Recombinant human slit1

A quarter piece of hydrogel (Hydrogel #SP PI5, MedGel) was used as a carrier. Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 µg/ml at a temperature of approximately 4 °C. Matrigel without the agents was used as the control. Each EB was adhered to the centre of a well in 24-well plates. Matrigel was absorbed by the piece of hydrogel, which was placed at the edge of each well of the 24-well plates, in front of the attached OVs on D29 (Fig. S2). On D31, 2 days after the administration of the respective agents, colonies were fixed and immunostained using neurofilament protein L (NFL) antibodies. Each experiment was repeated at least five times.

Affi-gel Blue beads (Bio-Rad) were used as carriers for the sustained release of chemical agents. Beads were washed in DPBS and then incubated for 1 h at room temperature (20 °C) in solutions containing chemical agents at different concentrations. Recombinant human NGF (R&D Systems) and SEMA3A (R&D Systems) diluted in DPBS were used at a concentration of 200 ng/ml, and recombinant human SLIT1 (R&D Systems) diluted in DPBS was used at a concentration of 5 μg/ml. Beads without the agents were used as the controls. Beads were transplanted on D28, one by one, at the site near the base of attached OVs using forceps (Inami) and fine needles (TERUMO) under a microscope (Olympus). Because the filopodia of axons are away from the attached OV on D29, i.e., the day of starting administration of the agents by means of hydrogel, we transplanted the beads on D28 when the filopodia are located close to the OVs. Each experiment was repeated at least five times.