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WO2025212920A1 - Organoïdes hépatiques à zones multiples - Google Patents

Organoïdes hépatiques à zones multiples

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Publication number
WO2025212920A1
WO2025212920A1 PCT/US2025/023001 US2025023001W WO2025212920A1 WO 2025212920 A1 WO2025212920 A1 WO 2025212920A1 US 2025023001 W US2025023001 W US 2025023001W WO 2025212920 A1 WO2025212920 A1 WO 2025212920A1
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Prior art keywords
zonal
liver
hlo
cells
hepatocyte
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Takanori TAKEBE
Hasan AL REZA
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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Publication of WO2025212920A1 publication Critical patent/WO2025212920A1/fr
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Definitions

  • liver organoids prepared from human pluripotent stem cells, methods of preparation, and compositions including the same, as well as uses thereof.
  • BACKGROUND [0004] The liver is a multi-faceted organ with a wide range of functions, such as glycolysis and gluconeogenesis, lipogenesis and fatty acid oxidation, protein synthesis, and xenobiotic catabolism. These divergent and complex functions are spatially segregated through compartmentalization into distinct regions, called zone 1, zone 2, and zone 3 hepatocytes, based on their proximity from the central vein to the portal vein.
  • the maintenance of nitrogen level in the liver is precisely balanced between the input of nutrients and the output as ammonia, which is metabolized by the urea cycle, nitric oxide cycle, and glutamine synthesis.
  • the urea cycle is primarily carried out in zone 1 and 2 hepatocytes, while glutamine synthesis takes place in zone 3 hepatocytes, and the nitric oxide cycle is primarily maintained in zone 2 and 3 hepatocytes.
  • Citrulline is necessary for these pathways and its levels are maintained by the nitric oxide cycle, which is augmented by glutathione.
  • metabolic diseases tend to manifest within the particular zone in which the derangement is most significant.
  • GLUL glutamine synthetase
  • CPS1 carbamoylphosphate synthetase
  • Embodiments of the disclosure include methods of producing multi-zonal liver organoids, the methods including: co-culturing one or more first human liver organoid (HLO) with one or more second HLO, wherein the second HLO comprises a bilirubin-treated HLO; contacting the co-cultured first HLO and the bilirubin-treated second HLO with bilirubin for a period of time to provide a liver organoid with at least one phenotypically distinct (e.g. structurally distinct, and/or functionally distinct, etc.) hepatocyte subpopulation.
  • the first HLO can be an ascorbate-treated HLO.
  • the first HLO can be a doxycycline-treated HLO.
  • the first HLO is a functional L-gulonolactone oxidase (GULO)-expressing HLO.
  • a zonal gradient can be created in the multi-zonal liver organoid.
  • the first HLO and/or the bilirubin-treated second HLO comprises an immature HLO.
  • the first HLO comprises an immature HLO.
  • the first HLO comprises an immature functional L-gulonolactone oxidase (GULO)-expressing HLO.
  • the bilirubin-treated second HLO comprises an immature HLO.
  • the first HLO comprises an immature HLO
  • the bilirubin-treated second HLO comprises an immature HLO.
  • the first HLO includes an ascorbate-enriched progenitor cell population
  • the bilirubin-treated second HLO includes a bilirubin-enriched progenitor cell population.
  • each phenotypically distinct hepatocyte subpopulation includes a zone 1 (Z1) or zone 1-like (Z1-like) hepatocyte subpopulation, a zone 2 (Z2) or zone 2- like (Z2-like) hepatocyte subpopulation, or a zone 3 (Z3) or zone 3-like (Z3-like) hepatocyte subpopulation.
  • the co-cultured first HLO e.g.
  • a functional GULO-expressing HLO optionally an immature HLO
  • the bilirubin-treated second HLO optionally another immature HLO
  • the co-cultured first HLO e.g. a functional GULO-expressing HLO
  • the bilirubin-treated second HLO are contacted with bilirubin for a period of time sufficient for the co-cultured HLOs to self-assemble.
  • the co-cultured first HLO e.g.
  • the bilirubin-treated second HLO are contacted with bilirubin for a period of time sufficient for the co-cultured HLOs to fuse into interconnected dual organoids.
  • the period of time sufficient to develop two or more phenotypically distinct hepatocyte subpopulations, to self-assemble into multizonal HLOs, and/or to fuse into interconnected dual organoids can be at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.
  • the first HLO e.g.
  • a functional GULO-expressing HLO includes a Z1 or Z1-like, Z2 or Z2-like, or Z3 or Z3-like hepatocyte subpopulation
  • the bilirubin-treated second HLO includes a Z1 or Z1-like, Z2 or Z2-like, or Z3 or Z3-like hepatocyte subpopulation.
  • the multi-zonal liver organoid includes two or more hepatocyte subpopulations.
  • the two or more hepatocyte subpopulations include a Z1 or Z1-like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte subpopulation; or a Z1 or Z1-like hepatocyte subpopulation and a Z2 or Z2-like hepatocyte subpopulation; or a Z2 or Z2-like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte subpopulation.
  • the first HLO e.g.
  • a functional GULO-expressing HLO includes a Z1 or Z1-like hepatocyte subpopulation
  • the bilirubin-treated second HLO includes a Z3 or Z3-like hepatocyte subpopulation.
  • the multi-zonal liver organoid includes three or more hepatocyte subpopulations.
  • the three or more hepatocyte subpopulations include a Z1 or Z1-like hepatocyte subpopulation, a Z2 or Z2-like hepatocyte subpopulation, and a Z3 or Z3-like hepatocyte subpopulation.
  • the liver organoid includes a tubular structure with a single lumen. In some embodiments, the liver organoid does not contain hematopoietic tissue and/or acquired immune cells. [0014] In some embodiments, during the co-culturing, the concentration of bilirubin can be maintained continuously. In some embodiments, during the co-culturing, the concentration of bilirubin can be refreshed through addition of exogenous bilirubin during every media change. In some embodiments, during the co-culturing, the concentration of bilirubin can be maintained continuously at a level less than or equal to about 5 mg/L.
  • the bilirubin concentration during the co-culturing step can be maintained continuously at about 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L, 2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, or 3 mg/L; preferably at about 1 mg/L.
  • the functional GULO-expressing HLO and bilirubin-treated HLO are co-cultured with bilirubin in a hepatocyte culture medium.
  • the hepatocyte culture medium includes HCM, hepatocyte growth factor, oncostatin M, and/or dexamethasone, or any combination thereof.
  • the first HLO e.g.
  • a functional GULO-expressing HLO and the bilirubin-treated second HLO are seeded for co-culturing at a density of greater than about 1 ⁇ 10 4 cells/well, greater than about 0.5 ⁇ 10 5 cells/well, greater than about 1 ⁇ 10 5 cells/well, greater than about 2 ⁇ 10 5 cells/well, greater than about 3 ⁇ 10 5 cells/well, greater than about 4 ⁇ 10 5 cells/well, greater than about 5 ⁇ 10 5 cells/well, or higher.
  • the first HLO e.g.
  • a functional GULO-expressing HLO and the bilirubin-treated second HLO are seeded for co- culturing at a density of greater than about 50-5000 organoids per well; preferably about 500-2000 organoids per well.
  • the first HLO e.g. a functional GULO-expressing HLO
  • the bilirubin-treated second HLO are seeded for co-culturing at a ratio of about 1:1; or 2:1, 3:1, 4:1, 5:1, or greater; or 1:2, 1:3, 1:4, 1:5, or greater; preferably at a ratio of about 1:1.
  • the functional GULO-expressing HLO can be produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of a heterologous expression system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells under conditions to induce expression from the heterologous expression system, to provide the functional GULO-expressing HLO.
  • the functional GULO-expressing HLO can be produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of an inducible system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with an induction agent, to provide the functional GULO-expressing HLO.
  • the functional GULO-expressing HLO can be produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of an tetracycline inducible system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with doxycycline, to provide the functional GULO-expressing HLO.
  • the functional GULO-expressing HLO produced from a genetically modified progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, is able to synthesize ascorbate.
  • the functional GULO protein is a Rodentia GULO, preferably a murine GULO (mGULO).
  • the culturing the posterior foregut cells under conditions to induce expression from the heterologous expression system occurs on or about day 17 of culture of the progenitor cell population.
  • the functional GULO-expressing HLO can be engineered with the gene that encodes for the functional GULO protein using CRISPR.
  • the gene or mRNA, or both, that encodes for the functional GULO protein can be introduced to the functional GULO- expressing HLO by transfection.
  • the bilirubin-treated HLO can be produced by: culturing a progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with bilirubin, to provide the bilirubin-treated HLO.
  • the multi-zonal liver organoid can have: a) expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; b) expression of one or more Z2-associated genes and/or expresses one or more Z2-associated proteins; c) expression of one or more Z3-associated genes and/or expresses one or more Z3- associated proteins; and/or d) expression of one or more pan-hepatocyte-associated genes and/or expresses one or more pan-hepatocyte-associated proteins.
  • the multi-zonal liver organoid can have expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; and expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins.
  • the multi-zonal liver organoid can have expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; expression of one or more Z2-associated genes and/or expresses one or more Z2- associated proteins; expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins; and expression of one or more pan-hepatocyte-associated genes and/or expresses one or more pan-hepatocyte-associated proteins.
  • the Z3- associated genes and/or Z3-associated proteins function in xenobiotic metabolism, WNT signaling, glycolysis, and/or lipogenesis.
  • the Z1-associated genes and/or Z1-associated proteins function in gluconeogenesis, lipid catabolism, glutamine catabolism, and/or reactive oxygen species (ROS) metabolism.
  • the Z2-associated genes and/or Z2-associated proteins function in DNA repair, amino acid metabolism, and/or cell growth.
  • the first HLO e.g.
  • a functional GULO-expressing HLO can have elevated expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; and/or wherein the bilirubin-treated second HLO can have elevated expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins.
