Erythropoietin epo

10 MPP3 and MPP4 cells were sorted into 96-well U-bottom plates. Cells were cultured for 10 d in Stempro-34 SFM (Gibco) supplemented with 10% StemPro Nutrient Supplement and cytokines SCF (50 ng/ml; Peprotech), IL-3 (5 ng/ml; Peprotech), Erythropoietin (EPO; 2 U/ml; BioLegend), IL-6 (10 ng/ml; Peprotech), and GM-CSF (10 ng/ml; Peprotech). Media with cytokines were refreshed on day 6. Differentiation was assessed by flow cytometry on days 10–12 (see Table S1 for antibody panel). Cultures were maintained at 37°C at 5% CO₂. Lineage-negative cells from Ebf1^WT and Ebf1^KO mice were transfected with DharmaFECT1 (Dharmacon), with 25 nM control non-targeting or Cebpa siRNA (D-001810-10-05, L-040561-00-0005; Dharmacon) together with 25 nM siGLO Green (Dharmacon). After 48–72 h, 10 MPP3 and MPP4 siGLO Green-positive cells were sorted into 96-well U-bottom plates as described above.

CSCs were isolated by magnet-activated cell sorting (MACS) from the hearts of male Sprague-Dawley rats as described previously (13 (link),21 (link)). Briefly, the heart was excised and the aorta was rapidly cannulated, followed by perfusion with Ca²⁺-free Tyrode solution for 10 min and then digestion with 0.5 mg/ml collagenase (Sigma, St. Louis, MO, USA) and 0.05 mg/ml trypsin (Difco, Kansas, MO, USA) at 37°C for 30 min. The heart tissue was sectioned and the resulting cell suspension was filtered with a strainer (Becton-Dickson, Franklin Lakes, NJ, USA). Cells were then incubated with a rabbit anti-c-Kit antibody (1:50; Santa Cruz Biotechnology, Inc., Texas, USA) and separated using immunomagnetic microbeads (Miltenyi Biotech, Bergish Gladbach, Germany). CSCs were then cultured in Dulbecco’s modified Eagle’s medium/Ham’s Nutrient Mixture F12 (1:1) (DMEM/F12) (Sigma-Aldrich) containing 15% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) (both from Sigma-Aldrich) and 2.5 μ/ml erythropoietin (EPO) (BioLegend, San Diego, CA, USA) at 37°C. After 28 days of culture, confluent CSCs were passaged.