Maturation of mammalian hepatocytes

Abstract

The present invention relates to directed differentiation and maturation of mammalian hepatocytes, such as human hepatocytes. The hepatocyte obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown. In particular, the present invention relates to exposure of mammalian hepatocytes, such as human hepatocytes, to at least one maturation factor selected from the group consisting of Src kinase inhibitors, vitamin D including precursors, metabolites and analogs thereof, hypoxia inducing compounds, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), PKC inhibitors, and combinations thereof.

Claims

1. A method for promoting the maturation of human hepatocytes, which have been derived in vitro from human pluripotent stem cells or human hepatic progenitor cells, the method comprising: exposing said hepatocytes to at least one Src kinase inhibitor selected from the group consisting of 4-Amino-1-tert-butyl-3-(4-methylphenyl)pyrazolo[3,4-d]pyrimidine (PP1), 4-Amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2), 4-Amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-d]pyrimidine (1-NA PP1), 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1), Src Inhibitor-1 (Src-I1), Src Kinase Inhibitor I, Src Kinase Inhibitor II, A-419529, A-770041, AZM 475271, bosutinib, CGP77675, Damnacanthal, dasatinib, dasatinib monohydrate, ER 27319 maleate, Fingolimod (FTY720), Geldanamycin, Herbimycin A, KB SRC 4, KX2-391, KX1-004, Lavendustin A, Lavendustin C, LCK inhibitor 2, Lyn peptide inhibitor, MLR-1023, MNS, N-Acetyl-O-phosphono-Tyr-Glu Dipentylamide, N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu, NVP-BHG712, PD 166285, PD173952, PD 180970, pp60 c-src, quercetin, radicicol from Diheterospora chlamydosporia solid, saracatinib, SU 6656, TC-S 7003, TG 100572, WH-4-023, ZM 306416, and combinations thereof, optionally in combination with at least one maturation factor selected from the group consisting of vitamin D including precursors, metabolites and analogs thereof, hypoxia inducing compounds, sphingosine and sphingosine derivatives, activators of peroxisome proliferator-activated receptors (PPARs), platelet-activating factor (PAF), and PKC inhibitors.

2. Method according to claim 1, wherein said at least one Src kinase inhibitor is PP1, PP2 or a combination thereof.

3. The method according to claim 1, wherein said human hepatocytes are exposed to at least one vitamin D, vitamin D precursor, vitamin D metabolite or vitamin D analog.

4. The method according to claim 3, wherein said at least one vitamin D is selected from the group consisting of cholecalciferol, calcifediol, calcitriol, and combinations thereof.

5. The method according to claim 1, wherein said human hepatocytes are exposed to at least one hypoxia inducing compound.

6. The method according to claim 1, wherein said human hepatocytes are exposed to at least one hypoxia inducing compound selected from the group consisting of RAR-related orphan receptor alpha (ROR-alpha) ligands, CoCl.sub.2, and NaN.sub.3.

7. The method according to claim 6, wherein said at least one RAR-related orphan receptor alpha (ROR-alpha) ligand is selected from the group consisting of CGP52608, a CGP52608 analog, melatonin, melatonin analogs, cholesterol, cholesterol derivatives, and combinations thereof.

8. The method according to claim 1, wherein said human hepatocytes are exposed to at least one sphingosine or sphingosine derivative.

9. The method according to claim 8, wherein said sphingosine is D-erythro-sphingosine.

10. The method according to claim 8, wherein said sphingosine derivative is sphingosine-1-phosphate or sphingolipid.

11. The method according to claim 10, wherein said sphingolipid is a ceramide or a ceramide analog.

12. The method according to claim 11, wherein said ceramide analog is L-erythro MAPP or D-erythro MAPP.

13. The method according to claim 1, wherein said human hepatocytes are exposed to at least one activator of peroxisome proliferator-activated receptors (PPARs).

14. The method according to claim 13, wherein said at least one activator of peroxisome proliferator-activated receptors (PPARs) is selected from the group consisting of thiazolidinediones, free fatty acids (FFAs), eicosanoids including eicosanoid precursors and eicosanoid analog, and combinations thereof.