  • the one or more Z1-associated genes can be selected from Fumarylacetoacetase (FAH), 4-Hydroxyphenylpyruvate dioxygenase (HPD), Stearoyl-CoA desaturase (SCD), Acyl- coenzyme A synthetase 2 (ACSS2), Argininosuccinate lyase (ASL), Carbamoyl phosphate synthetase I (CPS1), Ornithine transcarbamylase (OTC), Stem-loop binding protein (SLBP), Glutaminase (GLS), and Rho family GTPase 3 (RND3) genes; the one or more Z1-associated proteins are selected from CPS1 and ACSS2; the one or more Z2-associated genes can be selected from Glutathione synthetase (GSS), Telomerase reverse transcriptase (TERT), and Aldo-keto reductase family 1 member C1 (Fumarylace
  • the multi-zonal liver organoid can have hepatocyte proliferation, hepatocyte differentiation, urea cycle, ammonia removal, glycolysis, bilirubin removal, lipid catabolism, glutamine catabolism and biosynthesis, reactive oxygen species (ROS) catabolism and/or metabolism, oxygen response, xenobiotic metabolism, DNA repair, pigment metabolism, lipogenesis, gluconeogenesis, glutathione, and glutamine metabolic functionality.
  • the multi-zonal liver organoid can be enriched for Notch signaling and/or Wnt signaling.
  • the multi-zonal liver organoid includes hepatocytes and additionally includes one or more additional cell types selected from cholangiocytes, endothelial cells, macrophages, stellate cells, and mesenchyme cells. In some embodiments, the multi-zonal liver organoid includes hepatocytes and additionally includes cholangiocytes, endothelial cells, macrophages, stellate cells, and mesenchyme cells.
  • the multi-zonal liver organoid includes two or more cell types selected from pericentral or pericentral-like (Z3 or Z3- like) hepatocytes, periportal or periportal-like (Z1 or Z1-like) hepatocytes, and interzonal or interzonal-like (Z2 or Z2-like) hepatocytes.
  • the multi-zonal liver organoid includes: a) pericentral or pericentral-like (Z3 or Z3-like) hepatocytes, b) periportal or periportal- like (Z1 or Z1-like) hepatocytes, and c) interzonal or interzonal-like (Z2 or Z2-like) hepatocytes.
  • the multi-zonal liver organoid further includes hepatoblasts.
  • the hepatoblasts can be characterized as expressing fetal markers and/or growth mitogenic markers.
  • a functional GULO-expressing HLO) and/or the bilirubin-treated HLO has been made according to a method including: a) contacting definitive endoderm cells (DE) with an FGF signaling pathway activator and a Wnt signaling pathway activator for a first period of time; b) contacting the cells of step a) with the FGF signaling pathway activator, the Wnt signaling pathway activator, and a retinoic acid (RA) signaling pathway activator for a second period of time, thereby differentiating the DE to posterior foregut cells; and c) embedding the posterior foregut cells in a basement membrane matrix and culturing the posterior foregut spheroids for a third period of time to differentiate the posterior foregut cells to the first HLO (e.g.
  • the multi-zonal liver organoid does not contain hematopoietic tissue and/or acquired immune cells.
  • the multi-zonal liver organoid includes a hepatoblast population and at least two phenotypically distinct interzonal hepatocyte populations.
  • the multi-zonal liver organoid includes SERPINA1+ hepatocytes, KRT7+ cholangiocytes, PECAM1+ endothelial cells, LYZ+ macrophages, COL1A1+ stellate cells, and CD44+ mesenchyme.
  • the multi-zonal liver organoid includes a functional L-gulonolactone oxidase (GULO)-expressing cell population.
  • the multi-zonal liver organoid expresses one or more pan hepatocyte markers, one or more basal marker, and one or more apical marker; optionally wherein the one or more pan hepatocyte markers include ALB, HNF1A, A1AT, CEBPB, PROX1, HNF4A, and/or TUBA1A; and/or optionally wherein the one or more basal markers include CTNNB1; and/or optionally wherein the one or more apical markers include ZO-1.
  • the multi-zonal liver organoid expresses one or more periportal marker, one or more interzonal marker, and one or more pericentral zonal marker; optionally wherein said markers include TET1, GLS, GLS2, ALDH1A2, ALDH6A1, GHR, AR, CPS1, OTC, ACSS2, ARG1, GLUL, CYP2E1, and/or HIF1A.
  • the multi-zonal liver organoid has nitrogen, glucose and lipid metabolic activity; optionally wherein said activity includes urea cycle activity, glutathione S-transferase activity, and/or glutamine synthesis.
  • the three-dimensional artificial multi-zonal liver organoid does not contain hematopoietic tissue and/or acquired immune cells.
  • Further embodiments of the disclosure include ex vivo compositions including one or more three-dimensional multi-zonal liver organoids, including Z1-like (periportal-like) hepatocytes, and Z3-like (pericentral-like) hepatocytes.
  • the hepatocytes self-assemble into the three-dimensional artificial multi-zonal liver organoid.
  • the three-dimensional artificial multi-zonal liver organoid includes a tubular structure with a single lumen. In some embodiments, the three-dimensional artificial multi-zonal liver organoid does not contain hematopoietic tissue and/or acquired immune cells. [0030] In some embodiments, the multi-zonal liver organoids, artificial multi-zonal liver organoids, cell compositions, and/or ex vivo compositions further include Z2-like (interzonal-like, mid-lobular-like) hepatocytes. In some embodiments, the multi-zonal liver organoids, artificial multi-zonal liver organoids, cell compositions, and/or ex vivo compositions further include hepatoblasts.
  • the Z1-like hepatocytes can be engineered to express a heterologous functional GULO protein, and ascorbate is produced by the Z1-like hepatocytes.
  • the composition includes exogenously provided bilirubin at a concentration of about 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L
  • the multi-zonal liver organoids, artificial multi-zonal liver organoids, cell compositions, and/or ex vivo compositions include about 20-40% Z3-like (pericentral-like) cells, about 20-40% Z1-like (periportal-like) cells, about 20-40% hepatoblasts, and about 10-30% Z2-like (interzonal-like) cells.
  • the multi-zonal liver organoids, artificial multi-zonal liver organoids, cell compositions, and/or ex vivo compositions include greater than or equal to 10% Z2-like cells, and/or greater than or equal to 20% hepatoblasts.
  • the methods include transplanting including: a) ligating a bile duct in a subject; and b) transplanting the multi-zonal liver organoid at base of the liver.
  • the liver-related disease or disorder includes one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, Wilson’s disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, metabolic dysfunction–associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease,
  • MASLD metabolic dysfunction–associated liver disease
  • the liver dysfunction and/or failure includes hyperammonemia and/or hyperbilirubinemia.
  • the metabolic disease includes nonalcoholic fatty liver disease (NAFLD).
  • the nonalcoholic fatty liver disease (NAFLD) includes metabolic dysfunction-associated steatohepatitis (MASH).
  • the hepatitis includes hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis.
  • the subject has improvements one or more of the liver- related disease or disorders following transplantation.
  • Further embodiments of the disclosure include use of the aforementioned multi- zonal liver organoids, as an in vitro human model system for studying hepatocyte function and developmental divergence; studying liver-related disease; identifying therapeutic targets; and/or identifying therapeutic compounds and/or compositions effective in treating a liver-related disease or disorder, and uses of the aforementioned multi-zonal liver organoids for treating a liver-related disease or disorder.
  • Further embodiments of the disclosure include the aforementioned multi-zonal liver organoids, for use in the manufacture of a medicament for the treatment of a liver-related disease or disorder.
  • Fig.1D Cellular Antioxidant concentration in Dox treated Z1-HLOs compared to control HLOs.
  • Fig.1E ROS levels in Dox treated and extracellular ascorbate induced Z1-HLOs compared to control HLOs.
  • Fig.1F Heatmap of Zone 1 genes from RNAseq dataset for Dox treated Z1-HLOs compared to control.
  • Fig. 1G Heatmap of Zone 1 genes from RNAseq dataset for Dox treated Z1-HLOs compared to control.
  • Fig. 2C Brightfield images of Dox (100 ng/ml)-treated Z1-HLOs (left). RT-qPCR of ACSS2, ASL, CPS1 and OTC gene for Z1-HLOs compared
  • Fig. 2F Brightfield images (top) of progression of bilirubin induced fusion from Day 1 to Day 7.
  • Fig. 3C Brightfield image of Z3-HLOs treated with low dose bilirubin (1mg/L) compared to control, and luminal outline using ImageJ, arrows indicate luminal projections that are similar to bile canaliculi found in human liver. Scale bar indicates 200 ⁇ m.
  • Fig.3E Comparison of size and circularity of lumen of the control and 1 mg/L bilirubin treated Z3-HLOs.
  • Fig.3G Immunofluorescence images of Z3-HLOs for GLUL, NR3C1 and CDH1 compared to control HLOs and primary liver. Scale bar indicates 200 ⁇ m.
  • FIG.3H Heatmap of Z1- and Z3- HLOs depicting expression of zonal genes and lack of consensus expression of markers such as ARG1 and AKR1C1.
  • FIG. 3B and Fig. 3F data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper and lower whisker extends from the hinge to the largest and smallest value respectively no further than 1.5 ⁇ IQR from the hinge (where IQR is the inter-quartile range).
  • Figs. 3B and 3F use one-way ANOVA with multiple comparisons and Tukey’s correction.
  • Fig. 3B and 3F use one-way ANOVA with multiple comparisons and Tukey’s correction.
  • FIG. 4A Brightfield images of bilirubin induced fusion in high density HLOs compared to low density and no bilirubin treatment. Scale bar indicates 200 ⁇ m.
  • Fig. 4C Comparison of mean segment length in high density HLOs compared to low density and no bilirubin treatment.
  • Fig.4I Percentage of fused organoids for each type of organoid.
  • Fig.4E data are represented as boxplot where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper and lower whisker extends from the hinge to the largest and smallest value respectively no further than 1.5 ⁇ IQR from the hinge (where IQR is the inter-quartile range).
  • Figs.4B and 4D use Kruskal-Wallis with multiple comparisons and Dunn-Holland-Wolfe correction.
  • Fig. 4E uses unpaired two-tailed Student’s t-test.
  • Fig.4I uses one-way ANOVA with multiple comparisons and Tukey’s correction.
  • Fig. 5C
  • Fig. 6B Distinct expression profile all populations in mZ-HLOs. The size of the circle indicates the percentage of nuclei in each population expressing each gene. The color represents the average expression level for the indicated gene.
  • Fig.6C Heatmap showing scaled mean expression of all genes in each cluster. Top 10 marker genes in each cluster have been added as labels.
  • Fig. 6D Expression of known hepatoblast and zonal hepatocyte marker genes in each population.
  • FIG. 6E Violin plot for expression of AFP (hepatoblast gene), GSS (interzonal hepatocytes), GHR (pericentral hepatocyte), and GLS2 (periportal hepatocyte).
