A hepatocyte maturation composition, mature IPSC-derived hepatocytes and their use in drug-induced liver injury prediction

Abstract

Described herein is a hepatocyte maturation composition (Hepatocyte Differentiation Cocktail 6"(HDC6)) comprising a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. Methods for generating mature iPSC-derived hepatocytes and 3D liver spheroids are presented. The 3D liver spheroids described herein are suitable for predicting drug-induced liver injury and identifying a preferred therapeutic agent for an individual.

Description

P-643500-PCA HEPATOCYTE MATURATION COMPOSITION, MATURE IPSC-DERIVED HEPATOCYTES AND THEIR USE IN DRUG-INDUCED LIVER INJURY PREDICTIONFIELD OF THE DISCLOSURE

[0001] The invention described herein relates to the field of stem cell biology and pharmacology, specifically to the maturation of induced pluripotent stem cells (iPSC)-derived hepatocytes for the purpose of drug-induced liver injury (DILI) examination. This invention is particularly relevant to the development of in vitro models for toxicology studies and drug screening, aimed at improving the safety and efficacy of pharmaceutical compounds as well as for the purpose of precision medicine. This invention can also have applications in disease modeling and in vitro mechanistic studies.BACKGROUND

[0002] The differentiation of iPSCs into hepatocyte-like cells (HLCs) has been an active area of research, primarily due to the potential of these cells to serve as in vitro models for DILI and the assessment of other liver-related diseases. Traditionally, the maturation of iPSC-derived hepatocytes has relied on stepwise protocols that mimic embryonic liver development. These often include the use of growth factors and small molecules such as hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone (DEX). Despite these efforts, iPSC-derived hepatocytes often fall short of achieving full functional maturation and CYP450 enzyme activity comparable to Primary Human Hepatocytes (PHH), which is crucial for accurate DILI prediction.

[0003] Several approaches have been developed to enhance the maturation of iPSC-derived hepatocytes. For example, the use of chemical cocktails has been explored in recent years to improve hepatic expression and function. One such approach involves the use of a combination of small molecules that target specific signaling pathways involved in hepatocyte maturation. These molecules typically include components that modulate Wnt, TGF-P, and Notch signaling, which are key regulators of liver development and function. Another strategy involves the use of 3D culture systems or co-culture with non-parenchymal liver cells (NPCs) to better mimic the liver microenvironment and promote maturation.

[0004] However, while these strategies have shown some promise, they do not fully replicate the functional characteristics of PHHs, especially in the context of CYP450P-643500-PCenzyme activity and the ability to predict DILI accurately. This has necessitated the development of more effective methods to enhance the functional maturation of iPSC-derived hepatocytes.SUMMARY

[0005] In one aspect disclosed herein is a method for generating mature iPSC-derived hepatocytes comprising culturing immature iPSC-derived hepatocytes in a culture medium including a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes, wherein the hepatocyte maturation composition comprises TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0006] In a related aspect, the step of culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition is for a period of time of between about 5-10 days.

[0007] In a related aspect, the immature iPSC-derived hepatocytes are cryopreserved cells which are thawed before culturing.

[0008] In a related aspect, the immature iPSC-derived hepatocytes are non-frozen cells.

[0009] In a related aspect, the immature iPSC-derived hepatocytes are mammalian cells.

[0010] In a related aspect, the immature iPSC-derived hepatocytes are human cells

[0011] In a related aspect, the mature iPSC-derived hepatocytes are characterized by having one or more of the following:express one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and multi drug resistance-associated protein 2 (MRP2);albumin levels exceeding 40 pg / day / million cells;increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control;CYP3 A4 activity at levels of above 0.2 pmol / min / million cells;CYP1 A2 activity at levels of about 5 pmol / min / million cells; andreduced expression of AFP or FM01, compared to immature iPSC-derived hepatocytes.

[0012] In another aspect disclosed herein is a method for generating a 3D liver spheroid from iPSC-derived immature hepatocytes comprising (a) culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes; and (b) seeding the mature iPSC-derived hepatocytes of step (a) on a low attachment surface and culturing in hepatocyte maturation composition supplemented with Rho-associated kinase (ROCK) inhibitor, to obtain a 3D liver spheroid, wherein theP-643500-PCmaturation composition comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0013] In a related aspect, the step of culturing the mature iPSC-derived hepatocytes in hepatocyte maturation composition supplemented with a ROCK inhibitor is for a period of time of between about 12 to 18 hours followed by removal of ROCK inhibitor and continued culture in hepatocyte maturation composition in the absence of ROCK inhibitor for a period of time of between about 5-10 days.

[0014] In a related aspect, the ROCK inhibitor comprises Y-27632.

[0015] In a related aspect of the method for generating a 3D liver spheroid, the method comprises combining the mature iPSC-derived hepatocytes from step (a) with iPSC-derived Kupffer-like cells (iKCs), prior to seeding.

[0016] In another aspect disclosed herein is a 3D liver spheroid prepared according to the method described herein.

[0017] In a related aspect, the 3D liver spheroid is used for at least one purpose selected from the group consisting of: a model platform for liver disease testing; model platform for drug-induced liver injury (DILI) prediction; for idiosyncratic drug induced liver injury (iDILI) identification and studies; for disease modeling; in vitro mechanistic studies; food supplement induced liver injury prediction; drug-drug interaction (DDI) testing; and drugfood supplement interaction testing.

[0018] In another aspect disclosed herein is a hepatocyte maturation composition comprising a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0019] In a related aspect, the cAMP Activator is selected from the group consisting of forskolin or a derivative thereof, colforsin daropate, CW008, PACAP 1-38, 8-bromo-cAMP, dibutyryl-cAMP, NKH477, or mixtures or combinations thereof. In one aspect, the cAMP Activator is forskolin.

[0020] In a related aspect, the TGF-P Inhibitor is selected from the group consisting of SB431542, SB525334, Ki26894, LY364947, SD-208, SD-093, SM16, Ly2109761, Ly2157299, K02288, SB505124, LDN-193189, GW788388, Ly580276, EW-7203, EW-7195, EW-7197, YR-290, A 83-01, D4476, RepSox, R268712, or mixtures or combinations thereof. In one aspect, the TGF-P Inhibitor is SB431542.

[0021] In a related aspect, the Notch Inhibitor is selected from the group consisting of LY41 1575, MDL-28170, Compound E, RO4929097, DAPT, L- 685458, BMS-708163, BMS-299897, M -0752, YO-01027, MDL28170, LY41 1575, ELN-46719, PF-03084014,P-643500-PCSemagacestat, or mixtures or combinations thereof. In one aspect, the Notch Inhibitor is DAPT.

[0022] In a related aspect, the thyroid hormone is selected from the group consisting of Liothyronine, (S)-triiodothyronine, (S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoic acid, (S)-thyroxine, Levothyroxine (L-thyroxine), Liotrix, Tiratricol, or mixtures or combinations thereof. In one aspect, the thyroid hormone is Liothyronine.

[0023] In a related aspect, the PPAR activator comprises a PPAR-gamma (PPARy) activator, a PPAR-alpha (PPARa) activator, PPAR-delta (PPAR5) activator, a dual PPARa / 5 agonist, a pan PPAR activator, or any combination thereof. In a related aspect, the PPAR activator comprises a PPARa activator selected from the group consisting of Wyl4643, 15-HETE, 15-HpETE, Aleglitazar, Aluminium clofibrate, Arachidonic acid, Bezafibrate, Chiglitazar, Clofibrate, CP-775146, Daidzein, DHEA (prasterone), Elafibranor, Etomoxir, Fenofibrate, Genistein, Gemfibrozil, GW-7647, Lanifibranor, Leukotriene B4, LG-101506, LG-100754, Lobeglitazone, Muraglitazar, Oleyl ethanol ami de, Palmitoylethanolamide, Pemafibrate, Perfluor ononanoic acid, Perfluorooctanoic acid, Pioglitazone, Saroglitazar, Sodelglitazar, Tesaglitazar, Tetradecylthioacetic acid, Troglitazone, or mixtures or combinations thereof. In one aspect, the PPARa activator is Wy 14643.

[0024] In a related aspect, the Tryptophan Metabolite is selected from the group consisting of FICZ, indole-3 -aldehyde (lAld), indole-3 -acetic acid (IAA), indole-3 -propionic acid (IP A), indole-3 -lactic acid (ILA), kynurenine, kynurenic acid, xanthurenic acid, quinolinic acid, tryptamine, indole, skatole (3-methylindole), or mixtures or combinations thereof. In one aspect, the Tryptophan Metabolite is FICZ.

[0025] In another aspect disclosed herein is a method for predicting drug induced liver injury (DILI) comprising bringing a drug into contact with the 3D liver spheroid described herein.

[0026] In a related aspect, the toxicity of the drug is determined by assessing spheroid viability or spheroid morphology.

[0027] In another aspect disclosed herein is a method for identifying a preferred therapeutic agent for an individual comprising bringing a candidate compound into contact with the 3D liver spheroid described herein.BRIEF DESCRIPTION OF THE DRAWINGSP-643500-PC

[0028] Figure 1A Schematic illustration of the traditional stepwise differentiation protocol for generating iPSC-derived hepatocytes (iHeps), highlighting the key stages from pluripotent stem cell maintenance to hepatic cells. iPSCs were differentiated into hepatocytes through a stepwise protocol: iPSC to endoderm, hepatic progenitors, and finally, immature hepatocytes. 3D liver spheroids were assembled from hepatocytes at the late differentiation stage.

[0029] Figure IB Immunofluorescent staining of iPSC-derived hepatocytes (iHeps, left) and 3D spheroids (iHeps-3D, right) for Albumin (ALB), EpCAM, and HNF4aw. Scale bar: 100pm.

[0030] Figure 1C urea secretion (left) and CYP450 function as measured by CYP3A4 (middle) and CYP1 A2 (right) in primary human hepatocytes (PHHs-3D) and iPSC-derived hepatocytes (iHeps-3D). CYP3A4 and CYP1A2 functions remained significantly lower in iPSC-derived hepatocytes (iPSCs), indicating incomplete maturation. (iHeps-3D: n = 29; PHHs-3D: n = 9; unpaired two-tailed t-test, t = 1.888, df = 36), CYP3A4 activity (n = 7; t = 12.01, df = 12), and CYP1A2 activity (n = 7; t = 11.71, df= 12).

[0031] Figure ID Primary screening of individual small molecules targeting hepatic signaling pathways, including DAPT (Notch inhibitor), forskolin (cAMP activator), Yap inhibitor, IWR1 (Wnt inhibitor), LCA / VitK, TUDCA / VitK, and FICZ (tryptophan metabolite). CYP1A2 activity: n = 6, one-way ANOVA, F(2,15) = 10.58, P = 0.0014; CYP3A4 activity: n = 6, F(2,15) = 30.74, P < 0.0001.

[0032] Figure IE Secondary screening identifies a synergistic six-compound cocktail — SB431542 (TGF-P inhibitor), DAPT, forskolin, T3 (thyroid hormone), WY14643 (PPARa agonist), and FICZ, enhancing CYP1A2 and CYP3A4 activity. CYP1A2: n = 6, F(3,28) = 68.21, P < 0.0001; CYP3A4: n = 6, F(3, 28) = 11.74, P < 0.0001.

[0033] Figure IF Schematic of final maturation protocol: HDC6 treatment of immature iHeps in 2D for 10 days, followed by 3D spheroid assembly.

[0034] Figure 1G Phase-contrast images showing morphological maturation of iHeps over time post-HDC6 treatment. HDC6-treated spheroids (iM-Heps-3D) display uniform morphology similar to PHHs-3D. Scale bar, 100 pm.

[0035] Figure 1H Flow cytometry analysis of intracellular albumin in iPSCs, vehicle-treated iHeps, HDC6-treated iHeps (donors 750 and 744), and PHHs in 2D.

[0036] Figure II Immunofluorescence staining of ASGR1 and HNF4a in 2D cultures, and ASGR1, CYP1A2, and albumin in 3D spheroids from vehicle- or HDC6-treated iHepsP-643500-PC(donors 750 and 744) and PHHs. Scale bar, 100 pm. Representative of three independent differentiations.

[0037] Figure 2A Principal component analysis (PCA) of RNA-seq data from iPSCs, immature iHeps, vehicle-treated iHeps spheroids (iHeps-3DV), HDC6-treated iHeps spheroids (iM-Heps-3D), and PHH spheroids (PHHs-3D). PHHs-3D (mono) contained -2,000 PHHs per spheroid; PHHs-3D (co) contained -2,000 PHHs plus -200 non-parenchymal cells (NPCs) per spheroid. iM-Heps-3D clustered closely with PHHs-3D, indicating enhanced transcriptomic similarity. Percentages indicate variance explained by each principal component, n = 3 biological replicates per group.

[0038] Figure 2B Heatmaps showing normalized expression (Z-score) of representative genes in phase I and phase II drug-metabolizing enzymes, transporters, and canonical hepatic markers across iPSCs, immature iHeps, iHeps-3DV, iM-Heps-3D, and PHHs-3D.

[0039] Figure 3A Albumin secretion in 3D liver spheroids generated from five iPSC donor lines treated with vehicle or HDC6. PHHs spheroids served as a functional reference. Group sizes: donor 744 HDC6 (n = 8), donor 750 HDC6 (n = 9), all other groups (n = 4). One-way ANOVA, F(10,42) = 181.8, P < 0.0001.

[0040] Figure 3B Urea synthesis under basal and stimulated conditions (ammonium chloride + L-arginine) in vehicle- and HDC6-treated spheroids, with PHH spheroids as control. All groups: n = 8. Two-way ANOVA, F(10,154) = 30.12, P < 0.0001.

[0041] Figure 3C Functional activities of CYP3A4, CYP1A2, CYP2C8, CYP2B6, CYP2C9, and CYP2C19 measured by luminescence-based assays in vehicle- and HDC6-treated spheroids from multiple iPSC lines, with PHHs as control. CYP3A4: donors 744, 750, PHH (n = 11), others (n = 4), F(12,74) = 26.47, P < 0.0001; CYP1A2: donors 744, 750, PHH (n = 15), other groups vehicle (n = 4), HDC6 (n = 8), F(12,110) = 27.93, P < 0.0001; CYP2C8: donors 744, 750, PHH (n = 12), others (n = 4), F(12,79) = 47.98, P < 0.0001; CYP2B6: donors 744, 750, PHH (n = 11), others (n = 4), F(12,74) = 9.340, P < 0.0001; CYP2C9: donors 744, 750, PHH (n = 12), others (n = 4), F(12,79) = 20.44, P < 0.0001; CYP2C19: donors 744, 750, PHH (n = 7), others (n = 4), F(12,54) = 6.571, P < 0.0001.

[0042] Figure 3D LC-MS / MS quantification of metabolite formation from probe substrates in 744 iM-Heps spheroids and PHH spheroids: midazolam —> 1-OH-midazolam (CYP3A4, n = 8, unpaired two-tailed t-test, t = 6.040, df = 14), phenacetin acetaminophen (CYP1A2, n = 8, t = 15.85, df = 14), paclitaxel —> 6-OH-paclitaxelP-643500-PC(CYP2C8, 744: n = 4, PHH: n = 7, t = 1.159, df = 9), and 7-hydroxy coumarin 7-OH-coumarin sulfate (SULT, n = 4, t = 3.138, df = 6).

[0043] Figure 3E Live-cell imaging of bile canaliculi using DCFDA in immature iHeps and HDC6-treated iM-Heps (2D), with PHHs as control. Cyclosporin A (CsA) was applied as an MRP2 inhibitor. Scale bar, 100 pm. Representative of three independent experiments.

[0044] Figure 4A Heatmap of RNA-seq data showing expression of genes involved in the TCA cycle and oxidative phosphorylation across iPSCs, immature iHeps, vehicle-treated iHeps spheroids (iHeps-3DV), HDC6-treated iHeps spheroids (iM-Heps-3D), and PHH spheroids (mono and co-culture).

