1. Human induced Pluripotent Stem Cells: A new source for Brown Adipocytes
Christian Dani
Team “Stem Cells and Differentiation”
Faculty of Medicine, iBV, CNRS/Inserm, Nice, France
Targeting Diabetes and Novel Therapeutics 2015 1 1

2. The Energy Intake and Energy Expenditure Balance
Obesity
Type 2 Diabetes …
How to increase energy expenditure ? 2

3. Different Types of Adipocytes in Humans
Brown Adipocytes
White Adipocytes
Energy expenditure
Thermogenesis: UCP1
Energy storage
Obesity, lipoatrophy
Beige Adipocytes
Energy expenditure
UCP1
Studies in rodents have shown that activation of BAT has healthy metabolic consequences in regard to obesity and type 2 diabetes
How to expand BAT in Humans ? 3

4. The Beneficial Effects of Brown Fat Transplantation
Strategies for expanding BAT:
Genetic intervention to trigger BAT/Beige differentiation: Gene therapy difficult to apply in clinic
Stimulate differentiation pathways to drive a white to brown adipocyte transition, to activate mitochondria function, using drugs, nutrients,..
Three labs have reported that adult BAT transplantation in mice could reverse metabolic disorders:
Gunawardana et al.: Insulin-independent reversal of type 1 diabetes in non-obese diabetic mice with BAT transplant. Am J Physiol Endocrinol Metab 2014
Stanford et al. : BAT regulates glucose homeostasis and insulin sensitivity. J Clin. Inves. 2013
Liu et al.: BAT transplantation improves whole-body energy metabolism. Cell res. 2013
Liu et al.: BAT transplantation reverses obesity in Ob/Ob mice. Endocrinology 2015
BAT Transplantation is an alternative approach. Autologous BAT transplantation in Humans requires an unlimited source of brown adipocytes: human iPS cells.

5. Human induced Pluripotent Stem Cells
Somatic cell: Skin fibroblasts
Blastocyst
Inner cell
mass
Enucleated
donor oocyte
Oct4
Sox2
Klf4
c-myc
iPSCs/hESCs
Embryonic Stem cells
Obese patients
Diabetic patients
hiPSCs
hESCs 5 5

6. Generation of iPS Clones from Human Fibroblasts
hESC medium
Oct4, Sox2, Klf4, c-Myc
hiPSc clones
Clone 1
Clone 7
Clone 39
TRA1-60
TRA1-60
Oct4
Oct4 6

7. Adipocyte Differentiation of hiPS cells Adipocyte Progenitors
Day30
Day0
Day10
Day20
EB formation
Adipocyte Progenitors
Mesoderm
Ectoderm
Endoderm
D D30
Fibroblasts
Fibroblasts-iPSc
FABP4
Und. d8
d16
Leptin
Nanog
Brachyury (Meso.)
GAPDH
aFP (Endo.)
PAX6 (Ecto.)
Many other cell types
K5 (Ecto.)
ßActin 7

8. Adipocyte Differentiation of hiPS cells8

9. iPS cells are Pluripotent
Form Teratomas in Vivo
Human fibroblast-iPSCs
Human fibroblasts
SCID mice
Teratomas
Vimentin
Pancytokeratin
rosette
Epithelial cells
Neuro-ectodermal cells
mesenchymal cells

10. Derivation of APs from hiPSCs
EB formation
Outgrowths
Adipogenic medium
Day 3
Day 5
Day 10
Day 20
Day 30 EB
± Retinoic Acid (10-7M )
Derivation of CD73+ cells
from untreated or RA-treated iPSCs
BAPs (-RA)
WAPs (+RA)
FABP4
Tubulin
UCP1 10

11. Molecular Characterization of BAPs and WAPs
0.2
0.4
0.6
0.8 1
1.2
1.4
1.6
1.8
hiPSCS
hiPSC-BAPs
hiPSC-WAPs
Expression relative to TBP
Pax3
Dio2
HOXC8
BMP4
HOXA5
1.8
Adult Adipose tissue
1.6
hBAPs (Chin AT)
hWAPs (Knee AT)
1.4
1.2
Expression relative to TBP 1
0.8
0.6
0.4
0.2
Pax3
Dio2
HOXC8
BMP4
HOXA5

12. Weak Adipogenic Ability of hiPSC-APs
compared to Adult APs
hiPSC-APs
Adult adipose-tissue APs
Observed by several authors, using different approaches to derive APS from hiPSCs or hESCs
Limited to adipogenesis as hiPSCs/hESCs-APs are able to differentiate into chondrocytes and osteoblasts at high levels 12

13. Critical Role of the hiPSC Niche for Brown Adipocyte Differentiation
Adipocytes
in hiPSC cultures
BAP derivation
hiPSC-conditioned medium 13

14. Cytokines secreted by the AP “niche” during hiPSC differentiation
(RayBio Human growth Factor Antibody Array, containing 40 Abs)
Pos
Neg
FGF2
IGFBP-2
IGFBP-3
IGF-II
GCSF
GM-GCSF
EGF
PDGF AA AB BB
TGF-1
BAP “niche”
WAP “niche”

15. TGF Pathway Inhibitor and EGF
promote hiPS-AP Adipocyte Differentiation
hiPSC-APs
hiPSC-APs
hAT-APs
EGF (10ng/ml) +
SB (5 M) 15

16. Brown Adipocytes Derived from hiPSCs are Functionnal
hiPSCs-BAPs *
Insulin min min 1 2 3 4 5 5 10 15 20 25 30 35 40 **
Glycerol release (nmol/mg)
FSK
pAKT
Total AKT
pErk1/2
UCP1 Expression relative to un-stimulated
IRS1-pTyr
Total IRS1
Tubulin
FSK

17. Conclusions
There is a great potential for brown adipocyte progenitors derived from hiPSCs to correct the energy imbalance in obese/diabetics patients via autologous transplantation.
There are still many challenges to overcome prior to clinical applications
These include to investigate the safety of hiPSC-BAPs, the need of an effective transplantation method, immunogenicity…
Generation of iPSC and derivation of BAPs are time consuming, require several steps difficult to apply in clinic.
hiPSCs is an abundant and unlimited source for brown adipocytes that open up a new avenue to treat obesity and metabolic disorders.

18. Labex 18 18