  • Figure 7. Single cell analysis of multi-zonal human liver organoids (mZ-HLO).
  • Fig. 7A UMAP plot with the major populations (Hepatoblasts, Interzonal like hepatocytes, Pericentral like hepatocytes, and Periportal like hepatocytes) of parenchymal nuclei in mZ-HLOs. Velocyto force field showing the average differentiation trajectories (velocity) for nuclei located in different parts of the UMAP plot (left).
  • Fig. 7B Feature plots for pan liver makers: TTR and SERPINA1; Cholangiocyte marker: KRT7; Zone 1 marker: mCherry, GLS2, CPS1 and OTC; Zone 2 marker: GSS, TERT, and AKR1C1; and Zone 3 maker: GFP, GLUL, CYP2E1 and HIF1A.
  • Fig.7C UMAPs for human hepatocytes from PSC-derived liver organoid cell atlas colored by organoid source and cell type.
  • FIG. 8A Spatial plot for TAT (zone 1), HAMP (zone 2), and CYP3A4 (zone 3) markers in 10X Xenium healthy human liver dataset (publicly available dataset).
  • Fig. 8B UMAP plot of mZ-HLO with hepatocyte populations (top) and distribution of replicate data (bottom).
  • Fig.8C UMAP plot of mZ-HLO with hepatocyte populations (top) and distribution of replicate data (bottom).
  • Fig. 8D UMAP plot for zonal hepatocyte populations from primary liver (Andrews et al., 2022 Hepatology Communications 6, 821-840) and mZ-HLOs integrated together (top). UMAP plot depicting distribution for total hepatocyte populations from primary liver and mZ-HLOs integrated together (bottom).
  • Fig.8E Expression of known hepatoblast and zonal hepatocyte marker genes in mZ-HLOs benchmarked against the Andrews et al., 2022 snRNAseq dataset referenced above.
  • Fig.8F Expression of known hepatoblast and zonal hepatocyte marker genes in mZ-HLOs benchmarked against the Andrews et al., 2022 snRNAseq dataset referenced above.
  • Fig.8G Feature plot for GLS2 (zone 1), HAMP (zone 2), and GLUL (zone 3) markers.
  • Fig.9A Phase portrait of AFP, GLUL, and GLS2 depicting the dynamics of the gene splicing in the nuclei with the velocity and expression of AFP, GLUL, and GLS2 in nuclei as feature plots.
  • Fig. 9B Phase portrait of AFP, GLUL, and GLS2 depicting the dynamics of the gene splicing in the nuclei with the velocity and expression of AFP, GLUL, and GLS2 in nuclei as feature plots.
  • FIG. 9C Boxplot showing the pseudotime of each nuclei population in mZ- HLOs.
  • data are represented as boxplot where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper and lower whisker extends from the hinge to the largest and smallest value respectively no further than 1.5 ⁇ IQR from the hinge (where IQR is the inter-quartile range), while data beyond the end of the whiskers are outlying points that are plotted individually.
  • Figure 10 Figure 10.
  • Fig. 10A Peak density plots showing EP300 bound loci, a marker of active enhancers. Profile plot of all peaks are in the top panel.
  • Fig.10B Genome browser view of HNF4A (pan marker) showing the EP300 ChIPseq peak.
  • Fig.10C Genome browser view of CTNNB1 (pan marker) showing the EP300 ChIPseq peak.
  • Fig. 10D Genome browser view of SLBP (zone 1 gene) showing the EP300 ChIPseq peak.
  • Fig.10E Genome browser view of SLBP (zone 1 gene) showing the EP300 ChIPseq peak.
  • Fig. 10G Top 10 upregulated Gene Ontology terms (Biological Process) for the gene regulated bound by EP300 in the Z1-HLOs.
  • Fig. 10H Top 10 upregulated Gene Ontology terms (Biological Process) for the gene regulated bound by EP300 in the Z3-HLOs.
  • Fig.11A Fig.11A.
  • Genome browser view of ACSS2 (zone 1 gene) showing the EP300 ChIPseq peak.
  • Fig. 11B Genome browser view of HPR (zone 2 gene) showing the EP300 ChIPseq peak.
  • Fig. 11C Genome browser view of ALDH6A1 (zone 3 gene) showing the EP300 ChIPseq peak.
  • Fig. 11D Top 10 upregulated Gene Ontology terms (Biological Process) for the gene regulated bound by EP300 in the mZ-HLOs.
  • Fig. 11E Motif enrichment analysis of EP300 bound peaks analyzed by MEME-ChIP.
  • Fig.11F Motif enrichment analysis of EP300 bound peaks analyzed by MEME-ChIP.
  • the interconnected dual organoids (mZ-HLOs) formed from mixing respective organoid progenitors (e.g. immature organoids) from the mGULO-expressing iPSCs and normal iPSC lines in the presence of bilirubin can have a gradient between regions, such as between phenotypically distinct zones.
  • hepatocyte subpopulations including zone 1, zone 2, and zone 3 (or zone 1-like, zone 2-like, and zone 3-like) hepatocytes, is observed in this dual organoid assembly model.
  • zone 1, zone 2, and zone 3 or zone 1-like, zone 2-like, and zone 3-like hepatocytes
  • This self-assembled dual organoid system can exhibit zone-specific functions associated with urea cycle, glutathione synthesis and/or glutamate synthesis.
  • Single nucleus RNA sequencing analysis identified a hepatoblast differentiation trajectory towards interzonal-, periportal-, and pericentral-like cells.
  • epigenetic and transcriptomic analysis showed the zonal divergence is orchestrated by ascorbate- or bilirubin-induced preferential binding of histone acetyltransferase p300 (EP300) to methylcytosine dioxygenase TET1 or hypoxia-inducible factor 1-alpha (HIF1 ⁇ ).
  • Transplantation of the self-assembled zonally patterned human organoids can improve survival by ameliorating the hyperammonemia and hyperbilirubinemia caused by bile duct ligation in rats.
  • the multi-zonal organoid system can serve as an effective in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease and to study the functional ensemble across diverse hepatocytes in development and disease.
  • zone 1 hepatocytes represent the main target for metabolic diseases like NASH
  • zone 2 hepatocytes represent the main target for liver regeneration
  • zone 3 hepatocytes can allow for studying and addressing alcoholic disease and/or drug-induced cholestasis.
  • the disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
  • “a” or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
  • Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.
  • the term “plurality” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the term “set of” means one or more.
  • a set of items includes one or more items.
  • the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed.
  • the item may be a particular object, thing, step, operation, process, or category.
  • “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required.
  • “at least one of item A, item B, or item C” means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and C.
  • “at least one of item A, item B, or item C” means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.
  • a treatment can include executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease.
  • Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance.
  • “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition.
  • “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.
  • Treatment thus can cover any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. “Treatment” can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition.
  • terapéuticaally effective or “therapeutically effective amount” as used throughout this application can refer to an amount effective to achieve a desired and/or beneficial effect, and/or anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease.
  • An effective amount can be administered in one or more administrations.
  • a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life.
  • Such achievement can be measured by any suitable method, such as measurement of tumor size or blood cell count, or any other suitable measurement.
  • the terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to that amount of a recited composition or compound that, results in an observable effect.
  • Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that, is effective to achieve the desired response for a particular subject and/or application.
  • the selected dosage level wall depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
  • the term “disease state” as used herein, can generally refer to a condition that affects the structure or function of an organism. Disease states can include, for example, stages of a disease progression.
  • the term “assessing” can include any form of measurement, and includes determining if an element is present or not.
  • the terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” can be used interchangeably and can include quantitative and/or qualitative determinations.
  • the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.
  • marker can include a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.
  • cellular phenotype can refer to any determinable, observable, and/or measurable characteristic associated with a cell population.
  • a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof.
  • a model can be used in a process and/or applied to an assay, in accordance with various embodiments as disclosed herein.
  • a “process” can include one or more steps involving one or more features of one or more model as disclosed herein.
  • the terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to a biological, enzymatic, or therapeutic function.
  • inhibitor has its plain and ordinary meaning as understood in light of the specification, and can refer to the reduction or prevention of a biological activity.
  • the reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values.
  • delay has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected.
  • the delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values.
  • the terms inhibit and delay may not necessarily indicate a 100% inhibition or delay, A partial inhibition or delay may be realized.
  • isolated has its plain and ordinary meaning as understood in light of the specification, and can refer to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man.
  • Isolated substances and/or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and/or spanning the aforementioned values).
  • isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and/or spanning the aforementioned values).
  • a substance that is “isolated” may be “pure” (e.g., substantially free of other components).
  • isolated cell can refer to a cell not contained in a multi -cellular organism or tissue.
  • in vivo is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.
  • ex vivo is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method outside a living organism with little alteration of natural conditions.
  • in vitro is given its plain and ordinary' meaning as understood in light of the specification and can refer to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.
  • nucleic acid or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • oligonucleotides those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with statically and electronically similar structures, such as aza- sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramlidate, or phosphoramidate.
  • nucleic acid molecule also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g.
  • plasmid plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAG), yeast artificial chromosome (YAC), or human artificial chromosome (HAG)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems.
  • BAG bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • HAG human artificial chromosome
  • sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths.
  • nucleic acids described herein comprise nucleobases.
  • Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil.
  • Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.
  • peptide “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds.
  • the numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available.
  • nucleic acid template By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g.
  • upstream on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N- terminus of a subsequent sequence.
  • purity of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual abundance of the substance, compound, or material relative to the expected abundance.
  • the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between.
  • Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof.
  • the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents.
  • Purity' can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • Yield of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual overall amount of the substance, compound, or material relative to the expected overall amount.
  • the yield of the substance, compound, or material is is about, is at least, is at least about, is not more than, or is not more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including ail decimals in between.
  • Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.
  • “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity.
  • a “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts.
  • a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs.
  • the term diluent, excipient, and/or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
  • Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
  • Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.
  • the physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such
  • compositions can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.
  • Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals.
  • Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch.
  • Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum [FCS]) to enhance post-thawing survivability of the cells.
  • nutrients e.g. albumin, serum, bovine serum, fetal calf serum [FCS]
  • At least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.
  • Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, ure
  • pharmaceutically acceptable salts has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.
  • Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.
  • Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections.
  • Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
  • % w/w or “% wt/wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.
  • % v/v or “% vol/vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.
  • base membrane matrix or “extracellular matrix” as used herein has its plain and ordinary meaning in light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and/or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein.