15. The method according to claim 13, wherein said at least one activator of peroxisome proliferator-activated receptors is at least one unsaturated fatty acid selected from the group consisting of IOZ-heptadecenoic acid, arachidonic acid (AA), 9(Z),II(E)-Conjugated Linoleic Acid, eicosadienoic acid, eicosatrienoic acid (ETE), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, docosadiennoic acid, adrenic acid, mead acid, ricinoleic acid, docosatrienoic acid, and combinations thereof.

16. The method according to claim 15, wherein said at least one activator of peroxisome proliferator-activated receptors is at least one saturated fatty acid selected from the group consisting of dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, and combinations thereof.

17. The method according to claim 16, wherein said at least one PKC inhibitor is selected from the group consisting of Bisindolylmaleimide I, Bisindolylmaleimide II, Bisindolylmaleimide III, Bisindolylmaleimide V, Bisindolylmaleimide VI, Bisindolylmaleimide VII, Bisindolylmaleimide VIII, Bisindolylmaleimide X, HBDDE, Rottlerin, Palmitoyl-DL-carnitine, R-Stearoyl Carnitine Chloride, Piceatannol, p-PKC antibody (H-9), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, dihydrochloride (H-8), 1-(5-Isoquinolinesulfonyl)-3-methylpiperazine, HA-100 dihydrochloride, N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), 1-(5-Isoquinolinylsulfonyl)homopiperazine (HA-1077), 5-Iodotubericidin, Ro-32-0432, Ro-31-7549, Enzastaurin (LY317615), Sotrastaurin, Dequalinium Chloride, Go 6976, Go 6983, Go 7874, Myricitrin, 4-Hydroxy-Tamoxifen,N-Desmethyltamoxifen HCl, Safingol, Phloretin, UCN-01, 7-Oxostaurosporine, K-252a, K-252b, K-252c, Melittin, Hispidin, Calphostin C, Ellagic acid, PKC Inhibitor Peptide 19-31, PKC Inhibitor Peptide 19-36, PKC epsilon Translocation Inhibitor II, EGF-R Fragment 651-658, PKC beta inhibitor (CAS 257879-35-9), PKC 20-28, PKCII/EGFR Inhibitor (CAS 145915-60-2), PKC Pseudosubstrate Inhibitor, PKC/ Inhibitor, [Ala107]-MBP (104-118), [Ala113]-MBP (104-118), zeta-Pseudosubstrate inhibitory peptide (ZIP), 1-(5-Isoquinolinesulfonyl)-3-methylpiperazine (C-1), Bryostatin 1, LY 333531 hydrochloride, CGP 53353, Chelerythrine Chloride, TCS 21311, CID 755673, Gossypol, ET-18-OCH3, 1-O-Hexadecyl-2-O-methyl-rac-glycerol, NPC-15437 dihydrochloride, NGIC-I, MDL-27,032, DAPH-7, 7-Aminoindole, 5-Amino-2-methylindole, rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine, Copper bis-3,5-diisopropylsalicylate, D,L-3,4-Dihydroxymandelic Acid, rac-3-Octadecanamido-2-Methoxypropan-1-ol Phosphocholine, KRIBB3, Ilmofosine, rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine, and combinations thereof.

18. The method according to claim 15, wherein said at least one activator of peroxisome proliferator-activated receptors is at least one eicosanoid, eicosanoid precursor or eicosanoid analog.

19. The method according to claim 15, wherein said at least one activator of peroxisome proliferator-activated receptors is at least an eicosanoid, eicosanoid precursor or eicosanoid analog selected from the group consisting of Diacylglycerol, Eicosapentaenoic acid, Dihomo-gamma-linolenic acid, Arachidonic acid, ETYA (5,8,11,14-eicosatetraynoic acid), members of the hydroxyeicosatetraenoic acid (HETE) family, including 5-HETE and 15-HETE, members of the hydroxyoctadecadieonic acid (HODE) family, including 9-HODE and 13-HODE, classic eicosanoids, and non-classic eicosanoids.

20. The method according to claim 1, wherein said human hepatocytes are exposed to at least one platelet-activating factor (PAF).

