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Objective: This study aim to enhance endothelial differentiation of human embryonic stem cells (hESCs) by transduction of an adenovirus (Ad) vector expressing hVEGF165 gene (Ad-hVEGF165 ). Purified hESC-derived CD133+ endothelial progenitors were transplanted into a rat myocardial infarct model to assess their ability to contribute to heart regeneration. Methods: Optimal transduction efficiency with high cell viability was achieved by exposing differentiating hESCs to viral particles at a ratio of 1:500 for 4 h on three consecutive days. Results: Reverse transcription-PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF165 -transduced cells. Additionally, flow cytometric analysis of CD133, a cell surface marker, revealed an approximately fivefold increase of CD133 marker expression in Ad-hVEGF165 -transduced cells compared with the nontransduced control. Within a rat myocardial infarct model, transplanted CD133+ endothelial progenitor cells survived and participated, both actively and passively, in the regeneration of the infarcted myocardium, as seen by an approximately threefold increase in mature blood vessel density (13.62 ± 1.56 vs 5.11 ± 1.23; p < 0.01), as well as significantly reduced infarct size (28% ± 8.2% vs 76% ± 5.6%; p < 0.01) in the transplanted group compared with the culture medium-injected control. There was significant improvement in heart function 6 weeks post-transplantation, as confirmed by regional blood-flow analysis (1.72 ± 0.612 ml/min/g vs 0.8 ± 0.256 ml/min/g; p < 0.05), as well as echocardiography assessment of left ventricular ejection fraction (60.855% ± 7.7% vs 38.22 ± 8.6%; p < 0.05) and fractional shortening (38.63% ± 9.3% vs 25.2% ± 7.11%; p < 0.05). Conclusion: hESC-derived CD133+ endothelial progenitor cells can be utilized to regenerate the infarcted heart.

Endothelial progenitors derived from human embryonic stem cells (hESCs) hold much promise in clinical therapy. Conventionally, lineage-specific differentiation of hESCs is achieved through supplementation of various cytokines and chemical factors within the culture milieu. Nevertheless, this is a highly inefficient approach that is often limited by poor replicability. An alternative is through genetic modulation with recombinant DNA. Hence, this study investigated whether transduction of hESCs with an adenoviral vector expressing the human VEGF165 gene (Ad-hVEGF165) can enhance endothelial-lineage differentiation. The hESCs were induced to form embryoid bodies (EBs) by culturing them within low-attachment plates for 7 days, and were subsequently trypsinized into single cells, prior to transduction with Ad-hVEGF165. Optimal transduction efficiency with high cell viability was achieved by 4-h exposure of the differentiating hESCs to viral particles at a ratio of 1 : 500 for three consecutive days. ELISA results showed that Ad-hVEGF165-transduced cells secreted human vascular endothelial growth factor (hVEGF) for more than 30 days post-transduction, peaking on day 8, and the conditioned medium from the transduced cells stimulated extensive proliferation of HUVEC. Real-time PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF165-transduced cells. Additionally, flow cytometric analysis of CD133 cell surface marker revealed an approximately 5-fold increase in CD133 marker expression in Ad-hVEGF165-transduced cells compared to the non-transduced control. Hence, this study demonstrated that transduction of differentiating hESCs with Ad-hVEGF165 facilitated expression of the VEGF transgene, which in turn significantly enhanced endothelial differentiation of hESCs. Copyright © 2007 John Wiley & Sons, Ltd.

Very few proteins are capable to induce pluripotent stem (iPS) cells and their biochemical uniqueness remains unexplained. For example, Sox2 cooperates with other transcription factors to generate iPS cells, but Sox17, despite binding to similar DNA sequences, cannot. Here, we show that Sox2 and Sox17 exhibit inverse heterodimerization preferences with Oct4 on the canonical versus a newly identified compressed sox/oct motif. We can swap the cooperativity profiles of Sox2 and Sox17 by exchanging single amino acids at the Oct4 interaction interface resulting in Sox2KE and Sox17EK proteins. The reengineered Sox17EK now promotes reprogramming of somatic cells to iPS, whereas Sox2KE has lost this potential. Consistently, when Sox2KE is overexpressed in embryonic stem cells it forces endoderm differentiation similar to wild-type Sox17. Together, we demonstrate that strategic point mutations that facilitate Sox/Oct4 dimer formation on variant DNA motifs lead to a dramatic swap of the bioactivities of Sox2 and Sox17.