Vascular network formation and EPO delivery in vivo. (A) epoECFCs and MSCs were subcutaneously injected into nude mice. Macroscopic view of explant at day 10 is shown in the inset (scale bar indicates 2 mm). H&E and hCD31 immunohistochemistry showed formation of numerous blood vessels (yellow arrowheads; scale bar indicates 50 μm). Microvessel density was quantified at day 10 (n = 4). (B) Double immunofluorescent staining of microvessels using UEA binding (red) and an Ab against hEPO (green; scale bars indicate 50 μm). (C) HEPO detected by ELISA in plasma at day 10. (D) Hematocrit at day 10 (epoECFCs vs no implant, †P < .0001; epoECFCs vs laczECFCs, *P < .0001). (E) Macroscopic view of spleens at day 10 (scale bar indicates 10 mm). Spleens/body weight ratio (epoECFCs vs laczECFCs, †P < .005; epoECFCs vs no implant,*P < .005). Immunofluorescent staining using an Ab against Ki67 (green). Cell nuclei were stained with DAPI. Scale bars indicate 50 μm. (F) Hematocrit was monitored for 5 weeks; implants were surgically excised at 2 weeks. Sham-operated mice, and mice treated (IP) with rhEPO and saline served as control (all vs excision, *P < .001; all vs saline, †P < .001). (G) Quantitative RT-PCR analyses of endogenous mEPO expression in kidney, heart, and liver at 5 weeks (*P < .01).