Vascular endothelial growth factor (vegf)

H1 human embryonic stem cells (WiCell Research Institute, USA) were cultured in PSCeasy medium (Cellapy, China) in Matrigel-coated dishes (Corning, USA) and passaged with 0.5 mM EDTA at 70–80% confluence. At ~ 90% confluence, H1 cells were moved into CDM3 medium with different supplements to induce directional differentiation. CDM3 medium consists of RPMI 1640 medium (Thermo Fisher, USA), recombinant human albumin (Sigma-Aldrich, USA) and L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA). During days 0–2, the medium was supplemented with 6 μM CHIR99021 (Sigma-Aldrich, USA). On day 2, 50 ng/mL bFGF (Novoprotein, USA) was added. After that, 50 ng/mL VEGF (Novoprotein, USA), 25 ng/mL BMP4 (PeproTech, USA) and 25 ng/mL bFGF (Novoprotein, USA) were used for the next 3 days. The medium was changed daily. On day 6, CD34⁺ cells were enriched using anti-CD34 antibody-conjugated magnetic beads (Miltenyi, USA) according to the manufacturer’s instructions. The sorted cells were defined as human embryonic stem cell-derived endothelial cells (hESC-ECs).

The gelatin methacrylate (Engineering For Life, Suzhou, China) hydrogels were prepared following the instructions provided by the manufacturer. Briefly, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was completely dissolved in the phosphate-buffered saline (PBS) (HyClone, Logan, UT, USA) in a water bath at 60 °C for 30 min. Then, the GM solid was completely dissolved in a LAP solution to form a 5% (w/v) uncrosslinked GM precursor solution. Then, the solution was irradiated with an ultraviolet light source (405 nm, 3 W; 20 s) to initiate the cross-linking of GM and turn the solution into hydrogel for subsequent experiments. For the preparation of the GM hydrogels loaded with growth factors, namely, GM/GF, VEGF (Novoprotein, Suzhou, China), and NGF (Novoprotein, Suzhou, China) were added to an uncrosslinked GM hydrogel solution at a concentration of 100 ng/mL and irradiated as described above.