Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.
The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.
Sen
A
A
Zen-Bio, Inc., Research Triangle Park, North Carolina 27709, USA. asen@zen-bio.com
Lea-Currie
Y R
YR
Sujkowska
D
D
Franklin
D M
DM
Wilkison
W O
WO
Halvorsen
Y D
YD
Gimble
J M
JM
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D000328:Adult; D001391:Azo Compounds / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D020738:Leptin / Q000378:metabolism; D009363:Neoplasm Proteins; D009765:Obesity / Q000378:metabolism; D010078:Oxazines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins
D001391:Azo Compounds; D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes; D020738:Leptin; D009363:Neoplasm Proteins; D010078:Oxazines; D025521:Tumor Suppressor Proteins; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C011049:oil red O; C044808:nile red