For transcriptome analysis, RNA was prepared from 2D and 3D cultured hMSCs, DPBS-treated, 2D EV- or 3D EV-treated cells as well as Fenbendazole or Brompheniramine-treated cells. Treatment was performed for 6 hrs. All of the treatment was independently carried out in triplicate.
Growth protocol
For 2D culture, hWJ-MSCs were cultured in MEMα medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic solution (2D medium) under 5% CO2 and 37°C conditions, and the medium was changed every 3 days. When the cells reached 80% confluence, cells were passaged using 0.25% (w/v) trypsin-EDTA (Gibco). For 3D culture of hWJ-MSCs, 3D medium was prepared by supplementing MEMα medium described above with 0.05% (v/v) Cellhesion VP (Nissan Chemical Industries, Ltd., Tokyo, Japan) according to the manufacturer’s instruction. Cells were seeded at a density of 1 × 106 cells/flask in 10 mL of 3D medium in a 125 mL non-adherent flask (Corning, Corning, NY, USA) and incubated on a rocker (LABOGENE, Seoul, Korea) at 9 rpm with mechanical dissociation daily up to 15 days under 5% CO2 and 37°C conditions. The medium was changed every 3 days. Nine days after 3D culture, primed hMSCs were retrieved by transferring the cell-Cellhesion VP aggregates to a tissue culture plate. The aggregates were spin-down at 1,500 rpm for 3 min and the supernatant was discarded. The aggregates were resuspended by pipetting in 5 mL of 2D medium, transferred to the 100 mm tissue culture plate (Corning), and subjected to cell retrieval from the aggregates. The retrieved cells were maintained in 2D culture condition until used.
Extracted molecule
total RNA
Extraction protocol
Total RNA from hWJ-MSCs was prepared using a RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. During RNA preparation, DNase (Qiagen) was applied to prevent the genomic DNA contamination.
Label
biotin
Label protocol
The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit.
Hybridization protocol
Approximately 5.5 μg of labeled cDNA was hybridized to the Affymetrix GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) at 45 °C for 16 h.
Scan protocol
After hybridization, the arrays were scanned on a GCS3000 Scanner (Affymetrix) and data analyses were carried out with the GeneChip Command Console Software (Affymetrix).
Data processing
Expression data were normalized using the robust multi-array average (RMA) approach.