21. The method according to claim 1, wherein said human hepatocytes are exposed to at least one protein kinase C (PKC) inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1.

(2) A-U: Effect of treatment with 10Z-Heptadecenoic acid (A,B), Arachidonic acid (C,D), Calcifediol (E,F), Calcitriol (E,F), Cholecalciferol (G,H), CGP 52608 (I,J), Docosahexaenoic acid (K,L), PP1 (M,N,O,P), PP2 (Q,R), D-erythro-Sphingosine (S,T), and Tetradecanoic acid (U) on CYP activities of hiPSC-derived hepatocytes on day 29 and/or 36 of the differentiation protocol.

(3) Abbreviations: CGP=CGP 52608; CYP=Cytochrome P450; Sphingosine=D-erythro-Sphingosine.

(4) FIG. 2.

(5) A-D: Effect of treatment with 10Z-Heptadecenoic acid (A), Arachidonic acid (A), Calcitriol (C), Cholecalciferol (A), CGP 52608 (A), PP1 (A), D-erythro-Sphingosine (A), 13 cis-RA (B), L-erythro MAPP (B), PAF C16 (B), C16 Ceramide (B), and combinations of these compounds (A,C,D) on CYP activities of hiPSC-derived hepatocytes on day 29 and/or 36 of the differentiation protocol.

(6) E-G: Effect of treatment with 10Z-Heptadecenoic acid (E,F) and a combination of CGP 52608, PP1, Cholecalciferol, Arachidonic acid, 10Z-Heptadecenoic acid, and D-erythro-Sphingosine (G) on mRNA expression levels of the nuclear receptor PXR (E,G) and the transporter protein OATP1B1 (F).

(7) Abbreviations: 10Z=10Z-Heptadecenoic acid; 13 cis=13 cis retinoic acid; Ara=Arachidonic acid; Caltr=Calcitriol; Ceramide=C16 Ceramide; CGP=CGP 52608; Chole=Cholecalciferol; CYP=Cytochrome P450; MAPP=L-erythro MAPP; PAF=platelet activating factor; OATP1B1=organic anion-transporting polypeptide 1B1; PXR=pregnane X receptor; Sph=D-erythro-Sphingosine.

(8) FIG. 3.

(9) A,B: Effect of treatment with 10Z-Heptadecenoic acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-erythro-Sphingosine starting at different time points of the differentiation protocol on CYP activities of hiPSC-derived hepatocytes on day 29 (A) and 36 (B) of the differentiation protocol.

(10) Abbreviations: 10Z=10Z-Heptadecenoic acid; Ara=Arachidonic acid; Caltr=Calcitriol; CGP=CGP 52608; Chole=Cholecalciferol; CYP=Cytochrome P450; Sph=D-erythro-Sphingosine.

(11) FIG. 4.

(12) A-G: Effect of treatment of hepatocytes derived from the hiPS cell lines ChiPSC4 (A), ChiPSC6b (B), ChiPSC22 (C), P11015 (D), P11021 (E), P11032 (F), and the hES cell line SA121 (G) with 10Z-Heptadecenoic acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-erythro-Sphingosine on day 29 of the differentiation protocol.

(13) Abbreviations: 10Z=10Z-Heptadecenoic acid; Ara=Arachidonic acid; Caltr=Calcitriol; CGP=CGP 52608; Chole=Cholecalciferol; CYP=Cytochrome P450; Sph=D-erythro-Sphingosine.

(14) FIG. 5.

(15) A,B: Effect of treatment with 10Z-Heptadecenoic acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-erythro-Sphingosine, and with or without Oncostatin M and/or HGF on CYP1A, 3A, and 2C9 (A) and CYP2B6, 2D6, and 2C19 (B) activities of hiPSC-derived hepatocytes on day 31 of the differentiation protocol.

(16) Abbreviations: CYP=Cytochrome P450; HGF=hepatocyte growth factor; OSM=Oncostatin M.

EXAMPLES

(17) Examples of general culturing and passaging techniques are disclosed in applications WO2004/099394, WO2003/055992, WO/2007/042225, WO2007/140968 and WO2011116930.

