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Preadipocyte to adipocyte

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Series:IN_VITRO DIFFERENTIATION SERIES
Species:Human (Homo sapiens)
Genomic View:Zenbu
Expression table:TET
Link to TET:FILE
Sample providers :Peter Arner
Germ layer:ectoderm
Primary cells or cell line:primary cells
Time span:12 days
Number of time points:3


Overview

Adipose tissue can account for between 5% (lean athletes) and 60% (morbidly obese) of total body mass, making it one of the most plastic organs in the body [8]. In response to changed nutritional status, both adipocyte cell number and size change and even under stable conditions as much as 10% of the adipocytes are turned over annually [1,2]. Thus the birth of new adipocytes from precursor cells (adipogenesis) is central for a functional fat tissue.

References:
[1] Dynamics of fat cell turnover in humans. Spalding KL et al, Nature, 2008, vol 453, p783-787. PMID:18454136
[2] Adipocyte turnover:relevance to human adiopose tissue morphology- Arner E et al, Diabetes, 2010,59(1):105-9. PMID:19846802
[8] Cawthorn WP, Scheller EL, MacDougald OA., Adipose tissue stem cells meet preadipocyte commitment: going back to the future. J Lipid Res. 2012 Feb;53(2):227-46.

Sample description

We are using two different models of human adipogenesis in the FANTOM5 study. Both originate from the stromal-vascular fraction (SVF) of human subcutaneous adipose tissue. The first model, here called preadipocytes, consists of cells differentiated in vitro directly after SVF isolation from adipose tissue. This means only later stages of differentiation can be monitored as samples in the beginning contain other cell types, like immune and endothelial cells, that contaminates the results. These contaminants die off after approximately 3-4 days. For this model we have taken tissue from four donors and sampled differentiation at time-points 4, 8 and 12 days after differentiation start. We have also isolated mature adipocytes from the adipose tissue of these donors. For the other model system, here called human adipose-derived mesenchymal stem cells (hASC), we have propagated cells from the SVF in vitro thus selecting for a stem cell/progenitor population. These cells can be expanded and cultured for several passages. This gives us cells with a homogenous genetic background and since contaminants are removed from the beginning, differentiation can be monitored from start to finish. Here we have collected triplicate samples at 0, 15, 30, 45 min, 1 h, 1 h 20 min, 1 h 40 min, 2 h, 2 h 30 min, 3 h, 12 h and 1, 2, 4, 8, 12 and 14 days after differentiation start.



Figure 1. Sample collection scheme for the different model system. Sample collection time points are indicated as well as the removal of some components of the differentiation cocktail. IBMX=3-isobutyl-1-methylxanthine, Dexam.=dexamethasone, Rosig.=Rosiglitazone.

Quality control

A key aspect of adipocyte maturation is the accumulation of lipid in intracellular lipid droplets. The accumulation of lipids can be seen as morphological changes in the microscope at low magnification (10x) and can be further visualized using lipid stains such as bodipy (shown below for day 8 and 12).


Figure 2: Lipid accumulation during differentiation. hASC cells were stained with Hoechst (nuclei, blue) day 0, 8 and 12 and Bodipy (neutral lipids, green) at day 8 and 12 during differentiation. An overlay of phase contrast and fluorescent image is shown.

Marker gene expression:

Preadipocyte, CAGE:



hASC, CAGE:


hASC, qPCR:



Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples



13019-139D4Adipocyte differentiationday04donor1
13020-139D5Adipocyte differentiationday08donor1
13021-139D6Adipocyte differentiationday12donor1
13022-139D7Adipocyte differentiationday04donor2
13023-139D8Adipocyte differentiationday08donor2
13024-139D9Adipocyte differentiationday12donor2
13025-139E1Adipocyte differentiationday04donor3
13026-139E2Adipocyte differentiationday08donor3
13027-139E3Adipocyte differentiationday12donor3
13028-139E4Adipocyte differentiationday04donor4
13029-139E5Adipocyte differentiationday08donor4
13030-139E6Adipocyte differentiationday12donor4