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The Main Tea Eta Anton Alto Da Utama Anait THE MAIN TEA ETA USANTON 20170266145A1 ALTO DA UTAMA ANAIT ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2017/ 0266145 A1 NAHMIAS et al. (43 ) Pub . Date : Sep . 21, 2017 ( 54 ) METHODS OF INDUCING METABOLIC Publication Classification MATURATION OF HUMAN PLURIPOTENT (51 ) Int. CI. STEM CELLS -DERIVED HEPATOCYTES A61K 31 /201 (2006 . 01) ( 71 ) Applicant: Yissum Research Development GOIN 33 /50 (2006 . 01) Company of the Hebrew University of ( 52 ) U . S . Cl. CPC .. .. A61K 31/ 201 ( 2013. 01 ) ; GOIN 33 / 5014 Jerusalem Ltd ., Jerusalem ( IL ) ( 2013 .01 ) ( 72 ) Inventors : Yaakov NAHMIAS, Rishon LeZion (57 ) ABSTRACT ( IL ) ; Michal ZIMERMAN , Caesarea Provided are methods of increasing metabolic maturation of ( IL ) ; Gahl LEVY , Ramat- Gan ( IL ) ; an immature hepatocyte , by contacting an immature hepato Yishai AVIOR , Rosh HaAyin ( IL ) cyte which expresses alpha - fetoprotein ( AFP ) and albumin with an effective amount of a fatty acid or a small molecule ( 21 ) Appl . No . : 15 /459 , 139 selected from the group consisting of: an amphipathic car boxylic acid , Thiazolidinedione ( TZD ) , WY - 14643 (Pirin ( 22 ) Filed : Mar . 15 , 2017 ixic Acid ) , GW409544 , GW6471 , Leukotriene B4 , GW 7647 , Perfluorooctanesulfonic Acid , Perfluorooctanoic Acid , Related U . S . Application Data CP - 775146 , CP - 865520 , UNII - 999KY5ZIGB , and Gemfi (60 ) Provisional application No . 62 / 308 , 372 , filed on Mar . brozil . Also provided are isolated hepatocytes and uses 15 , 2016 . thereof. Patent Application Publication Sep . 21 , 2017 Sheet 1 of 11 US 2017 / 0266145 A1 Fig . A Differentiation Protocol Activin A , 323 00X AGM asuline 23 DEX OSM , FGF2 Insulin TATTETETTTTT + tutatatatatatatatatatatatatatet 777 Fig . 1B Days 2 1012 14 Day 0 Day 3 Day 7 Day 12 Day 16 Fig . 10 Ta Fetal Differentiated Hepatocytes Hepatocytes Oct4 Stem Cells Endoderm . Sox2 Phase fromfrontage.coza .. 0 4 8 12 16 Fig . 1D * Sox17 OCT4 -- - -- - Gata4 . * FOXA2 - HNF40 SOX17 111111111111111 RelativeMRNAExpression HAW w 0 4 8 12 16 Fig . 1E XXXN * -- - - -- AFP * mm. Albumin HNFAFOXA2GATA4 -- - * -- A1AT / 1.. : wwww k 4 8 12 16 Oleme Differentiation ( Days ) Fig . 1F Fig . 1G Fig . 1H Day 3 - Endoderin 3ay ? » Hepa??????? Day 16 - Hepatocytes * * ** * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * + + + + + + + + OC % 41 % 90% 83 % 13 % 83 % SOX17 HNF4A . INF4A 2 % 2 % 15 % 3 % 4060 1999999999999999999999999999999999999999 CXCR4 FOXA2 Albumin Patent Application Publication Sep . 21 , 2017 Sheet 2 of 11 US 2017 / 0266145 A1 Fig . 2A DPHH WESC -HD16 HepG2 Fig . 2B CPHH 3 ? ESC- H D16 :48pc RelativemRNA Expression * I I 888 0 CAR FXR PXR MORI MAPI CYP1A2CYP2C9 CYP2D6 CYS2E1 CYP3A4 HNFXa | PPARA Albumin ALAT Vuclear Receptors Transporters Prese ! Nulcear Receptors Plasma Proteins LLLLLLLLLL 01 thin Fig . 2E Fig . 2C Fig . 20100 SPYR PXR Lithocholic Acid SCAR CAR ExpressionLog) BCYP2C9 * * * CYP2C9 . ECYP3A4 CYP3A4 . Vitamin K , (MK4 ) RelativemRNA RelativemRNA Expression wat . .. 0 10 20 50 0 10 50 100 Fig . 2F { {{ p aís Acid { { } } Fig . 