Figure S1. Phenotypic characterization of transfected ECFC. (a) ECFC were transfected using a lentivirus with a vector encoding for either human EPO

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1 Figure S1. Phenotypic characterization of transfected ECFC. (a) ECFC were transfected using a lentivirus with a vector encoding for either human EPO (epoecfc) or LacZ (laczecfc) under control of a cytomegalovirus (CMV) promoter and selected using blasticidin. (b) LacZ staining of culture-expanded epoecfc, laczecfc, and non-transfected ECFC using x-gal. Immunofluorescent staining of cells in monolayer cultures using antibodies against human CD31 (green), human EPO (red) showed EPO expression only present in epoecfc but not in non-transfected ECFC. All cells expressed VE-Cadherin (green), and vwf (red), as expected in EC. Cell nuclei were stained with DAPI. Scale bars, 50 um. (c) Representative flow cytometry histograms showing positive CD31 and vwf expression and negative CD90 and CD45 expression by epoecfc, laczecfc, and non-transfected ECFC. Red line histograms represent cells stained with fluorescent antibodies against each cellular marker, while black line histograms correspond to the isotypematched controls.

2 Figure S2. Release of EPO into conditioned media. Human EPO was detected in conditioned medium (CM) from epoecfc using an ELISA kit specific for human EPO detection. EPO was not detected in CM from either laczecfc, MSC, or ECFC.

3 Figure S3. Functionality of secreted EPO. Secreted EPO was confirmed functional in hematopoietic colony-forming assays by culturing human umbilical cord blood-derived mononuclear cells in EPO-free methylcellulose medium supplemented with either ECFC-CM, epoecfc-cm, or rhepo. (a) Macroscopic view of the cultures. Only cultures with epoecfc-cm, or rhepo showed erythrocyte-containing (red color) colonies. (b) Presence of EPO-dependent erythrocyte-containing colonies such as BFU-E (burst forming units-erythroid) and multipotential colonies CFU-GEMM (granulocyte, erythrocytes, monocyte, megakaryocyte) were detected in cultures supplemented with either epoecfc-cm or rhepo, but not in cultures containing control ECFC-CM. EPO-independent colonies such as CFU-GM (granulocyte, monocyte), CFU-M, and CFU-G were detected in all cultures. Scale bars, 200 um. (c) Quantification of total number of BFU-E, CFU-GEMM, and CFU-GM colonies observed in duplicate cultures from two independent cord blood samples.

4 Figure S4. Preservation of EC phenotype in transfected ECFC. (a) Up-regulation of leukocyte adhesion molecules E-selectin and ICAM-1 in response to TNF-α. Blue line histograms represent cells stained with fluorescent antibodies against adhesion molecules, while red line histograms correspond to isotypematched controls. Bars represent the mean fluorescent value of the entire cell population. (b) Phase contrast pictures showing an increased number of bound HL-60 leukocytes after TNF-α treatment. Scale bar: 100 µm. Quantification of bound leukocytes (mean ± S.D.). (c) Sprouting ability was examined under phase contrast microscope; tube formation ability was evaluated with a florescence microscope after live/dead staining. Length (sprouting), branch number and length (tube formation) were quantified (mean ± S.D.). (d) Cell proliferative capacity was tested in basal medium containing either 10 ng/ml VEGF-A, 1 ng/ml bfgf, 10 ng/ ml EGF, and 10 ng/ml IGF for 48 hours (mean ± S.D.). Values were normalized to cell density obtained in basal medium for 24 hours.

5 Figure S5. Anti-apoptotic properties of transfected ECFC. Cell monolayers were challenged with or without 0.5 mmol/l H2O2 for 14 hours and analyzed with an Annexin V apoptosis detection kit. (a) Representative flow cytometry density plots showing gates for viable, necrotic, early and late apoptotic cells. (b) Quantification of percentage of viable and apoptotic cells. Values (mean ± S.D.) were obtained in triplicates.

6 Figure S6. Vascular network formation in immunodeficient mice. MSC were combined with either epoecfc or laczecfc in 200 ul of collagen-based gel and the mixture injected into the dorsal flank of athymic nude mice. (a) Macroscopic view of representative explants at day 10 (scale bar, 2 mm). (b) H&E staining of explant sections at day 10 revealed the presence of numerous blood vessels (yellow arrowheads) containing murine erythrocytes. scale bar: 50 µm. (c) Immunohistochemistry showed that microvessels stained positive for human CD31 (yellow arrowheads). Scale bar: 50 µm. (d) Double fluorescent staining using UEA (red) binding and antibody against human EPO (green); human microvessels in implants engineered with epoecfc expressed EPO. In contrast, EPO expression was absence in laczecfc-lined vessels (scale bars, 50 um).

7 Figure S7. Evidences of erythropoiesis in blood from implant-bearing mice. Automated complete blood count (CBC) test with differential analysis was carried out in whole blood samples drawn at day 10. The results from mice bearing epoecfc-based implants were compared to both naive control mice (* P<0.0001) and mice bearing laczecfc-based implants ( P<0.0001).

