Neuropilin-1 identifies endothelial precursors in human and murine embryonic stem cells before CD34 expression

Abstract

Background: In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells.

Methods and results: We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2(+)NRP-1(+) cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells.

Conclusions: The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.

Figure 1. NRP-1 Expression Coincides With Bry and VEGFR-2 in Differentiating Murine ESCs

Panel A: RT-PCR Time course analysis of embryoid bodies from Bry GFP murine ESCs in the absence and presence of BMP4 and bFGF. Results are representative of 3 experiments. The murine endothelialioma cell line bEnd.3: positive control for VEGFR-2, NRP-1, SCL/Tal-1, and LMO 2. Panel B: Time course analysis of murine ESC differentiation as embryoid bodies in serum free conditions. Percent positive cells determined by flow cytometry analysis Panel C: Representative flow cytometry plots of Bry GFP murine ESCs stained with antibodies to E-Cadherin, VEGFR-2, NRP-1, and CD34. Double staining experiments with NRP-1/E-cadherin and NRP-1/VEGFR-2 were performed in day 4 embryoid bodies in Rosa 26 mESCs. Quadrants set with IgG control. Data are representative of 5 experiments.

Figure 2. Bry+VEGFR-2+ and Bry+NRP-1+ Murine ESC derived cells differentiate to endothelium in vitro

Panel A: Differentiation of Bry+VEGFR-2+ and Bry+NRP-1+ murine ESC derived cells to endothelium. Specific antibodies indicated on each photo. Nuclei were counterstained with bis-benzamide in all photos. CD34 images shown at 4× magnification (scale bar=100 microns), CD31/AcLDL and VE-Cadherin/eNOS shown at 20× magnification (scale bar=20 microns). Panel B: Quantification of CD34+ cells derived from Bry+VEGFR-2+ and Bry+NRP-1+ cells. Means ± SEM of three independent experiments are shown.

Figure 3. Time Dependence of VEGFR-2 and NRP-1 Expression in Human ESCs Differentiated in Serum Free Conditions

Panel A: Representative flow cytometry analysis of day 5 human ESC EBs differentiated in the absence or presence of GF (BMP4 (10ng/mL), VEGF (10ng/mL), and bFGF (10ng/mL)). Quadrants are set to mouse IgG controls. Panel B: Time course analysis of VEGFR-2 and NRP-1 cell surface protein expression measured by flow cytometry analysis. Human ESCs (NIH Code UC06) were differentiated as embryoid bodies in the presence or absence of GF as described in the Methods and Materials. Quantity of VEGFR-2+ and NRP-1+ cells was determined by flow cytometry and presented as % positive cells. Significant differences between untreated and GF treated cells were assessed by a two-way ANOVA with Holm-Sidak post hock test, and indicated with * for each time point. Means ± SEM of four independent experiments are shown. Panel C: PCR time course analysis of mesoderm and vascular markers in differentiating human ESCs in the absence or presence of GF. HUVEC used as a positive control for vascular markers. Results are representative of >3 experiments. Panel D: Western blot analysis of VEGFR-2 and NRP-1 expression in day 5 embryoid bodies derived from human ESCs. Results are representative of four experiments.

Figure 4. VEGFR-2+NRP-1+ Cells Are CD31− and CD34−

Panel A: Representative flow cytometry plots of human ESC derived embryoid bodies grown in the absence or presence of GF for 5 days. Mouse IgG controls shown in the left-most panels. Quadrants set by mouse IgG control for each secondary combination. Panel B: Quantitative analysis of VEGFR-2, NRP-1, CD31+ and CD34+ cells in day 5 human ESC EBs. Means ± SEM of seven different human ESC lines (NIH Codes: WA07, WA13, WA14, UC01, UC06, BG02, TE06) are shown.

Figure 5. VEGFR-2+NRP-1+ Human ESC Derived Cells Differentiate to Endothelial-like Cells In Vitro

Panel A: RT-PCR analysis of sorted VEGFR-2+ NRP-1− and VEGFR-2+ NRP-1+ cells prior to endothelial differentiation. HUVECs used as a positive control. Panel B: Differentiation of VEGFR-2+NRP-1+ and VEGFR-2+NRP-1− cells in endothelial growth conditions. Phase contrast images shown at 10× magnification (scale bar=40 microns). Fluorescence images shown at 20× (scale bar=20 microns). Results are representative of four independent experiments. Panel C: Quantification of CD31+ and CD34+ cells derived from VEGFR-2+NRP-1+ and VEGFR-2+NRP-1− cells. Means ± SEM from four independent experiments are shown. Panel D: Matrigel in vitro angiogenesis assays in VEGFR-2+NRP-1+ and VEGFR-2+NRP-1− cells. Images shown are representative of three independent experiments. Scale bar=20 microns.

Figure 6. Effect of NRP1 Function Blocking Antibodies on Differentiation of Stem Cells to Endothelial Cells

Panel A–C: Photomicrographs of representative photomicrographs of murine stem cells differentiated to endothelial cells for seven days in the presence of mouse IgG (Panel A), NRP1A Ab (Panel B), NRP1B Ab (Panel C). Results are representative of three independent experiments. Scale bar=20 microns. Panel D: Summary of three independent experiments, means ± SEM are shown. Significant differences in a one-way ANOVA with Holm-Sidak post-hoc test for VE-Cadherin (*) and CD31 (**) at p>0.001.