Human induced pluripotent stem cell (IPSC)-derived fetal and neonatal hepatocyte-like cell model for drug and toxicant screening

Abstract

In a first embodiment, a method for producing fetal and / or neonatal hepatocyte-like cells (fnHLCs) is disclosed. The method includes producing a definitive endoderm cell population by a stage 1 process, producing a hepatoblast-like progenitor cell population from the definitive endoderm cell population by a stage 2 process, and producing a fetal and / or neonatal hepatocyte-like cell population from the hepatoblast-like progenitor cell population by a stage 3 process. In another embodiment a method for screening a target is disclosed. The method includes co-culturing and incubating the target with a fetal and / or neonatal hepatocyte-like cell (fnHLC), retrieving a sample of a fnHLC after the co-culturing and incubating and determining one or more characteristics of the fnHLC based on analysis of the sample of the fnHLC.

Description

PCT ApplicationAttorney Docket No. : CUBR-0007PCTitle: Human Induced Pluripotent Stem Cell (iPSC)-Derived Fetal and Neonatal Hepatocyte-like Cell Model for Drug and Toxicant ScreeningInventors: Jed Lampe; Emily GraceyCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 648,943, filed on May 17, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] The invention was made with government support under Grant No. R01 All 83687 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] Embodiments of the present disclosure generally relate to processes for producing fetal and / or neonatal hepatocyte-like cells (herein referred to as fnHLCs). Embodiments described herein also generally relate to chemical screening models that include the fnHLCs cells. Embodiments of the present disclosure also generally relate to processes for screening a candidate chemical for, e.g., metabolism and / or toxicity in the fnHLC systems.BACKGROUND

[0004] Drugs are routinely given off-label to neonates and developing infants despite never being tested for safety and efficacy in this vulnerable patient population. A primary determinant of drug safety and efficacy in the adult population is drug metabolism, which typically occurs in the liver. The neonate and developing infant liver have very different metabolic processing capabilities in comparison to the adult liver. The adult liver contains the enzyme cytochrome P450 family 3 subfamily A member 4 (CYP3 A4), a main drug processing enzyme. In contrast, the neonate / developing infant liver contains little to no CYP3 A4, but has a related enzyme known as cytochrome P450 family 3 subfamily A member 7 (CYP3A7), which metabolizes most drug substrates aboutlOO times slower than CYP3A4, leading to much slower drug clearance and potential drug toxicity if drug doses are not titrated carefully.

[0005] Typically, drug safety testing is conducted using either primary human hepatocytes or human liver microsomes. However, these are virtually non-existent for thePCT ApplicationAttorney Docket No. : CUBR-0007PC neonatal / developing infant patient population, meaning that it is not typically feasible to test drug or toxicant metabolism in a relevant model system.

[0006] There is a need for new processes for producing fetal and / or neonatal hepatocytelike cells. There is also a need for chemical screening models that include the fetal and / or neonatal hepatocyte-like cells. There is also a need for processes for screening a candidate chemical in these cell systems.SUMMARY

[0007] Embodiments of the present disclosure generally relate to processes for producing fnHLCs. Embodiments described herein also generally relate to chemical screening models that include the fnHLCs. Embodiments of the present disclosure also generally relate to processes for screening a candidate chemical for, e.g., metabolism and / or toxicity in the finHLC systems.

[0008] In a first embodiment, a method for producing fetal and / or neonatal hepatocytelike cells (fnHLCs) is disclosed. The method includes producing a definitive endoderm cell population by a stage 1 process, producing a hepatoblast-like progenitor cell population from the definitive endoderm cell population by a stage 2 process, and producing a fetal and / or neonatal hepatocyte-like cell population from the hepatoblast-like progenitor cell population by a stage 3 process. The stage 1 process including culturing a population of induced pluripotent stem cells (iPSCs) in a first medium comprising activin A (ActA) and Wingless- related integration site isoform 3a (Wnt3a). The stage 2 process including culturing the definitive endoderm cell population in a second medium comprising a bone morphogenetic protein (Bmp) and a fibroblast growth factor (Fgf) to produce the hepatoblast-like progenitor cell population. The stage 3 process including culturing the hepatoblast-like progenitor cell population in a third medium comprising a hepatocyte growth factor (Hgf) and culturing the hepatoblast-like progenitor cell population in a fourth medium comprising oncostatin M (Osm) and a glucocorticoid (GC) to produce the fetal and / or neonatal hepatocyte-like cell population.

[0009] In another embodiment, a chemical screening model adapted to reproduce metabolic properties of a neonatal and / or fetal liver is disclosed. The chemical screening model including a fetal and / or neonatal hepatocyte-like cell operable to reproduce a plurality of metabolic properties of a neonatal or fetal liver

[0010] In another embodiment a method for screening a target is disclosed. The method includes co-culturing and incubating the target with a fetal and / or neonatal hepatocyte-like cellPCT ApplicationAttorney Docket No. : CUBR-0007PC(fnHLC), retrieving a supernatant or a cell lysate of a fnHLC after the co-culturing and incubating and determining one or more characteristics of the fnHLC based on analysis of the supernatant or the cell lysate of the fnHLC.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

[0012] Figure 1A shows a differentiation schematic, according to certain embodiments. iPSCs differentiated through definitive endoderm (DE) to hepatoblast (HB) and final hepatocyte-like cells (finHLCs) using recombinant proteins mimicking in vivo fetal development. Differentiation 4 tests differentiation 5, and the final differentiation protocol is shown.

[0013] Figures IB, 1C, ID, and IE show representative bright-field microscopy images of adult primary human hepatocytes (PHHs) and neonatal PHHs 24 hours after plating as well as the final fnHLCs at day 26. The scale bar is 50 pm.

[0014] Figures 2A and 2B show the mRNA gene expression in differentiation 1 fetal fnHLCs, according to certain embodiments. qPCR results reported as average threshold value (Cq) of 4 biological replicates. Bars represent standard deviation. Relative mRNA expression compared to ACTB as reference gene, 4 biological replicates with bars as standard deviation.

[0015] Figures 3A, 3B, 3C, and 3D show that the fnHLCs from differentiation 2 display fetal and liver gene expression, but no CYP3A7-mediated metabolism of DHEA-S, according to certain embodiments. Gene expression of fnHLCs compared to undifferentiated iPSCs. CYP3 A7 expression in fnHLCs. CYP3 A4 expression in fnHLCs. No CYP3 A metabolism of DHEA-S seen in fnHLCs, as evidenced by no change in substrate peak area ratio up to four hours. Points represent biological replicates, two for 10 nM dexamethasone (DEX) and three for 100 nM Dex.

[0016] Figures 4 A, 4B, and 4C show the different definitive endoderm treatments show varying efficiencies of differentiation, according to certain embodiments. mRNA expression of SOX17, a definitive endoderm marker. mRNA expression of POU5F1, a pluripotencyPCT ApplicationAttorney Docket No. : CUBR-0007PC marker. CRatio of SOX17 to POU5F1 expression in definitive endoderm cells. ACTB as housekeeping gene for all qPCR experiments, run in biological duplicate.

[0017] Figures 5A and 5B show that the fnHLCs from differentiation 3 display a high mRNA expression of fetal and hepatic genes according to certain embodiments. mRNA fold change compared to undifferentiated iPSCs. Comparison of CYP3A7 and CYP3A4 mRNA fold change with about 67x higher expression of CYP3A7 than CYP3A4 in fnHLCs. Points represent biological duplicates.

[0018] Figure 6 shows the formation of 16aOH DHEA by differentiation 3 in fnHLCs after 0-4 hour incubations with 50 pM DHEA, according to certain embodiments. Points represent biological duplicates.

[0019] Figure 7 shows the comparison of relative mRNA expression in fnHLCs in differentiations 1, 2, and 3, according to certain embodiments. Relative mRNA fold change calculated via ACq method with ACTB as reference. Points represent biological duplicates or triplicates.

[0020] Figure 8 shows a gene expression of fnHLCs of varying maturation times compared to undifferentiated iPSC controls, according to certain embodiments. mRNA fold change of pluripotency marker POU5F1, fetal hepatocyte markers AFP and CYP3A7, general liver markers ALB and HNF4A, and adult hepatocyte marker CYP3A4 for fnHLCs differentiated between 18 and 30 days. Points represent biological triplicates, and bars indicate standard deviation. mRNA fold change calculated relative to reference gene beta actin.

[0021] Figure 9 shows the formation of 16aOH DHEA by HLCs, according to certain embodiments. A standard curve of 16aOH standard was used to quantify metabolite production by HLC incubations. Points represent biological triplicate, and bars indicate standard deviation.

[0022] Figure 10 shows the gene expression of fnHLCs differentiated with varying glucocorticoids compared to undifferentiated iPSC controls, according to certain embodiments. mRNA fold change of pluripotency marker POU5F1, fetal hepatocyte markers AFP and CYP3A7, general liver markers ALB and HNF4A, and adult hepatocyte marker CYP3 A4 for fnHLCs differentiated in basal media, prednisolone (pred), betamethasone (beta), or dexamethasone (dex), compared to iPSC controls. Points represent biological triplicates, and bars indicate standard deviation. mRNA fold change calculated relative to reference gene beta actin.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0023] Figure 11 shows the formation of 16aOH DHEA by fnHLCs, according to certain embodiments. A standard curve of 16aOH standard was used to quantify metabolite production by fnHLC incubations. Points represent biological triplicate, and bars indicate standard deviation.

[0024] Figure 12 shows the peak area ratios of 1’OH MDZ and 4OH MDZ (midazolam) from fnHLC incubations with 30 pM MDZ, compared to metabolite standards prepped at 0.3 pM for comparison, according to certain embodiments. Ratio of 1’OH to 4OH MDZ is close to one at each timepoint. Points represent biological triplicates.

[0025] Figures 13A, 13B, 13C, 13D, 13E, and 13F show qPCR analysis of relative gene expression of hepatic phase 1 metabolism genes, fetal hepatocyte / hepatoblast genes, and transcription factors (Figure 14A) and hepatic phase 2 metabolism genes and hepatic transporter genes (Figure 14B) in male and female fnHLCs and adult and neonatal PHHs. Relative gene expression was determined as the median log transformation of gene-of-interest mRNA compared to internal reference beta-actin. mRNA fold change of CYP3A7 (Figure 14C), CYP3A4 (Figure 14D), AFP (Figure 14E), and GSTP1 (Figure 14F) in male fnHLCs (inverted triangle), female fnHLCs (diamond), neonatal PHHs (triangle), and adult PHHs (circle). mRNA fold change was normalized to beta-actin, and fold change in expression was calculated compared to 1-day neonatal PHHs. Points represent biological replicates and error bars indicate standard deviations. Statistical significance was determined via one-way ANOVA with Tukey’s multiple comparison tests. Significance indicated by P values above brackets, nd = not detected.