(18) As laid out in the following examples, the starting material may comprise any hepatic progenitor cell type, particularly one derived through an initial differentiation towards a definitive or extraembryonic lineage from a mammalian pluripotent stem cell, such as a human pluripotent stem cell. The starting material may also be any cell of hepatic progenitor lineage.

Example 1: Maintenance of hPS Cell Types

(19) All hPS cells (as defined above) can be used as staring material for this invention. For the examples below in particular hepatocytes were derived in vitro from undifferentiated human embryonic stem cells (hESC) established on mEF feeder cells (Heins et al 2004) and maintained under feeder-free conditions. The cell lines used for this experiment could be, but are not limited to the hES cell lines SA121, SA167, SA181, SA461 (Cellartis AB, Gteborg, Sweden) and they can be propagated as described by Heins et al. 2004 and Caisander et al. 2006.

(20) Along with hPS obtained from hESC, hiPS (human induced pluripotent stem) cells have also been used for the derivation of hepatocytes for the examples of this invention. The hiPSC line ChiPSC4 used in this invention was derived as followed: Human dermal fibroblasts (CRL2429, ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 1 glutamax, 5 U/ml penicillin and 5 g/ml streptomycin at 37 C. in a humidified atmosphere of 5% CO.sub.2 in air. Fibroblasts were transduced with recombinant lentiviruses encoding mouse Oct4, Sox2, Klf4 and c-myc and cultured for 5 days. The transduced cells were then dispersed with trypsin and seeded onto mitomycin C treated human dermal fibroblast feeder cells at a density of 510.sup.3 cells/cm.sup.2 in their normal growth medium. After 24 hours the medium was replaced with knockout DMEM supplemented with 20% knockout serum replacement, 1 non-essential amino acids, 1 glutamax, 5 U/ml penicillin, 5 g/ml streptomycin, 100 M 2-mercaptoethanol and 30 ng/ml bFGF at 37 C. in a humidified atmosphere of 5% CO.sub.2 in air. Half of the volume of medium was replaced every day and colonies of iPS cells emerged after approximately 30 days. iPS colonies were picked, expanded in DEF-CS, and cell banks prepared. The banked cells were then characterised to check for the expression of endogenous Oct4, Sox2, Klf4 and c-Myc, silencing of transgenes, potential to differentiate into cell types representative of all three germ layers in vitro, and to confirm their authenticity by STR profiling and comparison with the parental fibroblast cell line (ATCC).

(21) The hiPSC line ChiPSC6b used in this invention was derived from fibroblast line P11031 (Cellectis SA) by transfecting with episomal vectors encoding OCT4, SOX2, KLF4, LIN28, and L-MYC using electroporation (Neon transfection system; Invitrogen).

(22) All other hiPSC lines used in this invention were obtained from Cellectis SA and were derived from either peripheral blood cells (P11021) or adult dermal fibroblasts (ChiPSC18, ChiPSC22, P11015, P11021, P11032) using either episomal (P11021) or retroviral reprogramming (ChiPSC18, ChiPSC22, P11015, P11021, P11032) with OCT4, SOX2, KLF4, LIN28, and L-MYC. These 6 hiPSC lines were initially established in a Matrigel-based culture system and subsequently transferred to the DEF-CS.

(23) Alternatively to reprogramming using lentivirus, retrovirus and episomal vectors, hiPSC lines can also be reprogrammed using Sendai virus, adenovirus, proteins and mRNAs or other techniques. Other suitable cell lines for use are those established by Chung et al. (2008), such as cell lines MA126, MA127, MA128 and MA129 (Advanced Cell Technology, Inc. Worcester, Mass., USA), which all are listed with the International stem cell registry. These cell lines have been derived (or obtained) without destruction of the human embryo by employing a single blastomere removal technique.

(24) All hPSC lines used in this invention were cultured under standard conditions in the DEF-CS with continuous passaging twice a week and were immuno-positive for OCT4, TRA1-60, TRA1-81, and SSEA-4, and immuno-negative for SSEA-1. Pluripotency was confirmed by in vitro differentiation. Karyotyping as described by Caisander et al. 2006 showed a normal chromosomal profile.

