hfMSCs (2×105 cells/well) were cultured in cell pellets. Pellets were formed by centrifugation of the cells at 1200 rpm for 4 minutes in U-shaped 96-well suspension culture plates (Greiner). To induce chondrogenesis the pellets were cultured at 37°C with 5% CO2 in 200 ul of serum-free chondrogenic medium consisting of high-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 40 ug/ml proline (Sigma), 100 ug/ml sodium pyruvate (Sigma, USA), 50 mg/ml ITS (insulin-transferrin-selenic acid) with Premix (BD Biosciences), 1% Glutamax (Gibco), 1% penicillin/streptavidin, 50 ug/ml ascorbate-2-phosphate (Sigma), 10-7 M dexamethasone (Sigma), 10 ng/ml transforming growth factor-beta 3 (TGF-beta 3; R&D Systems), 500 ng/ml bone morphogenetic protein 6 (BMP6) and antibiotic and antimycotic mix (0.06% polymixin, 0.2% kanamycin, 0.2% penicillin, 0.2% streptavidin, 0.02% nystatin and 0.5% amphotericin) as described by Sekiya et al., 2001. The medium was changed twice per week for 5 weeks.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolation was performed with an optimized method for RNA extraction from cartilage as described by Heinrichs et al. (1994) except that the protocol was started by homogenizing the sections in 1 ml guanidine thiocyanate solution. RNA extraction was followed by purification with a RNeasy kit according to the manufacturers protocol (Qiagen)
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
Scan protocol
Affymetrix GeneArray Scanner3000
Description
direct isolation
Data processing
The data were analyzed with a commercial software called JMP Genomics, version 3.1, from SAS. Gene expression profiling was performed using arrays of human genome 133 Plus 2.0 -type from Affymetrix. A Custom CDF Version 10 with Entrez based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization.
