Christian Dani Team “ Stem Cells and Differentiation ”

1. Human Adipocyte Progenitors: Proliferation and Differentiation Christian Dani Team “Stem Cells and Differentiation” Faculty of Medicine, iBV, CNRS/Inserm, Nice, France Diabetic Medications 2015. Las Vegas USA

2. The Energy Intake and Energy Expenditure Balance Obesity Type 2 Diabetes … Factors regulating adipocyte generation in an obese context?

3. Human Adipose Stem Cells Adipocyte fraction Adipocytes Collagenase A digestion Chondrocytes Stromal-vascular fraction Endothelial cells, immune cells, preadipocytes, multipotent stem cells (adipocyte progenitors, adipose-derived stem cells). Osteoblasts hMADS Cells (Self-renewal) Therapeutic Potential: mdx mice (Rodriguez et al. 2005, J Ex Med) White Adipocytes Brown Adipocytes

4. Human Adipose Stem Cells Stromal fraction of human adipose tissue Adherent cells plated at clonal density Efficiency of colony formation: 12% Adipocyte+Osteoblast differentiations Osteoblasts only 4% Adipocytes only 24% Adipocytes+Osteoblasts 48% No adipocyte/osteoblast 24%

5. Adipose Stem Cells Adipose Stem Cells Adipocytes miRNA 30, C/EBPs, PPAR,.. Osteoblats

6. Role of miRNA 30 in Commitment of Adipose Stem Cells towards the Adipogenic Lineage

7. Day 8 Adipo Day 8 Osteo Differential Expression of miR30 in Adipogenesis and Osteogenesis

8. Effects of miR30 Knock-down on Adipocyte Differentiation Anti-miR control Anti-miR 30

9. Effects of miR30 knock-down on Adipogenic/Osteogenic Differentiation Anti miR control Anti-miR control Anti-miR 30

10. PremiR ctr PremiR 30a PremiR 30d Runx2 Tubulin Expression of RunX2 in Adipocyte Progenitors overexpressing miR30

11. -2 0 2 6 13 -2 0 2 21 10 Adipogenesis Osteogenesis Days RunX2 PPARg b-actin Expression of Runx2 During Adipocyte Progenitor Differentiation miR30

12. Adipose Stem Cells Adipose Stem Cells Adipocytes miRNA 30, C/EBPs, PPAR,.. ? Osteoblats

13. Adipocyte Progenitor Proliferation is Required for Adipocyte Renewal Growth of white adipose tissue in obesity is the result of an increase in number adipocyte number. Given that adipocytes do not divide, this increase depends on the proliferation of a pool of adipocyte progenitors. A pool of adipocyte progenitors remains present in adipose tissue during adult life. This pool is responsible for regeneration of adipocytes and the potential of this tissue to expand in response to chronic energy excess. However, factors controlling proliferation of human adipocyte progenitors in obesity are largely unknown. Identification of regulators of self-renewal could provide a means to understand the mechanisms that lead to obesity and type 2 diabetes.

14. Regulators of human adipocyte progenitor proliferation : FGF2, Activin A and IER3.

15. Regulators of hMADS Cell Proliferation We analyzed the transcriptome of hMADS cells and we screened for genes differentially expressed in undifferentiated and differentiated cells: FGF2, Activin A, IER3 FGF2, Activin A Control

16. FGF2

17. FGFR-3 FGFR-1 FGFR-2 FGFR-4 -Actin Expression of FGF2 and its receptor in undifferentiated hMADS cells RT-PCR Secretion of FGF2 WB 0.8 0.6 FGF2 fg FGF2/cell 0.4 0.2 0 Tubulin Hypothesis : autocrine FGF signalling participates in proliferation

18. No effect of: EGF, PDGF, FGF10 + FGF2 (2 ng/ml) Effect of FGF2 on hMADS cell Propagation 4.5 FGF2 4 Tubulin 3.5 3 2.5 doubling time (days) 2 1.5 1 0.5 0 20 25 10 15 number of passages

19. PD173074 a specific inhibitor of FGFR Control PD173074 Inhibition of FGF signalling during proliferation Impairs subsequent adipocyte differentiation

20. FGFR1 FGF2 Autocrine/Paracrine Loop Regulating Proliferation and Differentiation of human Adipose-derived Stem Cells FGF2 ERK1/2 Proliferation, Adipogenesis Human adipose tissue is a source of FGF (Mapping of the fibroblast growth factors in human white adipose tissue. Mejhert et al. J Clin Endocrinol Metab 2010). Excess of FGF2 in adipose tissue of obese people could contribute to increase the size of adipose precursor pool. Factors regulating FGF2 expression?

21. Activin A

22. 3 2.5 2 1.5 1 0.5 0 Expression of Activin A during Human Adipocyte Differentiation Expression in hMADS cells Expression in Adipose Tissue 2 1.5 Activin A secreted levels compared to d-3 INHBA expression (A.U) 1 0.5 0 d+15 Adip. d0 d+3 Progenitors Days relative to adipogenic induction

23. Activin A Promotes Proliferation and Inhibits Adipocyte Differentiation 200 160 Activin A : 0 120 80 Activin A: 100 ng/ml 40 * Number of cells per well (x1000) * Control Activin A -Activin A

24. QPCR C/EBPb C/EBPa PPARg SREBP GPDH FABP4 FABP5 GLUT4 CD36 LPL FASN HSL PEPCK UCP2 PAI1 IL6 Activin A - + 38 kD CEBP Tubulin -3.0 0.0 3.0 Log2 Suppression of Anti-Adipogenic Effects of Activin A by C/EBP GFP CEBP + + Activin A - - FABP4 tubulin

25. ActRI ActRII Activin A is a Critical Regulator of Human Adipocyte ProgenitorProliferation and Differentiation Activin A Smad2 C/EBP Smad2 EX Differentiation Proliferation (Zaragosi et al. Diabetes, 2010)

26. 7 6 5 4 3 2 1 0 Lean Subjects Obese Patients * Activin A expression (A.U.) Lean Obese Macrophages Activin A, FGF2 Activin A, FGF2

27. control FGF2 inhba Activin A and FGF2 are Regulated by Macrophage-Secreted Factors hMADS cells * 6 5 4 Relative gene expression 3 ** 2 CD14+ Macrophages 1 0 Ctr ATM-CM Conditioned Media (ATM-CM)

28. Regulation of Adipocyte Progenitor Proliferation and Differentiation by Macrophage Factors. Macrophages TGFb1 Activin A, FGF2 Adipocyte Progenitors

29. IER3

30. IER3 is Preferentially Expressed in Adipocyte Progenitors and is Increased by an Obese Microenvironment

31. IER3 Expression is Regulated by Actvin A, Hypoxia and Macrophages Factors

32. IER3 mediates Hypoxic and Activin-A-induced proliferation

33. Activin A, FGF2 and Macrophages support Adipocyte Progenitor Expansion through IER3 expression ACTIVIN A FGF2 ERK1/2 Smad2 MACROPHAGES IER3 PROLIFERATION HIF1 Hypoxia ADIPOCYTE PROGENITOR

34. « Stem Cells and Differentiation » Team