8 Figure S8. Proliferating cells in spleen of implant-bearing mice. (a) Representative fluorescence staining using an antibody against Ki67 (green) in spleen, liver, and myocardium from implantbearing mice. Cell nuclei were stained with DAPI. Scale bars, 50 um. (b) Quantification (mean ± S.D.) of percentage of Ki67-positive cells. Four mice were used for each type of ECFC-based implant.

9 Figure S9. Secretion of EPO by explants in vitro. (a) For reversibility studies, surgical excision of implants were carried out two weeks after implantation in 4 mice. (b) CM was generated by culturing excised implants in EBM-2, 5% FBS for 24 hours. Human EPO was measured by Quantikine IVD ELISA Kit (mean ± S.D.). (c) Concentration of hepo in explant-cm correlated with hematocrit found in plasma at the time of implant excision.

10 Figure 10. Reversibility by surgical excision. Hematocrit, WBC, RBC, hemoglobin, spleen and body weights of implant-bearing mice at 5 weeks (n=4 each group). In the excision group (green bars), implants were surgically excised at two weeks, and mice left without implants for another 3 weeks. Shamoperated mice (orange bars) retained the implants for 5 weeks. Control included mice bearing no implants that were treated (i.p.) with either rhepo or saline (* P<0.001 compared to excision group at 5 weeks; P<0.001 compared to saline group at 5 weeks).

11 Figure S11. Importance of MSC support. Implants were generated with epoecfc in either the presence or absence of MSC. Hematocrit in implant-bearing mice was measured at weekly intervals. In the absence of MSC, the hematocrit increased only during the first week, but it went rapidly down to basal levels. Values are expressed as mean ± S.D. (n=4 each group; * P<0.05; ** P<0.01).

12 Figure S12. Detection of epoecfc in distant organs and tissues. The number of mrna copies per cell normalized to 18S rrna abundance was determined by Multi-Gene Transcriptional Profiling (MGTP). (a) hepo/18s ratio from cells in culture. (b) hcd90/18s ratio from cells in culture. (c) hepo/18s ratio from organs and tissues lysates harvested from either naive control mice or implant-bearing mice (n=4; bars are mean ± S.D.). Titrations (lines) were carried out with whole organ/tissue lysates from control mice. These lysates were spiked with RNA from known amount of epoecfc, and lines represent the mean value of hepo/ 18S ratio for each condition.

13 Figure S13. Regulation of erythropoiesis with doxycycline (Dox). The capacity to regulate EPO by the transfected ECFC (tetr-epoecfc) was assessed by administration (on) or absence (off) of doxycycline Dox. Implant-bearing mice were monitored for 6 weeks with different Dox regimen (Dox regimen switched at 4 weeks). (a) Body weight, (b) WBC, (c) RBC, (d) hemoglobin, (e) hematocrit were measured at weekly intervals ( P<0.05; * P<0.01; ** P<0.001). (f) Hematocrit it is unaltered in naive nude mice with continuous access to Dox.

14 Figure S14. Model of radiation-induced anemia. (a) Nude mice received sublethal doses of radiation (4 Gy) and afterwards were given (i.p.) either rhepo (60 IU/200 ul saline) or saline (200 ul) every 2-3days. (b) Hematocrit, RBC, and hemoglobin and (c) WBC and body weight were monitored for 5 weeks for both group of mice ( P<0.05; * P<0.01; ** P<0.001).

15 Figure S15. Effect of 5/6 nephrectomy on hematocrit. (a) Schematic diagram representing twostep 5/6-nephrectomy. (b) Hematocrit of 5/6-nephrectomized mice at 1 and 2 weeks post surgery was compared to hematocrit of naive control mice (* P<0.01; ** P<0.001).

16 Figure S16. Correction of anemia in a model of renal failure. (a) Mice were subjected to a twostep 5/6-nephrectomy. Two weeks after surgery, mice were injected with tetr-epoecfc-based implants and were maintained in either the presence or absence of Dox for 5 weeks (n=4 each group). Non-nephrectomized mice served as control. (b) Hematocrit, RBC, hemoglobin, WBC, and body weight were monitored for all group of mice at weekly intervals (mean ± S.D.).

17 Figure S17. Compensatory kidney growth. (a) Wet weight of kidneys excised from implantbearing mice 5 weeks after cell implantation. Kidneys from 5/6-nephrectomized mice were compared to those from non-nephrectomized control mice (n=4 each group). Independently of the Dox regime followed, left kidneys were significantly larger in nephrectomized than in control implant-bearing mice (* P<0.001). Also, left kidneys in nephrectomized mice in the absence of Dox were larger than when Dox was administered ( P<0.001). Wet weight of (b) spleens, (c) livers, and (d) hearts from the same group of mice showed no differences.