Purpose: Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet.
Methods: We studied PI3K-FOXO1A pathways during differentiation of H7 hESC towards endothelial lineage and on proliferation, maturation and cell death of hESC-derived endothelial cells (hESC-EC). We differentiated hESC towards endothelial lineage and used FACS to isolate CD31+ cells at day 13 after initiation of differentiation.
Results: During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodelling-related genes. PI3K inhibitor LY294002 (10 μM) activated FOXO1A as shown by real-time PCR and nuclear translocation assay, and induced formation of CD31+ hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by siRNA showed lower mRNA levels of CD31 and angiopoietin2. Similarly, overexpression of FOXO1A-eGFP construct in hESC-EC resulted in an increased angiopoietin2 expression (1.5-fold, p<0.5, n=3) as well as higher percentage of Topro3-positive necrotic cells (p<0.001). LY294002 decreased proliferative activity of purified hESC-EC, whilst FOXO1A siRNA increased their proliferation. LY294002 inhibited migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. Using a small-animal PET/MRI system along with gallium-labelled NOTA-based conjugates for in vivo multimodality imaging showed an active angiogenic activity of hESC-EC in athymic nude rats. After in vivo conditioning of cells for three weeks, cells retain their low FOXO1A expression levels.
Conclusions: PI3K/FOXO1A pathway is important for function and survival of hESC-EC as well as in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.