  • extracellular matrices include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, collagen, collagen IV, gelatin, fibronectin, vitronectin, laminin, laminin-511 elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan, fibrin, basement membrane, Matrigel®, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof.
  • a common basement membrane matrix that is used in laboratories are those isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells.
  • EHS Engelbreth-Holm-Swarm
  • these basement membrane matrices are derived from non-human animals and therefore contain xenogeneic components that prevent its use towards humans. They are also not defined, which can lead to variability in manufacturing, as well as potentially harbor pathogens.
  • the methods for culturing cells may involve the use of synthetic and/or defined alternatives to these xenogeneic basement membrane matrices.
  • the use of non-xenogeneic basement membrane matrices or mimetics or derivatives thereof enables manufacturing of biological products better suited for human use.
  • the cells may be passaged by taking a fraction of the cells and seeding this fraction onto a fresh surface (e.g., of a cell culture container) in culture medium. This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface, upon which this passaging can be repeated successively.
  • a fresh surface e.g., of a cell culture container
  • This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface, upon which this passaging can be repeated successively.
  • the microscopic architecture of the liver is made up of polygonal structures called “hepatic lobules”. Classically, these lobules take on a hexagonal structure, although other geometric shapes are observed depending on tissue specification.
  • Each lobule unit comprises plates or layers of hepatocytes surrounding an internal central vein and encapsulated by bundles of vessels called portal triads, which are made up of a portal vein, hepatic artery, and bile duct. Hepatic activity occurs as blood flows from the portal triads at the periphery, across the hepatocytes, and into the central vein to return to the circulatory system. Due to the asymmetric organization of these lobules, the layers of hepatocytes are divided into three zones.
  • zone 1 Cells in the “periportal zone” (zone 1) are closest to the portal triad and receive the most oxygenated blood, the pericentral zone (zone 3) are closest to the central vein and therefore receive the least amount of oxygenated blood, and the transition zone (zone 2) is in between zone 1 and 3. Due to this separation, each zone of hepatocytes exhibit differing activities. For example, zone 1 hepatocytes are involved in oxidative liver functions such as gluconeogenesis and oxidative metabolism of fatty acids, whereas zone 3 hepatocytes are involved in glycolysis, lipogenesis, and cytochrome P450-mediated detoxification.
  • the liver organoids disclosed herein exhibit a periportal-like identity resembling the tissue found in the periportal zone of liver lobules, including the functional and cellular marker characteristics of the periportal zone.
  • the term “bilirubin” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the naturally occurring metabolite created by normal catabolic degradation of heme. Bilirubin arises from the catalysis of biliverdin by biliverdin reductase. In the liver, bilirubin is conjugated with glucuronic acid by a family of enzymes called UDT-glucuronosyltransferases (UGTs).
  • UDT-glucuronosyltransferases UDT-glucuronosyltransferases
  • This conjugation renders bilirubin water soluble, enabling it to be carried in bile to the small intestine and colon, whereby it is further metabolized to waste products.
  • Dysfunctional bilirubin metabolism particularly due to abnormal function of UGTs preventing conjugation of bilirubin, leads to accumulation of bilirubin and is associated with various diseases characterized by hyperbilirubinemia.
  • bilirubin also has antioxidant capabilities and therefore may have beneficial effects in reducing oxidative damage in cells.
  • hypobilirubinemia has its plain and ordinary meaning as understood in light of the specification and refers to the condition of elevated levels of bilirubin, which is a natural product of heme catabolism. Bilirubin is filtered from the blood by the liver and is converted to water soluble intermediates, which are then released to the intestinal tract in bile, metabolized by microbiota, and excreted as waste. In neonates, bilirubin levels, which were originally cleared by the mother through the placenta, might not be adequately cleared by the immature liver. Excessive levels of bilirubin may potentially cause severe neurological damage (kernicterus).
  • vitamin C is an essential nutrient that is involved in many biological functions such as use as a cofactor for several important enzymes and as an antioxidant.
  • humans, as well as other haplorrhine primates, certain species of bats, and Guinea pigs have evolved to harbor a non-functional GULO gene. Therefore, these organisms are unable to synthesize ascorbate and require vitamin C intake from diet or supplementation, where a deficiency of vitamin C can lead to scurvy.
  • a “functional GULO protein” is a GULO protein that has L-gulonolactone catalytic activity to result in the production of ascorbate.
  • an “inactive” GULO protein or “non-functional” GULO protein is one that does not have the catalytic activity to produce ascorbate.
  • Humans and cells that are derived from humans comprise a non-functional GULO protein and do not have the ability to synthesize ascorbate.
  • human cells may be engineered to express a functional GULO protein to enable ascorbate synthesis ability.
  • These functional GULO proteins may be expressed in human cells (or other cells that are unable to normally synthesize ascorbate) through conventional methods of cloning, such as genetically engineering cells to have genetic sequences that encode for a functional GULO protein.
  • exogenous as used herein has its plain and ordinary meaning as understood in light of the specification and refers to external factors that originate outside of a biological specimen (e.g., a cell, population of cells, organoid, etc.), as opposed to being naturally occurring and/or produced by the biological specimen itself.
  • exogenous components, reagents, and/or conditions are components, reagents, and/or conditions that are added to compositions described herein, although this does not necessarily preclude the possibility of the same components, reagents, and/or conditions also being present through a function endogenous to a biological specimen.
  • liver organoid and “hepatocyte organoid” are used interchangeably herein, and refer to populations of cells differentiated in vitro to form self-organizing structures, which generally are three-dimensional (3D), and include one or more functional cell types.
  • Liver organoids differ from naturally occurring liver tissue in a number of ways. For example, as compared with naturally occurring liver tissue, liver organoids can have a structure having a single lumen and generally a spherical shape, and can include a basement membrane which is unnatural.
  • the single lumen of a liver organoid contains 3D tissues but generally does not make any hepatic lobular structure nor cord-like structure, as with naturally occurring liver tissue.
  • Liver organoids also generally do not contain hematopoietic tissue and acquired immune cell subsets, such as T cell lineages. Further, as compared with naturally occurring liver tissue, liver organoids can have different efflux mechanisms, as a liver organoid can have a three-dimensional structure with a luminal structure but no ejection mechanism. In addition, liver organoids generally cannot receive dietary inputs, as they lack a gut and connected vascular channel. Organoids differ from embryonic bodies (EBs) in that organoids are composed of a majority of endoderm-derivatives (more than 50%). [0236] The relative maturity of a liver organoid can be based on one or more of several factors, including key marker expressions, protein secretion, and functional enzyme activity.
  • EBs embryonic bodies
  • immature liver organoid refers, in general, to a population of organoids that produce a low amount of albumin, or a reduced amount of albumin as compared to a mature liver organoid.
  • An immature liver organoid also generally can have reduced expression of ALB, HNF4A, MRP2, BSEP, GLS2, and/or PCK, and increased expression of SOX9 and/or CDX2, as compared to a mature liver organoid.
  • an immature liver organoid can also have decreased drug metabolism (CYP450 activity), urea production, and bile acid synthesis and excretion, as compared to a mature liver organoid.
  • Liver organoids can be derived from pluripotent stem cells (PSCs), including at least embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Liver organoids may also be formed from liver-derived stem cells.
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Liver organoids may also be formed from liver-derived stem cells.
  • liver organoids can self-organize through cell sorting and spatially restricted lineage commitment in a manner similar to that which occurs in vivo, but as directed in vitro by thoughtful introduction of exogenous and/or endogenous differentiating factors and/or conditions as described herein, optionally through one or more directed steps, optionally involving introduction of one or more components.
  • a mature liver organoid may be characterized as expressing reduced levels of AFP, CDX2, and/or NANOG relative to liver organoids, and/or as expressing increased levels of ALB, SLC4A2 and/or HO-1 relative to liver organoids.
  • a mature liver organoid may be characterized as expressing CYP2E1, CYP7A1, PROX1, MRP3, MRP3, and/or OATP2.
  • a mature liver organoid may exhibit increased CYP3A4 and/or CYP1A2 protein levels and/or enzymatic activity relative to liver organoids.
  • tissue culture surface as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a substrate surface on which cells may aggregate and/or adhere to facilitate cell growth, differentiation, and/or function.
  • engineered refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In certain embodiments, a construct and/or vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector.
  • Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.
  • Z1-like Zone 1-like
  • peripheral-like hepatocytes are used interchangeably herein, and refer to cells that have developed expression and/or developmental phenotypes similar to and/or potentially not significantly distinguishable from, those observed in naturally occurring Z1 (periportal) hepatocytes.
  • These Z1-like cells may have oxidative liver functions such as gluconeogenesis and/or oxidative metabolism of fatty acids metabolic capacity.
  • Z1-like hepatocytes may be Z1 hepatocytes. Any mention of “zone 1” or “Z1” as used herein can also encompass “Z1-like.”
  • Z2-like”, “Zone 2-like”, and “interzonal-like” hepatocytes are used interchangeably herein, and refer to cells that have developed expression and/or developmental phenotypes similar to and/or potentially not significantly distinguishable from, those observed in naturally occurring Z2 (interzonal) hepatocytes.
  • Z2-like cells may produce hepatoblasts and/or be involved in proliferation and/or differentiation of hepatocytes.
  • Z2-like cells may express increased levels of Glutathione synthetase (GSS), Telomerase reverse transcriptase (TERT), Apolipoprotein M (APOM), and/or Aldo-keto reductase family 1 member C1 (AKR1C1) gene products (e.g., coding and/or non-coding transcripts, polypeptides, proteins, etc.) relative to other hepatocyte cell types.
  • GSS Glutathione synthetase
  • TERT Telomerase reverse transcriptase
  • APOM Apolipoprotein M
  • ARR1C1 Aldo-keto reductase family 1 member C1
  • Z2-like hepatocytes may be Z2 hepatocytes.
  • Z3-like, Zone 3-like, and “pericentral-like” hepatocytes are used interchangeably herein, and refer to cells that have developed expression and/or developmental phenotypes similar to and/or potentially not significantly distinguishable from, those observed in naturally occurring Z3 (pericentral) hepatocytes. These Z3-like cells may have glycolysis, lipogenesis, and/or cytochrome P450-mediated detoxification metabolic capacity.
  • mZ-LO may be derived from human cells, and referenced as mZ-HLO.
  • Multi-zonal liver organoids may also comprise and/or develop additional cell types, including cholangiocytes, endothelial cells, macrophages, stellate cells, mesenchyme cells, and/or hepatoblasts.
