differentiation and for maintenance of normal plasma triglyceride levels.

Project description:Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co-culture with neonatal rat cardiomyocytes. 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5- to 1.9-fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation; however, ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33-fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI and cardiac myosin heavy chain in CM-DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.

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Inhibition of ?-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

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Inhibition of β-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

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