Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous

Abstract

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.

Regulation of adiponectin release and demonstration of adiponectin mRNA as well as release by the non-fat cells of human omental adipose tissue

“…25,26 The SVcell pellet is suspended in omental preadipocyte medium (DMEM/F12, HEPES pH 7.4, FBS, biotin, pantothenate, insulin, dexamethasone and antibiotics) and plated in a culture flask for expansion. Preadipocytes are subcultured prior to reaching confluence and plated at a density of 6300 cells cm À2 for further expansion in preadipocyte media containing growth factors.…”

Human adipose tissue‐derived mesenchymal stem cells improve cognitive function and physical activity in ageing mice

“…ADMSCs can be easily harvested from patients by a simple and minimally invasive method and are more abundant than other sources such as bone marrow-derived MSCs (BMMSCs; Fraser et al, 2006;Parker and Katz, 2006). Adipose tissue contains hundreds of thousands of MSCs in each gram of fat (Sen et al, 2001), whereas BMMSCs in the bone marrow fraction constitute a mere 0.0001-0.01% of all nucleated cells (Pittenger et al, 1999). In particular, ADMSCs differentiate into several cell types (Constantin et al, 2009), and, unlike embryonic stem cells, are an ethically uncontroversial source for stem cell therapy (Díaz-Prado et al, 2011).…”

PPARγ activity in subcutaneous abdominal fat tissue and fat mass gain during short-term overfeeding

“…The study enclosed a 7-day baseline period (days 1-8) and a 14-day overfeeding period (days [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. During the baseline period subjects chose their diets from a variety of food items provided daily in weighed food packages, bringing back the leftovers the next day, for calculation of habitual energy intake.…”

“…AP2 mRNA levels are primary regulated by PPARg and are shown to be a quantitative measure of PPARg activity, 7,8 therefore PPARg mRNA levels did reflect PPARg activity in vivo. This suggests that the ability to increase PPARg activity might be involved in the susceptibility to gain weight during overfeeding.…”

“…The adipocyte-specific fatty acidbinding protein (aP2) is primary a PPARg response gene and is therefore commonly used as an adipocyte-specific marker in vitro. 7,8 In addition, PPARg is involved in the regulation of the uncoupling protein 2 (UCP2) as PPARg agonists are shown to increase UCP2 expression in adipose tissue. [9][10][11] UCP2 mRNA levels are reduced in adipose tissue from obese compared to lean persons, 12 which suggests a role for UCP2 in obesity by influencing energy metabolism although a specific uncoupling function has not been confirmed in humans.…”