Human induced Pluripotent Stem Cells:

A new source for Brown Adipocytes

Christian DaniTeam “Stem Cells and Differentiation”

Faculty of Medicine, iBV, CNRS/Inserm, Nice, France

Targeting Diabetes and Novel Therapeutics 2015

The Energy Intake and Energy Expenditure Balance

ObesityType 2 Diabetes…

How to increase energy expenditure ?

Different Types of Adipocytes in Humans

Energy storage

Obesity, lipoatrophy

White AdipocytesBrown Adipocytes

Energy expenditure

Thermogenesis: UCP1

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 ?

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.

Human induced Pluripotent Stem Cells

Somatic cell: Skin fibroblasts

Embryonic Stem cells

Enucleated donor oocyte

Blastocyst

Inner cellmass

iPSCs/hESCs

Oct4Sox2Klf4c-myc

hiPSCs hESCs

Obese patientsDiabetic patients

Generation of iPS Clones from Human Fibroblasts

Fibroblasts

Oct4, Sox2, Klf4, c-Myc

TRA1-60

Oct4

Clone 1 Clone 7

TRA1-60

Oct4

Clone 39

hiPSc clones

hESC medium

Day0 Day10 Day20 Day30EB formation

Leptin

FABP4

D0 D30

GAPDH

Adipocyte Differentiation of hiPS cells

Mesoderm

Ectoderm

Endoderm

Adipocyte Progenitors

d8 d16

Fibroblasts

ßActin

PAX6 (Ecto.)

aFP (Endo.)

Brachyury (Meso.)

Nanog

K5 (Ecto.)

Fibroblasts-iPSc

Und.

Many other cell types

Adipocyte Differentiation of hiPS cells

iPS cells are Pluripotent

Form Teratomas in Vivo

Pancytokeratin rosette Vimentin

Epithelial cells Neuro-ectodermal cells mesenchymal cells

SCID mice Teratomas

Human fibroblast-iPSCs

Human fibroblasts

EB

Derivation of APs from hiPSCs

Derivation of CD73+ cellsfrom untreated or RA-treated iPSCs

Day 10 Day 30

EB formation Outgrowths

Day 3 Day 5

Adipogenic mediumDay 20

± Retinoic Acid (10-7M )

FABP4

Tubulin

UCP1

BAPs (-RA) WAPs (+RA)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Pax3 Dio2 HOXC8 BMP4 HOXA5

hiPSC-BAPs

hiPSC-WAPs

Expr

essio

n re

lativ

e to

TBP

Expr

essio

n re

lativ

e to

TBP

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Pax3 Dio2 HOXC8 BMP4 HOXA5

hBAPs (Chin AT)

hWAPs (Knee AT)

Molecular Characterization of BAPs and WAPs

hiPSCS

Adult Adipose tissue

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

Adipocytes in hiPSC cultures

BAP derivation hiPSC-conditioned medium

Critical Role of the hiPSC Niche for Brown Adipocyte Differentiation

Pos Neg

FGF2

IGFBP-2 IGFBP-3 IGF-II

GCSF GM-GCSF

EGF PDGFAA

PDGFAB

PDGFBB

TGF-1

Pos Neg

PDGFAA

PDGFAB

PDGFBB

EGF

GCSF GM-GCSF

TGF-1

IGF-II

(RayBio Human growth Factor Antibody Array, containing 40 Abs)

Cytokines secreted by the AP “niche” during hiPSC differentiation

BAP “niche” WAP “niche”

hiPSC-APs

EGF (10ng/ml)+

SB431542 (5 M)

TGF Pathway Inhibitor and EGF promote hiPS-AP Adipocyte Differentiation

hiPSC-APs hAT-APs

hiPSCs-BAPs

pAKT

Total AKT

pErk1/2

Tubulin

IRS1-pTyr

Total IRS1

Insulin - 5min 10 min

UCP1

Exp

ress

ion

rela

tive

to u

n-s

timul

ated

0

1

2

3

4

5

- +FSK

*

Brown Adipocytes Derived from hiPSCs are Functionnal

0

5

10

15

20

25

30

35

40**

Gly

cero

l rel

eas

e (

nm

ol/m

g)

- +FSK

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.

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