[0045] Figure 4B Heatmap showing expression of genes associated with fatty acid metabolism across the same sample groups.

[0046] Figure 4C Gene set variation analysis (GSVA) enrichment scores for selected metabolic pathways, including [3-oxidation of very long-chain fatty acids, peroxisomal lipid metabolism, fatty acid metabolism, glycolysis / gluconeogenesis, TCA cycle, and oxidative phosphorylationc.

[0047] Figure 4D Quantification of mitochondrial mass by qPCR of mitochondrial gene MT-ND1 normalized to nuclear gene RNase P. Group sizes: 744 iM-Heps-3D, 750 iM-Heps-3D, PHHs-3D (n = 4); all other groups (n = 3). One-way ANOVA, F(8,21) = 7.030, P = 0.0002.

[0048] Figure 4E Seahorse Mito Stress Test assessing oxygen consumption rate (OCR). Top: time course following sequential injections of oligomycin, FCCP, and rotenone / antimycin A. Bottom: bar plots of maximal respiration (one-way ANOVA, F(6,21) = 29.62, P < 0.0001) and spare respiratory capacity (F(6,21) = 33.56, P < 0.0001). All groups: n = 4.

[0049] Figure 4F Seahorse Glycolytic Rate Assay measuring extracellular proton efflux rate (PER). Top: time course after rotenone / antimycin A and 2-deoxyglucose (2-DG) injections. Bottom: bar plots quantifying basal glycolysis (one-way ANOVA, F(6,21) = 137.0, P < 0.0001) and mitoOCR / Gly coPER ratio (F(6,21) = 53.52, P < 0.0001). All groups: n = 4.

[0050] Figure 5A Schematic of DILI testing workflow. Liver spheroids were generated on day -5 and exposed to compounds at multiple concentrations on days 1, 3, and 5. Cell viability was measured on day 7 using CellTiter-Glo.

[0051] Figure 5B Representative brightfield images of spheroids treated with known hepatotoxic compounds (tolcapone, chlorpromazine, troglitazone, diclofenac) in HDC6-P-643500-PCtreated iHeps spheroids (iM-Heps-3D) from donors 750 and 744, and PHH spheroids. Scale bar, 100 pm.

[0052] Figure 5C Dose-response curves for drugs shown in panel b. ICso values were derived from non-linear regression fits of biological replicates (n = 4).

[0053] Figure 5D Brightfield images showing the effects of trovafloxacin, entacapone, and atorvastatin on iM-Heps-3D and PHHs-3D. Scale bar, 100 pm.

[0054] Figure 5E Dose-response curves for drugs shown in panel d. ICso values calculated as in panel c (n = 4).

[0055] Figure 5F Margin of safety (MOS; ICso / Cmax) for 20 reference drugs spanning “most DILI,” “less DILI,” “no DILI,” and “ambiguous” FDA DILIrank categories. Data from iM-Heps-3D (donors 750 and 744) and PHHs-3D. Dashed line at MOS = 50 denotes classification threshold for DILI-positive vs. DILI-negative. ND, not detected. Data from three independent repeats.

[0056] Figure 6A Schematic of stepwise differentiation to generate iPSC-derived Kupffer-like cells (iKCs) via mesoderm and hematopoietic stem cell (HSCs) intermediates, followed by cytokine-guided maturation.

[0057] Figure 6B Brightfield images of iKCs during differentiation on days 7, 14, and 20. Scale bar, 100 pm.

[0058] Figure 6C Flow cytometry of Kupffer cell markers CD14 and CD163 in iPSC-derived HSCs, iKCs from donors 750 and 744, and primary human Kupffer cells (PKCs).

[0059] Figure 6D Brightfield images of liver spheroids as monocultures (iM-Heps only) or co-cultures with iKCs (5:1 iM-Heps:iKCs). Scale bar, 100 pm.

[0060] Figure 6E Comparison of CYP3A4 activity and albumin secretion (24 h) between mono- and co-culture spheroids. CYP3A4: n = 3, two-way ANOVA, F(l,8) = 1.649, P = 0.2350; albumin: n = 8, F(l, 28) = 0.4868, P = 0.4911.

[0061] Figure 6F Cell viability after treatment with DILI-associated drugs, with or without free fatty acid (FFA) pre-treatment. n = 4. Two-way ANOVA: tolcapone F(33,134) = 0.8964; entacapone F(33,142) = 3.139; diclofenac F(33,143) = 1.442; atorvastatin F(33,141) = 2.158; flutamide F(33,144) = 1.602; trovafloxacin F(33,144) = 3.574.

[0062] Figure 6G Heatmap (z-scores) of cytokine / chemokine release after LPS stimulation in spheroids from donors 750 and 744, as monocultures or co-cultures with matched iKCs. n = 4 biological replicates.P-643500-PC

[0063] Figure 6H Heatmap (mean z-scores) of cytokine responses to ipilimumab or theralizumab in donor 750 iM-Heps-3D, with or without iKC co-culture. n = 4 biological replicates per condition.

[0064] Figure 61 Quantification of GDF-15 and CCL2 after LPS or biologic treatment in mono- and co-culture spheroids, n = 4 biological replicates. Two-way ANOVA: GDF-15 F(11,72) = 4.198, P < 0.0001; CCL2 F(11,58) = 11.42, P < 0.0001.DETAILED DESCRIPTION

[0065] The composition and methods described herein solve the critical problem of immature hepatic function in iPSC-derived hepatocytes, particularly the insufficient expression and activity of cytochrome P-450 (CYP450) family of enzymes. While iPSC derived organ models have great potential for expanding the capabilities of in vitro testing by enabling better access to diverse genomic backgrounds and enabling personalized precision diagnostic approaches, current iPSC-derived hepatocytes fail to fully recapitulate the metabolic capabilities of primary human hepatocytes (PHH), limiting their utility in DILI assessment and drug metabolism studies. This immature state leads to inaccurate predictions of drug toxicity and metabolism in vitro, which poses a significant challenge for preclinical evaluation of pharmaceutical compounds and for individualized precision medicine.

[0066] Our novel, chemically defined cocktail is composed of specific compounds designed to induce CYP450 maturation and, therefore, significantly enhance the metabolic competence of iPSC-derived hepatocytes. By promoting the expression and activity of CYP450 enzymes, this cocktail enables these cells to more accurately mimic the metabolic functions of liver cells.

[0067] In some embodiments, the composition and methods described herein advance beyond existing methodologies for maturation of iPSC-derived hepatocytes, by employing a novel cocktail of six compounds, including a cyclic AMP (cAMP) activator, Transforming Growth Factor-beta (TGF-P) inhibitor, Notch inhibitor, thyroid hormone, Peroxisome Proliferator-Activated Receptor (PPAR) activator, and a tryptophan metabolite, which together significantly enhance the functional maturation of iPSC-derived hepatocytes to a level comparable to PHHs. Using iPSC-derived hepatocytes produced by this novel cocktail has also proven to accurately predict DILI risk of known pharmaceutical compounds.

[0068] The functionality of HDC6 matured iM-Heps was significantly superior compared to traditional differentiation of iPSCs to hepatocytes like cells, reaching and even surpassingP-643500-PCfunctionality of primary human hepatocytes, in all aspects: albumin and urea production, and enzymatic activity of major subtypes of CYP450 metabolizing enzymes.

[0069] In a predictive DILI screen, the HDC6 iM-Hep spheroid model achieved superior predictive performance (66.7% sensitivity, 81.8% specificity) compared to PHH spheroids (55.6% sensitivity, 72.3% specificity), consistent with prior reports of PHH predictivity. Importantly, iM-Heps spheroids correctly identified the hepatotoxicity of drugs such as trovafloxacin and atorvastatin, which were under-predicted by PHHs. These improvements are directly attributable to the enhanced metabolic competence and stress responsiveness of the matured iM-Heps.

[0070] An immune-competent co-culture model incorporating iPSC-derived Kupffer cells retained hepatocyte function and responded robustly to inflammatory triggers, including LPS and immune-stimulatory biologies such as ipilimumab and theralizumab. This immune responsiveness, absent in monocultures, highlights the necessity of including non-parenchymal cells for modeling inflammation-associated liver injury. Additionally, both mono- and co-culture models recapitulate the synergy between metabolic stress (e.g., steatosis) and drug toxicity, further enhancing clinical relevance.Compositions

[0071] In some embodiments, provided herein is a hepatocyte maturation composition comprising one or more compounds selected from the group consisting of a TGF-beta inhibitor, a Notch inhibitor, a cyclic AMP (cAMP) activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. In some embodiments, the hepatocyte maturation composition comprises at least two compounds selected from the group consisting of a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. In some embodiments, the hepatocyte maturation composition comprises at least three compounds selected from the group consisting of a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. In some embodiments, the hepatocyte maturation composition comprises at least four compounds selected from the group consisting of a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. In some embodiments, the hepatocyte maturation composition comprises at least five compounds selected from the group consisting of a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.P-643500-PC

[0072] In some embodiments, provided herein is a hepatocyte maturation composition comprising a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0073] In some embodiments, the hepatocyte maturation composition is a cocktail of six chemical compounds, termed herein “Hepatocyte Differentiation Cocktail 6” (HDC6).

[0074] In some embodiments, the hepatocyte maturation composition comprises a cAMP Activator. In some embodiments, the cAMP Activator enhances cAMP signaling, for promoting liver-specific functions such as gluconeogenesis and albumin production. In some embodiments, the cAMP Activator increases intracellular levels of cAMP. In some embodiments, the cAMP Activator raises cAMP levels. In some embodiments, the cAMP Activator stimulates adenylyl cyclase (AC) activity. In some embodiments, the cAMP Activator activates adenylyl cyclase (AC).

[0075] In some embodiments, the cAMP Activator comprises forskolin or forskolin derivatives. In some embodiments, the forskolin derivative comprises CW008, NKH477 or colforsin daropate.

[0076] In some embodiments, the cAMP Activator comprises CL 316243.

[0077] In some embodiments, the cAMP Activator comprises a cAMP Agonist. In some embodiments, the cAMP agonist comprises Pituitary Adenylate Cyclase Activating Polypeptide 38 (PACAP 1-38).

[0078] In some embodiments, the cAMP Activator comprises a cAMP analog. In some embodiments, the cAMP analog comprises 8-bromo-cAMP or dibutyryl-cAMP.

[0079] In some embodiments, the cAMP activator is selected from, but not limited to the group consisting of CL 316243, forskolin or a derivative thereof, colforsin daropate, CW008, PACAP 1-38, 8-bromo-cAMP, dibutyryl-cAMP, NKH477, or mixtures or combinations thereof.

[0080] In some embodiments, the hepatocyte maturation composition comprises forskolin, provided at a concentration range of between about 0.5pM to 20pM.

[0081] In some embodiments, the cAMP activator is CW008, provided at a concentration range of between about 0.1 pM to 20 pM .

[0082] In some embodiments, the cAMP activator is NKH477 or colforsin daropate, provided at a concentration range of between about 0.1 pM to 20pM.

[0083] In some embodiments, the cAMP activator comprises PACAP 1-38, provided at a concentration range of between about O.lnM to 1 pM.P-643500-PC

[0084] In some embodiments, the cAMP Activator is provided at a concentration range of between about 0.1nM-l pM, e.g., from about 0.5 pM to about 1 pM, e.g., between about 1 nM to about 1 pM, e.g., between about 5 nM to about 1 pM, e.g., between about 10 nM to about 1 pM, e.g., between about 50 nM to about 1 pM, e.g., between about 100 nM to about 1 pM, e.g., between about 200 nM to about 1 pM, e.g., between about 300 nM to about 1 pM, e.g., between about 400 nM to about 1 pM, e.g., between about 500 nM to about 1 pM, e.g., between about 600 nM to about 1 pM, e.g., between about 700 nM to about 1 pM, e.g., between about 800 nM to about 1 pM, e.g., between about 900 nM to about 1 pM, e.g. about 1 pM.

[0085] In some embodiments, the cAMP Activator is provided at a concentration range of between about 0.1-30 pM, e.g., from about 0.1 pM to about 25 pM, e.g., between about 0.2-25 pM, e.g., between about 0.3-20 pM, e.g., between about 0.4-20 pM, e.g., between about 0.5-20 pM, e.g., between about 0.6-20 pM, e.g., between about 0.7-20 pM, e.g., between about 0.8-20 pM, e.g., between about 0.9-20 pM, e.g., between about 1-20 pM, e.g., between about 1-15 pM, e.g., between about 1-10 pM, e.g., between about 2-10 pM, e.g., between about 3-10 pM, e.g., between about 4-10 pM, e.g. about 10 pM.

[0086] In some embodiments, the cAMP activator is a cAMP analog. In some embodiments, the cAMP analog comprises 8-bromo-cAMP, provided at a concentration range of between about 10 pM to 500 pM. In some embodiments, the cAMP analog comprises dibutyryl-cAMP, provided at a concentration range of between about 1 pM to 1000 pM .

[0087] In some embodiments, the cAMP Activator is provided at a concentration range of between about 1.0-2000 pM, e.g., from about 1 pMto about 1500 pM, e.g., between about 5-1500 pM, e.g., between about 5-1500 pM, e.g., between about 10-1500 pM, e.g., between about 20-1500 pM, e.g., between about 50-1500 pM, e.g., between about 100-1500 pM, e.g., between about 200-1500 pM, e.g., between about 300-1500 pM, e.g., between about 400-1500 pM, e.g., between about 500-1500 pM, e.g., between about 600-1500 pM, e.g., between about 700-1500 pM, e.g., between about 800-1500 pM, e.g., between about 900-1500 pM, e.g., about 1000 pM.

[0088] In some embodiments, the hepatocyte maturation composition comprises a TGF-P Inhibitor. In some embodiments, the TGF-P Inhibitor inhibits the TGF-P pathway, preventing fate determination of cholangiocytes. In some embodiments, the TGF-P Inhibitor promotes hepatocyte maturation. In some embodiments, the TGF-P Inhibitor inhibits epithelial-mesenchymal transition (EMT). In some embodiments, the TGF-PP-643500-PCInhibitor prevents TGF-P from attaching to its receptors. In some embodiments, the TGF-P Inhibitor blocks TGF-P receptor downstream signaling. In some embodiments, the TGF-P Inhibitor targets Smad proteins.

[0089] In some embodiments, the TGF-P Inhibitor comprises a small molecule. In some embodiments, the TGF-P Inhibitor comprises SB431542.

[0090] In some embodiments, the TGF-P inhibitor is selected from, but not limited to the group consisting of SB431542 (4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide), SB525334 (6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-lH-imidazol- 4-yl]quinoxaline), Ki26894, LY364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-quinoline), SD-208 (2-(5- Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), SD-093 (2-(2-fluorophenyl)-N- pyridin-4-ylpyrido[2,3-d]pyrimidi -4-amine), SMI 6 (4-(5-(benzo[d][l,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-lH-imidazol-2-yl)bicyclo[2.2.2]octane-l -carboxamide), Ly2109761 (4-[2-[4-(2-pyri din-2 -yl-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-yl)quinolin-7-yl]oxyethyl]morpholine), Ly2157299 (2-(6-methyl- pyri din-2 -yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro -4H-pyrrolo[l,2-b]pyrazole monohydrate), K02288 (3-[6-amino-5-(3,4,5-trimethoxy-phenyl)-pyridin-3-yl]-plienol), SB505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l, l-dimethylethyl)-lH-imidazol-5-yl]-6-methyl- pyridine), LDN-193189 ( 4-(6-(4-(piperazin-l-yl) phenyl) pyrazolo[l,5-a]pyrimidin-3- yl)quinoline hydrochloride), GW788388 (4-[4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-2- pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), Ly580276 (3-(4-fluorophenyl)-2-(6- methylpyri din-2 -yl)-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole), EW-7203 (3-((5- ([l,2,4]triazolo[l,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2-ylamino)methyl)benzonitrile), EW-7195 (3-[methyl-[5-(6-methylpyridin-2-yl)-4-([l,2,4]triazolo[l,5-a]pyridin-6-yl) H-imidazol-2-yl]amino]benzonitrile), EW-7197 (N-[[4-([l,2,4]triazolo[l,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-lH-imidazol2-yl]methyl]-2-fluoroaniline), YR-290 (N-phenylacetyl-l,3,4,9-tetrahydro-l H -beta-carboline), A 83-01 (3- (6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)- IH-pyrazole- 1 -carbothioamide), D4476 (4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide), RepSox[alternatively E-616452, SJN 2511] (2-(3-(6-Methylpyridine-2-yl)-lH-pyrazol-4-yl)- 1,5-naphthyridine), R268712 (4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-lH-pyrazol-4- yl]phenyl]-lH-pyrazole-l-ethanol), or mixtures or combinations thereof.