[0026] Figures 14A, 14B, 14C, 14D, 14E, and 14F show the metabolic activity of fnHLCs and PHHs towards endogenous and exogenous substrates. Metabolism schematic of endogenous hormone DHEA to the 16aOH metabolite, the major product of fetal / neonatal CYP3A7 with minor contributions from CYP3A4 (Figure 15 A). LC-MS / MS quantification of 16aOH DHEA formation in male and female fnHLCs, neonatal PHHs (n=4), and adult PHHs following DHEA incubations (Figure 15B). Metabolism schematic of substrate MDZ into LOH MDZ and 4OH MDZ by both CYP3A7 and CYP3A4 isoforms (Figure 15C). Ratio of 1’OH MDZ to 4OH MDZ formed by male and female fnHLCs and neonatal and adult PHHs (Figure 15D). LC-MS / MS quantification of 1’OH MDZ and 4OH MDZ in male and female fnHLCs (Figure 15E), and neonatal PHHs (n=4) and adult PHHs (Figure 15F) following MDZPCT ApplicationAttorney Docket No. : CUBR-0007PC incubations. Points represent biological replicates and bars indicate standard deviations. Statistical significance determined by one-way ANOVA with Tukey’s multiple comparison tests (Figure 15B and Figure 15D) and two-tailed student’ s t tests (Figure 15E and Figure 15F). Significance indicated by P values above brackets, ns = not significant.

[0027] Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, 151, and 15J show CYP3A induction and changes in enzymatic activity by rifampicin (RIF) and betamethasone (BMZ). CYP3A7 and CYP3A4 mRNA fold changes with 5 and 50 pM M RIF in male and female fnHLCs (Figure 16A) and neonatal and adult PHHs (Figure 16B). Changes in CYP3A MDZ metabolite production with 5 and 50 pM RIF in male and female fnHLCs (Figure 16C) and neonatal and adult PHHs (Figure 16D). 1’OH MDZ to 4OH MDZ metabolite ratios in male fnHLCs (inverted triangle), female fnHLCs (diamond), neonatal PHHs (triangle), and adult PHHs (circle) with RIF induction (Figure 16E). CYP3A7 and CYP3A4 mRNA fold changes with 50 and 500 nM BMZ in male and female fnHLCs (Figure 16F) and neonatal and adult PHHs (Figure 16G). Changes in CYP3A midazolam metabolite production with 50 and 500 nM BMZ in male and female fnHLCs (Figure 16H) and neonatal and adult PHHs (Figure 161). 1’OH MDZ to 4OH MDZ metabolite ratios in male fnHLCs (inverted triangle), female fnHLCs (diamond), neonatal PHHs (triangle), and adult PHHs (circle) with BMZ treatment (Figure 16J). All cells were induced over 72hr with solvent controls and two concentrations of inducer. Points represent biological replicates and bars indicate standard deviations. Statistical significance determined by two-way ANOVA with Tukey’s multiple comparison tests, ns = not significant.

[0028] Figure 16A, 16B, 16C, 16D, 16E, and 16F show CYP3A bioactivation of aflatoxin. Simplified schematic of aflatoxin Bi (AFBi) metabolism by CYP3A to detoxification species aflatoxin Qi (AFQi) and toxic epoxide AFBO (Figure 17A). Representative brightfield microscopy images of male and female fnHLCs, neonatal PHHs, and adult PHHs at 0, 1, 10, 100 pM for up to 48hr (Figure 17B). Representative UV chromatograms of AFBi metabolism products in adult PHHs after dosage with 10 pM after 24hr compared to DMSO control (Figure 17C). Peak area ratios of AFQi formed by male and female fnHLCs and neonatal and adult PHHs after 24hr and 100 pM AFBi with and without CYP inhibitor ABT (Figure 17D). Formation of AFQi over time after dosing with 100 pM AFBi in male and female fnHLCs (Figure 17E) and neonatal and adult PHHs (Figure 17F) over 48hr. Points represent biological replicates and bars indicate standard deviations.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0029] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0030] Embodiments of the present disclosure generally relate to processes for producing fetal and / or neonatal hepatocyte-like cells (fnHLCs). Embodiments described herein also generally relate to chemical screening models that include the fnHLCs. Embodiments of the present disclosure also generally relate to processes for screening a candidate chemical for, e.g., metabolism and / or toxicity in the fnHLC systems.

[0031] Fetuses and neonates have a varied metabolic profile, contributing to differences in overall xenobiotic clearance and toxicity when compared to adults. This is primarily due to the ontogeny of many xenobiotic metabolizing enzymes, wherein expression is low during the fetal period, and begins to increase directly after birth and into the neonatal and pediatric periods. CYP3A7 is the major cytochrome P450 (CYP) in the liver of the developing fetus and neonate, responsible for the metabolism of both endogenous and exogenous substrates. CYP3A7 metabolizes the adrenal steroid dehydroepiandrosterone sulfate (DHEA-S) to 16a- hydroxy (16aOH) DHEA-S, a precursor for estriol synthesis in the placenta. During the fetal period, CYP3A7 represents up to 50% of the CYP content in the liver and CYP3A7 activity can be detected as early as 50 days of gestation. The expression and activity of CYP3A7 is high during the fetal and neonatal periods, decreasing throughout the first year of life. Adult isoform CYP3 A4 is expressed at low levels in the fetal liver, and after birth increases to reach full adult levels after 12 months. CYP3A4 is the major CYP3A isoform in adults, constituting approximately 30% of CYP levels in the liver and metabolizing >30% of marketed drugs. CYP3A4 and CYP3A7 share 87% amino acid sequence identity and consequently display overlapping substrate specificity which can cause difficulty in determining differences in metabolism by each isoform. CYP3 A7 is integral to estriol biosynthesis, metabolizing adrenal steroids DHEA and DHEA-S to the 16aOH precursors utilized by the placenta to produce estriol. Estriol, in turn, regulates the timing of parturition by maintaining uterine quiescence until just before birth. Both CYP3 A4 and CYP3 A7 are able to metabolize DHEA-S, and the non-sulfated form DHEA, and produce metabolites with different hydroxylation patterns. CYP3A7 produces the major metabolite 16aOH DHEA(S) and the minor metabolite 7POHPCT ApplicationAttorney Docket No. : CUBR-0007PCDHEA(S), while CYP3A4 produces the major metabolite 7POH DHEA(S) with 16aOH DHEA(S) as the minor product.

[0032] Low estriol levels during pregnancy are associated with fetal growth restriction and risk for preterm labor. Therefore, any perturbation of this sensitive system, through xenobiotic inhibition or induction of CYP3A7, can have harmful outcomes for the fetus. Due to the scarcity of human fetal hepatocytes, a sustainably sourced finHLC model derived from induced pluripotent stem cells was developed (iPSCs) in order to study CYP3A7 in a cell-based, in vitro system.

[0033] The fnHLC differentiation protocol is conducted to increase the fetal and / or neonatal characteristics. iPSCs were differentiated through three stages: definitive endoderm (DE) (stage 1), hepatoblast-like cells (stage 2), and final fnHLCs (stage 3). For the differentiation step to DE, several sub-protocols were tested to maximize cell survival and differentiation efficiency. The fnHLCs produced by the disclosed methods can be used in chemical screening models. Further processes of screening a candidate chemical for metabolism and / or toxicity can be conducted in the disclosed cell systems.

[0034] Currently, no reliable model exists for metabolic safety testing of new drugs and chemicals that neonates and developing infants may be exposed to. This is a critical need due to the fact that neonates and developing infants are exposed to potentially harmful drugs in the clinic and / or chemicals that they may come in contact with. To address this need, a sustainably sourced fetal and / or neonatal hepatocyte-like cell model derived from induced pluripotent stem cells (iPSCs) is disclosed. This robust model recapitulates the metabolic features and drug metabolism characteristics of fetal and / or neonatal hepatocytes, including, but not limited to, enzyme / transporter expression profile, enzyme activity, and morphological phenotype. This model system may find use in both pre-clinical screening of drugs undergoing FDA approval that may be given to neonates and also toxicity testing of new chemicals that are undergoing EP A approval.

[0035] A reliable, robust, and reproducible fetal and / or neonatal hepatocyte-like cell systems that can accurately recapitulate the metabolic properties of the neonatal liver is disclosed herein. Embodiments described herein have myriad applications. For example, pharmaceutical research into developing new drugs that may be used in the neonatal and developing infant population, as well as researchers in the chemical industry who test the safety of chemical products produced that a neonate or developing infant may come in contact with.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0036] The term “cell culture medium” (also referred to herein as a “culture medium” or “medium”) includes a medium for culturing cells containing nutrients that maintain cell viability, support proliferation, and / or optionally differentiation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, vitamins etc.

[0037] As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof.

[0038] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use aspects of the present disclosure, and are not intended to limit the scope of aspects of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for.ExamplesExample: iPSC-Derived Hepatocyte-like Cells to Study Fetal / Neonatal Drug Metabolism (in fnHLCs)Materials and Methods1. Induced Pluripotent Stem Cell (iPSC) Culture

[0039] iPSCs (iC7-2 male and iC4-4 female lines) were obtained from the University of Colorado Anschutz Medical Campus Stem Cell Biobank Core and were maintained in mTeSR Plus median (StemCell Technologies) on Matrigel (Corning)-coated plates. For optimization experiments, differentiation was initiated when iPSCs were approximately 50-60% confluent one day after passaging with ReLeSR (StemCell Technologies). For comparison to PHHs, iPSCs were plated at about 4xl04to about 5xl04cells / cm2and differentiation was initiated one day after passaging as single cells with TrypLE Select (ThermoFisher).2. Differentiation to fnHLCs

[0040] Differentiation was initiated when iPSCs were approximately 50-70% confluent one day after passaging. Five differentiation protocols were tested resulting in a final differentiation protocol. Differentiation was conducted with a method including producing a definitive endoderm (DE) cell population by a stage 1 process, producing a hepatoblast-like (HB) progenitor cell population from the definitive endoderm cell population by a stage 2PCT ApplicationAttorney Docket No. : CUBR-0007PC process, and producing a fnHLC population from the hepatoblast-like progenitor cell population by a stage 3 process, as shown in Figure 1A for differentiation 4, differentiation 5, and the final differentiation. The protocol (e.g., the method) for differentiation 4, differentiation 5, and the final differentiation includes a plurality of stages (e.g., operations) over a timeline of days, described herein. For days 1-4 (stage 1), the cells were maintained in a first medium (e.g., Roswell Park Memorial Institute Medium (RPMI media)) including 1 mM L-glutamine, about 1-2% B27+ supplement, and 4: 1 ratio of activin A to Wingless-related integration site isoform 3a (Wnt3a), between about 90 ng / mL to about 100 ng / mL activin A and 22.5-25 ng / mL Wnt3a. For example, the first medium may include about 2% B27+ supplement, about 100 ng / mL activin A, and about 25 ng / mL Wnt3a. The Wnt3a was part of the first medium for the first two days of culturing only. The media was changed daily.

