Regulation of EPO expression with Dox. (A) ECFCs were transduced using lentivirus vectors encoding for a tetracycline operator element (TetO₂) upstream of the hEPO gene and TetR under the control of a CMV promoter. The capacity to regulate EPO by the transduced ECFCs (tet-epoECFCs) was assessed by administration (on) or absence (off) of Dox. (B) Double immunofluorescent staining for CD31 (green) and hEPO (red) showed progressive expression of EPO on administration of Dox (day 0-4). Thereafter, in the absence of Dox, EPO expression ceased progressively (day 4-7). Cell nuclei were stained with DAPI. Scale bars indicate 50 um. (C-D) Regulation of EPO with Dox was also demonstrated by analyzing CM from tet-epoECFCs with ELISA (C) and immunoblotting (D). (E) Regulation of the hematocrit in vivo was assessed in mice bearing tet-epoECFC–based implants subjected to 3 different Dox regimes for 4 weeks (n = 4 each group). The hematocrit in mice that did not receive Dox (off) was compared with those subjected to permanent administration of Dox (on vs off; *P < .01; **P < .001) and an intermittent Dox regime (off vs on/off; ‡P < .05; †P < .01).