2G Vitamin Ky - MK4 (UM ) * * EXE SUNF40 * * - CAR SPXR - HESC ( DO ) W CYP3A4 CYP2C9 NESC -FH (112 ) RelativemRNA voissadx3 hESC - H (016 ) TITIT FIL DESC - H (016 ) +LCAMK4 © . 0 10 20 30 40 50 heSC -+ (D16 ) + MK4 * LCA MK4 + LCA PXR Transcriptional Activity Fig . 2H Fig . 21 DESC - H (116 ) * * OFXR OHNF4A CAR DESC - H (116 ) * * * V * * * * * LCAMKA RNARe?v} OPXR PAH voissaidx3 OCYP3A4 WCYP2C9 0 % 50 % 100 % O 100100 200 Nuclear Localization Silibinin (UM ) Patent Application Publication Sep . 21 , 2017 Sheet 3 of 11 US 2017 / 0266145 A1 F} FH * F1-11 FHH " GPTI ASGRI CYP3A4 . PHKLOAMK4ControlFHH w PHHLCAMK4Coniro PHILCA/UK4Control PHHLOANAX4Control PHHLCAMK4Control mm DuCYP3A7 du RFC3 di 1200- 800 100 50 150 100 120 Fig.3F Fig.3E 0.50 710. www FHH Control 1200 Contro MK4! LCA 700 MK4/ LCA PHH 0 Fig.3D PHH 17 17 17 + ed .. f . s many from wwwww l fawn a 74444444444444444444 Differentiation(Days) The Differentiation(Days) . Differentiation(Days) 3111315 .9111315 *rm ?9111315 PL-secretion PHHsecretion n omwwwwww .00 7 Fig.30 en hau i o Ö D o 5 { wiajosd 6wwron ) (wiajoid Swwn) } Y???in ??????Lm???} { Albumin dy Ap08100 Fig.3A Fig.3B Patent Application Publication Sep . 21 , 2017 Sheet 4 of 11 US 2017 / 0266145 A1 Fig . 4A Fig . 48 Fig . 4C { F } Control LCAMK4 Omeprazole (72 hrs ) B Control Control Rifampicin (72 xs ) 0 32 % DMSO HepG2 0 plem{ngpratn} A -. 1 . CYP450Activity 0 CY7450Activity{???le??????mgpaten RelativemRNA Expression m EROD BFC MFC ?????? BFC MFC ( 1A ) (3A4 , 1A2) 251, 289 ) ( 1A ) (3A4 , 1A2 ) ( 281, 289 ) PXR CYP3.4.4 Fig . 40 CYP261 CYP2D6CYP1A2 Auntaisex Balerie Militars 34 10 value { 3386) Kigos Work IKKENA canta * * * * * * * * * * . ? ???????? Acetaminophen 3365 + 547 2061 + 2150 Casascos saavoit Front XQX* * * * * * * WISHA Diclofenac 11614144 32411313 Aflatoxin 81 16 + 7 34 + 27 Worx Troglitazone 13619 9262141 ? ?????????????? ???????????????????? AREX Cssxosdiosx Chlorpromazine 04 : 11 7575427 comybszörvae dok Artiodarossa WEESSIISKI Acetylsalicylic Acid 6155 + 1020 n 742421539 Amiodaronie 686257 20604630 mm KOOYEN Valproic Acid 269 # 113 14810197 cocodromu kwa Cox xotos: Ky X nenne Melatonin 699 + 223 1 0071585 Hemmin Barrotex : ??Y; } NA NA. innenennen Mannitoi NAM . A . MN .. A . * OMWA Coordancia Fig . 4E Fig . 4F . DPH 0000 . Aflatoxin B1 . Chlorpromazine LCAMK4 ** Control O **** * * Amiodarone 000 Q HepG2 **** * Troglitazone ** * (wsl)301.601 Diclofenac PrimaryHumanHepatocytes omtome an *** ** * 2013 *** * * 111 *** * * * ** * * * **** * * Valproic Acid .. Acetaminophien Control R2= 0 . 19 Acetylsalicylic Acid . OLCA /MK4 R2= 0 . 94 " . .. 10000 100000 1910 100 02 10 100 1000 hES - derived Hepatcytes Log TC ( UM ) log TCso (UM ) Patent Application Publication Sep . 21 , 2017 Sheet 5 of 11 US 2017 / 0266145 A1 Fig . 58 Fig . SA LipidroX / Moechst Control * Control Amiodarone Valoroic Acetylsalicilic Amiodarone Acid SE Acids Valproic Acid Acetylsalicylic Acid - - - - - - - - - - - - | y . 0 0 . 1 0 . 2 03 0 . 4 0 .5 0 . 6 intracellular Lipids ( r. 1 . Fig . SC fig , SD COFOA / Hoechst {Contact * ** * * * * Control Troglitazone Oyclosporine A Chlorpromazine Troglitazonie Cyclosporin A Chlorpromazine 0 0 . 2 0 . 4 0 . 6 0 . 