[0041] For days 5-9 (stage 2), the cells were maintained in a second medium, a hepatocyte culture media (HCM, Lonza), with about 18 ng / mL to about 20 ng / mL bone morphologic protein 4 (Bmp4) and about 27 ng / mL to about 30 ng / mL fibroblast growth factor 2 (Fgf2) to achieve a hepatoblast-like state (e.g., a hepatoblast-like progenitor cell), with daily media changes. For example, the second medium includes about 20 ng / mL Bmp4 and about 30 ng / mL Fgf2. For final fnHLCs, cells were maintained in a third medium that is a HCM with about 18 ng / mL to about 20 ng / mL hepatocyte growth factor (Hgf) for four days. For example, the final fnHLCs were maintained in a third medium that is a HCM with about 20 ng / mL of Hgf.

[0042] For days 5-30 (stage 3), the cells were maintained in a fourth medium for a maturation period of 5-30 days in HCM with about 9 ng / mL to about 10 ng / mL oncostatin M (Osm) and about 10 nM to about 100 nM glucocorticoid (GC), with media changes every other day. In one or more embodiments, the maturation period of 10-11 days. The glucocorticoids utilized includes prednisolone (PNL), betamethasone (BMZ), dexamethasone (DEX), or combinations thereof. Any suitable amount of the glucocorticoid may be utilized. For example, the fifth medium may include an amount of glucocorticoid that is from about 10 nanomolar (nM) to about 100 nM. In the tests, different maturation times and different glucocorticoid derivatives were tested. Glucocorticoid derivatives were introduced to the maturation period to increase CYP3A7 expression while minimizing CYP3A4 expression. For maturation time, a final stage length of 5, 9, 13, and 17 days were tested (overall protocol lengths of 18, 22, 26, and 30 days). To determine the impact of different glucocorticoids, prednisolone and betamethasone were tested at 10 nM, and dexamethasone was tested at 10 nM and 100 nM.PCT ApplicationAttorney Docket No. : CUBR-0007PCMonolayers were observed at each media change and microscopy images were obtained on an Olympus CKX53 microscope with an EP50 camera.

[0043] In one or more embodiments, the protocol for differentiation 1 includes four replicates and was 20 days total with 0.5% FBS in stage 1 and 100 nM Dex in stage 3. In one or more embodiments, the protocol for differentiation 2 includes 19 days total with 0.5% FBS in stage 1 and two replicates at 10 nM Dex and three replicates at 100 nM Dex during stage 3. In one or more embodiments, the protocol for differentiation 3 includes 6 replicates and was 20 days total with 2% B27+ in stage 1 and at 100 nM Dex in stage 3.

[0044] In one or more embodiments the final differentiation includes iPSCs differentiated to DE during stage 1 with RPMI media with 1 mM L-glutamine, 2% B27+, 100 ng / mL activin A, and 25 ng / mL WNT3A for two days, followed by two days in the same media omitting WNT3 A. For hepatoblast induction during stage 2, cells were maintained in hepatocyte culture media (HCM, Lonza) with 20 ng / mL BMP4 and 30 ng / mL FGF2 for five days. For the HLC stage (e.g., stage 3), cells were maintained in HCM with 20 ng / mL HGF for four days, followed by a maturation period of 5-17 days in HCM with 10 ng / mL OSM and 10-100 nM glucocorticoid derivative. The final differentiation consisted of a final maturation period of 13 days in HCM with 10 ng / mL OSM and 100 nM DEX (26 days total). Monolayers were observed at each media change and phase-contrast microscopy images were obtained on an Olympus CKX53 microscope with an EP50 camera.

[0045] Representative bright-field microscopy images were obtained at each stage of the fnHLC differentiation (as shown in Figure IB) and the final morphology of fnHLCs was compared to both adult and neonatal hepatocytes (as shown in Figures 1C, ID, and IE). fnHLCs were differentiated from two iPSC donors, one male and one female. Adult primary human hepatocytes (PHHs) originated from a pool of ten donors (10-64 years old), and neonatal PHHs were from a 1-day old donor. All cells appeared cuboidal and some with multiple nuclei, both hallmarks of hepatocyte morphology. Neonatal PHHs and fnHLCs were of similar size, slightly smaller than the adult PHHs. However, fnHLC wells were more heterogeneous in nature and borders between neighboring cells weren’t as bright as observed with PHHs.

[0046] The population of fnHLCs formed by the process may produce any suitable protein such as, for example, a cytochrome P450 protein. Illustrative, but non-limiting, cytochrome P450 proteins may include cytochrome P450 family 3 subfamily A member 7 (CYP3A7),PCT ApplicationAttorney Docket No. : CUBR-0007PC cytochrome P450 family 3 subfamily A member 4 (CYP3A4), Cytochrome P450 family 2 subfamily C member 9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6), cytochrome P450 family 7 subfamily A member 1 (CYP7A1), cytochrome P450 family 1 subfamily A member 2 (CYP1A2), cytochrome P450 family 2 subfamily B member 6 (CYP2B6), or combinations thereof.

[0047] In one or more embodiments, several different definitive endoderm sub-protocols were tested to determine cell survival and efficiency of stage 1 differentiation. The treatments are outlined in Table 1, all with RPMI media, 1 mM L-glutamine, 100 ng / mL activin A, and 25 ng / mL Wnt3a for the first two days. iPSCs were plated either as colonies or single cells on day 0, and the differentiation was initiated on day 1 with starting confluences around 80% or 95%. Cells were monitored daily under the microscope to observe confluency and attachment. On Day 5, RNA was extracted as described below, and qPCR was performed in duplicate for genes POU5F1 (pluripotency), SOX17 (definitive endoderm), and ACTB (reference).Table 1PCT ApplicationAttorney Docket No. : CUBR-0007PC3. Characterization of fnHLCs.

[0048] At the end of the differentiation protocol, fnHLCs were probed for fetal and liver characteristics at the gene expression and enzymatic activity levels. First, fnHLCs were washed with PBS and incubated with about 5 to about 250 pM dehydroepiandrosterone (DHEA) or 30 pM midazolam (MDZ) for up to four hours at 37°C and 5% CO2 to determine CYP3A activity. Supernatants (100-150 pL) were collected and stopped with an equal volume of cold methanol with internal standard (DHEA-d5 for DHEA incubations and aOH-MDZ-d4 for MDZ incubations). Precipitated proteins were collected by centrifugation for 20 min at 2000 x g and 4 °C and supernatants were analyzed by LC-MS / MS for formation of the 16aOH DHEA and E0H / 40H MDZ metabolites. The remaining fnHLCs were washed once with PBS, and RNA was extracted with the RNeasy Mini Kit (Qiagen) following manufacturer protocols with inclusion of the DNAse step. RNA concentration and purity were determined based on absorbance at 260 and 280 nm using the NanoQuant plate with the Tecan Infinite M Plex plate reader. cDNA was prepared with 1 pg purified RNA using the i Script Reverse Transcriptase Supermix kit (BioRad). qPCR was performed in technical duplicate on the BioRad CFX96 instrument using SYBR green primers (IDT) listed in Table 2. Fold change in mRNA was determined with the AACq methods with ACTB as the reference gene, compared to undifferentiated iPSC controls.Table 2PCT ApplicationAttorney Docket No. : CUBR-0007PC4. Evaluation of DHEA-S as a CYP3A7 Probe Substrate.

[0049] The specificity of DHEA-S as a probe substrate for CYP3A7 was evaluated with recombinant CYP3A7 and CYP3A4. Incubations (200 uL) containing 10 pmol / mL CYP3A (CYP3A4 supersomes, Coming or CYP3A7 bactosomes, Cypex) in 100 mM potassium phosphate buffer, 3.3 mM MgCh, and 5 pM DHEA-S (dissolved in methanol, 0.1% v / v) were pre-equilibrated at 37 °C for 3 minutes. Triplicate reactions were initiated by the addition of a NADPH regeneration system (1 mM NADP+, 10 mM glucose-6-phosphate, and 2 lU / mL glucose-6-phosphate dehydrogenase) and were incubated for 10 minutes at 37 °C. Reactions were stopped with the addition of an equal volume of cold methanol containing the internal standard DHEA-S-d5 (20 ng / mL). Precipitated proteins were collected by centrifugation at 2,500 x g and 4 °C for 20 minutes and supernatants were analyzed via LC-MS / MS for the formation of 16aOH DHEA-S. Triplicate controls with NADPH were included for each CYP3 A isoform.5. Analytical Method for Formulation and DHEA(S) Metabolites.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0050] Formation of 16aOH DHEA was determined by LC-MS / MS analysis on a Waters Acquity Ultra-Performance Liquid Chromatography (UPLC) system interfaced by electrospray ionization with a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer (Waters Corp). The following source conditions were applied in ESI positive mode: 3.5 kV for the capillary voltage, 350 °C for the desolvation temperature, and 650 L / h for the desolvation gas flow. The following source conditions were applied for ESI negative mode for DHEA(S) incubations: 0.5 kV for the capillary voltage, 450 °C for the desolvation temperature, and 900 L / h for the desolvation gas flow. Peaks were integrated via QuanLynx software and the analyte / internal standard area ratios were used for relative quantification.6. Analytical Method for DHEA-S Hydroxylation by fnHLCs (Differentiation 2) and Recombinant Enzyme.

[0051] MRM scan type in ESI negative mode was used to detect analytes at the following mass transitions: 367 > 97 for DHEA-S, 383 > 97 for 16aOH DHEA-S, 372 > 98 for DHEA- S-d5 (IS). Metabolites were separated on a Waters BEH C18 column (1.7 um, 2.1 x 50 mm), with water and methanol with 0.1% formic acid. The following gradient was used: 20% organic held for 0.5 min, increased to 98% over 3.5 min, and held at 98% for 0.9 min.7. Analytical Method for DHEA(S) Hydroxylation by fnHLCs (Differentiation 3).

[0052] MRM scan type in ESI positive mode was used to detect analytes at the following mass transitions: 289 > 253 for DHEA, 305 > 231 for 16aOH DHEA, 294 > 218 for DHEA- d5 (IS). Metabolites were separated on a Waters BEH C18 column (1.7 um, 2.1 x 100 mm), with water with 0.1% formic acid and acetonitrile with 0.1% formic acid. The following gradient was used: 20% organic for 0.5 min, increased to 70% over 3.5 min, increased to 98% over 1 min, and held at 98% for 1 min. MRM scan type in ESI negative mode was used to detect analytes at the following mass transitions: 367 > 97 for DHEA-S and 383 > 97 for 16aOH DHEA-S. Metabolites were separated on a Waters BEH C18 column (1.7 um, 2.1 x 100 mm), with 5 mM ammonium acetate in water and 5 mM ammonium acetate in methanol. The following gradient was used: 20% organic for 0.5 min, increased to 45% over 2.5 min, held at 45% for 1.5 min, increased to 98% over 1.5 min, and held at 98% for 1 min.8. Analytical Method for DHEA(S) Hydroxylation by HLXs (Differentiation 4 and 5).

