Therapeutic effects of hESC-derived CD34⁺ progenitor cells on neovasculogenesis in ischemic mouse hind limbs. (A) Comparison of perfusion rates and prognosis in ischemic limbs injected with CD34⁺, medium, or CD34⁻ cells. Left panel indicates indocyanine green perfusion maps obtained at 0, 3, and 7 days after surgery. The perfusion maps of day 0 show tissue perfusion distribution of the entire lower half of the body, including normal limbs. Right panel shows photographs of the hind limbs at 3 and 7 days after injection. (B) Average perfusion rates of ischemic hind limbs according to POD are indicated for each group. *P = .014 vs POD 0 (ANOVA F_2,24 = 5.311, P = .012). (C) Probability of necrosis; the relationship between the probability of regional tissue necrosis on POD 7 and the tissue-perfusion rate of the corresponding region estimated immediately after surgery. The X axis shows the regional perfusion rate; poor (lower than 15%/min), moderate (16%-120%/min), and (> 120%/min). ANOVA and Scheffe post-hoc test applied to the significant effect of groups on poor and moderate perfusion rate, (ANOVA F_2,3 = 27.993, P = .011. *P = .015 vs media-treated group and P = .02 vs SC34⁻ cells-treated group with poor perfusion rate; ANOVA F_2,9 = 18.872, P = .001. **P = .02 vs media-treated group and P = .001 vs SC34⁻ cells-treated group with a moderate perfusion rate. (D) Various types of hESC-derived CD34⁺ cells involved in neovasculogenesis in ischemic hind limbs. hESC-derived CD34⁺ cells could contribute indirectly (i), partially (ii-iii), or mainly (iv) to neovasculogenesis. Immunohistochemical analysis of ischemic hindlimb regions transplanted with hESC-derived CD34⁺ cells. (E) Expression of angiogenic genes in the ischemic region injected with hESC-derived CD34⁺ cells. Human- and mouse-specific primers were used for analyzing the expression of various angiogenic genes: (1) normal hind limb tissue, (2) ischemic hind-limb tissue injected with the hESC-derived CD34⁺ cells.