[0091] In some embodiments, the TGF-P inhibitor is selected from, but not limited to the group consisting of SB431542, SB525334, Ki26894, LY364947, SD-208, SD-093, SM16, Ly2109761, Ly2157299, K02288, SB505124, LDN-193189, GW788388, Ly580276, EW-P-643500-PC7203, EW-7195, EW-7197, YR-290, A 83-01, D4476, RepSox, R268712, or mixtures or combinations thereof.

[0092] In some embodiments, the TGF-P Inhibitor comprises an anti-TGF-P antibody. In some embodiments, the TGF-P Inhibitor comprises Fresolimumab.

[0093] In some embodiments, the hepatocyte maturation composition comprises SB431542, provided at a concentration range of between about 0.1 pM to 20 pM.

[0094] In some embodiments, the TGF-P inhibitor is provided at a concentration range of between about 0.5-70 pM, e.g., from about 1 pM to about 70 pM, e.g., between about 1-60 pM, e.g., between about 1-55 pM, e.g., between about 1-50 pM, e.g., between about 1-45 pM, e.g., between about 1-40 pM, e.g., between about 1-35 pM, e.g., between about 1-30 pM, e.g., between about 1-25 pM, e.g., between about 1-20 pM, e.g., between about 1-15 pM, e.g., between about 1-10 pM, e.g., between about 2-10 pM, e.g., between about 3-10 pM, e.g., between about 4-10 pM, e.g. about 5 pM.

[0095] In some embodiments, the hepatocyte maturation composition comprises a Notch Inhibitor. In some embodiments, the Notch Inhibitor blocks Notch signaling, which is involved in maintaining cholangiocyte fate determination and delaying hepatocyte maturation. In some embodiments, the Notch Inhibitor blocks the gamma-secretase enzyme complex, preventing the release of the active Notch intracellular domain (NICD). In some embodiments, the Notch Inhibitor comprises Gamma-Secretase Inhibitors (GSIs). In some embodiments, the Notch Inhibitor comprises an antibody which targets a specific Notch ligand.

[0096] In some embodiments, the Notch Inhibitor comprises DAPT.

[0097] In some embodiments, the Notch inhibitor is selected from, but not limited to the group consisting of LY41 1575, MDL-28170, RO4929097, DAPT (N-[(3,5-Difiuorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-l, 1 - dimethylethyl ester), L- 685458 ((5 S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy- (2R)benzylhexanoyl)- L-leu-L-phe-amide), BMS-708163 (Avagacestat), BMS-299897 (2-[(l R)-l -[[(4- Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5- fluorobenzenebutanoic acid), M -0752, YO-01027, MDL28170 (Sigma), LY41 1575 (N- 2((2S)-2-(3,5- difluorophenyl)-2-hydroxyethanoyl)-N 1 -((7S)-5-methyl-6-oxo-6,7- dihydro-5H- dibenzo[b,d]azepin-7-yl)-l-alaninamide), ELN-46719 (2-hydroxy- valeric acid amide analog of LY41 1575), PF-03084014 ((S)-2-((S)-5,7-difiuoro-l,2,3,4- tetrahydronaphthalen-3-ylamino)-N-(l -(2-methyl-1 -(neopentylamino)propan-2-yl)- 1 H- imidazol-4-yl)pentanamide), Compound E ((2S)-2- {[(3,5- Difhirophenyl)acetyl]amino}- N-[(3S)- 1 -methyl-2-oxo-5-phenyl-2,3-P-643500-PCdihydro- 1 H-1,4- benzodiazepin-3- yl]propanamide, Semagacestat (LY450139, (2S)-2-hydroxy-3- methyl-N-(( 1 S)-l - methyl-2- {[(1 S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-l H-3-benzazepin- 1 -yl]amino}-2- oxoethyl)butanamide), or mixtures or combinations thereof.

[0098] In some embodiments, the Notch inhibitor is selected from, but not limited to the group consisting of LY41 1575, MDL-28170, Compound E, RO4929097, DAPT, L-685458, BMS-708163, BMS-299897, M -0752, YO-01027, MDL28170, LY41 1575, ELN-46719, PF-03084014, Semagacestat, or mixtures or combinations thereof.

[0099] In some embodiments, the hepatocyte maturation composition comprises DAPT, provided at a concentration range of between about 0.1 pM to 20 pM.

[0100] In some embodiments, the Notch Inhibitor is provided at a concentration range of between about 0.5-70 pM, e.g., from about 1 pM to about 70 pM, e.g., between about 1-60 pM, e.g., between about 1-55 pM, e.g., between about 1-50 pM, e.g., between about 1-45 pM, e.g., between about 1-40 pM, e.g., between about 1-35 pM, e.g., between about 1-30 pM, e.g., between about 1-25 pM, e.g., between about 1-20 pM, e.g., between about 1-15 pM, e.g., between about 1-10 pM, e.g., between about 2-10 pM, e.g., between about 3-10 pM, e.g., between about 4-10 pM, e.g. about 10 pM.

[0101] In some embodiments, the hepatocyte maturation composition comprises a Thyroid Hormone. In some embodiments, the Thyroid Hormone stimulates metabolic processes, contributing to the overall functional maturity of hepatocytes. In some embodiments, the Thyroid Hormone comprises a triiodothyronine (T3) thyroid hormone. In some embodiments, the Thyroid Hormone comprises a thyroxine (T4) thyroid hormone.

[0102] In some embodiments, the thyroid hormone comprises thyroxine (T4), triiodothyronine (T3), an analog thereof, or any combination thereof.

[0103] In some embodiments, the thyroid hormone comprises Liothyronine.

[0104] In some embodiments, the thyroid hormone comprises (S)-triiodothyronine, liothyronine, (S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoic acid, or an ester, salt or solvate thereof.

[0105] In some embodiments, the thyroid hormone comprises (S)-thyroxine, or an ester, salt or solvate thereof. In some embodiments, the thyroid hormone comprises Levothyroxine (L-thyroxine).

[0106] In some embodiments, the thyroid hormone comprises Liotrix. In some embodiments, the thyroid hormone comprises Tiratricol.

[0107] In some embodiments, the thyroid hormone is selected from, but not limited to the group consisting of Liothyronine, (S)-triiodothyronine, (S)-2-amino-3-[4-(4-hydroxy-3-P-643500-PCiodophenoxy)-3,5-diiodophenyl]propanoic acid, (S)-thyroxine, Levothyroxine (L-thyroxine), Liotrix, Tiratricol, or mixtures or combinations thereof.

[0108] In some embodiments, the hepatocyte maturation composition comprises liothyronine, provided at a concentration range of between about 1 nM to 10 pM.

[0109] In some embodiments, the thyroid hormone is provided at a concentration range of between about 0.1nM-l pM, e.g., from about 0.5 pM to about 1 pM, e.g., between about 1 nM to about 1 pM, e.g., between about 5 nM to about 1 pM, e.g., between about 10 nM to about 1 pM, e.g., between about 50 nM to about 1 pM, e.g., between about 100 nM to about 1 pM, e.g., between about 200 nM to about 1 pM, e.g., between about 300 nM to about 1 pM, e.g., between about 400 nM to about 1 pM, e.g., between about 500 nM to about 1 pM, e.g., between about 600 nM to about 1 pM, e.g., between about 700 nM to about 1 pM, e.g., between about 800 nM to about 1 pM, e.g., between about 900 nM to about 1 pM, e.g. about 1 pM.

[0110] In some embodiments, the thyroid hormone is provided at a concentration range of between about 0.5-70 pM, e.g., from about 1 pM to about 70 pM, e.g., between about 1-60 pM, e.g., between about 1-55 pM, e.g., between about 1-50 pM, e.g., between about 1-45 pM, e.g., between about 1-40 pM, e.g., between about 1-35 pM, e.g., between about 1-30 pM, e.g., between about 1-25 pM, e.g., between about 1-20 pM, e.g., between about 1-15 pM, e.g., between about 1-10 pM, e.g., between about 2-10 pM, e.g., between about 3-10 pM, e.g., between about 4-10 pM, e.g. about 3 pM.

[0111] In some embodiments, the hepatocyte maturation composition comprises a PPAR Activator. In some embodiments, the PPAR activator activates peroxisome proliferator-activated receptors, which are key regulators of lipid metabolism and energy homeostasis in hepatocytes.

[0112] In some embodiments, the PPAR activator comprises Wyl4643 (WY-14643).

[0113] In some embodiments, the PPAR activator comprises a PPAR-gamma (PPARy) activator, a PPAR-alpha (PPARa) activator, PPAR-delta (PPAR5) activator, a dual PPARa / 5 agonist, a pan PPAR activator (acting on all three isoforms), or any combination thereof.

[0114] In some embodiments, the PPARy activator is selected from, but not limited to the group consisting of 5-Oxo-ETE, 5-Oxo-15-hydroxy-ETE, 15-Deoxy-A12 14-prostaglandin J2, 15-HETE, 15-HpETE, Aleglitazar, Arachidonic acid, Balaglitazone, Berberine, Bezafibrate, Cannabidiol, Cevoglitazar, Chiglitazar, Ciglitazone, Daidzein, Darglitazone, Edaglitazone, Efatutazone, Englitazone, Etalocib, Farglitazar, Genistein, GW-1929,P-643500-PCIbuprofen, Imiglitazar, Indeglitazar, Lanifibranor, LG-100268, LG-100754, LG-101506, Lobeglitazone, Muraglitazar (muroglitazar), nTZDpa, Naveglitazar, Netoglitazone, Oxeglitazar, Peliglitazar, Pemaglitazar, Perfluorononanoic acid, Pioglitazone, Prostaglandin J2, Ragaglitazar, Reglitazar, Rivoglitazone, Rosiglitazone, RS5444, Saroglitazar, Sipoglitazar, Sodelglitazar, Telmisartan, Tesaglitazar, Troglitazone, AMG131, or mixtures or combinations thereof.

[0115] In some embodiments, the PPARy activator comprises a selective PPAR modulator (SPPARM). In some embodiments, the SPPARM comprises BADGE, EPI-001, INT-131, MK-0533, or S26948.

[0116] In some embodiments, the PPARa activator is selected from, but not limited to the group consisting of Wyl4643, 15-HETE, 15-HpETE, Aleglitazar, Aluminium clofibrate, Arachidonic acid, Bezafibrate, Chiglitazar, Clofibrate, CP-775146, Daidzein, DHEA (prasterone), Elafibranor, Etomoxir, Fenofibrate, Genistein, Gemfibrozil, GW-7647, Lanifibranor, Leukotriene B4, LG-101506, LG-100754, Lobeglitazone, Muraglitazar, Oleyl ethanol ami de, Palmitoylethanolamide, Pemafibrate, Perfluorononanoic acid, Perfluorooctanoic acid, Pioglitazone, Saroglitazar, Sodelglitazar, Tesaglitazar, Tetradecylthioacetic acid, Troglitazone, or mixtures or combinations thereof.

[0117] In some embodiments, the pan PPAR activator is selected from, but not limited to the group consisting of Bezafibrate, lobeglitazone, chiclitazar, aleglitazar, elafibranor, saroglitazar, muraglitazar, tesaglitazar, Lanifibranor (IVA337), or mixtures or combinations thereof.

[0118] In some embodiments, the PPAR5 activator is selected from, but not limited to the group consisting of 15-HETE, 15-HpETE, Arachidonic acid, Bezafibrate, Daidzein, Elafibranor, Fonadelpar, Genistein, GW-0742, GW-501516, L-165,041, Lanifibranor, LG-101506, Seladelpar, Sodelglitazar, Tetradecylthioacetic acid, or mixtures or combinations thereof.

[0119] In some embodiments, the PPAR5 activator comprises a PPAR5 Antagonist. In some embodiments, the PPAR5 Antagonist comprises FH-535, GSK-0660, GSK-3787, or mixtures or combinations thereof.

[0120] In some embodiments, the dual PPARa / 5 agonist binds to both the a and y PPAR isoforms. In some embodiments, the dual PPARa / 5 agonist is selected from, but not limited to the group consisting of aleglitazar, muraglitazar, oxeglitazar, naveglitazar, tesaglitazar, saroglitazar, chiglitazar, elafibranor, or mixtures or combinations thereof.P-643500-PC

[0121] In some embodiments, the PPAR activator comprises a dual PPARy / 5 agonist. In some embodiments, the dual PPARy / 5 agonist comprises telmisartan.

[0122] In some embodiments, the hepatocyte maturation composition comprises WY14643, provided at a concentration range of between about 0.1 pM to 100 pM.

[0123] In some embodiments, the PPAR activator is provided at a concentration range of between about 0.01-100 pM, e.g., from about 0.05 pM to about 100 pM, e.g., from about 0.07 pM to about 100 pM, e.g., from about 1 pM to about 100 pM, e.g., between about 1-90 pM, e.g., between about 1-80 pM, e.g., between about 1-70 pM, e.g., between about 1- 60 pM, e.g., between about 1-55 pM, e.g., between about 1-50 pM, e.g., between about 1- 45 pM, e.g., between about 1-40 pM, e.g., between about 1-35 pM, e.g., between about 1- 30 pM, e.g., between about 1-25 pM, e.g., between about 1-20 pM, e.g., between about 1- 15 pM, e.g., between about 1-10 pM, e.g., between about 2-10 pM, e.g., between about 3- 10 pM, e.g., between about 4-10 pM, e.g. about 10 pM.

[0124] In some embodiments, the hepatocyte maturation composition comprises a Tryptophan Metabolite. In some embodiments, the Tryptophan Metabolite modulates the aryl hydrocarbon receptor (AhR) pathway. In some embodiments, the Tryptophan Metabolite comprises 6-Formylindolo[3,2-b]carbazole (FICZ).

[0125] In some embodiments, the tryptophan metabolite is selected from the group consisting of 6-Formylindolo[3,2-b]carbazole (FICZ), indole-3 -aldehyde (lAld), indole-3-acetic acid (IAA), indole-3 -propionic acid (IP A), indole-3 -lactic acid (ILA), kynurenine, kynurenic acid, xanthurenic acid, quinolinic acid, tryptamine, indole, skatole (3-methylindole), or mixtures or combinations thereof.

[0126] In some embodiments, the hepatocyte maturation composition comprises FICZ , provided at a concentration range of between about 0.1 pM to 5 pM.