Example 2: Differentiation of hPS Cell Types to Produce Hepatocytes

(25) Hepatocytes may be derived from hPS cells by employing the following exemplary basic protocols A and B:

(26) Protocol A:

(27) Undifferentiated hPS cells are dissociated and seeded directly in day 0-medium onto a Fibronectin-based coating. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase.

(28) Day 0

(29) Pre-treatment medium

(30) 3 M CHIR99021

(31) 5 M ROCK inhibitor

(32) Day 1

(33) RPMI 1640 (+0.1% PEST+1% Glutamax)

(34) 1B27

(35) 50 ng/ml Activin A

(36) 3 M CHIR99021

(37) 5 M LY294002

(38) 3 M CHIR99021

(39) Day 2

(40) RPMI 1640 (+0.1% PEST+1% Glutamax)

(41) 1B27

(42) 50 ng/ml Activin A

(43) 5 M LY294002

(44) 10 nM 5-aza-2-deoxycytidine

(45) Day 3

(46) RPMI 1640 (+0.1% PEST+1% Glutamax)

(47) 1B27

(48) 50 ng/ml Activin A

(49) Day 4-7

(50) RPMI 1640 (+0.1% PEST+1% Glutamax)

(51) 1B27

(52) 50 ng/ml Activin A

(53) The pre-treatment medium is available from Takara Bio Europe AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden) upon request.

(54) On day 7 the cells are passaged. The cells are incubated for 3-7 minutes with TrypLE Select at 37 C., the same volume of BasHES is added and the cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter, the cells are replated onto a Fibronectin-based coating at a cell density of 50 000-350 000 cells/cm.sup.2 such as e.g. 75 000-300 000 cells/cm.sup.2, preferably 100 000 cells/cm.sup.2. The Fibronectin-based coating has a concentration of 7.5 g Fibronectin per cm.sup.2 culture area. To prepare the coating, 50 l of a 1 mg/ml Fibronectin stock is added per ml DPBS, and 150 l of this coating solution is added per cm.sup.2 culture area.

(55) Day 7-14 (Pre-Hepatic)

(56) Knockout-DMEM+1% PEST+1% Glutamax

(57) 20% Knockout-Serum Replacement

(58) 1% non-essential amino acids (NEAA)

(59) 0.2% beta-mercaptoethanol

(60) 1% DMSO

(61) Day 14-45 (Differentiation and Maturation)

(62) WME+1% Glutamax+0.1% PEST

(63) 0.55 mg/mL BSA-FAF

(64) 0,025 mg/mL Ascorbic Acid

(65) 0.67 g/mL Hydrocortisone Hemisuccinate

(66) 10 g/mL Transferrin

(67) 5 g/mL Insulin

(68) 0.003 g/mL EGF

(69) 0.1 M DexM

(70) 10 ng/ml OsM

(71) 20 ng/ml HGF

(72) 0.5% DMSO

(73) 1.4 M BIO

(74) 0.5 M Kenpaullone

(75) 0.2 M 9cis retinoic acid

(76) On day 14 and 16, matrix overlays are performed. To this end, 53 l of a 1 mg/ml Fibronectin stock and 9 l of a 3 mg/ml Collagen I stock are added per ml day 14-45 medium (RT), the medium is mixed well and then a regular medium change is perfomed. The addition of the matrix components corresponds to the addition of 25 g Fibronectin and 12.5 g Collagen I per cm.sup.2 culture area per overlay addition.

(77) Protocol B:

(78) Undifferentiated hPS cells are dissociated and seeded directly in day 0-medium onto a Fibronectin-based coating. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase.

(79) Day 0

(80) Pre-treatment medium

(81) 3 M CHIR99021

(82) 5 M ROCK inhibitor

(83) Day 1

(84) RPMI 1640 (+0.1% PEST+1% Glutamax)

(85) 1B27

(86) 50 ng/ml Activin A

(87) 3 M CHIR99021

(88) 5 M LY294002

(89) 3 M CHIR99021

(90) Day 2

(91) RPMI 1640 (+0.1% PEST+1% Glutamax)

(92) 1B27

(93) 50 ng/ml Activin A

(94) 5 M LY294002

(95) 10 nM 5-aza-2-deoxycytidine

(96) Day 3

(97) RPMI 1640 (+0.1% PEST+1% Glutamax)

(98) 1B27

(99) 50 ng/ml Activin A

(100) Day 4-7

(101) RPMI 1640 (+0.1% PEST+1% Glutamax)

(102) 1B27

(103) 50 ng/ml Activin A

(104) The pre-treatment medium is available from Takara Bio Europe AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden) upon request.