  • Multi-zonal liver organoids may maintain a tubular structure and/or a continuous lumen, and may display differential expression of zone-specific liver markers, indicating the variable functionality of the various zones.
  • a multi-zonal liver organoid may develop functional capacity for hepatocyte proliferation, hepatocyte differentiation, urea cycle, ammonia removal, glycolysis, bilirubin removal, lipid catabolism, glutamine catabolism and biosynthesis, reactive oxygen species (ROS) catabolism and/or metabolism, oxygen response, xenobiotic metabolism, DNA repair, pigment metabolism, lipogenesis, gluconeogenesis, glutathione, and/or glutamine metabolism.
  • ROS reactive oxygen species
  • the hepatocytes are specialized into periportal hepatocytes (zone 1) located near the portal vein, pericentral hepatocytes (zone 3) near the central vein, and a small population of interzonal hepatocytes in the intermediate region.
  • the periportal hepatocytes develop in an oxygen- and nutrient-rich environment, and vice versa for pericentral hepatocytes.
  • the candidate signaling cascade responsible for proper zonation of the pericentral hepatocytes is the Wnt family molecules, while the Hedgehog (Hh) and Notch signaling pathway are linked to periportal hepatocytes and cholangiocytes.
  • Hh Hedgehog
  • Notch Notch signaling pathway
  • Ascorbic acid which is an essential antioxidant for hepatocyte development, regulates the expression of several Zone 1 specific liver genes. Periportal hepatocytes are principally responsible for functions, including gluconeogenesis, cholesterol synthesis, and fatty acid oxidation are potentiated by the activity of ascorbate, whereas lipogenesis, attributed to pericentral hepatocytes, is inhibited by ascorbic acid. Ascorbate is also known for the activation of Tet1 (Tet methylcytosine dioxygenase 1) in the liver, which in turn activates Hh signaling essential for the activation of periportally-enriched gene expression in late embryogenesis.
  • Tet1 Tet methylcytosine dioxygenase 1
  • bilirubin a metabolic waste product made from heme
  • bilirubin has a potential to enrich metabolic activities located in the pericentral areas.
  • bilirubin can promote the expression of Zone 3-enriched specific CYP enzymes directly or indirectly through Wnt signaling activation.
  • ascorbate and bilirubin can prime differential zone-specific programs. The Wnt activating role of bilirubin is explained by its pro-angiogenic properties, thereby activating the Akt-NOS3 signaling pathway.
  • bilirubin is known to activate both transcription and translation of HIF1 ⁇ in even normoxic conditions to emulate the after-effects on exposure to hypoxia. Given that GLUL+ pericentral hepatocytes are HIF1 ⁇ positive, bilirubin can be involved in sustaining the expression of Zone 3 specific programs. [0250] As described herein, in order to employ differential inductive conditions, the disclosure relates to dual organoid systems developed by combining ascorbate- and bilirubin- enriched progenitors (e.g. immature HLOs) derived from human induced pluripotent stem cells (hiPSCs).
  • ascorbate- and bilirubin- enriched progenitors e.g. immature HLOs
  • hiPSCs human induced pluripotent stem cells
  • Ascorbate-enriched progenitors can be derived by genetic manipulation, ascorbate supplementation, hedgehog signal activation, Notch signal activation, Wnt inhibition, Tet activation, hyperoxia any/or the like.
  • Bilirubin-enriched progenitors can be derived by exposure to bilirubin, genetic manipulation, hypoxia culture, Wnt supplementation, Hedgehog inhibition, Notch inhibition, manipulation of any gene listed in Fig.11, and/or the like.
  • an ascorbate-treated HLO can be prepared by manipulation of one or more genes listed in Fig.11;
  • a bilirubin-treated HLO can be prepared by manipulation of one or more genes listed in Fig.11.
  • periportal identity for the ascorbate-enriched progenitors can be evoked using a tetracycline (TET)-inducible active GULO knock-in hiPSC line (Reza, H. A. et al. Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids. Stem Cell Reports (2023)) or the like.
  • TET tetracycline
  • standard hiPSC lines can be exposed to bilirubin to prime into pericentral lineage for the bilirubin-enriched progenitors.
  • the transcriptomic, epigenetic and functional profile of the resulting generated organoids demonstrate multi-zonal phenotypes and functions.
  • the mZ-HLO organoid system allows for the study of development and disease affecting divergent hepatocyte subpopulations in humans.
  • Emerging single cell genomics approaches have revealed the high-resolution signatures that define the zonation pattern in the liver. For example, snRNAseq has been used to characterize iPSC-derived epithelial components in organoids.
  • the snRNAseq dataset showed divergent parenchymal populations including hepatoblasts, interzonal, pericentral, and periportal hepatocyte-like cells, which were annotated based on the aforementioned datasets and well-known genetic markers. Moreover, when compared to primary liver snRNAseq dataset, the zonal hepatocyte population are found to be highly concordant with subpopulations found in the mZ- HLO, with the exception of the hepatoblast population.
  • the genes and pathways activated in zonal subpopulations are largely in agreement with existing knowledge related to specialized molecular markers, though with lower expression of TTR, CEBPB, APOA1 and ARG1 when compared to publicly available primary liver-derived datasets, likely due to immaturity or differences between snRNAseq and scRNAseq.
  • the developmental lineage predictions supported the theory that zonal hepatocytes originate from hepatoblasts after differentiation through the interzonal hepatocyte fates.
  • the trajectory model of the mZ-HLOs depicted the development trajectory of early zonal liver development.
  • Adult hepatocytes have been reported to transdifferentiate into different zonal hepatocytes.
  • EP300 a histone acetyltransferase
  • EP300 is one such epigenetic modifier that acetylates enhancer regions and activates transcription leading to hepatoblast differentiation.
  • EP300 marks poised and active enhancers and activates expression of zonal genes.
  • periportal metabolic functions such as gluconeogenesis and beta- oxidation of fatty acids, can be activated by EP300.
  • EP300 can contribute to the upregulation of genes involved in glycolysis and lipogenesis, which are functions specific to pericentral hepatocytes.
  • EP300 can bind to enhancers upstream of zonal genes, such as ALDH6A1, ACSS2 and HPR, in a context-dependent manner to differentially activate gene expression.
  • zonal genes such as ALDH6A1, ACSS2 and HPR
  • the integrated RNAseq and ChIPseq data showed that the top targets were HIF1A and TET1 in the dox-treated Z1-HLOs and bilirubin-treated Z3-HLOs, respectively.
  • the Tet1 deletion impairs periportal identity and function in the liver, whilst repressing the pericentral characters regulated through Hedgehog signaling.
  • bilirubin possesses signaling properties that can activate HIF and WNT signaling cascades. which are important for pericentral hepatocytes.
  • Hepatocyte transplantation has been used to treat liver diseases, but the difficulty in obtaining compatible primary human hepatocytes makes this an impractical approach.
  • stem cell-derived tissues have been used to treat a multitude of hepatic diseases (Reza, H. A., Okabe, R. & Takebe, T. Organoid transplant approaches for the liver. Transplant International 34, 2031-2045 (2021)).
  • the most therapeutic proof-of-concept has aimed at correcting monogenic conditions by targeting one particular disease parameter.
  • the presently described mZ-HLO model endowed with the multi-zonal functionality, can ameliorate multiple aspects of liver failure for example, in vivo in a bile duct- ligated immunodeficient rat model.
  • zone-1 or zone-3 primed organoid transplants only offer either hyperammonemia or hyperbilirubinemia improvements resulting in reduced survival benefit.
  • mZ-HLO transplants offer improvements in both hyperammonemia and hyperbilirubinemia.
  • the mZ-HLOs integrate into the parenchyma of the rat liver in a tubular morphology while retaining their zonal characteristics as indicated by the expression of mCherry, GFP, ARG1, and GLUL.
  • the Z1-HLOs showed a preferential integration near the portal vein, while the Z3-HLOs exhibited a slightly higher affinity for the central vein region.
  • the disclosed mZ-HLOs are able to engraft into the resident liver and maintain zonal- specific functionality and augment the survival rate in rodents following biliary duct ligation.
  • the mZ-HLOs can be used as a tool to recapitulate zonal identity and function, which is critical in the study and treatment of hepatic disease.
  • Human Liver Organoids [0260] The microscopic architecture of the liver is made up of polygonal structures called “hepatic lobules”.
  • Each lobule unit comprises plates or layers of hepatocytes surrounding an internal central vein and encapsulated by bundles of vessels called portal triads, which are made up of a portal vein, hepatic artery, and bile duct. Hepatic activity occurs as blood flows from the portal triads at the periphery, across the hepatocytes, and into the central vein to return to the circulatory system. Due to the asymmetric organization of these lobules, the layers of hepatocytes are divided into three zones.
  • zone 1 Cells in the “periportal zone” (zone 1) are closest to the portal triad and receive the most oxygenated blood, the “pericentral zone” (zone 3) are closest to the central vein and therefore receive the least amount of oxygenated blood, and the “transition zone” (zone 2) is in between zone 1 and 3. Due to this separation, each zone of hepatocytes exhibit differing activities. For example, zone 1 hepatocytes are involved in oxidative liver functions such as gluconeogenesis and oxidative metabolism of fatty acids, whereas zone 3 hepatocytes are involved in glycolysis, lipogenesis, and cytochrome P450-mediated detoxification.
  • HLOs Human liver organoids
  • progenitor cells such as, for example, patient-derived induced pluripotent stem cells (iPSCs), where the patient can be healthy or having a diseased condition, and are identical in genetic content to the respective patient. They express most liver markers that are expressed in the pre-natal stages of development. Furthermore, they are clonal and therefore reacts similarly to external stimuli and biochemical perturbations.
  • iPSCs patient-derived induced pluripotent stem cells
  • HLOs are highly scalable and tractable, allowing screening approaches to test a vast array of drugs and small molecules.
  • HLOs are easy to work with as model systems and have very low variation across batches. Large batches of HLOs can be generated within a couple of weeks.
  • bilirubin In the liver, bilirubin is conjugated with glucuronic acid by a family of enzymes called UDT- glucuronosyltransferases (UGTs). This conjugation renders bilirubin water soluble, enabling it to be earned in bile to the small intestine and colon, whereby it is further metabolized to waste products. Dysfunctional bilirubin metabolism, particularly due to abnormal function of UGTs preventing conjugation of bilirubin, leads to accumulation of bilirubin and is associated with various diseases characterized by hyperbilirubinemia. Notably, however, while excessive bilirubin is detrimental, bilirubin also has antioxidant capabilities and therefore may have beneficial effects in reducing oxidative damage in cells.
