Driving murine embryonic stem cells toward endothelial phenotype for cell-based therapy

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.

To achieve pure ECs, we made two lentiviral-based constructs, wherein the antibiotic resistance and reporter genes were driven by specific promoters for ECs, namely vascular endothelial growth factor receptor type 2 (VEGFR-2) and vascular endothelial (VE)-cadherin. Pseudoviral particles were produced by assembling the main construct together with packaging and envelope plasmids on HEK 293T cell line, and were later used for transduction of target cells. Mouse endothelioma cells, as positive control, showed robust GFP expression. The positive ESCs clones, which had integrated the DNA into their genome, were screened by polymerase chain reaction. Subsequently, they were put into culture for differentiation and detection of GFP expression. The later step would be to select the desired cells according to antibiotic resistance and to maintain and propagate them for further therapeutic uses in animal models such as hypoxia-induced pulmonary hypertension and myocardial infarction.