Rencell cx cells

The SVGA cell line [33 (link)] (gifted by Dr. Avindra Nath, NIH) is derived from immortalized human foetal astrocytes, and was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin (HyClone, South Logan, UT, USA). ReNcell CX cells [34 (link)] (Millipore, Temecula, CA, USA) are immortalized human neural progenitor cells (HNPCs), and were maintained in a proprietary ReNcell neural stem cell medium (Millipore) supplemented with 20 ng/mL human epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA), 20 ng/mL human basic fibroblast growth factor (bFGF; Peprotech), and 1% Penicillin/Streptomycin. All cell lines were maintained in a humidified chamber containing 5% CO₂ at 37 °C.
SVGA cells were seeded into six-well plates and onto glass coverslips in twelve-well plates at a density of 300,000 cells/mL and 30,000 cells/mL, respectively, and grown for 24 h. To differentiate HNPCs into neurons, ReNcells were seeded in laminin (20 μg/mL; Millipore) coated six-well plates at a density of 50,000 cells/mL for 24 h. Adhered cells were rinsed with 1X PBS and allowed to differentiate in the presence of ReNcell medium lacking EGF and bFGF for two weeks. SVGAs and neurons were treated with 0.1, 0.5, 1, and 5 ng/mL doses of human IFNγ (PeproTech) for 24 h. Plated untreated cells were used as negative controls.

The human neural progenitor cell lines SK-N-SH cells (American Type Culture Collection, Manassas, VA, USA) and ReNcell CX cells (Millipore, Billerica, MA, USA) were cultured according to the manufacturer’s protocols and maintained in a 75 cm² flask at 37 °C and 5% CO₂. When cells were 75% confluent, the human neural progenitor cells were subcultured to a density of 1 × 10⁶ cells per 75 cm² flask for harvests 24 h post-transfection, and 5 × 10⁵ cells per 75 cm² flask for harvests 72 h post-transfection.

Immortalized human neural progenitor cells (ihNPCs, ReNcell CX cells) were obtained commercially from Millipore (Temecula, CA, USA). Cells were recovered and cultured on a 100 mm diameter dish pre-coated with laminin (Sigma-Aldrich, Milan, Italy), using ReNcell NSC maintenance medium (Millipore, Temecula, CA, USA) with fresh epidermal growth factor (EGF) (20 ng/mL; Millipore) and fibroblast growth factor 2 (FGF 2) (20 ng/mL; Millipore), as described previously [45 (link)].