[0053] MRM scan type was used to detect analytes at the following mass transitions: 289 > 253 for DHEA, 305 > 231 for 16aOH DHEA, 294 > 218 for DHEA-d5 (IS). Metabolites were separated on a Waters BEH C18 column (1.7 pm, 2.1 x 100 mm), with water with 0.1%PCT ApplicationAttorney Docket No. : CUBR-0007PC formic acid and acetonitrile with 0.1% formic acid. The following gradient was used: 20% organic for 0.5 min, increased to 70% over 3.5 min, increased to 98% over 1 min, and held at 98% for 1 min. Peaks were integrated via QuanLynx software and the analyte / internal standard areas were used to determine peak area ratio and metabolite standard curves were used to quantify metabolite production. For quantification of the 16aOH metabolite, a linear regression was performed based on the analyte / IS peak area ratios of the calibration standards, ranging from 0.2-4 pM, with ±20% error allowed at the lower limit of quantification.9. Analytical Method for MDZ Hydroxylation.

[0054] Formation of the 1’OH and 4OH MDZ metabolites was determined on the same system as described above in ESI positive mode. MRM scan type was used to detect analytes at the following mass transitions: 325 > 291 for MDZ, 342 > 203 for 1’OH MDZ, 342 > 234 for 4OH MDZ, and 345 > 203 for aOH-MDZ-d4 (IS). Metabolites were separated on a Waters BEH C 18 column (1.7 pm, 2.1 x 50 mm) with water with 0.1% formic acid and methanol with 0.1% formic acid. The following gradient was used: 10% organic for 0.5 min, increased to 98% over 2.5 min, and held at 98% for 1 min. Peaks were integrated via QuanLynx software and the analyte / internal standard areas were used to determine peak area ratio. Authentic standards of the 1’OH and 4OH MDZ metabolites were included at 0.3 pM as a comparison in initial testing.Non-limiting Results

[0055] As part of the analysis of the results, the gene expression of the finHLCs was compared to primary human hepatocytes of a one-day old donor. This time frame was chosen due to the fact that most fetal hepatocytes are typically obtained at or before 13 weeks of gestation, an age when liver functions have not yet fully developed. The one-day old donor neonatal hepatocytes represent a maturing liver on the continuum of development from the third trimester to the first weeks of life, when liver metabolic functions are more pronounced. The pattern of ontogenetic expression of many xenobiotic metabolizing enzymes was observed across the fnHLC model system and both sets of hepatocytes.1. Differentiation 1

[0056] While establishing protocols for iPSC culture, four wells of iPSCs were subject to differentiation 1. The mRNA expression of differentiation 1 finHLCs is shown in Figure 2A and 2B. qPCR results are presented in the form of average threshold value (Cq, Figure 2A).PCT ApplicationAttorney Docket No. : CUBR-0007PCThe data presentation allows for relative comparison of expression of different genes in the fnHLCs at a preliminary stage. fnHLCs showed high expression (low Cq values) of references ACTB and GAPDH, as well as liver marker ALB and fetal markers alpha fetoprotein (AFP) and CYP3A7. Liver marker hepatocyte-nuclear factor 4a (HNF4a) and adult hepatocyte marker CYP3A4 displayed moderate expression (moderate Cq values), and the pluripotency marker POU5F 1 was expressed at a very low level (high Cq value). The fetal isoform CYP3 A7 showed higher expression in fnHLCs compared to the adult isoform CYP3A4. Relative mRNA expression was calculated via the ACq method. Relative mRNA expression is a more robust comparison method than pure Cq values after being normalized to an endogenous control, although the trends remain the same (Figure 2B).2. Differentiation 2

[0057] Results for differentiation 1 are shown in Figures 3A, 3B, 3C, and 3D. Both 10 nM and 100 nM DEX were tested in the maturation phase to maximize CYP3A7 expression while limiting CYP3A4 expression. qPCRgene expression analysis is similar to differentiation 1 (as disclosed above), but now compared to undifferentiated iPSC controls (Figure 3A). Liver markers ALB and HNF4alpha were both increased over 100-fold compared to iPSCs, with little difference between 10 and 100 nM DEX. Fetal AFP was increased over 4 orders of magnitude compared to iPSCs. There were important differences in expression of the different CYP3A isoforms. CYP3A7 mRNA was increased to about 25 fold with 10 nM Dex and aboutl25 fold with 100 nM Dex, although there was large variation and only two data points at the latter concentration (Figure 3B). CYP3A4 did not see the same increases, and at 10 nM DEX was one third of the iPSC expression level, and slightly increased to 2.2 fold at 100 nM DEX (Figure 3C). For activity, the probe substrate tested was DHEA-S, as it is an established endogenous substrate of CYP3 A7. DHEA-S was tested in a recombinant system to determine individual contributions and CYP3A7 and CYP3A4 to 16aOH DHEA-S formation. CYP3A7 produced more 16aOH DHEA-S than CYP3A4. In fnHLCs for up to four hours, no measurable peak of the 16aOH metabolite was observed and no decrease in DHEA-S was observed, likely due to lack of substrate uptake in this cell line (Figure 3D).3. Definitive Endoderm Determination

[0058] Because less than half of the original wells in differentiation 2 survived stage 1, this stage was adjusted for cell survival and efficiency of differentiation from iPSCs to definitive endoderm. A B27+ supplement at different concentrations and timings compared toPCT ApplicationAttorney Docket No. : CUBR-0007PC the FBS control during stage 1 (Table 1). As shown in Figures 4A, 4B, and 4C, at day 5, DE cells were analyzed via qPCR for their SOX17 (DE marker, Figure 4A) and POU5F1 (pluripotency marker, Figure 4B) expression and the ratio of the two genes was calculated for each treatment (Figure 4C). Treatments A and E showed the highest expression of SOX17 and the lowest expression of POU5F1, indicating the most effective protocols for differentiation from iPSC to DE. Treatments A and E also had the highest confluency of cells at Day 5, indicating better cell survival. Because treatment E was able to achieve similar results to treatment A with a lower starting confluency and avoids the use of lot-to-lot variable FBS, treatment E was chosen for differentiations protocols.4. Differentiation 3

[0059] For differentiation 3, the differentiation protocol was similar, with changes to support cell survival and differentiation during stage 1 as disclosed above, and changes to the endpoint activity assay in order to maximize CYP3A7 activity. As shown in Figure 5A, fnHLCs from differentiation 3 show the highest mRNA expression of fetal and liver genes. In differentiation 3, liver markers ALB and HNF4A are highly expressed, 2-3 orders of magnitude over undifferentiated iPSCs. Fetal hepatocyte marker AFP was increased over 6 orders of magnitude compared to iPSCs. As shown in Figure 5B, there were notable differences in CYP3A isoform expression. CYP3A7 mRNA was increased compared to previous differentiations to approximately 4000-fold change compared to iPSCs. CYP3A4 mRNA also saw an increase compared to differentiation 2, up to about 65-fold increase but still almost two orders of magnitude less than CYP3 A7.

[0060] fnHLCs produced from differentiation 3 were able to metabolize DHEA to 16aOH DHEA, the major product of CYP3A7. Figure 6 shows the peak area ratios of the 16aOH DHEA metabolite compared to a synthetic standard. At 1 hour, the peak intensity was less than 3 times the background. After two-hour and four-hour DHEA incubations, there was an increase in peak area ratio from 0.123 to 0.248. For DHEA-S metabolism, the LC-MS method was updated to a more current method in order to see further separation of the 16aOH DHEA- S peaks. Even with the updated method, no peak matching the metabolite standard was observed at any timepoints or with either substrate concentration. For example, one sample of each was run as a test to determine differences in the ability of DHEA and DHEA-S to enter the fnHLCs. For DHEA, the ratio of supernatant to lysate signal intensity was about 28, andPCT ApplicationAttorney Docket No. : CUBR-0007PC for DHEA-S was aboutl27, indicating a difference in the ability of the steroid substrates to enter the cells.5. Relative mRNA Expression in Differentiations 1, 2, and 3.

[0061] The relative mRNA expression was compared between all three differentiations (100 nM dexamethasone samples only) to assess the reproducibility of the protocol. The results in Figure 7 show relatively similar mRNA expression for differentiations 1 and 3, and slight decreases in some hepatic genes for differentiation 2. One explanation is the massive cell death observed during differentiation 2, that could have impacted differentiation efficiency of the remaining cells. It is unclear why the same level of cell death was not observed during differentiation 1, but it could have to do with different starting confluences and different feed volumes in 6- vs 12-well plate formats affecting the overall differentiation efficiency.6. Differentiation 4

[0062] Once a general protocol was established that yielded fnHLCs with measurable CYP3A7 expression and activity in differentiation 3, maturation time in the final stage was tested to determine the suitable timing for fetal-like characteristics. The final stage maturation times of 5, 9, 13, and 17 days were tested, corresponding to total protocol lengths of 18, 22, 26, and 30 days. Figure 8 shows the gene expression analysis of iPSCs compared to fnHLCs of varying maturation times for a panel of six representative genes. The pluripotency marker POU5F1 showed high expression in iPSCs, and very low expression in all fnHLCs as expected. The two fetal hepatocyte markers AFP and CYP3A7 showed increasing expression with increasing maturation time, plateauing after 26 days. General liver markers ALB and HNF4A followed similar trends. Adult isoform CYP3 A4 showed a similar expression pattern to that of CYP3A7, but importantly resulted in >100-fold less expression than CYP3A7. At 26 days, CYP3A7 mRNA fold change was approximately 20,000-fold change compared to undifferentiated iPSCs, and only aboutl 50-fold for CYP3A4 mRNA.

[0063] fnHLCs from differentiation 4 were again able to metabolize endogenous substrate DHEA and produce the 16aOH DHEA metabolite, the major product of fetal / neonatal CYP3 A7. This time, the levels of metabolite were quantified with a standard curve to calculate metabolite formation in terms of pmol per minute per well of cells. The results are shown in Figure 9. The CYP3A7 activity mirrors that of the CYP3A7 gene expression data, with increasing activity from 18 days to 26 days, and plateauing from 26 to 30 days. Based on the gene expression and activity data, 26 days was selected as the best timepoint for futurePCT ApplicationAttorney Docket No. : CUBR-0007PC differentiations. At 26 days, maximum CYP3A7 expression and activity was observed while also limiting excess time in culture.7. Differentiation 5

[0064] The induction effect of different glucocorticoids (GCs) cause differential inductive effects of different GCs on CYP3A isomers in fetal liver cells. Prednisolone and betamethasone were tested against dexamethasone, which is the standard GC used in a majority of protocols. Figure 10 shows the gene expression results of the same six representative genes. Again, POU5F1 was included as a control for pluripotency and behaved as expected. For AFP, ALB, and HNF4A, there was some variability in the GC derivatives to induce these three genes, with 10 nM prednisolone, 10 nM betamethasone, and 100 nM dexamethasone leading to the highest expression. For the CYP3A isoforms, prednisolone did not induce much expression over the basal media with no GCs present. 10 nM betamethasone and 10 nM dexamethasone yielded similar results, about 2000 fold change in mRNA compared to undifferentiated iPSCs. However, 100 nM dexamethasone yielded the highest CYP3A7 expression. Again, a similar trend was observed for CYP3A4, about 1-2 orders of magnitude below CYP3A7.