[0127] In some embodiments, the Tryptophan Metabolite is provided at a concentration range of between about 0.1-100 pM, e.g., from about 0.1 pM to about 100 pM, e.g., between about 0.1-90 pM, e.g., between about 0.1-80 pM, e.g., between about 0.1-70 pM, e.g., between about 0.1-60 pM, e.g., between about 0.1-55 pM, e.g., between about 0.1-50 pM, e.g., between about 0.1-45 pM, e.g., between about 0.1-40 pM, e.g., between about 0.1-35 pM, e.g., between about 0.1-30 pM, e.g., between about 0.1-25 pM, e.g., between about 0.1-20 pM, e.g., between about 0.1-15 pM, e.g., between about 0.1-10 pM, e.g., between about 0.1-8 pM, e.g., between about 0.1-5 pM, e.g., between about 0.5-5 pM, e.g., between about 0.5-4 pM, e.g., between about 0.5-3 pM, e.g., between about 0.5-2 pM, e.g., about 1 pM.P-643500-PC

[0128] In some embodiments, the hepatocyte maturation composition comprises six distinct compounds: cAMP activator, TGF-P inhibitor, Notch inhibitor, thyroid hormone, PPAR activator, and a tryptophan metabolite, each chosen for its unique ability to modulate specific signaling pathways involved in hepatocyte maturation. In some embodiments of the hepatocyte maturation composition, the compounds work synergistically to enhance the iPSC-derived hepatocyte maturation process.

[0129] In some embodiments, the hepatocyte maturation composition is added to cell culturing media. In some embodiments, additional agents are added to the hepatocyte maturation composition described herein, these agents include, but are not limited to, growth factors, metabolic enhancers, lipids, antioxidants, at least one, at least two, at least three, at least four, at least five, at least six or more of the following agents:

[0130] Growth factors and cytokines. In some embodiments, the additional agent comprises a growth factor or cytokine selected from the group consisting of hepatocyte growth factor (HGF), epidermal growth factor (EGF), oncostatin M (OSM), fibroblast growth factor (FGF), insulin-like growth factor (IGF), bone morphogenetic proteins (BMPs), and combinations thereof.

[0131] Metabolic enhancers. In some embodiments, the additional agent comprises hormone or metabolic enhancers selected from the group consisting of insulin, glucagon, dexamethasone, cortisol, growth hormone, retinoic acid, nicotinamide, coenzyme Q10 ,and combinations thereof.

[0132] Lipids and Fatty acids. In some embodiments, the additional agent comprises a lipid or fatty acid selected from the group consisting of oleic acid, linoleic acid, palmitic acid, stearic acid, arachidonic acid, omega-3 fatty acids, ketone bodies, and combinations thereof.

[0133] Oxidative stress modulators. In some embodiments, the additional agent comprises an antioxidant or redox modulator selected from the group consisting of glutathione, N-acetylcysteine (NAC), vitamin C, vitamin E, selenium, and combinations thereof.

[0134] In some embodiments, the hepatocyte maturation composition described herein can be used to generate mature iPSC-derived hepatocytes.iPSC-derived mature hepatocytes

[0135] The hepatocyte maturation composition described herein can be used in the generation of mature hepatocytes from induced pluripotent stem cells (iPSCs). In some embodiments, the hepatocyte maturation composition used for generation matureP-643500-PChepatocytes comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0136] In some embodiments, the term “induced pluripotent stem cells (iPSCs)”, may encompass pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing expression of certain genes. In some embodiments, hiPSC refers to human iPSCs.

[0137] In some embodiments, the cells are any cells, e.g., prokaryotic or eukaryotic cells, e.g., primate cells, e.g., mammalian cells, e.g., human cells. According to some embodiments, the cells are pluripotent stem cells (PSCs), induced PSCs (iPSCs), primed pluripotent stem cells, non-naive pluripotent stem cells, naive pluripotent stem cells. In some embodiments, iPSC-derived hepatocytes comprise human iPSC-derived hepatocytes.

[0138] In some embodiments, immature iPSC-derived hepatocytes comprise hepatocytelike cells differentiated from induced pluripotent stem cells that exhibit reduced hepatic functional maturity relative to adult primary human hepatocytes (PHH).

[0139] In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP450 enzyme activity, including one or more of CYP3 A4, CYP2C9, CYP2D6, CYP1A2, CYP2C8, CYP2C19 or CYP2B6, at levels less than about 20% of the corresponding activity observed in PHH.

[0140] In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP450 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP3 A4 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP2C9 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP2D6 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP1 A2 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP2C8 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP2C19 enzyme activity. In some embodiments, immature iPSC-derived hepatocytes are characterized by reduced CYP2B6 enzyme activity.

[0141] In some embodiments, immature iPSC-derived hepatocytes reduced enzyme activity is at levels less than about 10% of the corresponding activity observed in PHH. In some embodiments, immature iPSC-derived hepatocytes reduced enzyme activity is at levels less than about 20% of the corresponding activity observed in PHH. In some embodiments, immature iPSC-derived hepatocytes reduced enzyme activity is at levels lessP-643500-PCthan about 30% of the corresponding activity observed in PHH. In some embodiments, immature iPSC-derived hepatocytes reduced enzyme activity is at levels less than about 15% of the corresponding activity observed in PHH.

[0142] In some embodiments, mature iPSC-derived hepatocytes have increased CYP450 enzyme activity. In some embodiments, mature iPSC-derived hepatocytes have a functional level comparable to that of Primary Human Hepatocytes (PHH).

[0143] In some embodiments, primary cells refer to cells that were obtained by direct removal from an organism, an organ or a tissue and put in culture. In some embodiments, Primary Human Hepatocytes (PHH) comprise primary cells from human liver. Hepatocytes make up about 70-80% of the liver's mass.

[0144] In some embodiments, mature iPSC-derived hepatocytes have increased CYP450 enzyme activity, comparable to that of PHHs. In some embodiments, mature iPSC-derived hepatocytes have increased expression of Phase I metabolizing enzymes including CYP3A4, CYP1A2, and CYP2C8, as well as Phase II and transporter genes, at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes have CYP3A4 expression at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes have CYP1A2 expression at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes have CYP2C8 expression at levels comparable to PHHs.

[0145] In some embodiments, the terms “improved”, “increased”, or “reduced” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same cells prior to the treatment described herein, or a measurement in a control cell (or multiple control cells) in the absence of the treatment described herein (untreated cells), e.g. PHH cells.

[0146] In some embodiments, mature iPSC-derived hepatocytes have Albumin (ALB) expression at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes have ASGR1 expression at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes have multidrug resistance-associated protein 2 (MRP2) expression at levels comparable to PHHs.

[0147] In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 40 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 35 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albuminP-643500-PClevels exceeding 30 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 28 pg / day / million cells.

[0148] In some embodiments, mature iPSC-derived hepatocytes are characterized in increased urea secretion. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 20 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 30 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 40 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 50 pg / day / million cells, comparable to PHH urea secretion levels. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 60 pg / day / million cells.

[0149] In some embodiments, mature iPSC-derived hepatocytes demonstrate functional urea cycle activity, as evidenced by a significant increase in urea production following exposure to ammonia and arginine, compared to untreated control. In some embodiments, untreated control conditions comprise mature iPSC derived hepatocytes which have not been treated with ammonium salts and arginine.

[0150] In some embodiments, mature iPSC derived hepatocytes exhibit at least 1.5 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions.

[0151] In some embodiments, mature iPSC derived hepatocytes exhibit at least 2 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions.

[0152] In some embodiments, mature iPSC derived hepatocytes exhibit at least 3 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions.

[0153] In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1A2 activity at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1A2 activity at levels of about 5 pmol / min / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1 A2 activity at levels of above 5 pmol / min / million cells.

[0154] In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3A4 activity at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3A4 activity at levels of about 0.2P-643500-PCpmol / min / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3 A4 activity at levels of above 0.2 pmol / min / million cells.

[0155] In some embodiments, mature iPSC-derived hepatocytes are characterized by having one or more of the following:expression of Phase I metabolizing enzymes, including CYP3A4, CYP1A2, and CYP2C8 at levels comparable to expression in PHHs;CYP1 A2 activity at levels comparable to activity levels in PHHs;CYP3 A4 activity at levels comparable to activity levels in PHHs;expression of ALB, ASGR1, or MRP2 at levels comparable to PHHs; albumin levels exceeding 30 pg / day / million cells; and- urea secretion levels comparable to secretion levels in PHHs.

[0156] In some embodiments, mature iPSC-derived hepatocytes have increased expression of Phase I metabolizing enzymes, compared to immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have increased expression of CYP3 A4 compared to immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have increased expression of CYP1A2 compared to immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have increased expression of CYP2C8 compared to immature iPSC-derived hepatocytes.

[0157] In some embodiments, mature iPSC-derived hepatocytes have reduced expression of fetal-associated genes such as AFP and FMO1, which are highly expressed in immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have reduced expression of AFP, compared to immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have reduced expression of FMO1 compared to immature iPSC-derived hepatocytes.

[0158] In some embodiments, mature iPSC-derived hepatocytes are characterized by having one or more of the following:increased expression of CYP3A4, CYP1A2, or CYP2C8, compared to immature iPSC-derived hepatocytes;reduced expression of fetal -associated genes such as AFP and FMO1, compared to immature iPSC-derived hepatocytes; andincreased albumin levels, compared to immature iPSC-derived hepatocytes.

[0159] In some embodiments, the mature iPSC-derived hepatocytes are characterized by having one or more of the following:enhanced metabolic and secretory function, comparable to PHH levels;P-643500-PCincreased the enzymatic activity of cytochrome P450 enzymes, compared to immature iPSC-derived hepatocytes; andincreased oxidative metabolism, including TCA cycle, OXPHOS, and fatty acid P- oxidation pathways, compared to immature iPSC-derived hepatocytes.

[0160] In some embodiments, the mature iPSC-derived hepatocytes are characterized by having one or more of the following:express one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and multi drug resistance-associated protein 2 (MRP2);albumin levels exceeding 40 pg / day / million cells;increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control;CYP3 A4 activity at levels of above 0.2 pmol / min / million cells;CYP1 A2 activity at levels of about 5 pmol / min / million cells; andreduced expression of AFP or FMO1, compared to immature iPSC-derived hepatocytes.Methods for generating iPSC-derived mature hepatocytes

[0161] According to some embodiments, provided herein is a method of generating mature iPSC-derived hepatocytes comprising culturing immature iPSC-derived hepatocytes in the hepatocyte maturation composition described herein, the hepatocyte maturation composition allowing generation of mature iPSC-derived hepatocytes.

[0162] In some embodiments, provided herein is a method for preparing mature iPSC-derived hepatocytes comprising adding the hepatocyte maturation composition described herein to immature iPSC-derived hepatocyte cells to obtain mature iPSC-derived hepatocytes.

[0163] In some embodiments, provided herein is a method for generating mature iPSC-derived hepatocytes comprising culturing immature iPSC-derived hepatocytes in a culture medium including a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes, wherein the hepatocyte maturation composition comprises TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0164] In some embodiments, a culture medium, cell culture medium, or cell culture medium may encompass a solution containing sufficient nutrients to promote the growth of cells in the culture. In some embodiments, these solutions contain essential amino acids,P-643500-PCnon-essential amino acids, vitamins, energy sources, lipids and / or trace elements. In some embodiments, the medium can also contain other adjuvants such as hormones, growth factors and growth inhibitors.

[0165] In some embodiments, the step of culturing iPSC-derived hepatocytes in a hepatocyte maturation composition comprises culturing iPSC-derived hepatocytes in a culture medium supplemented with the hepatocyte maturation composition described herein.

[0166] In some embodiments, the step of culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition is for a period of time of from about 1 day to about 50 days, e.g., between about 1-40 days, e.g., between about 1-30 days, e.g., between about 1-25 days, e.g., between about 1-20 days, e.g., between about 1-15 days, e.g., between about 1-10 days, e.g., between about 5-10 days, e.g. about 10 days.

[0167] In some embodiments, the step of culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition is for a period of time of between about 5-10 days.

[0168] In some embodiments, the immature iPSC-derived hepatocytes are cryopreserved cells which are thawed before culturing. In some embodiments, the immature iPSC-derived hepatocytes are non-frozen cells.

[0169] In some embodiments, the immature iPSC-derived hepatocytes are any cells, e.g., prokaryotic or eukaryotic cells, e.g., primate cells, e.g., mammalian cells, e.g., human cells. According to some embodiments, the immature iPSC-derived hepatocytes are mammalian cells. In some embodiments, the immature iPSC-derived hepatocytes are human cells.

[0170] In some embodiments, the mature iPSC-derived hepatocytes express one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and MRP2. In some embodiments, the mature iPSC-derived hepatocytes express CYP3A4. In some embodiments, the mature iPSC-derived hepatocytes express CYP1A2. In some embodiments, the mature iPSC-derived hepatocytes express CYP2C8. In some embodiments, the mature iPSC-derived hepatocytes express ALB. In some embodiments, the mature iPSC-derived hepatocytes express ASGR1. In some embodiments, the mature iPSC-derived hepatocytes express MRP2.

[0171] In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 40 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 35 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albuminP-643500-PClevels exceeding 30 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in albumin levels exceeding 28 pg / day / million cells.

[0172] In some embodiments, mature iPSC-derived hepatocytes are characterized in increased urea secretion. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 20 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 30 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 40 pg / day / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 50 pg / day / million cells, comparable to PHH urea secretion levels. In some embodiments, mature iPSC-derived hepatocytes are characterized in having urea secretion levels exceeding 60 pg / day / million cells.

[0173] In some embodiments, mature iPSC derived hepatocytes exhibit at least 1.5 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions. In some embodiments, untreated control conditions comprise mature iPSC derived hepatocytes which have not been treated with ammonium salts and arginine.

[0174] In some embodiments, mature iPSC derived hepatocytes exhibit at least 2 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions.

[0175] In some embodiments, mature iPSC derived hepatocytes exhibit at least 3 fold or greater increase in urea production following 24 hours treatment with ammonium salts and arginine, compared to untreated control conditions.

[0176] In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1A2 activity at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1A2 activity at levels of about 5 pmol / min / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP1 A2 activity at levels of above 5 pmol / min / million cells.

[0177] In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3A4 activity at levels comparable to PHHs. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3A4 activity at levels of about 0.2 pmol / min / million cells. In some embodiments, mature iPSC-derived hepatocytes are characterized in CYP3 A4 activity at levels of above 0.2 pmol / min / million cells.P-643500-PC

[0178] In some embodiments, mature iPSC-derived hepatocytes have reduced expression of fetal-associated genes such as AFP and FM01, which are highly expressed in immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have reduced expression of AFP, compared to immature iPSC-derived hepatocytes. In some embodiments, mature iPSC-derived hepatocytes have reduced expression of FM01, compared to immature iPSC-derived hepatocytes.

[0179] In some embodiments, the mature iPSC-derived hepatocytes are assessed for functional maturity using a variety of assays. Specifically, these include real-time quantitative PCR (RT-qPCR) for liver-specific markers (e.g., albumin, HNF4a, CYP450 enzymes, etc.), enzyme activity assays (e.g., CYP450s activity), and secretion levels of liver-specific proteins (e.g., albumin and alpha-l-antitrypsin).

[0180] In some embodiments, the mature iPSC-derived hepatocytes are characterized by having one or more of the following:express one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and MRP2;albumin levels exceeding 40 pg / day / million cells;increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control;CYP3 A4 activity at levels of above 0.2 pmol / min / million cells;CYP1 A2 activity at levels of about 5 pmol / min / million cells; andreduced expression of AFP or FMO1, compared to immature iPSC-derived hepatocytes.

[0181] In some embodiments, the mature iPSC-derived hepatocytes are characterized by having one or more of the following:enhanced metabolic and secretory function, comparable to PHH levels; increased the enzymatic activity of cytochrome P450 enzymes, compared to immature iPSC-derived hepatocytes; andincreased oxidative metabolism, including TCA cycle, OXPHOS, and fatty acid [3- oxidation pathways, compared to immature iPSC-derived hepatocytes.Methods for generating 3D liver spheroids

[0182] In some embodiments, provided herein is a method for generating a 3D liver spheroid from iPSC-derived immature hepatocytes comprising (a) culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition to obtain mature iPSC-P-643500-PCderived hepatocytes; and (b) seeding the mature iPSC-derived hepatocytes of step (a) on a low attachment surface and culturing in hepatocyte maturation composition supplemented with Rho-associated kinase (ROCK) inhibitor, to obtain a 3D liver spheroid.

