YAP inhibition promotes endothelial cell differentiation from pluripotent stem cell through EC master transcription factor FLI1
Graphical abstract
Introduction
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripoten stem cells (iPSCs), have unlimited proliferative potency and could differentiate into almost all somatic cells, including endothelial cells (ECs). Therefore, PSCs have great potential for disease modeling, tissue engineering, clinical regeneration medicine and drug screening, as well as molecular mechanism study for development in vitro [1,2]. Indeed, PSCs has become a great resource for EC generation by various differentiation approaches, which providing opportunities for EC application in vascular regeneration, as well as the molecular mechanism study of EC differentiation regulation [3,4]. Although the potential molecular mechanism of EC differentiation has been advanced recently, to identify the additional mechanisms is necessary to fully understand and control EC differentiation for its applications [[5], [6], [7]].
The Hippo pathway was first discovered in Drosophila melanogaster in 1995, involving many biological processes, including organ size control, development, cancer and apoptosis [[8], [9], [10]]. Accumulating evidence indicates that the Hippo pathway and its downstream transcriptional co-effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) could also determine the cell fate during tissue development and regeneration [11]. Hippo-YAP pathway not only regulated stem cell self-renewal but also influenced different type of cell differentiation [12]. For example, YAP has been reported to inhibit cardiac mesoderm lineage differentiation from hESCs by repressing WNT3 gene expression [13]. While inhibition of YAP enhances the differentiation of functional stem cell-derived insulin-producing β cells [14].
Hippo-YAP pathway also plays a critical role on cardiovascular development, diseases and regeneration [[15], [16], [17]]. For example, deletion of Salvador (Salv), a Hippo pathway component, in mouse heart led to heart enlargement or cardiomegaly during development, and deficiency of Salv could also reverses systolic heart failure after infarction in adult [18,19]. Hippo pathway is also involved in modulating endothelial cell proliferation, migration and therefore angiogenesis, as well as maintaining vascular barrier stability, and regulating vascular remodeling [20]. For example, Endothelial-specific deletion of YAP and TAZ leads to impaired vascularization and embryonic lethality [21]. Our previous study also suggested YAP promotes endothelial barrier repair after TNF-α-induced permeability increase [22]. However, whether Hippo-YAP pathway regulates EC differentiation from PSCs is still unknown. Because YAP lacks of a DNA binding domain, YAP usually need to bind to many transcription factors including TEAD family to regulate different functions. The TEAD family of proteins consist of four members, TEAD1-4 [23], which is known to be implicated in processes such as development, cell proliferation and stem cell functions [24]. Recent studies indicated that TEAD1 is essential for cardiovascular development and promotes cardiac myocytes and vascular smooth muscle differentiation [25,26]. Although many studies suggested TEAD could mediate the functional effects of YAP, TEAD could also regulate by other factors and may play independent roles in various cellular context and developmental stages. How TEAD regulates EC differentiation from PSCs also need to be elucidated.
In this study, we revealed that Hippo pathway is modified during mesoderm-mediated EC differentiation from PSCs. YAP expression is decreased during this process and inhibition of YAP promoted both mesoderm and EC differentiation, while overexpression of YAP prevented EC differentiation. Microarray data suggested that some of EC master transcriptional factors including FLI1 has elevated after YAP knockdown. Our results also indicated that YAP inhibition promoted FLI1 expression by cooperating with TEAD1 and overexpression of FLI1 could reverse the inhibitory effect of YAP on EC differentiation.
Section snippets
Materials
Culture medium and fetal bovine serum (FBS) were obtained from GIBICO (Thermo Scientific, Rockford, IL). All other chemicals and reagents were obtained from Sigma-Aldrich (St Louis, MO) unless otherwise indicated.
Human pluripotent stem cell culture and mesoderm-mediated EC differentiation
The H1 hESC lines were obtained from WiCell Research Institute (Madison, WI) and iPSCs were generated from BJ cells by episomal vector-mediated reprogramming in our lab. To maintain H1ESCs in a long-term undifferentiated status, H1ESCs (passages 60–80) were grown on Matrigel (Corning,
Mesoderm-mediated EC differentiation from H1ESCs
To effectively differentiate human pluripotent stem cells into endothelial cells, we applied two-step, small-molecule chemicals defined differentiation method as previous reported from Cowan's lab [27]. Using GSK3β inhibitor CP21R7 (1 μM), combined with BMP4 (25 ng/ml) to activate both WNT and BMP pathways to induce H1ESCs into mesoderm for 3 days, and then applying VEGF-A (200 ng/ml) and forskolin (2 μM) to further induce mesoderm cells into endothelial cells (ECs) for another 3–4 days (Fig. 1
Discussion
In this paper we found that Hippo pathway is activated during EC differentiation from H1ESCs. We demonstrated that YAP, the hippo pathway effector, is significantly downregulated in EC population that derived from H1ESCs. YAP knockdown promotes while YAP overexpression represses EC and mesoderm differentiation. The mechanism may be that YAP repressed the expression of some EC master transcription factors, such as FLI1 and therefore inhibited EC differentiation. TEAD1 is found to be contributed
Authors' contributions
YW, ML contributed to the conception, design and financial support of the study. YQ, XS, MH, HZ and JY performed the experiments, data analysis and interpretation. PJ, JM, JF, XF, and YG assisted with the experiments. YW and YQ analyzed the data and wrote the paper. YW and ML edit the paper. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgements
We would like to thank all the members in Institute of Hypoxia Medicine who contribute to this work. We appreciate the great help from Dr. Jiaxi Zhou (State Key Laboratory of Experimental Hematology in China), Dr. Bin Zhao (Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University) and Dr. Lei Zhang (Institute of Biochemistry and Cell Biology at Chinese Academy of Sciences) for this project. This work was supported by National Natural Science Foundation of
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These authors contributed equally to this work.