  • UGTs UDT- glucuronosyltransferases
  • L-gulonolactone oxidase catalyzes L-gulonolactone to produce L-xylo-hex-3-gulonolactone and hydrogen peroxide.
  • the L- xylo-hex-3-gulonolactone then spontaneously converts to ascorbate (vitamin C).
  • vitamin C is an essential nutrient that is involved in many biological functions such as use as a cofactor for several important enzymes and as an antioxidant.
  • humans, as well as other haplorrhine primates, certain species of bats, and Guinea pigs have evolved to harbor a non-functional GULO gene.
  • human cells may be engineered to express a functional GULO protein to enable ascorbate synthesis ability.
  • These functional GULO proteins may be expressed in human cells (or other cells that are unable to normally synthesize ascorbate) through conventional methods of cloning, such as genetically engineering cells to have genetic sequences that encode for a functional GULO protein.
  • iPSC-derived organoids expressing a functional L- gulonolactone oxidase (GULO), such as murine GULO (mGULO) have been generated.
  • mGULO organoids When the iPSCs and organoids are human in origin, the expression of the functional L-gulonolactone allows for ascorbate synthesis, which is normally inactive in humans. These mGULO organoids exhibit increased efficiency in conjugating bilirubin and exhibited improved viability when treated with bilirubin.
  • the production of ascorbate in mGULO organoids reduces oxidative stress in the organoids and drives expression of NRF2, which is a master regulator of cellular detoxification pathways and in turn promotes expression of UGT1A1, which catalyzes bilirubin conjugation.
  • NRF2 is a master regulator of cellular detoxification pathways and in turn promotes expression of UGT1A1, which catalyzes bilirubin conjugation.
  • HLOs have been shown to exhibit a specific hepatocyte subpopulation, such as periportal (zone 1) hepatocytes.
  • periportal (zone 1) hepatocytes there has been no evidence of HLOs with multi-zonal characteristics, such as HLOs which have, for example, periportal (zone 1) hepatocytes as well as pericentral (zone 3) hepatocytes and/or interzonal hepatocytes.
  • hepatocyte subpopulations Fusion of the organoids can be indicated by rearrangement and interaction of cytoskeleton proteins, increase in mean segment length. This fusion can occur by maintaining a continuous lumen. Fused organoids can expand canalicular connectivity. The fused organoid can include structures carrying bilirubin in the lumen. Activation of Notch signaling or Ezrin can increase fusion.
  • Notch activators, Ezrin activators, any of the like can be added to the co-culture; for example, such activators can be added once or more during day 15 through day 20 (D15-D20), optionally at 37Co.
  • DAPT is an exemplary Notch activator which can be used, at an appropriate concentration.
  • DAPT can be used at a concentration of 0.1 ⁇ M- 10 ⁇ M, or 0.01 ⁇ M-100 ⁇ M, or a larger range.
  • These liver organoids exhibit characteristics of two or more zones, including the genetic, functional, and cellular marker characteristics of the two or more zones.
  • the doxycycline-treated HLO can include a zone 1 hepatocyte subpopulation, while the bilirubin- treated HLO can include a zone 3 hepatocyte subpopulation.
  • the liver organoids disclosed herein can have a periportal-like identity resembling the tissue found in the periportal zone of liver lobules, including the genetic, functional, and cellular marker characteristics of the periportal zone, as well as a pericentral-like identity resembling the tissue found in the pericentral zone of liver lobules, including the genetic, functional, and cellular marker characteristics of the pericentral zone.
  • the liver organoids disclosed herein can have a periportal-like identity resembling the tissue found in the periportal zone of liver lobules, including the genetic, functional, and cellular marker characteristics of the periportal zone, as well as a pericentral-like identity resembling the tissue found in the pericentral zone of liver lobules, including the genetic, functional, and cellular marker characteristics of the pericentral zone, and a transition zone-like identity resembling the tissue found in the transition zone of liver lobules, including the genetic, functional, and cellular marker characteristics of the transition zone.
  • the multi-zonal liver organoids of the disclosure can include multiple cell types in addition to pericentral, periportal, and interzonal hepatocytes, such as cholangiocytes, endothelial cells, macrophages, stellate cells, mesenchyme cells, and hepatoblasts.
  • cholangiocytes cholangiocytes
  • endothelial cells a cell that is a cell that is associated with a multi-zonal liver organoid.
  • macrophages stellate cells
  • mesenchyme cells and hepatoblasts.
  • hepatoblasts hepatoblasts.
  • the benefits of having a multi-zonal liver organoid are demonstrated by the versatility of the system and broad applicability to liver disease models in general, as well as transplantation.
  • these multi-zonal liver organoids can have expression of zone 1-, zone 2-, and/or zone 3-associated genes, and/or can express zone 1-, zone 2-, and/or zone
  • these multi-zonal liver organoids can also have expression of pan-hepatocyte-associated genes and/or can express pan-hepatocyte-associated proteins.
  • these multi-zonal liver organoids can have expression of zone 1-, zone 2-, and zone 3-associated genes, and can express zone 1-, zone 2-, and zone 3-associated proteins, in addition to expression of pan-hepatocyte-associated genes and pan-hepatocyte-associated proteins. [0271] On account of their diverse zonal character, including diverse gene expression, these multi-zonal liver organoids have rich functionality.
  • liver organoids have been explored previously in, for example, Ouchi et al. “Modeling Steatobepatitis in Humans with Pluripotent Stem Cell-Derived Organoids” Cell Metabolism (2019) 30(2):374 ⁇ 384; Shinozawa et al.
  • Embodiments of methods for producing multi-zonal liver organoids are provided herein.
  • the methods include a) contacting definitive endoderm cells (DE) with an FGF signaling pathway activator and a Wnt signaling pathway activator for a first period of tune; b) contacting the cells of step a) with the FGF signaling pathway activator, the Wnt signaling pathway activator, and a retinoic acid (RA) signaling pathway activator for a second period of time, thereby differentiating the DE to posterior foregut cells; and c) embedding the posterior foregut cells in a basement membrane matrix and culturing the posterior foregut spheroids for a third period of time to differentiate the posterior foregut cells to the multi-zonal liver organoid.
  • DE definitive endoderm cells
  • RA retinoic acid
  • the third period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or a range defined by any two of the preceding values, for example 4-30, 10-30, 20-30, 4-17, 4-12, or 10-25 days.
  • the basement membrane matrix is Matrigel.
  • the liver organoid, DE, and/or pluripotent stem cells are derived from a patient.
  • direct deliver ⁇ ' of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification.
  • generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.
  • the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.
  • feeder cell as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface.
  • the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76/7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells.
  • conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or in combination with feeder cell co-culture.
  • feeder cells are not used during the proliferation of the target stem cells.
  • Differentiation of PSCs e.g., iPSCs or ESCs
  • pluripotent cells are derived from a morula.
  • pluripotent stem cells are stem cells.
  • Stem cells used in these methods can include, but are not limited to, embryonic stem cells or induced pluripotent stem cells.
  • Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges.
  • the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses.
  • the directed differentiation is done in a non- stepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time.
  • directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.
  • the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours, in some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added.
  • concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations. [0304] In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs are cultured in growth media that supports the growth of stem cells.
  • the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0,6%, 0.7%, 0.8%, 0,9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%.
  • the stem cell growth media does not contain xenogeneic components.
  • the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.
  • populations of cells enriched in definitive endoderm cells are used.
  • the definitive endoderm cells are isolated or substantially purified.
  • the isolated or substantially purified definitive endoderm cells express one or more (e.g. at least 1, 3) of SOX17, FOXA2, or CXRC4 markers to a greater extent than one or more (e.g. at least 1, 3, 5) of GCT4, AFP, I'M, SPARC, or SGX7 markers.
  • the stem cells are contacted with Activin A and BMP4.
  • activin-induced definitive endoderm can further undergo anterior endoderm pattering, foregut specification and morphogenesis, dependent on FGF, Wnt, or retinoic acid, or any combination thereof, or on FGF, Wnt, BMP, or retinoic acid, or any combination thereof, and a liver culture system that promotes liver growth, morphogenesis and cytodifferentiation.
  • human PSCs are efficiently directed to differentiate in vitro into liver epithelium and mesenchyme, it will be understood that molecules such as growth factors can be added to any stage of the development to promote a particular type of hepatic tissue formation.
  • iPSCs are expanded in cell culture.
  • pluripotent stem cells are expanded in a basement membrane matrix.
  • iPSCs are expanded in Matrigel,
  • the iPSCs are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632).
  • the iPSCs are differentiated into definitive endoderm cells. In the iPSCs are differentiated into definitive endoderm cells by- contacting the iPSCs with Activin A, BMP4, or both.
  • the iPSCs are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng/mL, 10 to 100 ng/mL, 100 to 200 ng/mL, or 50 to 150 ng/mL.
  • the pluripotent stem cells are contacted with Activin A at a concentration of 100 ng/mL or about 100 ng/mL.
  • the iPSCs are contacted with a concentration of BMP4 that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng/mL, or any concentration of BMP4 within a range defined by any two of the aforementioned concentrations, for example, 1 to 200 ng/mL, 1 to 100 ng/mL, 25 to 200 ng/mL, 1 to 80 ng/mL, or 25 to 100 ng/mL, In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration of 50 ng/mL or about 50 ng/mL.
  • the PSCs are differentiated into definitive endoderm cells. In some embodiments, the PSCs are differentiated into posterior foregut cells, in some embodiments, the PSCs are differentiated into a liver organoid.
  • any of the cells disclosed herein may be cryopreserved for later use. The cells can be cryopreserved according to methods generally known in the art, optionally including one or more cryoprotectants.
  • Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals.
  • Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3 -phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxy ethyl starch.
  • Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g.
  • Embodiments of the disclosure can include pharmaceutical compositions.
  • Such pharmaceutical compositions can include one or more additional pharmaceutically acceptable components, which can include carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity.
  • additional pharmaceutically acceptable components can include carriers, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity.
  • a “pharmaceutically acceptable” “diluent,” “excipient,” and/or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts.
  • Such pharmaceutical diluent, excipient, and/or earners can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
  • Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
  • Suitable pharmaceutical diluents and/or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.
  • the physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo
  • compositions can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like.
  • the formulation should suit the mode of administration.
  • Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, ure
  • excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b-propiolactone, gelatin, cell debris, nucleic acids, peptides, ammo acids, or growth medium components or any combination thereof.