[0065] The activity data for the GC derivatives mirrored that of the gene expression data, as seen in Figure 11. fnHLCs cultured in basal media with no GCs added in the final stage produced a small but measurable amount of 16aOH DHEA, with prednisolone producing a similar amount. Dexamethasone slightly outperformed betamethasone at 10 nM. Because neither of the GC derivatives led to higher activity than dexamethasone, 100 nM dexamethasone was selected for the final differentiation protocol.

[0066] Also included in differentiation 5 was a test of an additional CYP3A substrate midazolam (MDZ). MDZ is a benzodiazepine that is often used as a probe substrate for CYP3A activity. Both CYP3A4 and CYP3A7 can metabolize MDZ to two metabolites: 1’OH MDZ and 4OH MDZ. The ratio of 1’OH to 4OH MDZ is used to distinguish contributions from each isoform, with CYP3A4 producing a ratio of about 5-9 and CYP3A7 producing a ratio of about 1-1.4. When fnHLCs were incubated with 30 pM MDZ for up to 4 hours, the peak area ratio of both metabolites was detectable (Figure 12). Similar amounts of each metabolite were produced by the fnHLCs, with a 1’OH to 4OH MDZ ratio of 0.85, resembling the ratio of CYP3 A7.8. Final DifferentiationPCT ApplicationAttorney Docket No. : CUBR-0007PC

[0067] The testing of different protocols 1 through 5, as disclosed herein, resulted in the final differentiation protocol development. fnHLCs were compared to PHHs in terms of their gene expression of at least 25 genes involved in xenobiotic metabolism. Relative gene expression in the four cell types was determined via quantitative PCR (qPCR) and is depicted in heat map form for fetal hepatocyte / hepatoblast markers, hepatic phase 1 xenobiotic metabolizing enzymes, and transcription factors and hepatic phase 2 xenobiotic metabolizing enzymes and transporters, as shown in Figures 13A and 13B.

[0068] Phase 1 xenobiotic metabolizing enzymes, consisting mainly of CYPs and flavin monooxygenases (FMOs), presented varied expression profiles between cell types. CYP3 A7, the dominant CYP3A isoform in the fetus and neonate, was also the main CYP isoform expressed in fnHLCs. As shown in Figure 13C, the fold change in mRNA is calculated relative to neonatal PHHs. Female fnHLCs had approximately half the CYP3 A7 expression as neonatal PHHs at 0.49 ± 0.17 (mean fold change ± standard deviation), and male fnHLCs had an expression fold change of 0.07 ± 0.01. Male and female fnHLCs contained about 2-10 times more CYP3A7 than adult PHHs at a fold change of 0.045 ± 0.034. The opposite trend was observed for CYP3 A4, as shown in Figure 13D. Male fnHLCs, female fnHLCs, and neonatal PHHs were not statistically significant from each other, with CYP3 A4 mRNA fold changes of 0.045 ± 0.032, 0.325 ± 0.174, and 1.00 ± 0.65 respectively. Adult PHHs expressed significant amounts of CYP3A4 at a fold change of 15.33 ± 3.51 compared to neonatal PHHs. CYP isoforms 1A2 and 2B6 followed a similar pattern to CYP3A4, wherein there was low expression in fnHLCs, low to moderate expression in neonatal PHHs, and high expression in adult PHHs. For FMO expression, FM03 was only detected in PHHs, with moderate expression in neonatal and higher in adult samples. FM01 however, had similar expression across all cells, with the highest in neonatal PHHs closely followed by the fnHLCs.

[0069] In lieu of fetal hepatocyte samples, a few known fetal hepatocyte / hepatoblast markers (AFP, DLK1, KRT19) were tested and detected at high levels in fnHLCs. As shown in Figure 13E, AFP was highly expressed in fnHLCs, at 52.17 ±9.46 and 45.60 ± 17.84 foldchange relative to neonatal PHHs (1.00 ± 0.26). Adult PHHs had very low levels of AFP at 0.15 ± 0.11 relative fold change. Similarly, fnHLCs had high expression of DLK1 and KRT19, both involved in hepatocyte differentiation from progenitors. Four nuclear factors were tested for their gene expression in fnHLCs and PHHs: glucocorticoid receptor (GR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and hepatocyte-nuclear factor 4aPCT ApplicationAttorney Docket No. : CUBR-0007PC(HNF4a). In fnHLCs, the major transcription factor detected was GR, with similar levels in neonatal PHHs. PXR, CAR, and HNF4a were highly expressed in adult PHHs, with slightly less in neonatal PHHs and lower expression in fnHLCs.

[0070] In general, fnHLCs had lower expression of many phase 2 metabolizing enzymes compared to the neonatal PHHs, however one important exception was glutathione S- transferase Pl (GSTP1, shown in Figure 13F). GSTP1 was highly expressed in male and female fnHLCs and neonatal PHHs at fold changes of 0.81 ± 0.32, 1.54 ± 0.32, and 1.00 ± 0.69 respectively. On the contrary, GSTP1 was significantly lower in adult PHHs at a fold change of 0.020 ± 0.009. Transporter expression varied across all cell types, with the most prominent transporter genes expressed in fnHLCs being MRP4 and BCRP. Taken all together, the fnHLCs displayed a mix of fetal and neonatal gene expression and are generally more similar to the 1-day neonatal PHHs and distinct from the genetic expression profile of adult PHHs.Non-limiting Conclusions

[0071] Differentiation of iPSCs to definitive endoderm is the most crucial part of the protocol, and this stage was adjusted after observing massive cell death during differentiation 2. Treatment A (0.5% FBS) was successful after experiencing issues in past differentiations. This is likely due to the starting confluency of the iPSCs, which was increased to about 95%. Starting density of iPSCs varies widely throughout different protocols, with some citing specific cell densities (3,500-10,500 cells / cm2), and others stating and approximate confluencies. The B27+ supplement at 1% or 2% helped cells survive stage 1, but only if included for each of the four days. In one or more embodiments, the B27+ supplement included only on day 1 was sufficient to maintain cell survival. Plating the iPSCs as colonies was successful in terms of both differentiation efficiency and confluency at day 5 (treatment A compared to treatment F). Treatment E was decided as the best stage 1 sub-protocol moving forward, due to its high ratio of SOX17 to POU5F1 and cell survival and high confluency at day 5. Treatment E also avoids the use of FBS, which is ill-defined and can vary from lot-to- lot.

[0072] In differentiation 2, up to a 125-fold increase in CYP3A7 mRNA was observed, yet no metabolism of the CYP3A7 endogenous substrate DHEA-S. DHEA-S may not be entering the cells due to its polarity or the metabolite may not be exiting the cells. DHEA-S is transported via a few different transporters including OATPs and NCTP. It is unknown if thesePCT ApplicationAttorney Docket No. : CUBR-0007PC transporters are expressed in the disclosed model system. Sulfonated steroids may have difficulty entering the cells compared to the non-sulfonated forms, another possibility is that the substrate concentration was too low to observe any formation of the metabolite. In various CYP3 A assays, the fnHLCs are incubated at 1 mM testosterone in order to observe low levels of the 6P hydroxy metabolite.

[0073] Based on the results of differentiation 2, the goal of differentiation 3 was to maximize CYP3 A7 activity by adjusting the endpoint activity assay. Both DHEA and DHEA- S were included as a mixture in the endpoint incubation, to determine if transport of the probe substrate was an issue. Additionally, two replicates underwent cell lysis to determine the relative amounts of DHEA(S) inside the cells versus in the supernatant. The concentrations were also increased 10- and 25- fold from 5 pM to 50 pM and 250 pM to reach maximal rate of formation of the respective metabolites. DHEA proved to be a better substrate, with 16aOH peaks detected at 1, 2 and 4 hours, while no 16aOH DHEA-S peaks were seen at any timepoints. The increase in substrate concentration from 50 pM to 250 pM did not lead to an increase in the 16aOH DHEA formation. This could be due to reaching a plateau in substrate concentration, wherein the CYP3A7 present is already operating at its maximal velocity and additional substrate will have no effect. Another contributing factor could be variation between wells, with some wells displaying more fnHLC morphology than others, and therefore CYP3A7 protein expression could vary between wells. Supernatant samples vs. cell lysis samples gave an idea of the transport of DHEA(S) into the fnHLCs. The significant difference in peak intensity ratios of substrate found in supernatant vs lysate for DHEA and DHEA-S demonstrate that DHEA-S is not entering the cells to reach its target enzyme, but DHEA is.

[0074] The experiments disclosed herein led to a final differentiation protocol as noted in Figure 1A, including 100 nM dexamethasone in the final stage and an overall length of 26 days. This final protocol is used to generate fnHLCs from iPSCs from male and female donors. In one or more embodiments, the final protocol includes maintaining cells in a first medium (e.g., RPMI media) including 1 mM L-glutamine, 2% B27+ supplment, 100 ng / mL activin A, and 25 ng / mL Wingless-related integration site isoform 3a (Wnt3a) for four days. The Wnt3a is part of the first medium for the first two days of culturing only. For days 5-9, cells were maintained in a second medium that is a hepatocyte culture media (HCM, Lonza) with 20 ng / mL Bmp4 (bone morphologic protein 4) and 30 ng / mL Fgf2 (fibroblast growth factor 2) to achieve a hepatoblast-like state, with daily media changes. For final fnHLCs, cells werePCT ApplicationAttorney Docket No. : CUBR-0007PC maintained in a third medium that is a HCM with 20 ng / mL Hgf (hepatocyte growth factor) for four days, followed by a maturation period of 5-17 days in HCM with 10 ng / mL Osm (oncostatin M) and 10 or 100 nM glucocorticoid (GC), with media changes every other day. In one or more embodiments, the maturation period if 10-11 days. In one or more embodiments, the maturation time at the third stage included a length of 5, 9, 13, or 17 days (overall protocol lengths of 18, 22, 26, or 30 days)

[0075] The fnHLCs were characterized for fetal and liver markers via qPCR. Fold change in mRNA expression was calculated compared to undifferentiated iPSCs via the AACq method with ACTB as a reference (or via the ACq method for relative comparison to ACTB).

[0076] CYP3A7 was the most predominant CYP expressed in the fnHLCs, as in the fetal / neonatal liver. Furthermore, CYP3A4, CYP2B6, CYP1A2, and FMO3 followed previously described enzyme ontogeny patterns, where expression is low to negligible in the fetal period, minor increases occurring over the first week of life, and finally increasing to near adult levels after 1-2 years of age. Flavin-containing monooxygenases (FMOs) catalyze many of the same reactions as CYPs and an ontogenetic switch also occurs between these isoforms.

