Adipocyte differentiation

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Human adipose-derived stem cell and preadipocyte differentiation to adipocyte

Time course ID: human_mesenchymal_stem_cells_adipose_derived, human_Adipocyte_differentiation

Sample provider: Peter Arner, Niklas Mejhert, Anna Ehrlund, Jurga Laurencikiene, Erik Arner

Introduction

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.

Samples

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.


HAdipogenesis timeline.png

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.

Growth conditions

SVF preadipocytes to adipocytes:

Cells were isolated from human adipose tissue as decribed in [3,4,5].

Culture medium used:
Day 1-6: DMEM (1mg/ml glucose), PEST, Fungizone, 15 mM HEPES, 100 nM Cortisol, 66 nM Insulin, 10 µg/ml Transferrin, 33 µM Biotin, 17 µM Panthothenate, 1 nM T3, 10 µM Rosiglitazone
Day 6-12: Same as above but without Rosiglitazone


hASC (human Adipose-derived mesenchymal stem cells):

Cells were isolated, expanded and differentiatied as described in [5,6,7].

Culture medium used:

Proliferation medium: DMEM (1 mg/ml glucose), PEST, 10% FBS, 15 mM HEPES, 2 mM Glutamine, 2.5 ng/ml FGF2
Differentiation start, day 0-3: DMEM/F12 (1:1), PEST, 5 µg/ml Insulin, 10 µg/ml Transferrin, 0.2 nM T3, 100 µM IBMX, 1 µM Dexamethasone, 1 µM Rosiglitazone
Differentiation day 3-9: DMEM/F12 (1:1), PEST, 5 µg/ml Insulin, 10 µg/ml Transferrin, 0.2 nM T3, 1 µM Rosiglitazone
Differentiation day 9-14: DMEM/F12 (1:1), PEST, 5 µg/ml Insulin, 10 µg/ml Transferrin, 0.2 nM T3


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).

HADSC dif bodipy.jpg





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:

Human Adipocyte differentiation marker genes.jpg

hASC, CAGE:

HADSC marker genes.jpg

hASC, qPCR

HADSCqPCRvalid.jpg


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

Primary preadipocyte isolation and differentiation

[3] van Harmelen V, Skurk T, Hauner H (2005) Primary culture and differentiation of human adipocyte precursor cells. Methods Mol Med 107:125–135

[4] Dicker A, Le Blanc K, Astrom G et al (2005) Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308:283–290

[5] Pettersson AM, Stenson BM, Lorente-Cebrián S, Andersson DP, Mejhert N, Krätzel J, Aström G, Dahlman I, Chibalin AV, Arner P, Laurencikiene J. LXR is a negative regulator of glucose uptake in human adipocytes. Diabetologia. 2013 Sep;56(9):2044-54.

Isolation, propagation and differentiation of hASCs

[5] Pettersson AM, Stenson BM, Lorente-Cebrián S, Andersson DP, Mejhert N, Krätzel J, Aström G, Dahlman I, Chibalin AV, Arner P, Laurencikiene J. LXR is a negative regulator of glucose uptake in human adipocytes. Diabetologia. 2013 Sep;56(9):2044-54.

[6] Rodriguez AM , Pisani D , Dechesne CA , et al.. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med. 2005;201:1397–1405.

[7] Zaragosi LE , Ailhaud G , Dani C. Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells. Stem Cells. 2006;24:2412–2419.

References on adipocyte differentiation in general

[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.

[9] Forming functional fat: a growing understanding of adipocyte differentiation. Cristancho AG, Lazar MA. Nat Rev Mol Cell Biol. 2011 Sep 28;12(11):722-34. PMID:21952300

[10] Propagation of adipogenic signals through an epigenomic transition state. Steger DJ, Grant GR, Schupp M, Tomaru T, Lefterova MI, Schug J, Manduchi E, Stoeckert CJ Jr, Lazar MA. Genes Dev. 2010 May 15;24(10):1035-44. PMID:20478996























































































Beginning of non-public section


QC of sequencing data:

Quality control of the hCAGE data have been performed at RIKEN by Erik Arner. In short, data was RLE normalised and the subjected to hierarchial clustering and principal component analysis to check that sample clustered according to differentiation time points. K-means clustering (k=10) was also performed to detect any "abnormal" clusters. A few replicats were filtered away after this before continueing further with analysis. Some adipogenic marker genes, including for example PPARgamma, the C/EBPs, FABP4 and PerilipinA, were also checked to confirm an expected expression pattern during differentiation.

Related samples

We have an extensive collection of data from obese and lean patients. These include gene expression from microarrays, clinical parameters related to metabolism including insulin sensitivity, adipokine release and lipolysis measurements.



Bioinformatics collaborators

  • Erik Arner


Data

hCAGE sequencing:

hCAGE Expression tables

Short RNA expression

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_mesenchymal_stem_cells_adipose_derived/miRNA/

Zenbu configurations and status

MARA based network results

MARA

ISMARA analysis results for Adipose-derived mesenchymal stem cells

All samples: http://ismara.unibas.ch/timecourses/adipocyte/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/adipogenic-induction-avgd/averaged_report/index.html

ISMARA analysis results for Preadipocytes

All samples: http://ismara.unibas.ch/timecourses/adipocyte_differentiation/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/adipocyte-diff-avgd/averaged_report/index.html


Paper outline

This section is included as a starting point for discussions and is of course a work-in-progress. Suggestions are welcome!

Tentative title:

Transcriptional dynamics during human adipogenesis

Figure 1.
a) Description of system and time points: Anna
b) Profile of adipogenic marker gene expression (to show proper differentiation), CAGE + qPCR: Anna

c) Microscope (staining)

d) Expression dynamics during time course - K-means cluster - CAGE, miRNA and motif activity: Erik

Supp fig 1

Correlation plots, maybe PCA

Figure 2.
Early events.
In-depth analysis of the first 3 h.
Analysis of "early" K-means cluster. (Pathways and such)
Early governors (MARA)

Pathways?

Which/how many genes/TFs/miRNAs/enhancers/lncRNAs are expressed early?

How many are induced?

Expression of epigenes and connection to enhancers

Motif enrichment in enhancers

Figure 3.
Transient events.
Analysis of "transient" K-means cluster. (Pathways and such)
Transient governors (MARA)

GR, cAMP response (ChIP from mouse also available)

Network analysis: pathway analysis of predicted edges that overlap ENCODE

Figure 4.
Late events
As above

Rosen's data - PPARG and CEBPA

Figure 5.
Other:

miRNA

TSS swiching

lncRNA

Figure 6.
Overlap with genes changed in obesity.

GWAS SNPs in enhancers/promoters?
Map pathways if possible
Conclusions

Figure 7.
Model/pathway cartoon with mechanisms


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