Endothelial cells regulate cardiomyocyte development from embryonic stem cells.

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.

Does nonylphenol promote the growth of uterine fibroids?

OBJECTIVE: To study the effect and mechanism of action of nonylphenol (NP), an environmental oestrogen, on uterine leiomyoma (UL) cells. METHODS: Primary culture and subculture of human UL cells, identified as smooth muscle cells by immunocytochemical staining with a monoclonal anti-α-smooth muscle actin antibody, were performed. The viability of cells treated with various concentrations of NP for 24, 48 and 72h was determined by CCK-8 assay. mRNA expression of oestrogen receptor α (ERα), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was detected using real-time quantitative polymerase chain reaction, and protein expression was detected using Western blot analysis for all groups. RESULTS: NP promoted the growth of UL cells and expression of ERα, IGF-1 and VEGF; this was positively correlated with the concentration and duration of NP treatment. CONCLUSION: NP promotes the growth of UL cells. The mechanism of action appears to be over-expression of IGF-1 and VEGF, up-regulated by ERα, resulting in the growth of UL cells.

An evidence in vitro for the influence of bisphenol A on uterine leiomyoma.

OBJECTIVE: To study the effect and mechanism of environmental estrogens bisphenol A (BPA) on uterine leiomyoma (UL) cells. METHODS: Primary cultures and subcultures of human UL cells, which were identified by immunocytochemical staining with a monoclonal anti-α-smooth muscle actin antibody, were performed. The viability of cells in different concentration of bisphenol A of 24h, 48h and 72h were analyzed by CCK-8. The expressions of mRNA of ERα, IGF-1 and VEGF in all groups were detected by real-time quantitative PCR assay, and then the expressions of proteins detected by Western blot assay. RESULTS: BPA promoted the growth of UL cells and the expressions of ERα, IGF-1 and VEGF, which had positive correlation with the concentration and action time of BPA treatment. CONCLUSION: BPA promotes the growth of leiomyoma cells. The expressions of IGF-1, VEGF can be up-regulated by ERα, which may be possible mechanism of BPA promote the growth of leiomyoma cells.

The balance of positive and negative effects of TGF-ß signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells.

Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34(+)CD31(+)VE-cadherin(+) (VEC(+)) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34(+)CD31(+)VEC(+) cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-ß) 1 protein is increased during hPSC differentiation, and that TGF-ß signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-ß to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34(+)CD31(+)VEC(+) cells. An addition of TGF-ß inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34(+)CD31(+)VEC(+) progenitor cells. However, inhibition of TGF-ß signaling after mesoderm induction increases CD34(+)CD31(+)VEC(+) progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-ß signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

Bcl-xL enhances single-cell survival and expansion of human embryonic stem cells without affecting self-renewal.

Robust expansion and genetic manipulation of human embryonic stem cells (hESCs) and induced-pluripotent stem (iPS) cells are limited by poor cell survival after enzymatic dissociation into single cells. Although inhibition of apoptosis is implicated for the single-cell survival of hESCs, the protective role of attenuation of apoptosis in hESC survival has not been elucidated. Bcl-xL is one of several anti-apoptotic proteins, which are members of the Bcl-2 family of proteins. Using an inducible system, we ectopically expressed Bcl-xL gene in hESCs, and found a significant increase of hESC colonies in the single-cell suspension cultures. Overexpression of Bcl-xL in hESCs decreased apoptotic caspase-3(+) cells, suggesting attenuation of apoptosis in hESCs. Without altering the kinetics of pluripotent gene expression, the efficiency to generate embryoid bodies (EBs) in vitro and the formation of teratoma in vivo were significantly increased in Bcl-xL-overexpressing hESCs after single-cell dissociation. Interestingly, the number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Several genes of extracellular matrix and adhesion molecules were upregulated by Bcl-xL in hESCs without single-cell dissociation, suggesting that Bcl-xL regulates adhesion molecular expression independent of cell dissociation. In addition, the gene expressions of FAS and several TNF signaling mediators were downregulated by Bcl-xL. These data support a model in which Bcl-xL promotes cell survival and increases cloning efficiency of dissociated hESCs without altering hESC self-renewal by i) attenuation of apoptosis, and ii) upregulation of adhesion molecules to facilitate cell-cell or cell-matrix interactions.