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

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

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

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 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

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

Adipocyte Differentiation of hiPS cells Adipocyte Progenitors Day30 Day0 Day10 Day20 EB formation Adipocyte Progenitors Mesoderm Ectoderm Endoderm D0 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

Adipocyte Differentiation of hiPS cells 8

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

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

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

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

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

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”

TGF Pathway Inhibitor and EGF promote hiPS-AP Adipocyte Differentiation hiPSC-APs hiPSC-APs hAT-APs EGF (10ng/ml) + SB431542 (5 M) 15

Brown Adipocytes Derived from hiPSCs are Functionnal hiPSCs-BAPs * Insulin - 5min 10 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

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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