Fc005

hiPSC-NPC was seeded on top of sterile scaffolds in 1 cm² and incubated for DIV 15 under experimental conditions. The cells were then enzymatically dissociated from the biomaterials with 0.125% trypsin-EDTA and 25 mM of sodium citrate in PBS at 37 °C under the agitation of 25 rpm for 10 min. FBS was added for trypsin neutralization. The cells were washed three times by adding PBS and centrifuging at 600 g for 5 min. The cells were then fixed with flow cytometry fixation buffer (FC004, R&D Systems, Minneapolis, MN, USA) and incubated for 30 min at room temperature, then washed and resuspended in flow cytometry permeabilization/wash buffer I (1X) (FC005, R&D Systems, Minneapolis, MN, USA). Marker antibodies, TUJ1 (60052, Stemcell Technologies, Vancouver, BC, Canada) and GFAP (60048, Stemcell Technologies, Vancouver, BC, Canada), and isotype controls, mouse IgG2A PerCP-conjugated Isotype control (IC003C, R&D Systems, Minneapolis, MN, USA), were added per manufacturer’s instructions and incubated for 1 h at 4 °C in the dark. The cells were then washed twice with PBS, resuspended in washing buffer and the marker expression data was collected using a Guava EasyCyte HT flow cytometer (Millipore, Burlington, MA, USA).

Aggregate samples containing 1E6 cells were removed from the bioreactor culture and added into microcentrifuge tubes (Cat#10011-724, VWR). Aggregates were rinsed twice with PBS and resuspended in 0.5 mL of fixation buffer (Cat#FC001, R&D Systems) to be incubated for 1 h at room temperature. Aggregate samples were then rinsed twice with PBS and resuspended in 200-μL permeabilization buffer (Cat#FC005, R&D Systems) with 1 μg/10⁶ cells antibody stain and 1 μM/10⁶ cells nuclei stain and incubated for 3 h at room temperature. Conjugated antibody stains for SSEA-4 (Cat#FAB1435F, R&D Systems), TRA-1-60 (Cat# FAB4770P, R&D Systems), and Nanog (Cat#MABD24A4, Millipore Sigma) were used along with the nuclei stain To-Pro-3 Iodide Nucleic Acid Stain (Cat#T3605, Thermo Fisher). Cells were then rinsed twice with PBS and imaged using a Carl Zeiss Laser Scanning Microscope 700 with lasers at 488 nm and 639 nm and corresponding filter sets.