  • the amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w/w or any percentage by weight in a range defined by any two of the aforementioned numbers.
  • compositions can include one or more “pharmaceutically acceptable salts”, which can include relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; ammo acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl ammoethane.
  • Proper formulation is dependent upon the route of administration chosen.
  • the dosage can be about 0.5 mg/kg human body weight or about 6.5 mg/kg human body weight.
  • some subjects e.g., mammals, mice, rabbits, feline, porcine, or canine
  • a dose or a therapeutically effective dose of a compound disclosed herein will be that which is sufficient to achieve a plasma concentration of the compound or its active metabolite(s) within a range set forth herein, e.g., about 1-10 nM, 10- 100 nM, 0.1-1 ⁇ M, 1-10 ⁇ M, 10-100 ⁇ M, 100-200 ⁇ M, 200-500 ⁇ M, or even 500-1000 ⁇ M, preferably about 1-10 nM, 10-100 nM, or 0.1-1 ⁇ M.
  • a treatment can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.
  • the compounds and pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses can be used for treatment of a subject.
  • higher or lower daily doses can be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.
  • a treatment can be administered locally or systemically in a therapeutically effective dose.
  • Amounts effective for this use will, of course, depend on the severity of the disease or disorder and the weight and general state of the subject. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders. [0331] Various considerations are described, e. g. , in Langer, 1990, Science, 249: 1527; Goodman and Gilman's (eds.), 1990, Id., each of which is herein incorporated by reference and for all purposes. Dosages for parenteral administration of active pharmaceutical agents can be converted into corresponding dosages for oral administration by multiplying parenteral dosages by appropriate conversion factors.
  • the parenteral dosage in mg/mL times 1.8 the corresponding oral dosage in milligrams (“mg”).
  • the parenteral dosage in mg/mL times 1.6 the corresponding oral dosage in mg.
  • An average adult weighs about 70 kg. See e.g., Miller-Keane, 1992, Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 5th Ed., (W. B. Saunders Co.), pp.1708 and 1651.
  • the administration can include a unit dose of one or more treatments in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients.
  • the carrier, vehicle or excipient can facilitate administration, delivery and/or improve preservation of the composition.
  • the one or more carriers include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate- buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose.
  • Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • Nontoxic auxiliary substances such as wetting agents, buffers, or emulsifiers may also be added to the composition.
  • Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.
  • the quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component.
  • the composition can, if desired, also contain other compatible therapeutic agents.
  • a treatment can be administered to subjects by any number of suitable administration routes or formulations.
  • the treatment such as an immunotherapy, can also be used to treat subjects for a variety of diseases.
  • Subjects include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats.
  • the subject is a human.
  • the route of administration of the compounds of the treatments described herein can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route.
  • administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration.
  • the choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.
  • compositions for performing any of the methods disclosed herein.
  • cell compositions in the form of a three- dimensional artificial multi-zonal liver organoid comprising Z1-like (periportal-like) hepatocytes, and Z3-like (pericentral-like) hepatocytes.
  • compositions comprising a three-dimensional multi-zonal liver organoid, comprising Z1-like (periportal-like) hepatocytes, and Z3-like (pericentral-like) hepatocytes.
  • compositions such as cell compositions and/or multi-zonal liver organoids, that further comprise Z2-like (interzonal-like, mid-lobular- like) hepatocytes.
  • compositions provided herein comprise exogenously added and/or transgenically produced ascorbate (vitamin C), and/or exogenously provided bilirubin.
  • compositions comprising Z1-like hepatocytes that are engineered to express a heterologous functional GULO protein, and ascorbate is produced by the Z1-like hepatocytes.
  • compositions comprising exogenously provided bilirubin at a concentration of about 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L, 2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, or 3 mg/L.
  • compositions comprising exogenously provided bilirubin at a concentration of about 1 mg/L.
  • compositions comprising multiple cell types, including at least Z3-like hepatocytes, Z1-like hepatocytes, Z2-like hepatocytes, and hepatoblasts.
  • compositions provided herein comprise greater than or equal to, exactly or about, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, or any range derivable therein, Z1-like hepatocytes.
  • compositions provided herein comprise greater than or equal to, exactly or about, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, or any range derivable therein, Z3-like hepatocytes.
  • compositions provided herein comprise greater than or equal to, exactly or about, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%, or any range derivable therein, Z2-like hepatocytes.
  • compositions provided herein comprise less than or equal to 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5%, or any range derivable therein, cholangiocytes.
  • compositions provided herein are in vitro compositions, created outside of a multicellular living organism.
  • compositions provided herein may be introduced into a multicellular living organism.
  • compositions provided herein comprise exogenously provided components, reagents, and/or conditions.
  • a basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells, is not Matrigel®, is not Cultrex®, and/or is not Geltrex®.
  • a basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and/or hydrogel.
  • an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of A83-01, RepSox, LY365947, and/or SB431542.
  • an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of TGF-b pathway inhibitor A83-01.
  • a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations.
  • a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 500 nM.
  • a composition comprises an exogenous FGF pathway activator that comprises, consists essentially of, or consists of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and/or FGF23.
  • an exogenous FGF pathway activator comprises, consists essentially of, or consists of FGF2.
  • a composition comprises a FGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a FGF pathway activator at a concentration of, or of about 5 ng/mL. [0348] In some embodiments, provided herein are compositions that include an exogenous Wnt pathway activator.
  • a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and/or TWS119.
  • a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists of CHIR99021.
  • a composition comprises a Wnt pathway activator at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 ⁇ M, or any concentration within a range defined by any two of the aforementioned concentrations.
  • a composition comprises a Wnt pathway activator at a concentration of, or of about, 3 ⁇ M.
  • a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF and/or GS4012. In some embodiments, a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 10 ng/mL.
  • compositions that include an exogenous EGF. In some embodiments, provided herein are compositions that do not include an exogenous EGF. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 20 ng/mL. [0351] In some embodiments, provided herein are compositions that include exogenous and/or transgenically produced ascorbic acid.
  • compositions that do not include exogenous and/or transgenically produced ascorbic acid.
  • compositions comprising ascorbic acid at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ⁇ g/mL or any concentration within a range defined by any two of the aforementioned concentrations.
  • compositions comprising ascorbic acid at a concentration of, or of about, 50 ⁇ g/mL.
  • provided herein are compositions that include a ROCK inhibitor. In some embodiments, provided herein are compositions that do not include a ROCK inhibitor.
  • a ROCK inhibitor comprises, consists essentially of, or consists of Y-27632.
  • compositions comprising a ROCK inhibitor at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ⁇ M, or any concentration within a range defined by any two of the aforementioned concentrations.
  • compositions comprising a ROCK inhibitor at a concentration of, or of about, 10 ⁇ M.
  • compositions comprising liver organoids that have and/or that are being differentiated from stem cells.
  • compositions comprising liver organoids that have and/or that are being differentiated from induced pluripotent stem cells. In some embodiments, provided herein are compositions comprising liver organoids comprising cells that have been passaged 1 time, 2 times, or 3 times. In some embodiments, provided herein are compositions comprising liver organoids comprising cells that have been passaged less than 4 times. [0354] In some embodiments, provided herein are compositions comprising A83-01, FGF2, CHIR99021, VEGF, and/or Y-27632, optionally further comprising iPSCs, PSCs, and/or posterior foregut cells and/or posterior foregut endoderm cells.
  • compositions comprising: a) posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids, and b) a medium, wherein the medium optionally comprises hepatocyte culture medium and is optionally supplemented with a cMET tyrosine kinase receptor agonist, an IL-6 family cytokine, and a corticosteroid, and wherein the composition optionally additionally comprises c) a retinoic acid pathway activator.
  • compositions provided herein comprise a cMET tyrosine kinase receptor agonist.
  • compositions provided herein comprise a cMET tyrosine kinase receptor agonist that comprises, consists essentially of, or consists of hepatocyte growth factor (HGF), PG-001, fosgonimeton, terevalefim, recombinant InlB321 protein, and/or an agonist c-Met antibody (e.g., LMH85).
  • HGF hepatocyte growth factor
  • PG-001 hepatocyte growth factor
  • fosgonimeton terevalefim
  • recombinant InlB321 protein e.g., LMH85
  • an agonist c-Met antibody e.g., LMH85
  • an IL-6 family cytokine comprises, consists essentially of, or consists of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and/or cardiotrophin-like cytokine (CLC).
  • OSM Oncostatin M
  • LIF leukemia inhibitory factor
  • CLC cardiotrophin-like cytokine
  • a corticosteroid comprises, consists essentially of, or consists of dexamethasone, beclometasone, betamethasone, fluocortolone, halometasone, and/or mometasone.
  • compositions comprising a hepatocyte culture media supplemented with HGF, OSM, and/or dexamethasone. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with dexamethasone. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with HGF. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with OSM. [0359] In some embodiments, provided herein are compositions comprising a retinoic acid pathway activator.
  • a retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and/or AM580.
  • a retinoic acid pathway activator comprises, consists essentially of, or consists of retinoic acid.
  • compositions comprise a retinoic acid pathway activator at a concentration of, or of about, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 ⁇ M, or any concentration within a range defined by any two of the aforementioned concentrations.
  • compositions comprise a retinoic acid pathway activator at a concentration of, or of about, 2.0 ⁇ M.
  • compositions comprise HGF.
  • compositions comprise HGF at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise HGF at a concentration of, or of about 10 ng/mL. [0361] In some embodiments, compositions comprise OSM. In some embodiments, compositions comprise OSM at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng/mL, or any concentration within a range defined by any two of the aforementioned concentrations.
  • compositions comprise OSM at a concentration of, or of about 20 ng/mL.
  • compositions comprise dexamethasone.
  • compositions comprise dexamethasone at concentration of, or of about, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, or any concentration within a range defined by any two of the aforementioned concentrations.
  • compositions comprise dexamethasone at a concentration of, or of about 100 nM.
  • compositions comprise exogenous bilirubin.
  • compositions comprise both exogenous bilirubin and endogenous bilirubin. In some embodiments, compositions comprise a low concentration of exogenous bilirubin. In some embodiments, a low concentration of exogenous bilirubin is at or near a human fetal physiological concentration of bilirubin. Human fetal bilirubin levels are thought to be generally around 1 mg/L (0.1 mg/dL), which rises rapidly to 3-10 mg/L (0.3-1.0 mg/dL) 24 hours after birth.
