Fig 2: CD157 expressed in vascular endothelial cells (ECs) of large retinal vessels. (A) CD31 (blue) and CD157 (red) were stained using the retinal flat mount of a wild-type mouse. CD157-positive ECs were located in the large veins in the retina, but not in the capillaries. Scale bar represents 100 µm. (B) CD157 positive ECs were not detected in the retinas of CD157-KO (CD157−/−) mouse, indicating that the CD157 antibody used in A specifically recognized CD157 in retinal vasculature. Scale bar represents 100 µm.

Fig 3: FACS gating strategy for sorting retinal CD157-positive VESCs. Representative FACS plots of the percentage of parent gates for each population. The retinal cell population was selected (A), dead cells were excluded (B), and doublets were excluded (C, D). (E) CD31-BV421-A versus CD45-FITC-A dot plot was generated to identify vascular endothelial cells (ECs) positive for the endothelial marker CD31 and negative for the hematopoietic marker CD45 (CD31+CD45−) in the retina. The proportion of retinal ECs shown in red squares was 3.3% in this experiment. The orange square shows hematopoietic cells (CD31+CD45−), and the blue squares indicate others (CD31−CD45−). (F) CD31-BV421-A versus CD157-APC-A dot plot was generated to detect CD157-positive vascular endothelial stem cells (VESCs). The proportion of VESCs was 1.6% among all retinal ECs.

Fig 4: In vitro culture of CD157-positive VESCs showing proliferation and endothelial network formation. (A) CD157-positive VESCs and CD157-negative non-VESCs from the retina were cultured on OP9 feeder cells for 10 days. The cells were stained with an anti-CD31 antibody. Scale bars, 1 mm. (B) CD157-positive VESCs and CD157-negative non-VESCs from the choroid were cultured on OP9 feeder cells for 10 days. The cells were stained with an anti-CD31 antibody. Scale bars, 1 mm. (C) Quantification of the EC network area. **P < 0.01, Student's t-test. Data are mean ± SEM (n = 4).

Fig 5: CD157 expressed in the vascular endothelial cells (ECs) of large choroidal vessels. (A) CD31 (white) and CD157 (red) were stained using the choroidal flat mount of a wild-type mouse. CD157-positive ECs were located in the large vessels in the choroid, but not in the capillaries. Scale bars represents 100 µm. (B) CD157 positive ECs were not detected in the choroid of CD157-KO (CD157−/−) mice, indicating that the CD157 antibody used in A specifically recognized CD157 in choroidal vasculature. Scale bars represents 100 µm.