[0183] In some embodiments, provided herein is a method for generating a 3D liver spheroid from iPSC-derived immature hepatocytes comprising (a) culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes; and (b) seeding the mature iPSC-derived hepatocytes of step (a) on a low attachment surface and culturing in hepatocyte maturation composition supplemented with Rho-associated kinase (ROCK) inhibitor, to obtain a 3D liver spheroid, wherein the hepatocyte maturation composition comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0184] In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Fasudil. In some embodiments, the ROCK inhibitor comprises Ripasudil.

[0185] In some embodiments, the ROCK inhibitor is provided at a concentration range of between about 0.1-100 pM, e.g., from about 0.1 pM to about 100 pM, e.g., between about 0.1-90 pM, e.g., between about 0.1-80 pM, e.g., between about 0.1-70 pM, e.g., between about 0.1-60 pM, e.g., between about 0.1-55 pM, e.g., between about 0.1-50 pM, e.g., between about 0.1-45 pM, e.g., between about 0.1-40 pM, e.g., between about 0.1-35 pM, e.g., between about 0.1-30 pM, e.g., between about 0.1-25 pM, e.g., between about 0.1-20 pM, e.g., between about 0.1-15 pM, e.g., between about 0.1-10 pM, e.g. about 10 pM.

[0186] In some embodiments, the step of culturing the mature iPSC-derived hepatocytes in hepatocyte maturation composition supplemented with a ROCK inhibitor is for a period of time of from about 1 day to about 50 days, e.g., between about 1-40 days, e.g., between about 1-30 days, e.g., between about 1-25 days, e.g., between about 1-20 days, e.g., between about 1-15 days, e.g., between about 1-10 days, e.g., between about 5-10 days, e.g. about 10 days.

[0187] In some embodiments, the step of culturing the mature iPSC-derived hepatocytes in hepatocyte maturation composition supplemented with a ROCK inhibitor is for a period of time of between about 12 to 18 hours followed by removal of ROCK inhibitor and continued culture in hepatocyte maturation composition in the absence of ROCK inhibitor for a period of time of between about 5-10 days.P-643500-PC

[0188] In some embodiments of the method of generating a 3D liver spheroid, the method further comprises combining the mature iPSC-derived hepatocytes from step (a) with iPSC-derived Kupffer-like cells (iKCs), prior to seeding. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 5:1.

[0189] In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 1:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 2: 1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 3:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 4:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 5:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 6:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 7:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 8:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 9:1. In some embodiments, the ratio of iPSC-derived hepatocytes to Kupffer cells is 10:1.

[0190] In some embodiments, the low attachment surface may encompass cell culture plates with surfaces that prevent cells from sticking, forcing them to clump together via cell-to-cell contact into spherical, tumor-like structures (spheroids). In some embodiments, the low attachment surface may encompass "ultra-low attachment" (ULA) surfaces which are chemically treated to be hydrophilic and neutrally charged, supporting scaffold-free 3D growth.3D liver spheroids

[0191] In some embodiments, provided herein is a 3D liver spheroid prepared according to the method described herein. In some embodiments, the 3D liver spheroid is prepared by (a) culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes; and (b) seeding the mature iPSC-derived hepatocytes of step (a) on a low attachment surface and culturing in hepatocyte maturation composition supplemented with Rho-associated kinase (ROCK) inhibitor, to obtain a 3D liver spheroid, wherein the maturation composition comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

[0192] In some embodiments, the 3D liver spheroid is characterized by having one or more of the following:P-643500-PCexpress one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and MRP2;albumin levels exceeding 40 pg / day / million cells;increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control;CYP3 A4 activity at levels of above 0.2 pmol / min / million cells;CYP1 A2 activity at levels of about 5 pmol / min / million cells; andreduced expression of AFP or FMO1, compared to immature iPSC-derived hepatocytes.

[0193] In some embodiments, the 3D liver spheroid is characterized by expressing one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and MRP2. In some embodiments, the 3D liver spheroid is characterized by albumin levels exceeding 40 pg / day / million cells. In some embodiments, the 3D liver spheroid is characterized by increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control. In some embodiments, the 3D liver spheroid is characterized by CYP3 A4 activity at levels of above 0.2 pmol / min / million cells. In some embodiments, the 3D liver spheroid is characterized by CYP1A2 activity at levels of about 5 pmol / min / million cells. In some embodiments, the 3D liver spheroid is characterized by reduced expression of AFP or FMO1, compared to immature iPSC-derived hepatocytes.

[0194] In some embodiments, the 3D liver spheroid is characterized by enhanced metabolic and secretory function, comparable to PHH levels. In some embodiments, the 3D liver spheroid is characterized by increased the enzymatic activity of cytochrome P450 enzymes, compared to immature iPSC-derived hepatocytes. In some embodiments, the 3D liver spheroid is characterized by increased oxidative metabolism, including TCA cycle, OXPHOS, and fatty acid [3-oxidation pathways, compared to immature iPSC-derived hepatocytes.

[0195] In some embodiments, the 3D liver spheroid is characterized by having one or more of the following:enhanced metabolic and secretory function, comparable to PHH levels; increased the enzymatic activity of cytochrome P450 enzymes, compared to immature iPSC-derived hepatocytes; andincreased oxidative metabolism, including TCA cycle, OXPHOS, and fatty acid [3- oxidation pathways, compared to immature iPSC-derived hepatocytes.P-643500-PC

[0196] In some embodiments, the 3D liver spheroid is used for at least one purpose selected from the group consisting of: a model platform for liver disease testing; model platform for drug-induced liver injury (DILI) prediction; for idiosyncratic drug induced liver injury (iDILI) identification and studies; for disease modeling; in vitro mechanistic studies; food supplement induced liver injury prediction; drug-drug interaction (DDI) testing; and drugfood supplement interaction testing.

[0197] In some embodiments, the 3D liver spheroid is used as a model platform for liver disease testing. In some embodiments, the 3D liver spheroid is used as a model platform for drug-induced liver injury (DILI) prediction. In some embodiments, the 3D liver spheroid is used for idiosyncratic drug induced liver injury (iDILI) identification and studies. In some embodiments, the 3D liver spheroid is used for disease modeling. In some embodiments, the 3D liver spheroid is used for in vitro mechanistic studies. In some embodiments, the 3D liver spheroid is used for food supplement induced liver injury prediction. In some embodiments, the 3D liver spheroid is used for drug-drug interaction (DDI) testing. In some embodiments, the 3D liver spheroid is used for drug-food supplement interaction testing.

[0198] In some embodiments, the 3D liver spheroid is characterized by enhanced CYP450 enzyme activity. These enzymes are crucial for drug metabolism, and their relevant representation in vitro is essential for predicting DILI.Methods of use

[0199] In some embodiments, disclosed herein is a method for predicting drug induced liver injury (DILI) comprising bringing a drug into contact with the 3D liver spheroid described herein. In some embodiments, the toxicity of the drug is determined by assessing spheroid viability or spheroid morphology.

[0200] In some embodiments, the toxicity of a drug is determined by measuring a parameter selected from mitochondria membrane potential, measurement of ROS, swelling of liver mitochondria, and combinations thereof.

[0201] In some embodiments, Drug-induced liver injury (DILI) is an injury of the liver that may be caused by drugs, medicinal herbs, plants, and as well as nutritional supplements. In some embodiments, DILI may be the result of direct toxicity from an administered drug or its metabolite, or injury may result from indirect immune-mediated mechanisms.

[0202] In some embodiments, disclosed herein is a method for identifying a preferred therapeutic agent for an individual comprising bringing a candidate compound into contactP-643500-PCwith the 3D liver spheroid described herein. In some embodiments, the preferred therapeutic agent is determined by assessing spheroid viability or spheroid morphology.

[0203] In some embodiments, a “candidate compound” or “drug” may encompass any compound whose toxicity properties are of interest. In some embodiments, the candidate compound includes, but is not limited to carcinogens, environmental pollutants, and therapeutic agents (drugs), including: anti-neoplastics, immuno-suppressants, immune-stimulants, anti-proliferatives, anti-thrombins, anti -platelet, anti-lipid, anti-inflammatory, antibiotics, angiogenics, anti-angiogenics, vitamins, ACE inhibitors, vasoactive substances, anti-mitotics, metello-proteinase inhibitors, NO donors, estradiols, anti-sclerosing agents, hormones, free radical scavengers, toxins, alkylating agents, either alone or in combination.

[0204] In some embodiments, the candidate compound may also include, biologic agents, such as: peptides, lipids, protein drugs, protein conjugates drugs, antibodies, enzymes, oligonucleotides, ribozymes, genetic material, prions, viruses, and bacteria.

[0205] In some embodiments, the 3D liver spheroid is derived from the same ethnic background of the individual, or further, wherein the 3D liver spheroid is derived from the individual.

[0206] In some embodiments, the 3D liver spheroid demonstrated improved DILI prediction. The enhanced functional maturity enables iPSC-derived hepatocytes to serve as a more reliable source for in vitro models investigating DILI predictions. This improvement not only advances the utility of iPSC-derived hepatocytes in drug testing and toxicology studies but also provides a more ethical and cost-effective alternative to PHH and animal models.

[0207] In some embodiments, the 3D liver spheroid demonstrated improved DILI prediction accuracy. One of the primary challenges in drug development is the reliable prediction of DILI using in vitro models. The enhanced functional maturity of hepatocytes shown herein improves the accuracy of these models in predicting DILI, thereby reducing the risk of late-stage drug failures and adverse effects in clinical trials. It also opens up opportunities for improved study population stratification in clinical studies by enabling more feasible access to genomic diversity and better representation of different ethnic, gender and age groups.

[0208] In some embodiments, the 3D liver spheroid may be used in precision personalized medicine. 3D liver spheroids derived from individuals for in vitro diagnostics may provide unprecedented opportunities for individualized precision medicine and diagnostic toolsP-643500-PCwhere a simple blood sample as starting material can be utilized for multiple in vitro testing eliminating the need for more invasive sampling such as biopsy.

[0209] Further, the use of PHH is limited by cost, availability, and variability between donors. Similarly, personalized toxicology studies cannot be easily achieved using patient PHH ex vivo. In contrast, mature iPSC-derived hepatocytes are more readily available and can be produced in large quantities to support studies. This scalability is particularly advantageous for high-throughput screening applications in drug discovery (i.e. DILI investigation).

[0210] A skilled artisan would appreciate that the use of the term comprising throughout, may in certain embodiments be replace by the use of the term consisting essentially of or consisting of. The skilled artisan would appreciate that the term “comprising” is intended to mean that the composition includes the recited elements, but not excluding others which may be optional. For example, a composition which comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone. Further, the term “consisting essentially of’ may encompass a composition that includes the recited elements, for example, a hepatocyte maturation composition consisting essentially of a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone, but excludes other elements that may have an essential significant effect on the performance of the composition. Thus, such a composition may still include a buffer or culturing medium. Further, “consisting of’ encompasses excluding more than traces of other elements.

[0211] In some embodiments, methods as disclosed herein may be represented as uses of the compositions as described herein.

[0212] In some embodiments, the compositions and methods as disclosed herein comprise the various components or steps. However, in another embodiment, the compositions and methods as disclosed herein consist essentially of the various components or steps, where other components or steps may be included. In another embodiment, the compositions and methods as disclosed herein consist of the various components or steps.

[0213] A skilled artisan would appreciate that the term “about”, may encompass a deviance of between 0.0001-5% from the indicated number or range of numbers. Further, it may encompass a deviance of between 1 -10% from the indicated number or range of numbers. In addition, it may encompass a deviance of up to 25% from the indicated number or range of numbers.P-643500-PC

[0214] A skilled artisan would appreciate that the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" or "at least an agent" may include a plurality of agents, including mixtures thereof.

[0215] Throughout this application, various embodiments disclosed herein may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicated number and a second indicated number and “ranging / ranges from” a first indicated number “to” a second indicated number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0216] It should be understood that the disclosure presented herein is not limited to the particular methodologies, protocols and reagents, and examples described herein. The terminology and examples used herein is for the purpose of describing particular embodiments only, for the intent and purpose of providing guidance to the skilled artisan, and is not intended to limit the scope of the disclosure presented herein.EXAMPLESExample 1: Materials and Methods

[0217] Human iPSC Culture and Maintenance

[0218] Human iPSC lines 744 and 750 were purchased from I PEACE, Inc. (Palo Alto, CA). Human iPSC lines 393, 399, 408, and 477 were purchased from iXCells Biotechnologies (San Diego, CA). All iPSC lines were maintained in a feeder-free condition on Matrigel-coated plates (Coming, Cat. #354277) in mTeSR™ Plus medium (STEMCELL Technologies, Cat. #100-0276), following manufacturer-recommended protocols. Cells were routinely passaged at 70-90% confluency using Accutase (Gibco, Cat. Al 110501) and maintained at 37 °C in a humidified incubator with 5% CO2.

[0219] Stepwise Hepatocyte Differentiation and HDC6 Based MaturationP-643500-PC

[0220] Hepatocyte initial differentiation was performed by an optimized stepwise protocol described previously (Tian et al., Int J Biol Sci 12, 1052-1062, 2016). Briefly, iPSCs at 60-80% confluency were treated with Activin A (50-100 ng / mL; R&D Systems, Cat. #338-AC) in RPMI-1640 medium (Gibco, Cat. #61870036) for 3 days to generate definitive endoderm. These cells were then directed into hepatic progenitors by culture for 3-4 days in RPMI medium containing Activin A (50-100 ng / mL), HGF (10 ng / mL; R&D Systems, Cat. #294-HGN), and FGF4 (10 ng / mL; R&D Systems, Cat. #7460-F4). For basal differentiation, the progenitors were cultured for 10-15 days in Williams' E Medium (Gibco, Cat. #A1217601) supplemented with 1% ITS (Coming, Cat. #25-800-CR), HGF (10 ng / mL), Oncostatin M (OSM, 20 ng / mL; R&D Systems, Cat. #295-OM), and dexamethasone (0.1 pM).

[0221] To induce functional maturation, these immature hepatocytes were dissociated using Accutase, replated on collagen Lcoated plates (Coming, Cat. #354236) and cultured for an additional 5-10 days in maturation medium consisting of Williams' E Medium supplemented with the six-compound HDC6 cocktail: SB431542 (5 pM; MedChemExpress, Cat. #HY-10431), DAPT (10 pM; MedChemExpress, Cat. #HY-13027), forskolin (10 pM; MedChemExpress, Cat. #HY-15371), FICZ (1 pM; MedChemExpress, Cat. #HY-12451), WY14643 (50 pM; MedChemExpress, Cat. #HY-16995), and liothyronine (T3, 3 pM; MedChemExpress, Cat. #HY-A0070A).

[0222] Kupffer cell (iKC) Differentiation

[0223] iKCs were generated from iPSCs using a three-stage, 3D suspension-based differentiation protocol. Initially, iPSCs were aggregated into embryoid bodies (EBs) by culture in ultra-low attachment dishes for 3 days in APEL2 medium (STEMCELL Technologies, Cat. #05275) supplemented with bFGF (10 ng / mL; R&D Systems, Cat. #3718-F4), BMP4 agonist SB4 (200 nM; Tocris, Cat. #6881), VEGF (100 ng / mL; Thermo Fisher Scientific, Cat. #PHC9394), and CHIR99021 (3 pM; Tocris, Cat. #4423). To generate hematopoietic stem cells, these EBs were then transitioned to APEL2 medium containing bFGF (10 ng / mL), Flt-3L (10 ng / mL; Thermo Fisher, Cat. #300-19), VEGF (20 ng / mL), TPO (30 ng / mL; Thermo Fisher, Cat. #300-18), and SCF (40 ng / mL; Thermo Fisher, Cat. #300-07) for 7-12 days. Floating HSCs were collected from the culture supernatant and plated in SFEM II medium (STEMCELL Technologies, Cat. #09605) supplemented with M-CSF (100 ng / mL; Thermo Fisher, Cat. #300-25) and IL-3 (25 ng / mL; Thermo Fisher, Cat. #200-03) for 7-10 days to promote differentiation into macrophage-P-643500-PClike Kupffer cells. Cells were harvested and characterized via flow cytometry for CD14 and CD 163 expressions to confirm identity and purity.

