No abstract

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1/cs-CD34/cs-CD146 ASCs, ~7.5% cs-DLK1/cs-CD34/cs-CD146 and ~7.5% cs-DLK1/cs-CD34/cs-CD146 cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146 endothelial cells (CD31/CD34/CD146) and pericytes (CD31/CD34/CD146 ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34 and contained low amounts of intracellular CD146 protein (CD31/CD34/CD146). Additionally, we detected wavy CD34/CD146 and CD34/CD146 cells. CD34/CD146 cells were slightly more bulky than CD34/CD146 cells. Both CD34/CD146 and CD34/CD146 cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34/CD146 and CD34/CD146 cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1/cs-CD34/cs-CD146 ASCs.

Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells.  Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells  into functional adipocytes.