[0077] FMO3, the main hepatic form found in adults, was not detected in fnHLCs, but was increased in expression from neonatal PHHs to adult PHHs. The fnHLCs did not express measurable levels of PXR mRNA, and therefore were more reliant on GR (glucocorticoid receptor) and CAR (constitutive androstane receptor) for induction pathways. Fetal hepatocyte / hepatoblast markers AFP (alpha-fetoprotein), DLK1 and KRT19 were all highly expressed in the fnHLCs. AFP is the major hepatic protein secreted during the fetal period, replaced by albumin (ALB) in the adult, and is commonly used to determine hepatocyte or HLC maturity. DLK1 and KRT19 are both involved in hepatocyte commitment from bipotential hepatoblast precursors. GSTP1, expressed at high levels in the fnHLCs, is not only involved in detoxification reactions via glutathione conjugation, but has also been reported to regulate hepatocyte proliferation during liver regeneration40. Transporter MRP4 expression was comparable between fnHLCs and neonatal PHHs, and lower in adult PHHs, in agreement with studies citing higher MRP4 mRNA expression in fetal samples compared to adults. Altogether, the gene expression profile recapitulates well what we would expect from a liver in a developmental stage shortly before or after birth indicating the successful differentiation of the fnHLCs.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0078] In one or more embodiments, the CYP3A7 enzymatic activity was assessed via incubations with 50-250 pM DHEA(S) at 37 °C for up to 4 hours, and formation of the 16aOH metabolites was measured via LC-MS / MS. Inclusion of the supplement B27+ increased cell survival through DE differentiation, while also achieving high mRNA expression of the DE marker SOX17. fnHLCs displayed high levels of hepatic and fetal genes. In the most recent differentiation, liver markers ALB and HNF4A displayed mRNA fold changes over 1800- and 410- compared to undifferentiated iPSCs, and fetal AFP was increased over six orders of magnitude. There were important differences in expression of the CYP3A isoforms: fetal CYP3 A7 mRNA was increased 4320-fold over iPSCs, while adult CYP3 A4 mRNA was only increased about 65-fold. CYP3A7 activity was highest with the substrate DHEA, showing increasing formation of the 16aOH metabolite with time.

[0079] The one or more characteristics of the fetal and / or neonatal HLC that may be determined based on the analysis may include an enzyme expression profile, a transporter expression profile, an enzyme activity, a morphological phenotype, or combinations thereof. Other characteristics that may be determined are contemplated.

[0080] The one or more proteins interacting with the candidate chemical may include any suitable protein such as, for example, a cytochrome P450 protein, such as CYP3 A7, CYP3 A4, CYP2C9, CYP2D6, CYP7A1, CYP1A2, CYP2B6, or combinations thereof.Example: Chemical Screening Model and Process for Screening a Target for Metabolism and / or Toxicity.

[0081] In another embodiment is provided a chemical screening model. The chemical screening model may be adapted to reproduce metabolic properties of a neonatal or fetal liver. The chemical screening model may include a population of fnHLCs described herein produced by any of the disclosed protocols. For example, the final differentiation method as described herein is used to produce a population of fnHLCs. The fnHLCs metabolize both endobiotic substrates (DHEA) and drugs (midazolam, MDZ) in a fashion more similar to neonatal PHHs when compared to adult PHHs. Further, the induction of xenobiotic metabolizing genes in fnHLCs was tested by representative drugs and aflatoxin bioactivation in order to demonstrate the fnHLCs utility as a model system to study developmental hepatic response and replace scarce fnHLCs. However, it should be understood that the fnHLCs xenobiotic metabolizing genes may be induced by other representative drugs. For example, the finHLC system (e.g., the fnHLC model) is operable to screen for metabolism and / or toxicity of a target. The targetPCT ApplicationAttorney Docket No. : CUBR-0007PC includes a pharmaceutical (e.g., FDA-approved drugs or drug candidates for research purposes), a chemical, a toxin, a toxicant, a hepatic specific toxicant, an endogenous steroid, an antibiotic, a polyfluoroalkyl substance, or combinations thereof, or any other target that may have an effect on a hepatic cell (e.g., the finHLCs).

[0082] The chemical screening model may be utilized to test a metabolic property, an activity, a toxicity, or other attribute of a chemical. Such chemicals may include a pharmaceutical, a toxicant, a polyfluoroalkyl substance, or combinations thereof, though other chemicals are contemplated.

[0083] In another embodiment a process for screening a candidate chemical for metabolism and / or toxicity is provided. The process for screening the candidate chemical may include co-culturing and incubating a candidate chemical with fnHLC produced by processes described herein. The process for screening the candidate chemical may further include retrieving a supernatant or a cell lysate of the fnHLC after the co-culturing and incubating. The process for screening a candidate chemical for metabolism and / or toxicity enables the ability to screen for authentic drug metabolites in the fnHLC system, for, e.g., pre-clinical drug development.Materials and Methods

[0084] Generally, the fnHLCs were dosed with a target (e.g., a pharmaceutical (e.g., FDA- approved drugs or drug candidates for research purposes), a chemical, a toxin, a toxicant, a hepatic specific toxicant, an endogenous steroid, an antibiotic, a polyfluoroalkyl substance, or combinations thereof) and cultured in media. For example, the fnHLC model was incubated with DHEA, MDZ, BMZ, RIF, or Aflatoxin Bi, as described below to test the fnHLCs as a screening model. The fnHLCs may be used as a screening model to test any targets that affect hepatic cells.

[0085] DHEA and MDZ Incubations. fnHLCs and PHHs were incubated with 50 pM DHEA or 30 pM MDZ for about 0-4 hours at 37 °C and 5% CO2 to determine CYP3 A activity. A sample from the culture was collected. In one or more embodiments, the samples are supernatants (about 100-150 pL), which were collected and stopped with an equal volume of cold methanol with internal standard (DHEA-d5 for DHEA incubations and aOH-MDZ-d4 for MDZ incubations). Precipitated proteins were collected by centrifugation for 20 min at 2000 xg and 4 °C and supernatants were analyzed by LC-MS / MS for formation of the 16aOH DHEA and L0H / 40H MDZ metabolites.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0086] Xenobiotic Induction of CYP3A7. fnHLCs were differentiated and PHHs were cultured as described. BMZ, a glucocorticoid, and RIF, an antibiotic, were dissolved in DMSO and added to HCM at 0.1%. Phenobarbital (PB) was obtained as a certified reference solution at 1 mg / mL in MeOH, and added to HCM at 2%. Cells were dosed with 50 and 500 nM BMZ, 5 and 50 pM RIF, 8.6 and 86 pM PB, with DMSO and MeOH solvent controls. Media changes were performed daily for 72hr, prepped fresh each day. A sample from the culture was collected. In one or more embodiments, after 72hr, cells were rinsed with PBS and underwent MDZ incubations, followed by RNA extraction, cDNA synthesis and qPCR.

[0087] Aflatoxin Bi (AFBi), a mycotoxin, in fnHLCs and PHHs. HLCs were differentiated and PHHs were cultured as described. AFBi was dissolved in DMSO and added to HCM at a final volume of 0.2%. Cells were dosed with 0.1, 1, 10 and 100 pM AFBi and 100 pM AFBi + 1 mM ABT for 48hr. A sample from the culture was collected. In one or more embodiments, the samples are supernatants, which were stopped with cold acetonitrile (CAN) with aflatoxin G2 (AFG2) internal standard at 0, 4, 24, and 48hr for LC-MS / MS analysis of metabolite production, and brightfield microscopy images were obtained at 48hr post-dose.Results

[0088] fnHLCs and PHHs were compared in terms of their metabolic capabilities towards endogenous and exogenous substrates. First, the endogenous hormone DHEA was incubated with fnHLCs and PHHs and formation of the 16aOH metabolite was determined, as shown in Figure 15A and 15B. In general, fnHLCs produced about 3 to 6 times more 16aOH DHEA per min (1.17 ± 0.17 and 1.88 ± 0.58 pmol / min / well for male and female fnHLCs respectively) than adult PHHs (0.41 ± 0.07 pmol / min / well). Neonatal PHHs had the highest production of 16aOH DHEA at 10.96 ± 0.51 pmol / min / well. 16aOH DHEA is mainly formed by CYP3A7, but CYP3A4 can have minor contributions. fnHLCs and PHHs were also incubated with the drug substrate MDZ and the ratio of metabolites produced (1’OH MDZ / 4OH MDZ) can help determine the relative contributions from each CYP3A isoform, as shown in Figure 15C. Male and female fnHLCs had LOH MDZ / 40H MDZ ratios of 1.07 ± 0.16 and 1.00 ± 0.14 respectively, similar to that of the neonatal PHHs at 1.82 ± 0.03, as shown in Figure 15D. Adult PHHs had a significantly higher ratio of 5.97 ± 0.04. In terms of overall metabolism, fnHLCs were less active compared to both sets of PHHs yet recapitulate well the metabolite ratios of neonatal PHHs, as shown in Figure 14E and Figure 14F.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0089] Induction of xenobiotic metabolizing enzymes, mainly in the form of CYP expression increases, can have a significant impact on xenobiotic metabolism and can lead to undesired toxicities and / or DDL Representative drugs were screened for their potential to induce CYP3A mRNA expression and activity in fnHLCs and PHHs: rifampicin (RIF) and BMZ. RIF is a CYP3 A4 inducer through the nuclear hormone receptor PXR. In both fnHLCs and neonatal PHHs, there were no statistically significant changes in CYP3A7 expression at either 5 or 50 pM RIF compared to the respective solvent controls, as shown in Figure 15A and Figure 16B. The same was true for CYP3A4 expression in fnHLCs. Adult and neonatal PHHs were very responsive to RIF, with CYP3A4 mRNA fold changes between 30 and 48 compared to solvent controls, as shown in Figure 15B. Similar results are reflected in the CYP3A activity changes with RIF. fnHLCs had no significant changes in production of either 1’OH MDZ or 4OH MDZ, correlating well with the lack of change in expression of either CYP3A isoform, as shown in Figure 15C. Neonatal PHHs, with some CYP3A4 present, had some changes in CYP3A activity, as shown in Figure 16D. Fold changes in 4OH MDZ formation were 3.25 ± 0.19 and 3.88 ± 0.33 at 5 and 50 pM RIF respectively, and 6.81 ± 0.52 and 8.53 ± 0.73 at 5 and 50 pM RIF for 1’OH MDZ formation. Adult PHHs, with the highest CYP3A4 and PXR expression, had the greatest CYP3A enzymatic activity increases. Fold changes in 4OH MDZ formation were 22.10 ± 0.34 and 19.72 ± 0.89 at 5 and 50 pM RIF respectively, and 10.19 ± 0.07 and 8.59 ± 0.49 at 5 and 50 pM RIF for 1’OH MDZ formation. These various changes in activity are reflected in the 1’OH MDZ / 40H MDZ ratio with no changes for fnHLCs as CYP3A7 was not significantly impacted, as shown in Figure 16E. However, CYP3A4 was very responsive to RIF induction, and the 1’OH MDZ / 40H MDZ ratio for neonatal PHHs changed from 0.93 ± 0.01 at 0 pM to 1.96 ± 0.05 and 2.05 ± 0.06 at 5 and 50 pM RIF respectively. In adult PHHs, the ratio decreased from 5.32 ± 0.04 at 0 pM to 2.49 ± 0.05 and 2.35 ± 0.05 at 5 and 50 pM RIF respectively.

