Fig. 1: Endothelial stem/progenitor marker-positive cells are abundant in optic nerve vessels.

a Diagram illustrating the connection between the retina and the optic nerve. b Flat-mount immunostaining for CD31 on retina and optic nerve (8-week-old C57Bl6J mouse). Half of the retina has been removed. c–e Flow cytometric analysis of CD45^negCD31⁺ endothelial cells (ECs) in retina, retina plus optic nerve (ON) and ON. f–h Flow cytometric analysis for CD201 and CD157 in ECs from (c–e, respectively). i Quantification of percentage of CD157⁺ ECs, CD201⁺ECs and CD157⁺ CD201⁺ ECs from (f–h). Data are pooled from 3 independent experiments (each using 3 mice per experiment) and displayed as mean ± SD. j Immunostaining for CD31 (green) and CD157 (red) and staining with DAPI (blue) in ON section from 8-week-old C57Bl6J mice. Four mice were used in this experiment. k Immunostaining for CD31 (green) and CD201 (red) and staining with DAPI (blue) in ON sections from 8-week-old C57Bl6J mice. l, m Colony forming assay using ECs form retina or ON. Immunostaining for CD31 (green) and with phalloidin (red) and DAPI (blue) (l). ECs from ON potentiate, producing more ECs (m). Data are pooled from 3 independent experiments (each using 10 mice per experiment) and displayed as mean ± SEM. n, o Colony forming assay using CD157^+/neg CD201^+/neg ECs. Immunostaining for CD31 (green) and staining with phalloidin (red) and DAPI (blue) (n). Quantification of colony forming assay demonstrates CD157⁺ CD201⁺ ECs from retina plus ON potentiate, producing more ECs (o). Data are pooled from 3 independent experiments (each using 20 mice per experiment) and displayed as mean ± SEM. p Illustrative representation of the approach for FACS analysis of cultured endothelial colony-forming cells originating from CD157 + CD201+ ECs. q Re-analysis of ECs from colony-forming assays shows that the majority of cells in colonies formed by CD157⁺CD201⁺ VESCs were composed of CD157^negCD201⁺ endothelial cells or CD157^negCD201^neg endothelial cells. Hearts or livers from two mice were used in this experiment. r Diagrammatic illustration of the hierarchical model of vascular endothelial cells in the retina. Significance was determined using two-tailed unpaired t test in m and a one-way ANOVA with Tukey’s multiple comparisons test in (i, o). Each image are from representative of at least two independent experiments. Scale bars, 100 μm (500 μm in b).