Web-based supplemental material, Spees et al.

Preparation of human and rat serum. Five hundred mL of whole blood was taken from each of 5 consenting donors who had previously donated bone marrowfor preparation of hMSCs. The protocol was approved by an Institutional Review Board. The blood was recovered into 600 mL blood bags (Baxter Fenwall, Deerfield, IL) in the absence of anti-coagulants and allowed to clot for 4 hr at room temperature. The serum (100 to 150 mL) was aspirated from the clot and centrifuged at 500 g for 20 min. The supernatant was then centrifuged for a further 20 min at 2,000 g. The cleared serum was incubated at 56oC for 20 min to deactivate complement and stored at –80oC. Medium containing the human serum was filtered through a 0.22 μm membrane (Stericup, Millipore) before use. For the production of rat serum, blood was drawn from adult Sprague Dawley rats by cardiac puncture. The serum was prepared from whole blood as described above but the final centrifugation step was 20 min at 4,000 g.

Combinatorial assays of serum free media. The baseline medium in all samples was α-MEM. In each experiment, a stack of three 12-well plates was used. In the first experiment, 10 ng mL-1 EGF and 10 ng mL-1 bFGF was added to all 36 wells. Then transferrin at 3, 6 or 9 μg mL-1 was added to wells in the y-axis; 2, 4, 6, or 8 mg mL-1 of linoleic acid was added in the x-axis; and 2, 4 or 6 mg mL-1 of human serum albumin (HSA) in the z-axis. Few viable cells were seen by microscopy after 12 to 14 days. In a second experiment, 2 mg mL-1 of HSA were added to the α-MEM in all 36 wells and the same components in the same concentrations were added in the y-axis and the x-axis; the z-axis varied to contain (a) 10 ng mL-1 insulin-like growth factor; (b) 10 ng mL-1 each of IGF, EGF and bFGF; and (c) 10 ng mL-1 EGF, 10 ng mL-1 bFGF, and 5 ng mL-1 platelet-driven growth factor-BB. Few viable cells were seen after 14 days. In a third experiment, 2 mg mL-1 of HSA were added to the α-MEM in all 36 wells and the same components in the same concentrations were added in the y-axis and the x-axis; the z-axis varied to contain 5, 7.5 or 10 ng mL-1 of stem cell factor. Again, few viable cells were seen at 14 days. All reagents were from Sigma Aldrich except for stem cell factor that was acquired from Chemicon, Temecula, CA.

Microarray analysis. Microarray assays were performed according to the manufacturer’s recommendations (Affymetrix GeneChip Expression Analysis Technical Manual; Affymetrix, Santa Clara, CA). In brief, 8 μg of total RNA was used to synthesize double-stranded DNA (Superscript Choice System Life Technologies, Rockville, MD). The DNA was purified by phenol/chloroform and concentrated by ethanol precipitation. In vitro transcription of biotin-labeled cRNA was performed using a commercial kit(BioArray HighYield RNA Transcription Labeling Kit; Enzo Diagnostics, Farmingdale, NY) and labeled cRNA was cleaned (RNeasy Mini Kit; Qiagen, Valencia, CA). Twenty-five μg of labeled cRNA was fragmented to 50 to 200 nucleotides and hybridized for 16 hr at 45˚C to an array (HG-U133A), which contains approximately 22,200 human genes. After washing, the array was probed with streptavidin-phycoerythrin (Molecular Probes), amplified by biotinylated anti-streptavidin (Vector Laboratories) and re-probed with streptavidin-phycoerythrin. The chip was then scanned (Hewlett-Packard GeneArray Scanner). The raw data were analyzed using Affymetrix MicroArray Suite v5.0 and Affymetrix Data Mining Tool v3.0. Signal intensities of all probe sets were scaled to the target value of 2,500.

Table 1: Comparison of differentially transcribed mRNAs from hMSCs grown in either standard medium containing 20% FCS or AHS+. Values are shown for both donors tested and represent the fold change in signal intensity for cells grown in AHS+ compared with the same cells grown in standard medium.

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