Co-culture of BM Sca-1⁺ cells with EPDCs under hypoxia conditions increased proliferation and migration of EPDCs. Epicardial-derived cells (EPDCs, abbreviated as E) were isolated and co-cultured with bone marrow (BM) Sca-1⁺ cells or Sca-1^- cells under normoxia and hypoxia (0.1% O₂) conditions. (A) Representative images of EPDCs and immunofluorescent labeling of WT1. (B) Cell proliferation was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. (C, D) Immunofluorescent staining of BrdU and quantification of BrdU⁺ (proliferating epicardial cells) cells. EPDCs, co-cultured with BM Sca-1⁺ cells or Sca-1^- cells under normoxia (Nor) and hypoxia (Hypo) conditions for 72 h, were pulse-chased with BrdU (10 µM) for labeling of proliferative cells. Cell migration was evaluated by the transwell (E, F) and wound-scratch (G, H) assays after co-culture for 24 h. n=6/group, mean ± SD; **P<0.01. BrdU: bromodeoxyuridine.

BM Sca-1⁺ cells increased proliferation and migration of EPDCs through TGF-β1 signaling. Epicardial-derived cells (EPDCs, abbreviated as E), bone marrow (BM) Sca-1⁺ and Sca-1^- cells were isolated and subjected to normoxia and hypoxia (0.1% O₂) conditions. (A) Higher expression levels of TGF-β1 mRNA were found in BM Sca-1⁺ cells than in EPDCs and Sca-1^- cells under hypoxia conditions for 72 h. (B) TGF-β1 homodimer secretion in EPDCs, BM Sca-1⁺ and Sca-1^- cell lysate and culture medium under normoxia and hypoxia conditions for 72 h was quantified by ELISA. (C) TGF-β1 mRNA expression was measured in the Y(sca-1⁺)-O and Y(sca-1^-)-O chimeric hearts at baseline and in the infarcted area at 3 days post-MI. Epicardial-derived cells (EPDCs, abbreviated as E) were co-cultured with BM Sca-1^- cells (S^-), Sca-1⁺ cells (S⁺), TGF-β1 (T, 5 ng/mL), Sca-1⁺ cells with TGF-β1 blocking antibody (S⁺+BL, 1 µg/mL) or Sca-1^- cells with TGF-β1 (S^-+T) under hypoxia conditions. The following assays were conducted in EPDCs: (D) MTT assay measured cell proliferation; (E) EPDCs, co-cultured with BM Sca-1⁺ cells or Sca-1^- cells under normoxia and hypoxia conditions for 72 h, were pulse-chased with BrdU (10 µM) for labeling of proliferative cells; (F) transwell and (G) wound-scratch assays measured migration areas and number of EPDCs after co-culture for 24 h, respectively. Insufficiency of Sca1^- cells on EPDC migration can be rescued by TGF-β1, and EPDC migration was restored to a level comparable with that of the Sca-1⁺ cell- or TGF-β1-treated groups. n=6/group, mean ± SD; **P<0.01. BrdU: bromodeoxyuridine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.