Abstract 720: Neuropilin 1 Identifies Endothelial Precursors in Murine and Human Embryonic Stem Cells Prior to CD34 Expression
Human embryonic stem cells (ESCs) are a model to identify the mechanisms controlling human vasculogenesis. The identity of endothelial precursors from human ESCs is unknown. In murine ESCs, vascular endothelial growth factor receptor 2 (VEGFR2) identifies endothelial precursors. In contrast, undifferentiated human ESC lines express the endothelial and progenitor markers VEGFR2 (70±4%, p<0.001), CD133 (74±10%, p<0.02), CD146 (96±1%, p<0.0002), and expression of these three markers persists upon differentiation. The objective of our study is to identify endothelial precursors from both murine and human ESCs. Here we report that expression of the VEGF co-receptor Neuropilin 1 (Npl1) identifies endothelial precursors in murine and human ESCs. In murine ESCs, Npl 1 expression peaks in day 4 embryoid bodies, is co-expressed with VEGFR2, and the mesoderm defining transcription factor Brachyury. Brachyury+Npl 1+ cells are induced in serum-free conditions with BMP4 and bFGF (22±5%, p<0.05), and differentiate to endothelial-like cells in vitro. In human ESCs, Npl 1 is co-expressed with VEGFR2 five days after differentiation in BMP4 and bFGF (VEGFR2+Npl 1+ 34±6% vs. 8±2%, ± growth factors, n=5, p<0.009). VEGFR2+Npl 1+ cells do not express CD31 or CD34 consistent with a precursor population. VEGFR2+ Npl 1+ cells are obtained from seven different human ESC lines (27.2±7.6% vs. 6.1±1.9%, ± growth factors, p<0.02) indicating the findings are reproducible in multiple human ES cell lines. When sorted and differentiated in vitro, VEGFR2+Npl 1+ cells form endothelial colonies that express CD31 and CD34 five fold more efficiently than VEGFR2+Npl 1- cells. VEGFR2+Npl 1+ cells absorb LDL and form cords in Matrigel. Collectively, the onset of Npl 1 expression identifies endothelial precursors from murine and human ESCs prior to CD31 or CD34 expression. The findings define a model to understand the mechanisms controlling human vascular differentiation.