Basic fibroblast growth factor (bfgf)

To initiate hematopoietic differentiation^{42 (link)}, the hESCs were passaged with dispase (2 mg mL⁻¹) onto matrigel-coated 12-well plates. These cells were cultured in E6 medium^{46 (link)} plus ACTIVIN A and BMP4 (DMEM/F12 (Hyclone), 64 mg L⁻¹ L­ascorbic acid (Sigma), NaCl (Sigma, adjusting the osmolarity to 340 mOsm), ITS –G (Gibco, 100×) and 50 ng mL⁻¹ ACTIVIN A (Sino biological, 10429-HNAH-50), 50 ng mL⁻¹ BMP4 (Peprotech, 120-05ET)) for 2 days. And then cells were cultured in E6 medium plus 40 ng mL⁻¹ VEGF (Sino biological, 10008-HNAB-50) and 50 ng mL⁻¹ bFGF (Sino biological, 10014-HNAE-50) for next two days. For next 3 days, these cells were cultured in E6 medium plus 40 ng mL⁻¹ VEGF, 50 ng mL⁻¹ bFGF, 10 μM SB431542 (Selleck). After 7 days, these cells were collected for extracting total RNA and FACS analysis for CD34 expression. CD34 antibody conjugated with PerCP-Cy5.5 were used according to the manufacturer’s recommendations.

Media used in these experiments, designated “T1”, was LCDMV (used for culturing PC-iPS) minus LIF. Experiments were conducted in 12-well plates (Nunc; 150628), and NANOG tdTomato PC-iPS cells were passaged three times to ensure that NANOG tdTomato was stably expressed. To test the response of each cell line to individual cytokines, the T1 culture medium was supplemented with LIF (Peprotech, 300–05-1) (5 ng/mL, 10 ng/mL), IL-6 (R&D, 206-IL) (50 ng/mL, 100 ng/mL), IGF1 (Peprotech, 100-11-1000) (50 ng/mL, 100 ng/mL), bFGF (Peprotech, D2017) (5 ng/mL, 10 ng/mL), Activin A (Peprotech, 1017) (5 ng/mL, 10 ng/mL), or BMP4 (Peprotech, 120-05ET) (5 ng/mL, 10 ng/mL). The corresponding signal pathway inhibitors were as follows: 10 μM ruxolitinib (INCB018424) (Selleck, S1378) (Jak-STAT pathway inhibitor), 10 μM Ly294002 (S1737 1 mg) (PIP3-AKT pathway inhibitor), 10 μM AZD4547 (Selleck, S2801) (bFGF-ERK pathway inhibitor), 10 μM SB431542 (Selleck, S1067) (TGF-β/Activin A pathway inhibitor), and Noggin (R&D, 6057-NG-025) (antagonist of BMP4) (50 ng/mL and 100 ng/mL). Fluorescence microscopy, flow cytometry, and RT-PCR were done to detect expression of NANOG tdTomato.