EPO-R is a progenitor marker of human BMMSCs and mediates bone marrow organization in vivo. (A) Flow cytometric analysis revealed that a small percentage (5.54%) of BMMSCs expressed EPO-R. (B) RT-PCR analysis confirmed EPO-R gene expression in BMMSCs. Jurkat cells were used as a positive control for EPO-R expression. (C) Western blot analysis further confirmed that BMMSCs express EPO-R at passages 1, 5, and 10 (P1, P5, and P10). (D) rhEPO treatment (0.1 U/mL) for the indicated time (minutes) induced a significantly up-regulated expression of phospho-Stat5 in BMMSCs at 30 minutes compared with either β-actin or Stat5 (n = 3). However, expression level of Stat5 showed no significant change (n = 3). (E) STRO-1⁺, CD146⁺, and CD166⁺ BMMSCs were significantly increased in the rhEPO treatment group (EPO⁺) compared with the untreated group (EPO⁻; n = 3). (F) rhEPO (0.1 U/mL)–treated BMMSCs (EPO⁺) were capable of inducing active hematopoietic marrow formation (arrows) when transplanted into immunocompromised mice with HA/TCP (HA) as assessed by hematoxylin and eosin staining. B indicates bone. Original magnification ×400. Bars represent SD (EPO⁺, n = 3; EPO⁻, n = 3). Semiquantitative analysis showed that EPO treatment resulted in a significantly increased bone marrow formation in the BMMSC transplants compared with untreated control BMMSCs. (G) Western blotting analysis confirmed a significant inhibition of EPO-R and Stat5 expression in BMMSCs transfected with siRNA targeting EPO-R and Stat5, respectively. (H) Loss of function of EPO-R and Stat5 resulted in inhibition of bone marrow (arrow) formation in 8-week-old BMMSC transplants compared with the nonspecific siRNA-transfected transplant (Control). HA indicates HA/TCP. Original magnification ×200. Bars represent SD (Control, n = 3; EPO-R, n = 3; Stat5, n = 3; ***P < .005 vs Control; #P < .05 vs EPO-R; ###P < .005 vs EPO-R).