(105) On day 7 the cells are passaged. The cells are incubated for 3-7 minutes with TrypLE Select at 37 C., the same volume of BasHES is added and the cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter, the cells are replated onto a Collagen I-Fibronectin-based coating at a cell density of 50 000-350 000 cells/cm.sup.2 such as e.g. 75 000-300 000 cells/cm.sup.2, preferably 100 000 cells/cm.sup.2. The Collagen I-Fibronectin-based coating has a concentration of 2 g Fibronectin and 10 g Collagen I per cm.sup.2 culture area. To prepare the coating, 12 l of a 1 mg/ml Fibronectin stock and 21.5 l of a 3 mg/ml Collagen I stock is added per ml DPBS, and 150 l of this coating solution is added per cm.sup.2 culture area.

(106) Day 7-14 (Pre-Hepatic)

(107) Knockout-DMEM+1% PEST+1% Glutamax

(108) 20% Knockout-Serum Replacement

(109) 1% non-essential amino acids (NEAA)

(110) 0.2% beta-mercaptoethanol

(111) 1% DMSO

(112) Day 14-45 (Differentiation and Maturation)

(113) WME+1% Glutamax+0.1% PEST

(114) 0.55 mg/mL BSA-FAF

(115) 0,025 mg/mL Ascorbic Acid

(116) 0.67 g/mL Hydrocortisone Hemisuccinate

(117) 10 g/mL Transferrin

(118) 5 g/mL Insulin

(119) 0.003 g/mL EGF

(120) 0.1 M DexM

(121) 10 ng/ml OsM

(122) 20 ng/ml HGF

(123) 0.5% DMSO

(124) 1.4 M BIO

(125) 0.5 M Kenpaullone

(126) 0.2 M 9cis retinoic acid

(127) On day 14 and 16, matrix overlays are performed. To this end, 12 l of a 1 mg/ml Fibronectin stock solution and 21.5 L of a 3 mg/ml Collagen I stock solution are added per ml day 14-45 medium (RT), the medium is mixed well and then a regular medium change is perfomed. The addition of the matrix components corresponds to the addition of 6 g Fibronectin and 31.25 g Collagen I per cm.sup.2 culture area per overlay addition.

Example 3: Effect of Treatment of hiPSC-Derived Hepatocytes with 10Z-Heptadecenoic Acid, Arachidonic Acid, Calcifediol, Calcitriol, Cholecalciferol, CGP 52608, Docosahexaenoic Acid, PP1, PP2, D-Erythro-Sphingosine, and Tetradecanoic Acid

(128) Procedure:

(129) Following the basic protocol A, hiPS cell derived hepatocytes cultured on a Fibronectin-based coating were treated with 0.5 or 5 M 10Z-Heptadecenoic acid, 0.5 or 5 M Arachidonic acid, 5 M Calcifediol, 5 M Calcitriol, 0.2 or 0.5 M Cholecalciferol, 5 or 50 M CGP 52608, 0.5 or 5 M Docosahexaenoic acid, 0.5, 1.25, 2.5, 5, or 10 M PP1, 0.5 or 5 M PP2, 0.5 or 5 M D-erythro-Sphingosine, or 0.5 or 5 M Tetradecanoic acid from day 21 of the differentiation protocol and onwards (FIG. 1).

(130) On day 29 and 36 of the differentiation protocol, the cell cultures are subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams's medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10 M Phenacetin (model substrate for CYP1A), 10 M Bupropion (model substrate for 2B6), 10 M Diclofenac (model substrate for CYP2C9), 10 M Bufuralol (model substrate for 2D6), and 5 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 110 l/cm.sup.2) and incubated for 16 hr at 37 C. Then 100 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A.