[0224] 3D Liver Spheroid Assembly

[0225] HDC6-matured iM-Heps were dissociated using Accutase and resuspended in maturation medium supplemented with the ROCK inhibitor Y-27632 (10 pM; Tocris, Cat. #1254) at a final density of 2 * 104cells / mL. Aliquots of 100 pL (containing 2,000 cells) were seeded into each well of a 96-well ultra-low attachment Akura plate (InSphero, Switzerland). Plates were centrifuged at 100 x g for 30 seconds and incubated at 37 °C in 5% CO2 to promote spheroid formation. For co-culture spheroids, iM-Heps were combined with iPSC-derived Kupffer cells at a 5:1 ratio (2,000 iM-Heps to 400 iKCs per well) prior to seeding. Spheroids were maintained in HDC6 maturation medium, with medium changes every 2 days. Control spheroids composed of primary human hepatocytes were obtained from InSphero and consisted of 2,000 PHHs with or without non parenchymal fraction per spheroid.

[0226] Immunofluorescence Staining

[0227] Cells were fixed in 4% paraformaldehyde (PF A) in PBS for 20 minutes at room temperature. After three washes with 1 X PBS, cells were permeabilized and blocked for 1 hour at room temperature in blocking buffer, 1 X PBS with 0.3% BSA and 0.15% Triton X-100. Samples were then incubated with primary antibodies diluted in blocking buffer at 4°C overnight. The primary antibodies, including anti-albumin (Dako, Cat. #F011702-2), anti-EpCAM (R&D Systems, Cat. #AF960), anti-HNF4a (Invitrogen, Cat. #MA1-199), anti-ASGRl (Proteintech, Cat. #11739-1-AP), anti-CYPlA2 (Invitrogen, Cat. #MA3-037), and anti-MRP2 (Abeam, Cat. #ab3373), were used at 1:100 dilution. On the next day, cells were washed three times with 1 X PBS and incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies (1:400 diluted in PBS; Invitrogen) for 1 hour at room temperature. Nuclei were counterstained with DAPI (Invitrogen). Images were captured with a Leica STELLARIS 5 confocal microscope equipped with a 20X objective.

[0228] Flow Cytometry

[0229] To assess albumin expression, iPSC derived hepatocytes at various differentiation stages were harvested from 2D culture using 0.05% Trypsin-EDTA to generate a singlecell suspension. Cells were fixed in 4% PFA for 20 minutes at room temperature, then permeabilized and stained for 30 minutes with FITC-conjugated anti-albumin antibody (1:200; Dako, Cat. #F011702-2) in PBS containing 0.3% BSA and 0.15% Triton X-100.P-643500-PCAfter washing with PBS, samples were analyzed on a NovoCyte Penteon flow cytometer (Agilent Technologies).

[0230] For live-cell surface staining of iPSC-derived Kupffer cells, cells were harvested at various differentiation stages and processed as single-cell suspensions. To exclude dead cells, suspensions were incubated with Zombie Aqua™ Fixable Viability Dye (1:1000; BioLegend, Cat. #423101) for 15 minutes at room temperature. After washing, cells were blocked with TruStain FcX™ Fc receptor blocking reagent (BioLegend, Cat. #422302) for 10 minutes at 4°C. Without additional washing, fluorophore-conjugated antibodies against CD 14 and CD 163 were added and incubated for 30 minutes at 4°C in FACS buffer. Following a final wash, cells were analyzed on a NovoCyte Penteon flow cytometer.

[0231] RNA Sequencing and Bioinformatic Analysis

[0232] RNA sequencing was performed by Azenta Life Sciences (Chelmsford, MA). Total RNA was extracted from cell pellets using TRIzol Reagent according to the manufacturer’ s instructions. Messenger RNA was enriched via poly(A) selection, and sequencing libraries were prepared following standard protocols. Libraries were sequenced to a depth of ~20 million paired-end reads per sample.

[0233] Bioinformatic analysis was performed by Tharkka (Cambridge, MA). For principal component analysis (PCA), the principal axes were computed using only iPSC-derived samples (including iPSCs, iHeps, vehicle-treated iHeps, and HDC6-treated iM-Heps) to isolate transcriptional variance associated with differentiation and maturation. PHH samples were then projected onto this pre-defined PCA space, enabling direct visualization and comparison without influencing the dimensionality reduction process. Normalized gene expression data were used to generate heatmaps for selected gene sets. Z-scores were calculated per gene across samples to visualize relative expression levels. Functional enrichment analyses were performed using Gene Ontology and Gene Set Variation Analysis (GSVA) based on curated pathway databases.

[0234] RNA Extraction and Real-time PCR analysis

[0235] Total RNA was extracted using TRIzol Reagent and reverse-transcribed using the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad). Quantitative PCR was performed on a QuantStudio™ 6 Pro Real-Time PCR System (Applied Biosystems).

[0236] Each 10 pL PCR reaction consisted of 10 ng of cDNA template, IX TaqMan™ Fast Advanced Master Mix (Applied Biosystems), and IX pre-designed, multiplex-ready TaqMan probes for a target gene (F AM-labeled) and PPIG housekeeping gene (VIC-labeled). The TaqMan Gene Expression Assays (Applied Biosystems) used were: PPIGP-643500-PC(Hs01081188_gH, housekeeping), ALB (Hs0060941 l_ml), ASGR1 (Hs01005019_ml), CYP3A4 (Hs00604506_ml), CYP1A2 (Hs00167927_ml), CYP2C8 (Hs00426387_ml), CYP2C19 (Hs04401150_ml), CYP2C9 (Hs04260376_ml), and ABCC2 (MRP2, Hs00960489_ml). Gene expression levels were normalized to PPIG, and relative quantification (RQ) was calculated using the AACt method.

[0237] Urea Analysis

[0238] Urea secretion was quantified using a colorimetric assay kit (BioAssay Systems, Cat. #DIUR-100) according to the manufacturer’s instructions. To evaluate urea cycle functionality, liver spheroids were incubated overnight at 37°C in a humidified 5% CO2 incubator in basal medium consisting of Williams' E Medium supplemented with 1* GlutaMAX, and 1* ITS. For functional stimulation, 5 mM ammonium chloride and 5 mM L-arginine were added to the medium. Following incubation, 25 pL of the culture supernatant was mixed with 100 pL of assay reagent (prepared by combining equal volumes of Reagents A and B). The mixture was incubated for 30 minutes at room temperature, and absorbance was measured at 430 nm using a Hl BioTek Synergy microplate reader (Agilent). Urea concentrations were calculated based on a standard curve included with the kit.

[0239] Albumin Quantification by ELISA

[0240] Secreted albumin was measured in spheroid culture supernatants using a human albumin ELISA kit (Fortis Life Sciences, Cat. #E88-129). Supernatants were diluted 1:20 in the assay dilution buffer, and 100 pL of each diluted sample was transferred to antibody-coated wells. After a 1-hour incubation at room temperature, plates were washed, and 100 pL of HRP-conjugated detection antibody was added to each well. Following another 1-hour incubation, the wells were washed again and incubated sequentially with 100 pL of HRP solution and 100 pL of TMB substrate. The colorimetric reaction was terminated with 100 pL of stop solution, and absorbance was read at 450 nm using a Hl BioTek Synergy microplate reader (Agilent). Albumin concentrations were determined by comparison to a standard curve generated using provided albumin standards.

[0241] Luminescence Based CYP450 Activity Assay

[0242] Cytochrome P450 enzyme activity was quantified using isoform-specific P450-Glo™ Assay kits (Promega), following the manufacturer’s instructions. Liver spheroids were incubated at 37 °C for 3 hours in 50 pL of culture medium containing luminogenic substrates for individual CYP450 isoforms. The substrates used were: Luciferin-IPA (for CYP3A4), Luciferin-1A2 (for CYP1A2), Luciferin-2B6 (for CYP2B6), Luciferin-ME (forP-643500-PCCYP2C8), Luciferin-H (for CYP2C9), and Luciferin-H EGE (for CYP2C19), at concentrations specified by the manufacturer. After incubation, 35 pL of the supernatant were transferred to an assay plate and mixed with 35 pL of the Luciferin Detection Reagent. The plate was incubated for 20 minutes in the dark, and luminescence was measured with a Hl BioTek Synergy plate reader (Agilent). CYP450 activity was determined by luciferin standard curve and normalized to either cell number (pmol / min / million cells) or total cellular ATP (pmol / min / nmol ATP).

[0243] LC / MS / MS Based Metabolic Activity Assay

[0244] To validate metabolic activity, iPSC- and PHH-derived liver spheroids were incubated for 6 hours at 37°C with a cocktail of probe substrates in culture medium. The cocktail contained phenacetin (10 pM), paclitaxel (5 pM), midazolam (5 pM), and 7-hydroxycoumarin (100 pM) to assess the respective activities of CYP1A2, CYP2C8, CYP3A4, and Phase II sulfotransferase. After incubation, the culture medium, containing the metabolites acetaminophen, 6a-hydroxypaclitaxel, 1 -hydroxymidazolam, and 7-hydroxycoumarin sulfate, was collected and quantified by a validated LC / MS / MS method by BioAgilytix.

[0245] Cell Viability ATP Assay

[0246] Cell viability following drug treatment was assessed by quantifying intracellular ATP content using the CellTiter-Glo® 3D Luminescent Cell Viability Assay (Promega, Cat. #G9681), according to the manufacturer’s protocol. An equal volume of CellTiter-Glo reagent was added to each well containing a liver spheroid. Plates were shaken on an orbital shaker for 10 minutes to ensure complete cell lysis, followed by a 15-minute incubation at room temperature to stabilize the luminescent signal. Luminescence was measured using a multimode Hl BioTek Synergy microplate reader. ATP concentrations were determined using a standard curve and reported either as absolute values (nmol / spheroid) or normalized for comparative analysis. Viability was expressed as a percentage relative to untreated control spheroids.

[0247] Functional Bile Canaliculi Assay

[0248] The formation and functional activity of bile canaliculi were evaluated using 2', 7'-di chlorodihydrofluorescein diacetate (DCFDA; Invitrogen, Cat. #C369), a fluorescent substrate transported by MRP2. iPSC derived hepatocytes cultured in 2D monolayers were washed with PBS and incubated with 10 pM DCFDA in culture medium for 30 minutes at 37°C in the dark. Following incubation, cells were washed again with PBS, and nuclei were counterstained with Hoechst 33342 (5 pg / mL) for 5 minutes.P-643500-PC

[0249] Live-cell images were acquired using ECHO fluorescence microscope (ECHO Model: RVL2-K2). For inhibition studies, cells were pre-treated with cyclosporin A (10 pM; a known MRP2 inhibitor) for 30 minutes prior to and during DCFDA incubation.

[0250] Seahorse XF Metabolic Flux Analysis

[0251] Mitochondrial respiration and glycolytic activity were assessed using a Seahorse XFe96 Analyzer (Agilent Technologies, Santa Clara, CA) at the Beth Israel Deaconess Medical Center Metabolism Core Facility. For the Mito Stress Test, cells were incubated in Seahorse XF assay medium supplemented with glucose (10 mM), pyruvate (1 mM), and glutamine (2 mM). Oxygen consumption rate was measured in real time following sequential injections of: Oligomycin (1.5 pM), FCCP (1.0 pM), Rotenone / antimycin A (0.5 pM each). For the Glycolysis Rate Assay, cells were assayed in XF medium supplemented with 2 mM glutamine. Proton efflux rate and OCR were measured after sequential injections of: Rotenone / antimycin A (0.5 pM), 2-deoxy -D-glucose (2-DG, 50 mM). All measurements were normalized to total protein (quantified using BCA assay), and data were analyzed using Wave software (Agilent).

[0252] Cytokine and Chemokine Profiling by nELISA

[0253] Inflammatory cytokines and chemokines secreted into spheroid culture supernatants were profiled using a high-throughput proximity extension-based multiplex immunoassay (nELISA), performed by Nomic Bio (Montreal, QC, Canada). Data were visualized as heatmaps generated in GraphPad Prism (version 10). Z-scores were calculated for each analyte across all samples by standardizing to the mean and standard deviation of each cytokine’s expression, allowing comparative interpretation of cytokine induction across experimental conditions.

[0254] Statistical Analysis

[0255] All quantitative data are presented as the mean ± standard deviation (SD). Statistical comparisons were performed using one-way or two-way ANOVA with post-hoc tests for multiple comparisons, as appropriate. A p-value < 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism software.Example 2: Identification of a Six-Compound Cocktail to Enhance the Functional Maturation of iPSC-Derived Hepatocytes

[0256] Aim: identify optimal differentiation cocktail.

[0257] Method: assess CYP450 activity following treatment of iPSC derived liver spheroids with various pathway modulators and compounds.P-643500-PC

[0258] Results:

[0259] iPSCs derived hepatocyte-like cells (iHeps) were generated using a differentiation protocol (Fig. 1A) (Tian et al., Int J Biol Sci 12, 1052-1062, 2016). iHeps expressed canonical hepatic markers such as Albumin and HNF4a (Fig. IB), and readily assembled into 3D spheroids that secreted urea at levels comparable to PHH spheroids (Fig. 1C).However, iPSCs derived hepatocyte-like cells remained functionally immature, displaying significantly reduced CYP450 activity compared to PHHs in 3D culture (Fig. 1C).Specifically, CYP3 A4 activity in PHHs-3D was >2 times higher than its activity in iHeps-3D, whereas CYP1A2 activity was nearly undetectable in iHep-3D group.

[0260] A systematic screen of small-molecule modulators targeting key developmental and metabolic signaling pathways was performed. Co-treatment with Notch inhibitor DAPT and cAMP activator Forskolin moderately increased CYP1A2 activity but had minimal effect on CYP3A4 (Fig. ID). YAP inhibitor and bile acid derivatives, showed limited or inconsistent effects on CYP450 activities during maturation. The Wnt inhibitor IWR1 had a negative impact on CYP450s. The bile acid metabolites LCA and TUDCA showed only marginal effects. The tryptophan metabolite FICZ significantly upregulated CYP1A2 activity but concomitantly suppressed CYP3 A4 activity (Fig. ID).

[0261] To identify synergistic combinations that could simultaneously induce CYP3 A4 and CYP1A2, the thyroid hormone (T3) was incorporated into the DAPT / forskolin / TGF-P inhibitor (SB431542) backbone, which significantly boosted CYP3A4 activity. Further addition of FICZ and the PPARa agonist WY14643 significantly increased CYP1A2 expression, achieving levels exceeding 7 pmol / min / 106cells while maintaining high CYP3 A4 activity (Fig. IE). Inhibition of Wnt signaling with IWR-1 reduced both CYP1 A2 and CYP3 A4 activities, suggesting that persistent or untimely suppression of Wnt signaling may disrupt the complex regulatory balance required for hepatic functional maturation (Fig. IE).

[0262] These findings led to the formulation of a six-component maturation cocktail, designated HDC6, comprising a TGF-P inhibitor, Notch inhibitor, cAMP activator, thyroid hormone, PPARa agonist, and FICZ (Fig. IF, Table 1).

