VE-Cadherin as a Specific Promoter for Genetic Labelling and Selection of Pure Embryonic Stem Cell-Derived Endothelial Cells for Therapeutic Purposes

Rationale: Endothelial cells (ECs) play a central role in many pathological conditions related to cardiovascular diseases, including coronary artery disease, smoking, and pulmonary hypertension. One of the therapeutic modalities for diseases with underlying endothelial dysfunction is cell-based therapy. In this study, we took advantage of embryonic stem cells (ESCs), owing to their unlimited self-renewal capacity and potential to develop into functional cell types for further use in animal disease models.

Methods: To label and purify embryonic stem cell-derived endothelial cells, a lentiviral-based construct was synthesized, wherein the antibiotic resistance gene and reporter gene were driven by vascular endothelial (VE)-cadherin promoter. After proving the efficiency and accuracy of transduction, a high-throughput screening strategy was applied to select the positive clones that had integrated the transgene, and were positive for GFP-positive, vessel-like structures upon differentiation.

Results: In further characterization, the GFP positive clones, which comprised roughly 10% of the whole clones, showed a robust expression of endothelial markers. The cells were sorted according to GFP expression. They were cultured and characterized by RT-PCR for markers of progenitor-type cells at different time points of differentiation. In parallel, a feeder- and serum-free culture method was applied to eliminate the confounding effect added by culture conditions, and to increase the yield of endothelial cell formaion.

Conclusions: In conclusion, lentiviral system is highly efficient for transduction of embryonic stem cells, and labeling of embryonic stem cell-derived endothelial cells. Serum-free culture of the positive clones is also plausible, by reducing the confounding factors and possibly increasing the yield of endothelial cells. Due to their potential risks, lentiviral system may not be the optimal option for treatment purposes. Therefore, after the system is established both in vitro and in vivo, the non-viral gene transfer approaches with high efficiency and low hazard will be applied for further cellular therapy approaches.