Adipose-derived stromal/stem cells (ASC)

Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our recent experience isolating and immunophenotypically characterizing ASCs from >60 human subjects Total RNA was isolated from ASCs obtained from three individual donors and maintained under uninduced or adipogenic induced culture conditions for 9 days following confluence. The RNA was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), cDNA synthesized using a Superscript cDNA Synthesis kit (Invitrogen, Carlsbad, CA) and a T7-(dT)24 primer. Biotinylated cRNA was transcribed in vitro using a GeneChip IVT Labeling kit (Affymetrix, Santa Clara, CA), purified using a GeneChip Sample Cleanup Module, incubated in fragmentation buffer (200 mM Tris-acetate pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94oC for 35 min, chilled on ice, and hybridized to a Human U133 Plus 2.0 Array of ~47,400 transcripts and variants, including 38,500 well-characterized genes. Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000. Pixel intensities were measured, expression signals were globally scaled to a target intensity value of 2500, and features extracted using GeneChip Operating Software v1.2 (Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool v.3 (Affymetrix) algorithms. The absolute call (present, marginal, absent) of each gene expression, the direction of change, and the fold change were identified with the above software.