[0263] Further protocol optimization revealed that treatment of immature iPSCs derived hepatocyte-like cells with HDC6 for 10 days enhances recapitulation of key PHH morphological features, including polygonal cell shape, prominent nucleoli, and dense epithelial organization, in 2D monolayer cultures (Fig. 1G). Upon assembly into 3D spheres, HDC6-treated cells readily formed compact and uniform spheroids with structuralP-643500-PCintegrity comparable to PHH-derived spheroids. Flow cytometry analysis revealed that HDC6 treatment increased the proportion of ALB+cells from -65% in vehicle-treated controls to over 86%, exceeding the purity observed in PHHs populations (Fig. 1H). This effect was consistent across multiple iPSC donor lines. Immunofluorescence staining further confirmed the enhanced expression of mature hepatic markers following HDC6 exposure. In 2D culture, iM-Heps exhibited robust upregulation of ASGR1 and HNF4a, both at lower expression or absent in vehicle-treated controls. In 3D spheroid culture, HDC6 treatment led to strong expression of ASGR1, CYP1A2, and Albumin, closely resembling the spatial pattern and observed in PHH spheroids (Fig. II). Together, these results demonstrate that HDC6 significantly promotes hepatic functional maturation resembling that of PHHs, in both monolayer and 3D spheroid contexts.

[0264] Table 1: selected compounds for maturation enhancement of iPSC-derived hepatocytesExample 3: Transcriptomic profiling reveals features of mature, PHHs-like gene signatures in HDC6-treated iM-Heps

[0265] Aim: characterize genome wide transcriptional changes induced by HDC6 in iM-Heps.

[0266] Method: bulk RNA sequencing was performed on iPSCs derived hepatocyte-like cells at key check point stages of differentiation and compared them to PHHs.

[0267] Results:

[0268] Principal component analysis (PCA) revealed clear clustering by developmental stage and treatment condition (Fig. 2A). Undifferentiated iPSCs, immature iPSCs derived hepatocyte-like cells (iHeps), and vehicle-treated spheroids (iHeps-3DV) segregated distinctly, while HDC6-treated spheroids (iM-Heps-3D) clustered closely with PHHsP-643500-PCspheroids, indicating a transcriptomic shift toward a more mature hepatic profile following HDC6 treatment.

[0269] The expression of genes governing hepatic functionally in multiple categories, including drug metabolizing enzymes, membranal transporters, and key liver-specific markers including transcription factors, secreted carrier proteins and serum glycoprotein homeostasis transmembrane proteins was evaluated (Fig. 2B). iHeps at immature or vehicle-treated stages exhibited moderate expression of hepatocyte lineage markers such as ALB, TTR, and HNF4a, but lacked robust expression of cytochrome P450 enzymes, Phase II conjugation enzymes (e.g., UGT1A1, SULT1A1), and major hepatic transporters (e.g., SLCO1B1, ABCC2, ABCB4). In contrast, HDC6-treated iM-Heps-3D exhibited strong induction of Phase I metabolizing enzymes including CYP3A4, CYP1 A2, and CYP2C8, as well as Phase II and transporter genes at levels comparable to PHHs. While expression of certain CYPs (e.g., CYP2B6, CYP2D6) remained lower than PHHs, the global transcriptional profile of iM-Heps-3D reflected marked functional enhancement.

[0270] In addition to upregulating adult hepatic markers, HDC6 suppressed fetal-associated genes such as AFP and FM01, which are highly expressed in immature iHeps but minimally detected in adult PHHs. These marked genome wide transcriptional changes were further validated by qPCR, confirming upregulation of key mature markers including ALB, ASGR1, CYP450s, CYP1 A2, and MRP2 to levels comparable with PHH spheroids.

[0271] Gene Ontology (GO) enrichment analysis of genes upregulated >4-fold following HDC6 treatment identified significant overrepresentation of hepatic metabolic and detoxification pathways, including steroid metabolism, fatty acid oxidation, and P450-related xenobiotic clearance. Protein-protein interaction network analysis of HDC6-induced genes revealed densely interconnected modules enriched for liver-specific metabolic functions, underscoring the coordinated induction of hepatic programs.

[0272] Together, these transcriptomic findings demonstrate that HDC6 drives a comprehensive maturation program in iPSCs derived hepatocytes, inducing key hepatic functionality related gene networks and enhancing alignment with the molecular signature of primary human hepatocytes. The increased CYP450 activity demonstrated for iM-Heps-3D marks a substantial advancement over existing methods, where iPSC-derived hepatocytes often exhibit sub-optimal enzyme functions.Example 4: HDC6 treatment enhances metabolic and secretory function in iPSC-derived hepatocytesP-643500-PC

[0273] Aim: determine whether the transcriptomic enhancements induced by HDC6 translated into actual improved hepatic function.

[0274] Method: functional assays in iM-Heps derived from multiple independent donor iPSC lines.

[0275] Results:

[0276] Albumin secretion, a hallmark of hepatic protein synthesis, was significantly elevated in HDC6-treated iM-Heps-3D compared to vehicle-treated controls across all lines (Fig. 3A). Notably, three donor lines reached albumin levels exceeding 40 pg / day / million cells, substantially higher than the ~28 pg / day / million cells measured in PHH spheroids.

[0277] Ammonia detoxification was assessed through the urea cycle. Basal urea production remained similar across all donors; however, upon stimulation with substrates for the Urea cycle- NH4Q and L-arginine, HDC6-treated iM-Heps-3D exhibited a significant increase in urea secretion, achieving levels comparable to PHHs-3D, which was not observed in vehicle treated groups (Fig. 3B). These data confirm that the urea cycle is functionally inducible in HDC6-matured iM-Heps.

[0278] To evaluate one of the key functionalities of the liver related to drug safety-xenobiotic metabolism, the activity of key cytochrome P450 enzymes was measured. Remarkably, HDC6 treatment significantly increased the enzymatic activity of multiple major drug-metabolizing CYPs, including CYP3A4 and CYP1 A2 together responsible for metabolism of 50% - 70% of all marketed drugs, as well as CYP2C8, across all donor lines (Fig. 3C) CYP2C9 and CYP2B6 activities were also significantly upregulated, although they didn’t pass the statistical significance test in all donors due to higher assay variability. CYP2C19 activity remained unchanged, consistent with the modest baseline expression observed in both iPSCs derived hepatocytes and PHHs.

[0279] To further validate CYP450 enzymatic activity with more precision, drug-specific metabolites, metabolized by 3 major CYP450 enzymes were quantified via LC-MS / MS (Fig. 3D). HDC6-treated iM-Heps-3D demonstrated CYP3 A4-dependent hydroxylation of midazolam and CYP2C8-dependent hydroxylation of paclitaxel at levels comparable to PHHs-3D. Strikingly, CYP1A2 activity, measured by phenacetin O-deethylation to acetaminophen conversion, was significantly higher in HDC6-treated iM-Heps spheroids than in PHHs spheroids, reaching 25-35 pmol / min / million cells, a level that is close to freshly isolated PHHs. Sulfotransferase activity (conversion of 7-hydroxy coumarin to sulfate conjugate) was also significantly elevated in HDC6-treated iM-Hep spheroids compared to PHH spheroids.P-643500-PC

[0280] Finally, bile canaliculi formation, an important feature of mature liver functionality dependent on multidrug resistance-associated protein 2 (MRP2) expression and activity was assessed. In HDC6-treated iM-Heps (750 iM-Heps), fluorescent DCF, a substrate of MRP2 accumulated in discrete canalicular-like structures, phenocopying patterns observed in PHHs (Fig. 3E). In contrast, vehicle-treated iHeps (750 iHeps) displayed diffuse intracellular staining, highlighting non-existing canalicular organization. The canalicular signal was abolished upon treatment with cyclosporin A, a known MRP2 inhibitor, confirming the specificity of transporter-mediated excretion.

[0281] Together, these results demonstrate that HDC6-treated iM-Heps exhibit functional maturity across multiple key hepatic processes, including protein synthesis, ammonia detoxification, drug metabolism, and bile canaliculi formation, achieving performance levels comparable to or exceeding those of PHHs.Example 5: HDC6 treatment reprograms cellular energy metabolism toward mitochondrial oxidative respiration

[0282] Aim: investigate whether the functional maturation induced by HDC6 is accompanied by a metabolic shift in energy production.

[0283] Methods: transcriptomic and functional profiling of pathways involved in mitochondrial metabolism and glycolysis.

[0284] Results:

[0285] Undifferentiated iPSCs display low expression of genes associated with the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and fatty acid [3-oxidation (Fig. 4A and Fig. 4B). Differentiation alone led to modest induction of these pathways; however, HDC6 treatment elicited a pronounced upregulation of mitochondrial and lipid metabolic gene programs, yielding transcriptional profiles that closely resembled those of primary human hepatocytes.

[0286] Gene set variation analysis (GSVA) revealed distinct pathway-level transitions. While iPSCs were enriched for glycolytic gene signatures, HDC6-treated iM-Heps-3D demonstrated strong enrichment in oxidative metabolism, including TCA cycle, OXPHOS, and fatty acid P-oxidation pathways, reaching levels comparable to PHH spheroids (Fig.4C). Consistent with this transcriptomic reprogramming, HDC6 treatment increased the mitochondrial DNA content in iM-Heps-3D to a level comparable to PHH spheroids, as measured by the ratio of mitochondrial ND1 to nuclear RNaseP DNA, across both donor lines tested (Fig. 4D).P-643500-PC

[0287] To directly assess mitochondrial function, Mito Stress Tests were performed, using the Seahorse XF platform. HDC6-treated iM-Heps from donor 750 (750 iM-Heps) showed significantly elevated oxygen consumption rates (OCR), including maximal respiration and spare respiratory capacity compared to vehicle control (750 iHeps-V). In donor 744, both vehicle- and HDC6-treated iM-Heps (744 iHeps-V and 744 iM-Heps) displayed similarly high levels of maximal and spare respiration, with no significant differences between them (Fig. 4E). This case highlights potential donor to donor variability and the importance of using the HDC6 maturation step to ensure functionality of all iPSC differentiated cultures. Nevertheless, both HDC6 treated groups (744 and 750) reached OCR levels comparable to primary human hepatocytes, while undifferentiated iPSCs exhibited minimal OCR and lacked spare capacity, consistent with the immature metabolic state in iPSCs.

[0288] Seahorse XF Glycolysis Rate Assays (Fig. 4F) revealed that HDC6 treatment significantly suppressed glycolysis in both donors compared to vehicle controls. iPSCs from both donors showed the highest glycolytic rates, consistent with a glycolysis-dominant energy metabolism. The ratio of mitochondrial OCR to glycolytic PER (OCR / PER) highlighted distinct metabolic shifts. In donor 744, HDC6 treatment significantly increased the OCR / PER ratio, reflecting a clear transition from glycolytic to oxidative metabolism. In contrast, both 750 iHeps-v and 750 iM-Heps exhibited already high OCR / PER ratios with no significant difference, suggesting that oxidative phosphorylation was already dominant in the 750 vehicle condition.

[0289] Together, these findings demonstrate that HDC6 drives a profound metabolic reprogramming in iM-Heps, supporting their transition from a glycolysis-dependent to a mitochondria-dominant energy state characteristic of mature hepatocytes.Example 6: HDC6-matured iM-Heps spheroids reliably predict drug-induced liver injury (DILI)

[0290] Aim: assess the utility of HDC6-treated iM-Heps spheroids for DILI prediction.

[0291] Method: evaluate HDC6-matured iM-Heps spheroids response to a 20-compound panel encompassing drugs with known clinical hepatotoxicity risk profiles.

[0292] Results:

[0293] The compounds tested had a clinical hepatotoxicity risk profile determined by the FDA, ranging from No DILI Concern to Most DILI Concern (Table 2). The dosing regimen consisted of repeated drug exposure every other day over six days followed by viability and morphological assessment on day 7 (Fig. 5A). Representative hepatotoxins such asP-643500-PCtolcapone, troglitazone, diclofenac, and chlorpromazine, all known to cause significant hepatic injury and labeled as Most DILI Concern except chlorpromazine (less DILI Concern), induced dose-dependent cytotoxicity in both HDC6-treated iM-Heps and PHH spheroids, as evidenced by structural disintegration and loss of viability (Fig. 5B and Fig.5C). Importantly, the ICso values obtained for these compounds in both iPSC derived and PHH models were comparable and closely aligned with their reported clinical Cmax, indicating physiologically relevant sensitivity across all models.

[0294] Table 2: DILI profiling of 20 reference drugs in HDC6-matured iPSC-derived hepatocyte spheroids (iM-Heps-3D) and primary human hepatocyte spheroids (PHHs-3D).DILI IC50 IC50 Group Label Cm ax IC50 IC50 IC50 IC50 / Drugs (jiM) ( / Cma / Cm (DILI Section uM) (uM) (pM) Cm axx ax rank)Most- 21.75 34.88 0.73 Tolcapo Box 14.87 0.46± 0.31±Concern 47.60 ±11.9 ±30.7 ±0.6 ne Warning ±1.46 0.25 0.03(8) 2 1 5 Most- 38.47 45.63 8.45 Flutami Box 16.11 7.12± 2.98±Concern 5.4 ±25.4 ±19.4 ±3.6 de warning ±3.12 4.72 0.58(8) 8 3 0 Most- 18.43 19.24 3.01 Troglita 13.21 2.06± 2.88±Concern Withdrawn 6.4 ±10.9 ±12.9 ±2.0 zone ±2.11 0.33 1.70(8) 0 1 2 Most- 90.66 51.08 18.06Trovafl 10.17Concern Withdrawn 5.02 ±70.1 ±32.3 ND ±13.9 ND oxacin ±6.45(8) 2 6 7Most- Warning 134.7 181.0 106.7 17.92 10.5 Diclofe 13.34Concern and 10.1 8±77. 1±188 0±48. ±18.6 6±4. nac ±7.68(8) Precautions 60 .21 86 3 84 Most- Stavudi BoxConcern 3.46 ND ND ND ND ND ND ne warning(8)Most- Warning 51.00 849.9 104.5 Atorvast 6.27±Concern and 0.06 ±50.0 ND 2±834 4±85. ND atin 5.15(5) Precautions 7 .50 78 Most- WarningAcetamiConcern 165.4 ND ND ND ND ND ND andnophen(5) PrecautionsLess- 30.0 Paroxeti 4.63± 6.00± 21.25 23.13Concem Adverse „?4.25± 0±14 ne Reaction 0.88 2.97 ±3.89 ±4.42s 0.78.85 (8)Warning Less- PioglitaConcem and 2.95 ND ND ND ND ND ND zonePrecautions (3)Warning Less- MeloxicConcem and 5.21 ND ND ND ND ND ND amPrecautions (3)Warning Less- NifedipiConcem and 0.3 ND ND ND ND ND ND nePrecautions (3)P-643500-PCLess-Dili- 9.20 Chlorpr Adverse 7.34± 5.47± 8.65± 7.81± 5.82±Concem 0.94 ±1.5 omazine Reactions 1.29 0.56 1.44 1.37 0.60(2) 3 Less-Dili- 148.1 45.2 EntacapConcem No match 3.27 ND ND 0±3.3 ND ND 9±1. one(0) 9 04 No-DILI- Streptoconcem No match 64.5 ND ND ND ND ND ND mycin(0)No-DILI- Flumazeconcem No match 1.12 ND ND ND ND ND ND nil(0)Chlorph No-DILI- enirami concem No match 0.044 ND ND ND ND ND ND ne (0)No-DILI- 20.40 8.98 Benztro 19.30 12.03 5.30± 32.71concem No match 0.585 ±15.3 ±10. pine ±3.82 ±9.05 5.94 ±6.47(0) 4 07 AmbiguoAmbrise use DILI- Adverse0.79 ND ND ND ND ND ND ntan Concem reactions(7)AmbiguoBuspiro use DILI- Adverse0.01 ND ND ND ND ND ND ne Concem Reactions

[0295] In several cases, the HDC6 iM-Heps-3D outperformed PHHs spheroids in predictive fidelity (Fig. 5D and Fig. 5E). Interestingly Trovafloxacin, a drug withdrawn from the market due to hepatotoxicity, was accurately classified as toxic by iM-Heps-3D from both donor lines, whereas PHH spheroids exhibited limited response only at higher doses and no IC50 value could be determined. Similarly, atorvastatin induced toxicity in iM-Heps-3D at high concentrations but was falsely classified as non-toxic in PHHs-3D. Conversely, entacapone, which has a low DILI concern clinically, triggered an apparent toxic response in PHHs spheroids but not in iM-Heps spheroids. These discrepancies suggest that iPSC-derived models better reflect human DILI outcomes in specific contexts.