[0090] BMZ was also tested in all cell types, as glucocorticoids have CYP3A induction potential, especially through the GR. The male fnHLCs were semi-responsive to induction by BMZ, with CYP3A7 mRNA fold changes up to 4.5-fold at 500 nM BMZ, although not statistically significant from the control, as shown in Figure 15F. Female fnHLCs noted no significant difference in CYP3A7 or CYP3A4 mRNA compared to solvent controls. In neonatal PHHs, there was an observed increase in CYP3A4 mRNA, but not CYP3A7, as shown in Figure 15G. Adult PHHs displayed increases in both CYP3 A7 and CYP3 A4 mRNAPCT ApplicationAttorney Docket No. : CUBR-0007PC(fold changes of 7.52 ± 5.11 and 11.93 ± 8.28 respectively), noted at 50 nM BMZ and not 500 nM. Moderate, statistically significant changes in both 4OH and 1’OH MDZ formation were observed in both fnHLCs compared to solvent controls, as shown in Figure 16H. At 500 nM BMZ 4OH MDZ fold changes were 2.36 ± 0.43 in male fnHLCs and 2.30 ± 0.60 in female fnHLCs, and 1’OH MDZ fold changes were 2.11 ± 0.44 and 2.10 ± 0.54 respectively. This closely mirrors the results from the neonatal PHHs with a 4OH MDZ fold change of 1.71 ± 0.14 and a 1’OH MDZ fold change of 2.30 ± 0.22 at 500 nM BMZ, as shown in Figure 161. At 500 nM BMZ, adult PHHs favored the 4OH MDZ metabolite slightly at 1.86 ± 0.19 fold change compared to the 1’OH MDZ at 1.55 ± 0.12. The LOH / 4OH MDZ ratio did not change significantly for any cell type with BMZ treatment, however the ratios remain very distinct for adult PHHs (ranging from 4.48 to 5.3) compared to male and female fnHLCs (0.86-0.89; 0.82- 0.86) and neonatal PHHs (0.93-1.23), as shown in Figure 15J.

[0091] The bioactivation of the fungal -produced aflatoxin Bl (AFBi) has been well characterized and in this study fnHLCs were dosed with AFBi to determine their utility as a toxicological model compared to PHHs. AFBi can be metabolized through various pathways to toxic and non-toxic metabolites, with a simplified scheme presented in Figure 16A. AFBi is metabolized through the CYP3A isoforms into both the non-reactive aflatoxin Qi (AFQi) detoxification species and the toxic species AFBi-endo / exo-epoxide (AFBO) that can adduct proteins and DNA, leading to cell death. Monitoring for the AFQi metabolite can act as a proxy for enzymatic bioactivation, since AFBi-protein and AFBi-DNA adducts are not commercially available for usage as LC-MS standards. PHHs and fnHLCs dosed with 1-100 pM AFBi for up to 48hr had varying cytotoxic responses, as captured by brightfield microscopy images of the monolayers over time, as shown in Figure 16B. In general, adult PHHs were the most sensitive to AFBi bioactivation, showing signs of cell death and monolayer disruption at 1 pM, and 100% death at 10 pM and 100 pM. Neonatal PHHs behaved similar to adult PHHs, with more cells in the monolayer at 1 pM, however still experienced full cytotoxicity at 10 pM and 100 pM. fnHLCs in general were more robust, with concentrations up to 100 pM needed to visualize death and detachment of cells. As shown in Figure 16C, representative UV chromatograms show peaks of metabolite AFQi, substrate AFBi and internal standard (IS) AFG2. At 24hr and 100 pM AFBi, metabolite AFQI was measurable in all cell types, as shown in Figure 17D. AFQi peak area ratios for male andPCT ApplicationAttorney Docket No. : CUBR-0007PC female fnHLCs were 0.299 ± 0.126 and 2.243 ± 0.626 respectively. For neonatal and adult PHHs, AFQi peak area ratios were 30.42 ± 4.21 andl0.62 ± 1.22 respectively. All cells were also dosed with the pan-CYP inhibitor 1 -aminobenzotriazole (ABT) to block CYP-mediated metabolism. In all cell types, 1 mM ABT inhibited CYP activity and therefore halted AFQi production, with minimal peak area ratios of 0-0.629 in all cells, as shown in Figure 16D. AFQI formation over time is shown in Figure 16E for fnHLCs and Figure 16F for PHHs dosed with 100 pM AFBi. The CYP bioactivation pathway was active in both fnHLCs and PHHs. As seen previously, female fnHLCs were more active than male fnHLCs, with both fnHLCs still producing AFQi up to 48hr. Both neonatal and adult PHHs were more active in metabolizing AFBi, and interestingly the neonatal PHHs produced the highest amounts of AFQi. Both PHHs peaked in AFQi formation at 4hr, with cell death following afterwards.Non-Limiting Conclusions

[0092] Because xenobiotic metabolism is such an important determinant of xenobiotic toxicity to the developing fetus and neonate, we sought to characterize the ability of the fnHLCs to recapitulate the metabolic profiles observed with human neonatal hepatocytes. Four characteristic parameters were examined in this regard: metabolism of a representative endogenous hormone (DHEA), metabolism of a commonly used drug (MDZ), CYP enzyme induction, and metabolism of a well-known hepatotoxicant (aflatoxin). Fetal CYP3A7 activity is essential to steroid homeostasis, wherein >90% of the estriol produced during pregnancy comes from the fetal precursor 16aOH DHEA(S)41. Because the majority of estriol production depends on active CYP3 A7 in the fetal liver, it is often used as a marker for normal fetal health and development. The fnHLC model was capable of oxidizing DHEA into the CYP3A7 major metabolite 16aOH DHEA, as quantified via LC-MS / MS. Due to their high sequence similarity and overlapping substrate specificity, isolating metabolic contributions from CYP3A7 and CYP3A4 can prove challenging. The benzodiazepine MDZ is a CYP3A substrate that is hydroxylated at two different positions and is commonly utilized to determine CYP3 A activity. At concentrations below 30 pM, CYP3A4 produces about 5-8 times more LOH MDZ than 4OH MDZ, while CYP3A7 produces approximately equal amounts of both metabolites. Therefore the LOH MDZ / 40H MDZ ratio can provide insight into individual CYP3A contributions. Additionally, MDZ is a clinically relevant substrate as it is often used in the NICU for term and pre-term neonates, despite the lack of safety data in pediatric patients. fnHLCs were capable of producing both the LOH MDZ and 4OH MDZ metabolites, at a ratioPCT ApplicationAttorney Docket No. : CUBR-0007PC very similar to that of the neonatal PHHs (about 1-1.8). Adult PHHs produced much more 1’OH MDZ, and, therefore, the ratio was closer to 6. finHLCs recapitulated the metabolic profile of neonatal PHHs of both endogenous and exogenous substrates, as determined by specific LC-MS / MS assays. The induction of xenobiotic metabolizing enzymes, especially CYPs, is a considerable cause of adverse drug reactions and hepatotoxicity due to drug-drug interaction (DDI) and toxicant bioactivation. The induction potential of candidate drugs on CYP3A4, CYP2B6, and CYP1A2 expression is typically evaluated during the drug development process as these CYPs represent examples of enzymes regulated by different nuclear receptor sub-types in the adult. CYP3A4 expression is controlled by many factors, however PXR and CAR nuclear hormone receptors are the main driving forces behind gene induction. In contrast, the mechanisms controlling the regulation of CYP3 A7 in the fetus and developing neonate and its ontogenetic switch to CYP3 A4 are not fully understood, although the GR is highly correlated with CYP3A7 expression. Rifampin (RIF), a broad-spectrum antibiotic often used in neonatal and pediatric populations, is known for its CYP3 A induction through PXR (pregnane x receptor) activation and it is a classic positive control for CYP3 A4 induction in PHH experiments. In HepG2 cells, 12 pM RIF treatment led to >2-fold increase in CYP3A7 mRNA after 5 days. However, HepG2 cells generally have low CYP expression and are a poor model for drug metabolism or induction studies due to the fact that they are an immortalized cell line. In fetal hepatocytes, 40 pM RIF led to no change in CYP3 A7 mRNA after three days. The fnHLCs produced by the final differentiation protocol mimicked the results observed in the fetal hepatocytes, wherein about 5 pM to about 50 pM RIF did not lead to a significant change in CYP3 A7 gene expression or functional activity, indicating PXR does not have a major role in CYP3 A7 induction in the embodiments described herein. For decades, the glucocorticoids BMZ and DEX have been administered to pregnant women at risk of preterm labor to accelerate fetal lung maturation and reduce the risk of respiratory distress syndrome in the neonate. It was reported that BMZ at 10 nM increased CYP3A7 mRNA 4.3- fold in fetal hepatocytes. Due to the use of DEX in the final differentiation protocol, CYP3A7 was tested for induction potential with BMZ. All cell types showed up to 2-fold increases CYP3 A activity, again with distinctive MDZ metabolite ratios indicative of CYP3 A7 activity. In general, CYP3A7 was more resistant to induction compared to the more easily-inducible CYP3A4. The differences in CYP3A isoform induction between various cell types offers insight into the complicated and poorly understood transition from CYP3A7 to CYP3A4PCT ApplicationAttorney Docket No. : CUBR-0007PC triggered by parturition The candidate chemical may include a pharmaceutical, a toxicant, a polyfluoroalkyl substance, or combinations thereof.

[0093] Some xenobiotics that the liver encounters are non-toxic until they are enzymatically bioactivated into a reactive electrophilic species. An example of hepatic bioactivation is the metabolism of the fungal secondary metabolite AFBi. AFBi often contaminates crops such as grains and nuts, and is itself an inert compound. Metabolism to the reactive 8-9-epoxide (AFBO), specifically by the CYP3A isoforms, is implicated in AFBi’s carcinogenic, mutagenic, and teratogenic effects as it adducts cellular proteins and DNA. In the disclosed model system, finHLCs were more resistant to AFBi bioactivation compared to adult PHHs. Both CYP3A4 and CYP3A7 have been implicated in AFBI conversion to the detoxification product AFQi and the reactive AFBO, although display varying affinities and reaction velocities. Reported Kmvalues for CYP3 A isoforms and AFQi and AFBO formation are in the high micromolar range and vary approximately two-fold across isoforms and metabolic products (3A4 / AFQ1 139-324 pM; 3A7 / AFQ1 204 pM; 3A4 / AFBO 130-133 pM; 3A7 / AFBO 121 pM)60,61. Important differences are noted in the reported Vmax values, where CYP3A7 is less catalytically active than CYP3A4, a trend noted for many substrates. Vmax for AFBO formation is about 6 times greater with CYP3A4 than CYP3A7, and Vmax for AFQI formation is about 2 times greater with CYP3A4 than CYP3A7. In the disclosed model system, the lack of CYP3A4 protected the cells from damage, as CYP3A7 was the main isoform present and less active in producing the reactive epoxide. AFQI formation, measured in all cell types as a proxy for CYP3 A activity, was similar in male and female fnHLCs, and about 10 to about 100 times more in neonatal and adult PHHs. CYP1 A2 has also been implicated in AFBi metabolism, producing AFBO and another hydroxylated detoxification product AFMi, although pathways are species-dependent. In the fnHLCs and neonatal PHHs, CYP1A2 mRNA was low to non-detectable indicating CYP3 A isoforms were the main CYPs involved in bioactivation in a fetal / neonatal setting.

