Isolation of mouse heart cells and CD34+ cells was performed as previously described [29 (link), 30 (link)]. Then, cells were sorted with anti-CD34 magnetic beads (Miltenyi Biotec) according to manufacturer’s instructions. The cells were cultured as specified previously [31 (link)]. The purified CD34+ cells were seeded onto dishes with 0.1% gelatin (Sigma, G1393) and maintained in complete stem cell culture medium, which consists of DMEM, 1% FBS, 2% chick embryo extract (MP Biomedical), 100 nM retinoic acid (Sigma-Aldrich), 50 nM 2-mercaptoethanol (Sigma-Aldrich), 2% B27 (Invitrogen), 1% N2 (Invitrogen), 20 ng /ml bFGF (R&D Systems) and 1% P/S.
For cell differentiation experiments, CD34 + cells were cultured on gelatin-coated plates and maintained in complete stem cell culture with 50 ng/mL CTGF (Recombinant Human Connective Tissue Growth Factor, PeproTech, 120–19-20), which is sufficient to differentiate MSCs into fibroblast cells [32 (link)]. In the presence of recombinant TGFβ1 (5 ng/mL) (R&D systems, 7666-MB-005), cells were also treated with inhibitors including SB525334 (a selective TGFβ1 receptor inhibitor) (2 μM).
For cell differentiation experiments, CD34 + cells were cultured on gelatin-coated plates and maintained in complete stem cell culture with 50 ng/mL CTGF (Recombinant Human Connective Tissue Growth Factor, PeproTech, 120–19-20), which is sufficient to differentiate MSCs into fibroblast cells [32 (link)]. In the presence of recombinant TGFβ1 (5 ng/mL) (R&D systems, 7666-MB-005), cells were also treated with inhibitors including SB525334 (a selective TGFβ1 receptor inhibitor) (2 μM).
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