MSC differentiation: After expansion, hES-MSCs were seeded in T75 culture flasks for RNA extraction and in 12-well plates for staining during differentiation of adipocytes, and osteocytes, and in pellet form in 15ml polypropylene tubes for chondrocytes differentiation. Adipocytes and osteocytes grown in 12-well plates were used for Oil Red O and Von Kossa Staining respectively, while Alcian blue staining was done for chondrocytes pellet grown in polypropylene tube. MSCs were seeded at 1.8 x 104 cells per cm2 for adipogenic differentiation, 0.27 x 104 cells per cm2 for osteogenic differentiation, and in pellet size of 50 x 104 cells for chondrogenic differentiation. Trilineage differentiation was done using hMSC adipogenic, osteogenic, and chondrogenic differentiation BulletKits with recombinant human TGF-B3 (Lonza, MD, USA), according to manufacturer’s protocol. Media was changed every 2-4 days.
Growth protocol
Cell culture: Human embryonic stem cell derived MSCs (hES-MSCs) were a gift from Choo A and Lim SK. Cells were received at passage 11 and expanded for 2 passages in MSCGM BulletKit (Lonza, MD, USA). The hES-MSCs were seeded at 5000 cells per cm2 in polystyrene cell culture flask (NUNC, NY, USA) and grown in 5% CO2, 370C incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA of the undifferentiated MSC, adipocytes, and osteocytes of Flb-MSCs was harvested using 1ml of Trizol (Invitrogen, CA, USA)/ 10 cm2 area. Chondrocyte pellets were treated with 0.25% of collagenase type II in DMEM high glucose medium (Invitrogen, CA, USA) for 1-2 hr at 370C, centrifuged at 1200g for 5min, and the supernatant replaced with 300ul of Trizol per pellet. All samples were stored in Trizol at -800C until RNA extraction. RNA extraction from samples in Trizol was done using chloroform and purified with RNeasy kit (Qiagen, Netherlands). Concentration of RNA was determined using Nanodrop (Agilent Technologies, CA, USA) and the RNA integrity measured using RNA Nano LabChip on Agilent Bioanalyzer (Agilent Technologies, CA, USA). Only high quality total RNA samples of OD260/OD280 >1.8 and RNA Integrity Number (RIN) of >8, were used for microarray.
Label
biotin
Label protocol
RNA amplification was done using Illumina TotalPrepTM RNA amplification kit (Ambion, TX, USA), according to manufacturer’s instructions. 750ng of biotin-labelled cRNA for hybridization was randomly loaded on to Illumina Sentrix BeadChip Array human Ref-8v3 bead chips (Illumina, San Diego, CA)
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
After 16hr of hybridization, the Beadchips were washed, Cy3-labeled, and scanned with an Illumina Bead array reader confocal scanner.
Data processing
The data was then uploaded to GenomeStudio (Illumina, CA, USA) for background subtraction and conversion into Partek file for data analysis on Partek Genomic Suite. The data was then quantile normalised for further downstream analysis.
Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but are Transcriptionally and Biologically Different [I]
Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but are Transcriptionally and Biologically Different