(131) Results:

(132) FIG. 1 A-U) 10Z-Heptadecenoic acid, Arachidonic acid, Calcifediol, Calcitriol, Cholecalciferol, CGP 52608, Docosahexaenoic acid, PP1, PP2, D-erythro-Sphingosine, and Tetradecanoic acid increase CYP activity levels in hiPS cell-derived hepatocytes both on day 29 and 36 of the differentiation protocol. In some cases, 2B6 activity levels were below the detection level.

(133) These eleven compounds increase CYP activity levels in hiPS cell-derived hepatocytes. Therefore the skilled person wishing to improve CYP activity may select from these compounds according to their interest.

Example 4: Effect of Treatment of hiPSC-Derived Hepatocytes with 10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, D-Erythro-Sphingosine, 13 Cis-RA, L-Erythro MAPP, PAF C16, C16 Ceramide, and Combinations of these Compounds

(134) Procedure:

(135) Following the basic protocol B, ChiPSC4-derived hepatocytes cultured on a Collagen I-Fibronectin-based coating were treated with 0.5 M 10Z-Heptadecenoic acid, 0.5 M Arachidonic acid, 0.5 M Calcitriol, 5 M CGP 52608, 0.2 M Cholecalciferol, 5 M PP1, 0.5 M D-erythro-Sphingosine, 0.2 M 13 cis-RA, 0.5 M L-erythro MAPP, 0.5 M PAF C16, 0.5 M C16 Ceramide, or different combinations of these compounds from day 21 of the differentiation protocol and onwards (FIG. 2).

(136) On day 29 of the differentiation protocol, the cell cultures are subjected to a CYP activity assay according to the protocol described in Example 3. In some experiments, 50 M Mephenytoin (model substrate for 2C19) was included additionally to the substrates mentioned above and then the formation of the metabolite OH-Mephenytoin was measured by LC/MS in order to determine CYP2C19 activity.

(137) Results:

(138) FIG. 2 A-D) 10Z-Heptadecenoic acid (10Z), Arachidonic acid (Ara), Calcitriol (Caltr), Cholecalciferol (Chole), CGP 52608 (CGP), PP1, D-erythro-Sphingosine (Sph), 13 cis-RA (13 cis), L-erythro MAPP (MAPP), PAF C16 (PAF), C16 Ceramide (Cera), or different combinations of these compounds increase CYP activity levels in hiPS cell-derived hepatocytes on day 29 of the differentiation protocol. In some cases, 2B6 activity levels were below the detection level.

(139) FIG. 2 E,F) Protocol B and protocol B plus 10Z-Heptadecenoic acid increase OATP1B1 and PXR mRNA levels in hiPS cell-derived hepatocytes on day 36 of the differentiation protocol compared to protocol A.

(140) FIG. 2 G) Protocol B plus CGP 52608, PP1, Cholecalciferol, Arachidonic acid, 10Z-Heptadecenoic acid, and Sphingosine increase PXR mRNA levels in hiPS cell-derived hepatocytes on day 29 and 36 of the differentiation protocol compared to protocol A.

(141) These eleven compounds increase CYP activity levels in hiPS cell-derived hepatocytes. Therefore the skilled person wishing to improve CYP activity may select from these compounds according to their interest.

Example 5: Effect of Treatment of hiPSC-Derived Hepatocytes with 10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-Erythro-Sphingosine Starting at Different Time Points of the Differentiation Protocol

(142) Procedure:

(143) Following the basic protocol B, ChiPSC4-derived hepatocytes cultured on a Collagen I-Fibronectin-based coating were treated with 0.5 M 10Z-Heptadecenoic acid, 0.5 M Arachidonic acid, 0.5 M Calcitriol, 5 M CGP 52608, 0.2 M Cholecalciferol, 5 M PP1, and 0.5 M D-erythro-Sphingosine starting on day 11, 14, 21, 25 and 28, respectively, of the differentiation protocol and onwards (FIG. 3).

(144) On day 29 and 36 of the differentiation protocol, the cell cultures are subjected to a CYP activity assay according to the following described in Example 3.