[0094] Overall, the fnHLCs formed by the process discloses herein will yield a sustainable fetal / neonatal hepatocyte model which can be utilized in areas of drug metabolism, enzyme ontogeny, liver development, hepatotoxicity, and more. For example, the fnHLCs produced by the disclosed method can be used as a chemical screening model or in a process for screening a target for metabolism and / or toxicity. fnHLCs represent a powerful model systemPCT ApplicationAttorney Docket No. : CUBR-0007PC to understand fetal / neonatal drug and toxicant metabolism during a critical developmental time frame.Embodiments Listing

[0095] The present disclosure provides, among others, the following aspects, each of which may be considered as optionally including any alternate embodiments:

[0096] Clause 1. A method for producing fetal and / or neonatal hepatocyte-like cells (finHLCs), the method comprising: producing a definitive endoderm cell population by a stage 1 process comprising: culturing a population of induced pluripotent stem cells (iPSCs) in a first medium comprising activin A (ActA) and Wingless-related integration site isoform 3a (Wnt3a); producing a hepatoblast-like progenitor cell population from the definitive endoderm cell population by a stage 2 process comprising: culturing the definitive endoderm cell population in a second medium comprising a bone morphogenetic protein 4 (Bmp4) and a fibroblast growth factor 2 (Fgf2) to produce the hepatoblast-like progenitor cell population; and producing a fetal and / or neonatal hepatocyte-like cell population from the hepatoblast-like progenitor cell population by a stage 3 process comprising: culturing the hepatoblast-like progenitor cell population in a third medium comprising a hepatocyte growth factor (Hgf); and culturing the hepatoblast-like progenitor cell population in a fourth medium comprising oncostatin M (Osm) and a glucocorticoid (GC) to produce the fetal and / or neonatal hepatocytelike cell population.

[0097] Clause 2. The method of Clause 1, wherein the first medium further comprises L- glutamine and B27+ supplement.

[0098] Clause 3. The method of Clause 2, wherein the first medium includes about ImM L-glutamine, 2% B27+ supplement, 90 ng / mL to about 100 ng / mL activin A, and about 22.5 ng / mL to about 25 ng / mL Wnt3a.

[0099] Clause 4. The method of Clauses 1-3, wherein the stage 1 process comprises about4 days of culturing.

[0100] Clause 5. The method of Clause 4, wherein the first medium comprises Wnt3a for2 days of culturing.

[0101] Clause 6. The method of Clauses 1-5, wherein the second medium includes about18 ng / mL to about 20 ng / mL Bmp4 and about 27 ng / mL to about 30 ng / mL of Fgf2.

[0102] Clause 7. The method of Clauses 1-6, wherein the stage 2 process comprises about 5 to about 9 days of culturing.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0103] Clause 8. The method of Clauses 1-7, wherein the third medium comprises about 18 ng / mL to about 20 ng / mL Hgf.

[0104] Clause 9. The method of Clauses 1-8, wherein the stage 3 process in the third medium comprises about 4 days of culturing.

[0105] Clause 10. The method of Clauses 1-9, wherein the fourth medium comprises about 9 ng / mL to about 10 ng / mL Osm and about 10 nM to about 100 nM GC.

[0106] Clause 11. The method of Clauses 1-10, wherein the stage 3 process comprises a maturation period of about 5 days to 17 days in the fourth medium.

[0107] Clause 12. The method of Clauses 1-11, wherein the glucocorticoid (GC) comprises prednisolone, betamethasone, dexamethasone (DEX), or combinations thereof.

[0108] Clause 13. The method of any one of Clauses 1-12, wherein the fetal and / or neonatal hepatocyte-like cells produces more CYP3 A7 protein than CYP3 A4 protein.

[0109] Clause 14. A chemical screening model adapted to reproduce metabolic properties of a neonatal and / or fetal liver, the chemical screening model comprising: a fetal and / or neonatal hepatocyte-like cell operable to reproduce a plurality of metabolic properties of a neonatal or fetal liver.

[0110] Clause 15. A method for screening a target, the method comprising: co-culturing and incubating the target with a fetal and / or neonatal hepatocyte-like cell (fnHLC); retrieving a sample of a fnHLC after the co-culturing and incubating; and determining one or more characteristics of the fnHLC based on analysis of the sample of the fnHLC.[OHl] Clause 16. The method of Clause 15, wherein the one or more characteristics of the fnHLC comprises enzyme expression profile, transporter expression profile, enzyme activity, morphological phenotype, mRNA expression, or combinations thereof.

[0112] Clause 17. The method of Clause 15 or Clause 16, wherein the determining the one or more characteristics of the fnHLC comprises measuring an interaction between one or more proteins present in the fnHLC and the target.

[0113] Clause 18. The method of Clauses 15-17, wherein the one or more proteins comprises CYP3A7, CYP3A4, CYP2C9, CYP2D6, CYP7A1, CYP1A2, CYP2B6, or combinations thereof.

[0114] Clause 19. The method of Clauses 15-18, wherein, the target is a pharmaceutical, a chemical, a toxin, a toxicant, a hepatic specific toxicant, an endogenous steroid, an antibiotic, a polyfluoroalkyl substance, or combinations thereof.PCT ApplicationAttorney Docket No. : CUBR-0007PC

[0115] Clause 20. The method of any one of Clauses 15-19, wherein the target comprises a mycotoxin, a glucocorticoid, an antibiotic, or combinations thereof.

[0116] All documents described herein are incorporated by reference herein, including any priority documents and / or testing procedures to the extent they are not inconsistent with this text. As is apparent from the foregoing general description and the specific embodiments, while forms of the embodiments have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element, a group of elements, or a method is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition, method, or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, elements, or method, and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0117] In the foregoing, reference is made to embodiments of the disclosure. However, it should be understood that the disclosure is not limited to specific described embodiments. Instead, any combination of the following features and elements, whether related to different embodiments or not, is contemplated to implement and practice the disclosure. Furthermore, although embodiments of the disclosure may achieve advantages over other possible solutions and / or over the prior art, whether or not a particular advantage is achieved by a given embodiment is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s).

[0118] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, onlyPCT ApplicationAttorney Docket No. : CUBR-0007PC certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0119] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, embodiments comprising “a cell” includes embodiments comprising one, two, or more cells, unless specified to the contrary or the context clearly indicates only one cell is included.

[0120] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

PCT ApplicationAttorney Docket No. : CUBR-0007PCCLAIMSWhat is claimed is:

1. A method for producing fetal and / or neonatal hepatocyte-like cells (fnHLCs), the method comprising: producing a definitive endoderm cell population by a stage 1 process comprising: culturing a population of induced pluripotent stem cells (iPSCs) in a first medium comprising activin A (ActA) and Wingless-related integration site isoform 3a (Wnt3a); producing a hepatoblast-like progenitor cell population from the definitive endoderm cell population by a stage 2 process comprising: culturing the definitive endoderm cell population in a second medium comprising a bone morphogenetic protein 4 (Bmp4) and a fibroblast growth factor 2 (Fgf2) to produce the hepatoblast-like progenitor cell population; and producing a fetal and / or neonatal hepatocyte-like cell population from the hepatoblast- like progenitor cell population by a stage 3 process comprising: culturing the hepatoblast-like progenitor cell population in a third medium comprising a hepatocyte growth factor (Hgf); and culturing the hepatoblast-like progenitor cell population in a fourth medium comprising oncostatin M (Osm) and a glucocorticoid (GC) to produce the fetal and / or neonatal hepatocyte-like cell population.

2. The method of claim 1, wherein the first medium further comprises L-glutamine and B27+ supplement.

3. The method of claim 2, wherein the first medium includes about ImM L-glutamine, 2% B27+ supplement, about 90 ng / mL to about 100 ng / mL activin A, and about 22.5 ng / mL to about 25 ng / mL Wnt3a.

4. The method of claim 1, wherein the stage 1 process comprises about 4 days of culturing.PCT ApplicationAttorney Docket No. : CUBR-0007PC5. The method of claim 4, wherein the first medium comprises Wnt3a for 2 days of culturing.

6. The method of claim 1, wherein the second medium includes about 18 ng / mL to about 20 ng / mL Bmp4 and about 27 ng / mL to about 30 ng / mL of Fgf2.

7. The method of claim 1, wherein the stage 2 process comprises about 5 to about 9 days of culturing.

8. The method of claim 1, wherein the third medium comprises about 18 ng / mL to about 20 ng / mL Hgf.

9. The method of claim 1, wherein the stage 3 process in the third medium comprises about 4 days of culturing.

10. The method of claim 1, wherein the fourth medium comprises about 9 ng / mL to about 10 ng / mL Osm and about 10 nM to about 100 nM GC.

11. The method of claim 1, wherein the stage 3 process comprises a maturation period of about 5 days to 17 days in the fourth medium.

12. The method of claim 1, wherein the glucocorticoid (GC) comprises prednisolone, betamethasone, dexamethasone (DEX), or combinations thereof.

13. The method of any one of claim 1, wherein the fetal and / or neonatal hepatocyte-like cells produces more CYP3 A7 protein than CYP3 A4 protein.

14. A chemical screening model adapted to reproduce metabolic properties of a neonatal and / or fetal liver, the chemical screening model comprising: a fetal and / or neonatal hepatocyte-like cell operable to reproduce a plurality of metabolic properties of a neonatal or fetal liver.

15. A method for screening a target, the method comprising: co-culturing and incubating a target with a fetal and / or neonatal hepatocyte-like cell (fnHLC); retrieving a sample of a fnHLC after the co-culturing and incubating; and determining one or more characteristics of the fnHLC based on analysis of the sample of the fnHLC.

16. The method of claim 15, wherein the one or more characteristics of the fnHLC comprises enzyme expression profile, transporter expression profile, enzyme activity, morphological phenotype, mRNA expression, or combinations thereof.

17. The method of any one of claims 15, wherein the determining the one or more characteristics of the fnHLC comprises measuring an interaction between one or more proteins present in the fnHLC and the target.

18. The method of claim 17, wherein the one or more proteins comprises CYP3A7, CYP3A4, CYP2C9, CYP2D6, CYP7A1, CYP1A2, CYP2B6, or combinations thereof.

19. The method of any one of claims 15, wherein, the target is a pharmaceutical, a chemical, a toxin, a toxicant, a hepatic specific toxicant, an endogenous steroid, an antibiotic, a polyfluoroalkyl substance, or combinations thereof.

20. The method of any one of claims 15, wherein the target comprises a mycotoxin, a glucocorticoid, an antibiotic, or combinations thereof.

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