8 1 Bile Secretion (ru . } Fig . SF TUNEL Hoechst Fig . Se ? . ????? . " ControlContro ! W Control | Aflatoxin - B , Diclofenac si Acetaminophen Aflatoxin B1 * Diclofenac w * Acetaminophen * 0 % 10 % 20 % 30 % 40 % Apoptotic Index ( % ) Fig . 51 LipidroX * ! COFO / Fig . SG Contro Hoechst Hoechst Melatonin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 0 0traceluar . 2 0 . 4 Lipids0 . 5 0 . 9 Fig . SH Control Melatonin 0 . 5 1 1. 5 2 Site Secretion Patent Application Publication Sep . 21 , 2017 Sheet 6 of 11 US 2017 / 0266145 A1 100 30 AFS PositiveCelis(%) ApoptoticNuclei1%} GW9662LA+OA QA11A 50 1020 3 Control Salurin proteins roling yg ( Albumin EROD (1.4) 0 Fig.60 6 - Fig.6G ) UM( LA + OA 0 UM LA+ OA AFP PlasmaProteins 8FC (A) Abunin 80A-LA RHepatocytes (281,209) C#11a MIC Control 20A-SOLA *HepG2 SOA+LAGW9682 Q0A+LA LipidMetabolism 9 Fig.6CAllumin/AprMoects: Control FFAR { {{ {{ { ng? } . , , , , , , , , , , , , Activity CYP450 20. Fig.6J in Ô p E Expression Gene Relative Fig.6 CYP3A4 ? * I CPTIA A? * CYP3A4 BAAT CYP2C9 262UMOA+LA 2500MCALA VOA-9CLA+GW9862 Control &125umCALA PARA LXRCI FXR EXR Control W0A+LA 09+90A PXR : . 4: NY * * i Expression. N Expressius coo pression wo Expressioni Uos Batejax Fig.6B Gene Relative Ce???? Gone Relative ? ?? ????? 62125250 62125250 621ZS250 12125250 QA+LA(UM) * GW9562+9CLA 0A 0 0 0 0 . : . e N come to o o o o o o O ?????LA+DA Activity CHE Reative Activity LKRE Relative Activity PXRE Relative Activity FXPE Relative Fig.6H Control Fig.GA Localization Muclear Control OA GATE BIANO PXR Kelativa Patent Application Publication Sep . 21 , 2017 Sheet 7 of 11 US 2017 / 0266145 A1 SOA+LAOW966? OASCLA Control CA+LA KOA+9CLA FIS1 Pro-Fission MFN? OALA * Pro-Fusion Control Poci? Biogenesis 2.0 0. Fig.71 101 ) % ( Nuclei Apoptotic Expressiosi Gene Relative 1.2 1.2 1. 1.5 * ).1 ? 75 ? * OA+LA Hoechst * MinormitochondriaAxisfoldchange) Eccentricity(Foldchange) E!0. 0.8 Fig.19MajorkitochondriaAxis(faidchange) 0.50.5 Actinhoechst 0.651 }{$3ReativeExpression 0.606 0.9 0 LAOA+ OA+LA ortro OA+9CLA OA+LA 7DFig. OA+90L4 Control Control 500OA+LA 04+9CLA Control CA+9CLA i Contiol +9CLAGW9662 Fig.7H Fig.7 Hig OA+9CLAGW9662 OA+9CLAGW9662 Bin - 500 * - Celluiarare3 . - 400 *Nucleararea#Nucleararea 400 - - MitochondriaDiameter(mm) - 200300 Area(um2) 100200300 - - - 100 - 0 0 Fig.7B Fig.7C Contro! OAand 9C.A Control OAand OAand SCLA OAandLA Fig.7A Patent Application Publication Sep . 21, 2017 Sheet 8 of 11 US 2017 /0266145 A1 80 * Control OA+LA MOA+OCLA -Hepatocytes 70 60 MaximalRespiration Rotenone& AntimycinA * 304050 Time(min) FCCP ATPproduction Oligomycin 20 * 10 JAVCLA Fig.8A Fig.8B 80A+LA Hepatocytes BasalRespiration 0 100=6Control Ô ? ) cells 104 / min/pmol ( OCR ) cells 104 / min/ pmol ( OCR Patent Application Publication Sep . 21 , 2017 Sheet 9 of 11 US 2017 / 0266145 A1 Fig.9 U21 hipsCs 12F2 * * * * * * * * * * * * HUES8 H9 HESCs DAFP Albumin OCYP3A4 13 o Expression mRNA Relative Patent Application Publication Sep . 21 , 2017 Sheet 10 of 11 US 2017 / 0266145 A1 10 / 11 . Fig.10 ! biogenesis andanothing Mitochondria developmentandfusion PGC1 stationenir ritmo
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