(145) Results:

(146) FIG. 3 A,B) Treatment with 10Z-Heptadecenoic acid (10Z), Arachidonic acid (Ara), Calcitriol (Caltr), Cholecalciferol (Chole), CGP 52608 (CGP), PP1, and D-erythro-Sphingosine (Sph) starting between day 11 and 28 of the differentiation protocol increases CYP activity levels in hiPS cell-derived hepatocytes on day 29 and 36 of the differentiation protocol.

(147) The treatment with the seven compounds starting between day 11 and 28 of the differentiation protocol increases CYP activity levels in hiPS cell-derived hepatocytes. Therefore the skilled person wishing to improve CYP activity may select from these different time points to start the treatment according to their interest.

Example 6: Effect of Treatment of Hepatocytes Derived from Several hiPS and hES Cell Lines with 10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-Erythro-Sphingosine

(148) Procedure:

(149) Following the basic protocol B, hepatocytes derived from the hiPS cell lines ChiPSC4, ChiPSC6b, ChiPSC22, P11015, P11021, P11032 and the hES cell line SA121 were cultured on a Collagen I-Fibronectin-based coating and treated with a combination of 0.5 M 10Z-Heptadecenoic acid, 0.5 M Arachidonic acid, 0.5 M Calcitriol, 5 M CGP 52608, 0.2 M Cholecalciferol, 5 M PP1, and 0.5 M D-erythro-Sphingosine from day 21 of the differentiation protocol and onwards (FIG. 4).

(150) On day 29 of the differentiation protocol, the cell cultures are subjected to a CYP activity assay according to the following protocol described in Example 3. In some experiments, 50 M Mephenytoin (model substrate for 2C19) was included additionally to the substrates mentioned above and then the formation of the metabolite OH-Mephenytoin was measured by LC/MS in order to determine CYP2C19 activity.

(151) Results:

(152) FIG. 4 A-G) The treatment with 10Z-Heptadecenoic acid (10Z), Arachidonic acid (Ara), Calcitriol (Caltr), Cholecalciferol (Chole), CGP 52608 (CGP), PP1, and D-erythro-Sphingosine (Sph) increases CYP activity levels in hepatocytes derived from the hiPS cell lines ChiPSC4 (A), ChiPSC6b (B), ChiPSC22 (C), P11015 (D), P11021 (E), P11032 (F) and the hES cell line SA121 (G) on day 29 of the protocol.

(153) These seven compounds increase CYP activity levels in both hiPS and hES cell-derived hepatocytes. Therefore the skilled person wishing to improve CYP activity may select from these compounds according to their interest.

Example 7: Effect of Treatment of hiPSC-Derived Hepatocytes with 10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-Erythro-Sphingosine with or without the Addition of Oncostatin M and/or HGF

(154) Procedure:

(155) Following the basic protocol B, hepatocytes derived from the hiPS cell line ChiPSC18 were cultured on a Collagen I-Fibronectin-based coating and treated with a combination of 0.5 M 10Z-Heptadecenoic acid, 0.5 M Arachidonic acid, 0.5 M Calcitriol, 5 M CGP 52608, 0.2 M Cholecalciferol, 5 M PP1, and 0.5 M D-erythro-Sphingosine and with or without Oncostatin M and/or HGF from day 21 of the differentiation protocol and onwards (FIG. 5).

(156) On day 31 of the differentiation protocol, the cell cultures are subjected to a CYP activity assay according to the following protocol described in Example 3. 50 M Mephenytoin (model substrate for 2C19) was included additionally to the substrates mentioned above and the formation of the metabolite OH-Mephenytoin was measured in order to determine CYP2C19 activity.

(157) Results:

(158) FIG. 5 A,B) The treatment with 10Z-Heptadecenoic acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and D-erythro-Sphingosine, and with or without Oncostatin M and/or HGF results in similar CYP activity levels in hepatocytes derived from the hiPS cell line ChiPSC18 on day 31 of the protocol.

(159) These seven compounds increase CYP activity levels in both hiPS and hES cell-derived hepatocytes independent of the presence of Oncostatin M and/or HGF. Therefore, the skilled person wishing to improve CYP activity may select from these compounds according to their interest and include or exclude Oncostatin M and/or HGF.

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