[0296] A comprehensive margin of safety (MOS) analysis across the entire drug panel revealed strong alignment between iM-Heps-3D and known clinical classifications (Fig.5F). Of the nine high-risk DILI compounds, both 750 and 744 spheroids correctly identified six as hepatotoxic — comparable to, or exceeding, the performance of the PHH spheroid model (Table 3). Among the eleven compounds classified as low- or no-risk, the iM-Heps-3D correctly predicted nine as non-toxic, while PHHs accurately predicted eight.

[0297] Overall, HDC6-matured iM-Heps achieved 66.7% sensitivity and 81.8% specificity, outperforming PHHs (55.6% sensitivity and 72.3% specificity; Table 4). These results wereP-643500-PCconsistent across two donor lines. Additionally, comparable toxicity profiles were observed in HDC6 iM-Heps derived from multiple independent donors in response to most-DILI-concem drugs, underscoring the reproducibility and robustness of the HDC6 matured model.

[0298] Collectively, these findings demonstrate that the HDC6-treated iPSC-derived liver spheroid platform offers a scalable, genetically diverse, and highly predictive in vitro system for preclinical hepatotoxicity screening.

[0299] Table 3: Correct and incorrect DILI classifications for each model relative to known DILI risk (based on MOS threshold of 50).

[0300] Table 4: Sensitivity and specificity values for DILI prediction by iM-Heps-3D (donors 750 and 744) and PHHs-3D.Example 7: Addition of iPSC Derived Kupffer Cells Recapitulates Liver Inflammation Responses

[0301] Aim: model immune-mediated features of DILI.

[0302] Method: establish an immune-competent enhanced liver spheroid system by coculturing HDC6-treated iM-Heps with iPSC-derived Kupffer-like cells (iKCs).

[0303] Results:

[0304] iKCs were differentiated via a stepwise protocol that sequentially directed iPSCs through mesodermal and hematopoietic progenitor stages (Fig. 6A and Fig. 6B). Flow cytometry confirmed robust acquisition of Kupffer cell identity, with >60% of cells co-P-643500-PCexpressing CD14 and CD163, comparable to levels observed in primary human Kupffer cells (Fig. 6C).

[0305] For co-culture assembly, iM-Heps and iKCs were combined at a 5:1 ratio respectively, forming compact, uniform spheroids that were morphologically indistinguishable from monocultures (Fig. 6D). Importantly, the addition of iKCs did not compromise hepatocyte function: co-culture spheroids maintained comparable CYP450s activities, as well as albumin and urea secretion, relative to iM-Hep-only spheroids across multiple donor lines (Fig. 6E), confirming compatibility and functional stability of the immune-integrated system.

[0306] The model’s response to small molecular hepatotoxic compounds was tested. Upon treatment with high-risk DILI drugs (e.g., tolcapone, diclofenac, atorvastatin, flutamide, trovafloxacin), co-culture spheroids exhibited similar dose-dependent reductions in viability as monocultures, indicating that Kupffer cells are not essential for modeling direct small molecular drug cytotoxicity (Fig. 6F). Both models also tolerated entacapone, a low-DILI-risk compound, at high doses. However, pre-treatment with a free fatty acid (FFA) mixture of palmitic and oleic acids, used to mimic hepatic steatosis sensitized spheroids to entacapone, unmasking latent hepatotoxicity. This steatosis-induced sensitization was observed in both mono- and co-culture formats, demonstrating the system's utility in modeling metabolic comorbidities.

[0307] The functional role of the immune component was assessed under inflammatory stimulation. Multiplexed Nano-ELISA (nELISA, Nomic Bio) analysis of secreted proteins, revealed that lipopolysaccharide (LPS) triggered strong release of proinflammatory mediators, including IL-6, IL-8, TNF-a, and CCL2, exclusively in HDC6 iM-Heps / iKCs co-culture spheroids, while monocultures remained unresponsive (Fig. 6G). Similarly, biologic immunomodulators ipilimumab (antibody drugs marketed as Yervoy) and theralizumab (antibody drug that failed severely in clinical studies), elicited robust upregulation of key inflammatory markers such as GDF-15 and CCL2, but only in the presence of iKCs (Fig.6H and Fig.61). These immune-mediated responses were consistent across donors and drug classes, underscoring the critical role of Kupffer cells in mediating inflammation-driven liver injury.

[0308] Collectively, these results establish the HDC6-iM-Hep / iKC co-culture model as a robust and responsive platform for studying immune-related hepatotoxicity, with potential utility in the preclinical evaluation of biologic drugs and complex inflammatory liver injury mechanisms.

Claims

P-643500-PCCLAIMS1. A method for generating mature iPSC-derived hepatocytes comprising culturing immature iPSC-derived hepatocytes in a culture medium including a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes, wherein said hepatocyte maturation composition comprises TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

2. The method of any of claim 1, wherein the step of culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition is for a period of time of between about 5-10 days.

3. The method of claim 1 or 2, wherein said immature iPSC-derived hepatocytes are cryopreserved cells which are thawed before culturing.

4. The method of claim 1 or 2, wherein said immature iPSC-derived hepatocytes are nonfrozen cells.

5. The method of any of the preceding claims, wherein said immature iPSC-derived hepatocytes are mammalian cells.

6. The method of claim 5, wherein said immature iPSC-derived hepatocytes are human cells.

7. The method of any of the preceding claims, wherein said mature iPSC-derived hepatocytes are characterized by having one or more of the following:express one or more genes selected from CYP3A4, CYP1A2, CYP2C8, ALB, ASGR1, and multi drug resistance-associated protein 2 (MRP2);albumin levels exceeding 40 pg / day / million cells;increased urea production following 24 hours exposure to ammonia and arginine, compared to untreated control;CYP3 A4 activity at levels of above 0.2 pmol / min / million cells;CYP1 A2 activity at levels of about 5 pmol / min / million cells; andreduced expression of AFP or FM01, compared to immature iPSC-derived hepatocytes.

8. A method for generating a 3D liver spheroid from iPSC-derived immature hepatocytes comprising (a) culturing immature iPSC-derived hepatocytes in a hepatocyte maturation composition to obtain mature iPSC-derived hepatocytes; and (b) seeding the mature iPSC-derived hepatocytes of step (a) on a low attachment surface andP-643500-PCculturing in a culture medium including a hepatocyte maturation composition supplemented with Rho-associated kinase (ROCK) inhibitor, to obtain a 3D liver spheroid, wherein the maturation composition comprises a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

9. The method of claim 8, wherein the step of culturing the mature iPSC-derived hepatocytes in hepatocyte maturation composition supplemented with a ROCK inhibitor is for a period of time of between about 12 to 18 hours followed by removal of ROCK inhibitor and continued culture in hepatocyte maturation composition in the absence of ROCK inhibitor for a period of time of between about 5-10 days.

10. The method of claim 9, wherein said ROCK inhibitor comprises Y-27632.

11. The method of claim 8 further comprising combining the mature iPSC-derived hepatocytes from step (a) with iPSC-derived Kupffer-like cells (iKCs), prior to seeding.

12. The method of claim 1 or 8, wherein said cAMP Activator is selected from the group consisting of forskolin or a derivative thereof, colforsin daropate, CW008, PACAP 1- 38, 8-bromo-cAMP, dibutyryl-cAMP, NKH477, or mixtures or combinations thereof.

13. The method of claim 1 or 8, wherein said TGF-P Inhibitor is selected from the group consisting of SB431542, SB525334, Ki26894, LY364947, SD-208, SD-093, SM16, Ly2109761, Ly2157299, K02288, SB505124, LDN-193189, GW788388, Ly580276, EW-7203, EW-7195, EW-7197, YR-290, A 83-01, D4476, RepSox, R268712, or mixtures or combinations thereof.

14. The method of claim 1 or 8, wherein said Notch Inhibitor is selected from the group consisting of LY41 1575, MDL-28170, Compound E, RO4929097, DAPT, L- 685458, BMS-708163, BMS-299897, M -0752, YO-01027, MDL28170, LY41 1575, ELN- 46719, PF-03084014, Semagacestat, or mixtures or combinations thereof.

15. The method of claim 1 or 8, wherein said thyroid hormone is selected from the group consisting of Liothyronine, (S)-triiodothyronine, (S)-2-amino-3-[4-(4-hydroxy-3- iodophenoxy)-3,5-diiodophenyl]propanoic acid, (S)-thyroxine, Levothyroxine (L- thyroxine), Liotrix, Tiratricol, or mixtures or combinations thereof.

16. The method of claim 1 or 8, wherein said PPAR activator comprises a PPAR-gamma (PPARy) activator, a PPAR-alpha (PPARa) activator, PPAR-delta (PPAR5) activator, a dual PPARa / 5 agonist, a pan PPAR activator, or any combination thereof.

17. The method of claim 16, wherein said PPAR activator comprises a PPARa activator selected from the group consisting of Wyl4643, 15-HETE, 15-HpETE, Aleglitazar,P-643500-PCAluminium clofibrate, Arachidonic acid, Bezafibrate, Chiglitazar, Clofibrate, CP- 775146, Daidzein, DHEA (prasterone), Elafibranor, Etomoxir, Fenofibrate, Genistein, Gemfibrozil, GW-7647, Lanifibranor, Leukotriene B4, LG-101506, LG-100754, Lobeglitazone, Muraglitazar, Oleyl ethanol ami de, Palmitoylethanolamide, Pemafibrate, Perfluorononanoic acid, Perfluorooctanoic acid, Pioglitazone, Saroglitazar, Sodelglitazar, Tesaglitazar, Tetradecylthioacetic acid, Troglitazone, or mixtures or combinations thereof.

18. The method of claim 1 or 8, wherein said Tryptophan Metabolite is selected from the group consisting of 6-Formylindolo[3,2-b]carbazole (FICZ), indole-3 -aldehyde (lAld), indole-3 -acetic acid (IAA), indole-3 -propionic acid (IP A), indole-3 -lactic acid (ILA), kynurenine, kynurenic acid, xanthurenic acid, quinolinic acid, tryptamine, indole, skatole (3-methylindole), or mixtures or combinations thereof.

19. The method of claim 12, wherein said cAMP Activator is forskolin.

20. The method of claim 13, wherein said TGF-P Inhibitor is SB431542.

21. The method of claim 14, wherein said Notch Inhibitor is DAPT.

22. The method of claim 15, wherein said thyroid hormone is Liothyronine.

23. The method of claim 17, wherein said PPARa activator is Wyl4643.

24. The method of claim 18, wherein said Tryptophan Metabolite is FICZ.

25. A 3D liver spheroid prepared according to the method of any of claims 8 to 24.

26. The 3D liver spheroid of claim 25 used for at least one purpose selected from the group consisting of a model platform for liver disease testing; model platform for drug- induced liver injury (DILI) prediction; for idiosyncratic drug induced liver injury (iDILI) identification and studies; for disease modeling; in vitro mechanistic studies; food supplement induced liver injury prediction; drug-drug interaction (DDI) testing; and drug-food supplement interaction testing.

27. A hepatocyte maturation composition comprising a TGF-beta inhibitor, a Notch inhibitor, a cAMP activator, a tryptophan pathway metabolite, a PPAR activator, and a thyroid hormone.

28. The hepatocyte maturation composition of claim 27, wherein said cAMP Activator is selected from the group consisting of forskolin or a derivative thereof, colforsin daropate, CW008, PACAP 1-38, 8-bromo-cAMP, dibutyryl-cAMP, NKH477, or mixtures or combinations thereof.

29. The hepatocyte maturation composition of claim 27, wherein said TGF-P Inhibitor is selected from the group consisting of SB431542, SB525334, Ki26894, LY364947, SD-P-643500-PC208, SD-093, SM16, Ly2109761, Ly2157299, K02288, SB505124, LDN-193189, GW788388, Ly580276, EW-7203, EW-7195, EW-7197, YR-290, A 83-01, D4476, RepSox, R268712, or mixtures or combinations thereof.

30. The hepatocyte maturation composition of claim 27, wherein said Notch Inhibitor is selected from the group consisting of LY41 1575, MDL-28170, Compound E, RO4929097, DAPT, L- 685458, BMS-708163, BMS-299897, M -0752, YO-01027, MDL28170, LY41 1575, ELN-46719, PF-03084014, Semagacestat, or mixtures or combinations thereof.

31. The hepatocyte maturation composition of claim 27, wherein said thyroid hormone is selected from the group consisting of Liothyronine, (S)-triiodothyronine, (S)-2-amino- 3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoic acid, (S)-thyroxine, Levothyroxine (L-thyroxine), Liotrix, Tiratricol, or mixtures or combinations thereof.

32. The hepatocyte maturation composition of claim 27, wherein said PPAR activator comprises a PPAR-gamma (PPARy) activator, a PPAR-alpha (PPARa) activator, PPAR-delta (PPAR5) activator, a dual PPARa / 5 agonist, a pan PPAR activator, or any combination thereof.

33. The hepatocyte maturation composition of claim 32, wherein said PPAR activator comprises a PPARa activator selected from the group consisting of Wy 14643, 15- HETE, 15-HpETE, Aleglitazar, Aluminium clofibrate, Arachidonic acid, Bezafibrate, Chiglitazar, Clofibrate, CP-775146, Daidzein, DHEA (prasterone), Elafibranor, Etomoxir, Fenofibrate, Genistein, Gemfibrozil, GW-7647, Lanifibranor, Leukotriene B4, LG-101506, LG-100754, Lobeglitazone, Muraglitazar, Oleyl ethanol ami de, Palmitoylethanolamide, Pemafibrate, Perfluorononanoic acid, Perfluorooctanoic acid, Pioglitazone, Saroglitazar, Sodelglitazar, Tesaglitazar, Tetradecylthioacetic acid, Troglitazone, or mixtures or combinations thereof.

34. The hepatocyte maturation composition of claim 27, wherein said Tryptophan Metabolite is selected from the group consisting of 6-Formylindolo[3,2-b]carbazole (FICZ), indole-3 -aldehyde (lAld), indole-3 -acetic acid (IAA), indole-3 -propionic acid (IP A), indole-3 -lactic acid (ILA), kynurenine, kynurenic acid, xanthurenic acid, quinolinic acid, tryptamine, indole, skatole (3-methylindole), or mixtures or combinations thereof.

35. The hepatocyte maturation composition of claim 28, wherein said cAMP Activator comprises Forskolin.P-643500-PC36. The hepatocyte maturation composition of claim 29, wherein said TGF-P Inhibitor comprises SB431542.

37. The hepatocyte maturation composition of claim 30, wherein said Notch Inhibitor comprises DAPT.

38. The hepatocyte maturation composition of claim 31, wherein said thyroid hormone comprises Liothyronine.

39. The hepatocyte maturation composition of claim 33, wherein said PPARa activator comprises Wyl4643.

40. The hepatocyte maturation composition of claim 34, wherein said Tryptophan Metabolite comprises FICZ.

41. A method for predicting drug induced liver injury (DILI) comprising bringing a drug into contact with the 3D liver spheroid according to claim 25.

42. The method of claim 41, wherein the toxicity of said drug is determined by assessing spheroid viability or spheroid morphology.

43. A method for identifying a preferred therapeutic agent for an individual comprising bringing a candidate compound into contact with the 3D liver spheroid according to claim 25.

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