Neuronal Differentiation of miPB-IPC

To determine miPB-IPC’s multipotency and their neuronal cell differentiation (Figure S1), miPB-IPC were treated with 100 ng/mL neuronal growth factor (NGF, R & D Systems) + human neuronal stem cell growth medium (iXCells Biotechnologies, San Diego, CA, USA) for 3–5 days, in 24-well tissue culture-treated plates, at 37 °C in 5% CO₂. The differentiated cells were characterized by immunocytochemistry [29 (link)] with mouse anti-human tyrosine hydroxylase monoclonal Ab (mAb, Clone LNC1, Catalogue # MAB 318, at 1:100 dilution) and rabbit anti-Synapsin I polyclonal Ab (Catalogue # AB1543, at 1:100 dilution) (EMD Millipore, Temecula, CA, USA). The FITC-conjugated AffiniPure donkey anti-mouse 2nd Ab and Cy3-conjugated AffiniPure donkey anti-rabbit 2nd Ab were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Isotype-matched IgG served as negative control for immunostaining. After covering with Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), cells were photographed with Nikon A1R confocal microscope on Nikon Eclipse Ti2 inverted base.