Figure 6.
Functionally Intact in Endothelial Cells from KR-mESCs In Vitro and In Vivo after Light Exposure
(A) Tubule formation and (B) Ac-LDL (red) uptake assay performed 24 hr after light exposure. The scale bars represent 200 μm (A) and 100 μm (B), not significant: n/s. The results represent one of the experiments performed thrice.
(C) Cell death in ECs 24 hr after light exposure determined by immunostaining for cleaved caspase-3 and DAPI nuclear counterstaining. The scale bar represents 100 μm.
(D) Representative images of serial laser Doppler perfusion images (red to yellow) (top). Quantitative analysis of blood perfusion ratio of ischemic to non-ischemic hindlimb at indicative days is shown as a line graph (bottom). Data are mean ± SEM.
(E) Physiological status of ischemic limbs 24 days after PBS or cell transplantation. Standard scoring image of hindlimb of Limb salvage, Foot necrosis, Limb loss (top), and the recording number of cases for each condition with or without cell transplantation was summarized as a table (bottom).
(F) Histological and immunohistochemical analyses of normal limbs and ischemic limbs retrieved 24 days after treatment. Tissue regeneration and attenuated fibrosis were determined by H&E and trichrome Masson’s staining and detection of capillary formation was by anti-PECAM in serial-sectioned tissues. The scale bar represents 200 μm (Normal and EC-KR-mESCs) and 500 μm (PBS).
(G) Testis injection of KR-mESCs (1 × 106 cells containing 50% of KR-mESCs and 50% of EC-KR-mESCs) 24 hr after light exposure, 8 weeks after transplantation, representative images of teratoma mass (top) and normal testis (bottom) from mice transplanted with a cell mixture.
(H) Teratomas were counted and these tumor sizes were measured 51 days after injection of cells into total 20 mice (10 mice for each group) (∗∗∗p < 0.001).
(I) Fluorescent images of DiI (red) and DAPI (blue) for identifying ECs and nucleus, respectively. The squares with dotted lines were shown in the separate images with high-magnification field of each area (right and left). The scale bar represents 100 μm.