Wnt signaling promotes early SEP proliferation, and the expression of canonical Wnts is inhibited by Epo signaling. (A) ELISA analysis of serum Epo (right y-axis) and Wnt2b and Wnt8a mRNA expression (left y-axis) in spleen cells on indicated days after BMT. For each time point, n = 3 mice per group. (B) Wnt2b and Wnt8a mRNA expression in sorted murine spleen F4/80⁺ macrophages with or without Epo for 24 hours (left). mRNA expression of WNT2b and WNT8a in unfractionated human bone marrow cells cultured in SEEM for 5 days and then treated with or without Epo for 24 hours (right). (C) Representative flow cytometry analysis of CD34 and CD133 expression on Kit⁺Sca1⁺ SEPs from murine bone marrow cells cultured in SEEM with or without 1 μM BIO and with or without Epo. (D) Stress BFU-E colony assay of in vitro cultured SEPs treated with or without Epo and with or without BIO, as shown in panel C. (E) Stress BFU-E colony assay of SEPs expanded in SEEM with or without BIO. The SEPs were then switched into SEDM without BIO, and stress BFU-Es were assayed after 3 days. (F) Stress BFU-E colony assay of SEPs expanded in SEEM with or without Epo, with or without 100 ng/mL WIF1, or treated with Epo with or without WIF1. Student t test (2-tailed). Data represent means ± SEM. *P < .05; **P < .01; ****P < .0001.