Mesenchymal stem cell expansion medium

Human BMSCs were purchased from Roosterbio (USA). CHIKV (181/25 clone) was purchased from BEI Resources.
BMSCs were infected at an MOI of 1 and incubated for 1 h at 37°C, 5% CO₂ (Fig. 1A). At 1 h postinfection (hpi), cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Corning, USA), replenished with Rooster Basal MSC (RBM) medium (RoosterBio, USA), and incubated at 37°C, 5% CO₂ for 24 h.
For differentiation studies, BMSCs were treated with osteogenic medium containing mesenchymal stem cell expansion medium (Millipore, USA) and supplemented with 10 mM beta glycerophosphate (Sigma, USA), 0.1 μM dexamethasone (Sigma, USA), and 200 μM ascorbic acid (Sigma, USA) to differentiate into osteogenic cells. The osteogenic cells were infected at 7 or 14 dpd, at an MOI of 1, and incubated for 1 h at 37°C, 5% CO₂ (Fig. 1B). After 1 hpi, cells were washed with DPBS, replenished with osteogenic differentiation medium and incubated for 48 h at 37°C, 5% CO₂. For these experiments, mock-infected undifferentiated cells were negative controls, while mock-infected differentiated cells were positive controls.

Human neuroblastoma SH-SY5Y cells (ATCC, Manassas, VA) were grown in DMEM containing 10% FBS (vol/vol) and antibiotics in a humidified 5% CO2/95% air atmosphere at 37°C. Adipose derived mesenchymal stromal cells (hAD-MSCs) [41 ] were grown in Mesenchymal Stem cell Expansion medium (Millipore, Billerica, MA, USA), and the culture media was replaced every 3 days. X-tremeGENE HP transfection reagent (Roche) was used for transient transfections according to the manufacturer's instructions. Unless otherwise indicated, lysates were prepared 48 h after transfection. For the luciferase assay, SH-SY5Y cells were transiently transfected with pGL3-Basic, pGL3-PGC-1α promoter-Luciferase, CRE deletion mutant luciferase constructs for firefly Luciferase assay, and 10 ng pRL-TK vector (Promega) for Renilla luciferase control.