  • compositions comprise bilirubin, exogenous and/or endogenous, that is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/
  • compositions comprise exogenous bilirubin at a concentration that is, is about, is less than, or is less than about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75 or 3.0 mg/L, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 3 mg/L, 0.5 to 2.0 mg/L, 0.5 to 1.5 mg/L, 0.3 to 2.5 mg/L, or 0.5 to 1.75 mg/L; or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.1 to 1 mg/L, 0.1 to 0.5 mg/L, 0.5 to 1 mg/L, 0.3 to 0.7 mg/L, or 0.4 to 0.6 mg/L.
  • compositions comprising mature liver organoids.
  • compositions comprising mature liver organoids that were produced through contact with a exposure to exogenous bilirubin are provided herein.
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that have been engineered to comprise a functional L-gulonolactone oxidase (GULO) protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids are able to synthesize ascorbate.
  • GUILO L-gulonolactone oxidase
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids engineered to express functional GULO protein, wherein the functional GULO protein is murine GULO (mGULO).
  • mGULO murine GULO
  • a gene that encodes for a functional GULO protein is conditionally expressed.
  • a gene that encodes for a functional GULO protein is constitutively expressed.
  • a gene that encodes for a functional GULO protein is conditionally expressed using a tetracycline inducible system.
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using CRISPR mediated knock-in.
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids comprising a functional GULO encoding gene or mRNA, or both, that encodes for a functional GULO protein, wherein the functional gene was introduced to the posterior foregut cells and/or posterior foregut endoderm cells, liver organoids, mature liver organoids, and/or precursor cells by transfection.
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using adenovirus mediated gene transfection.
  • compositions comprising posterior foregut cells and/or posterior foregut endoderm cells, liver organoids and/or mature liver organoids that are engineered to comprise a gene that encodes for a functional GULO protein using adeno-associated virus mediated gene transfection.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit reduced caspase-3 activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein express increased levels of ALB relative to liver organoids and/or mature liver organoids that do not comprise the functional GULO protein.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein resemble periportal liver tissue and/or express periportal liver markers.
  • periportal liver markers comprise or consist of FAH, ALB, PAH, CPS1, HGD, or any combination thereof.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased CYP3A4 and/or CYP1A2 protein levels and/or enzymatic activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased bilirubin conjugation activity relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein.
  • liver organoids and/or mature liver organoids comprising a functional GULO protein exhibit increased viability in culture relative to liver organoids and/or mature liver organoids that do not comprise a functional GULO protein.
  • liver organoids and/or mature liver organoids have been differentiated from pluripotent stem cells comprising a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, whereby the pluripotent stem cells are able to synthesize ascorbate.
  • Kits [0368] In some embodiments, also disclosed herein are kits providing means for performing any of the methods described herein. In some embodiments, also disclosed herein are kits comprising any of the compositions or means of producing the compositions described herein. [0369] In some embodiments, a kit can be prepared from readily available components and reagents.
  • kits can comprise any one or more of the following components and/or reagents: enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies, cell culture media, differentiation induction reagents, amino acid mixtures/supplements, engineered constructs and/or polynucleotides, transcription induction agents, bilirubin, ascorbic acid, ascorbate, retinoic acid pathway activators, corticosteroids, cMET tyrosine kinase receptor agonists, IL-6 family cytokines, TGF-b pathway inhibitors, FGF pathway activators, Wnt pathway activators, VEGF pathway activators, ROCK inhibitors, organoids, and/or cells.
  • enzymes enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies, cell culture media, differentiation induction reagents, amino acid mixtures/supplements, engineered constructs and/or polynucleotides, transcription induction agents, bilirubin, ascorbic acid, ascorbat
  • kits may include components and reagents concentrated above the working concentrations disclosed herein, or at the working concentrations provided herein.
  • individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components.
  • concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.
  • a kit may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
  • kits are housed in a container. Kits may further comprise instructions for using the kit for assessing expression and/or differentiation of cells. Agents in a kit for measuring expression and/or determining differentiation may comprise a plurality of PCR probes and/or primers for qRT-PCR and/or a plurality of antibody or fragments thereof for assessing expression of biomarkers appropriate for classifying cell states. [0371] In some embodiments, kits are created using and comply with good manufacturing practice (GMP). [0372] Having described the embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the embodiments defined in the appended claims.
  • GMP good manufacturing practice
  • EXAMPLE 1 Materials and Methods Animals [0374] All animal experiments were conducted with the approval of the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) of the Cincinnati Children's Hospital Medical Center.
  • IACUC Institutional Animal Care and Use Committee
  • Adult Il2rg-deficient, Rag1-deficient RRG (SD/Crl) rats (breeding pairs, 9-12 weeks old) were housed in standard rat cages with paper bedding and maintained at a temperature of 20-24 °C and relative humidity of 45-55%, under a 12 h:12 h light:dark cycle. All animals had ad libitum access to dox chow before study. All animals were treated in accordance with the guidelines and regulations of the institution.
  • ODS/Shi Jcl-od/od rats were purchased from CLEA Japan, Inc.(Tokyo, Japan). They were housed in individual cages and maintained at temperature and humidity with 12 hours of light exposure each day from 7 a.m. to 7p.m. They were given free access to water and a purified diet.
  • the compositions of the diet (AsA 0mg/kg, AsA-free diet) and with or without 2% AsA (Wako-Fujifilm, Japan) contained water. After 1 week or 2 weeks of feeding, they were anesthetized with isoflurane and sampling liver with a perfusion fix of 4% Paraformaldehyde (Nacalai, Japan).
  • TMDU Animal care and experimental procedures were approved by the Animal Research Committee of TMDU (approval number A2023554).
  • Human samples [0375] All human samples including human foreskin fibroblasts and human liver samples were collected with informed consent from the corresponding patients and the approval of the Institutional Review Board (IRB) of the Cincinnati Children's Hospital Medical Center.
  • mGULO editing [0376] The murine GULO (L-gulonolactone oxidase) cDNA sequence was retrieved from NCBI. The 5′ linker and Kozak sequence were added to the start of the sequence, with HA tags to the end of the sequence. Additionally, a P2A-mCherry was added after the HA tag and a 3′ linker to the very end.
  • the custom gene was then synthesized and cloned into the pAAVS1-NDi- CRISPRi (Gen1) PCSF#117 vector using the restriction sites AflII and AgeI.
  • the vector has a TetON system and a Neor selectable marker was then inserted using the Gateway technology.
  • mGULO iPSC generation and general iPSC maintenance [0377] Experiments using iPSCs were approved by the Ethics Committees of Cincinnati Children's Hospital Medical Center. The 1383D6 used in this study was kindly provided by CiRA, Kyoto University.
  • the iPSCs 72.3, and 72.3-GFP were obtained from patient foreskin fibroblasts and reprogrammed into iPSC by Cincinnati Children’s Hospital Medical Center pluripotent stem cell core.
  • the PCSF#117 vector with the modified mGULO sequence was then inserted into the AAVS1 locus of the 72.3 iPSC cell line using a lentiviral mediated CRISPR/Cas9.
  • the correct clones were then selected using G418.
  • the surviving clones were then verified for correct insertion, random insertion and copy number using PCR, and verified by DNA sequencing.
  • the iPSCs were then maintained on Laminin iMatrix-511 Silk (REPROCELL USA Inc.) coated cell culture plates and maintained with StemFit Basic04 Complete Type (Ajinomoto Company) media with Y-45127632 (Stem Cell Technologies). The cells were passaged every 7 days with Accutase (Sigma-Aldrich) until passage 40 (p40). Organoid generation [0378] The p40 cells were plated on a 24 well plate coated with Laminin iMatrix-511 Silk at a density of 2 ⁇ 105 cells/well and maintained with Stemfit media with Y-27632. On Day 2, the media was replaced with fresh Stemfit.
  • the cells were treated with RPMI 1640 (Gibco) media mixed with Activin A (Shenandoah Biotechnology) and BMP4 (R&D Systems) to generate definitive endoderm.
  • RPMI 1640 Gibco
  • Activin A Shaenandoah Biotechnology
  • BMP4 R&D Systems
  • the media was replaced with RPMI, Activin A and 0.2% dFBS (HyClone) which was changed to 2% dFBS on day 5.
  • the cells were fed with FGF-4 (Shenandoah Biotechnology) and CHIR99021 (PeproTech) in Adv.
  • DMEM Advanced DMEM/F-12 (Gibco) with B27 (Gibco), N2 (Gibco), 10mM HEPES (Gibco), 2 mM L-glutamine (Gibco), and GA-1000 (Lonza)
  • the cells were dissociated into a single cell suspension using Accutase treatment. This single cell suspension was then mixed with 50% Matrigel and 50% EP media and plated as 50 ul drops in a 6-well plate. These cells were fed with EP media every 48 hrs for 4 days to generate organoids. These organoids were then treated with Adv.
  • DMEM and 2 ⁇ M RA (Sigma-Aldrich) every 48 hours for 4 days to specify the hepatic lineage.
  • the organoids were then fed with HCM (Lonza), HGF (PeproTech), Oncostatin M (PeproTech) and Dexamethasone (Sigma-Aldrich) every 3-4 days to generate HLOs and passaged as necessary.
  • HCM Nonza
  • HGF PeproTech
  • Oncostatin M PeproTech
  • Dexamethasone Sigma-Aldrich
  • the bilirubin treatment was maintained with every media change onwards by keeping the cells at 37°C in 5% CO2 with 95% air.
  • the Z1-HLOs (Zone 1) were maintained with Dox starting at Day 17 and co- cultured on Day 22 with the bilirubin treated 72.3-GFP (GFP+) Z3-HLOs (Zone 3) in a 1:1 ratio at higher density, i.e.2 ⁇ the number of organoids with continuous bilirubin and Dox treatment in HCM to obtain chimeric organoids that had dual zonal characteristics.
  • These mZ-HLOs and HLOs were visualized by using fluorescent microscopy BZ-X810 (Keyence, Osaka, Japan) and harvested for downstream analysis.
  • Live cell imaging and functional assay [0380] For live imaging of organoids, the CellDiscoverer 7 (Zeiss) was used to image every 30 min for 7 days. Observing organoid fusion necessitated looking at the cytoskeleton, the HLOs were incubated with 2 drops/ml NucBlue (Hoechst 33342) (Invitrogen, R37605) and 1 ⁇ M Cytoskeleton Kit (SiR-Actin and SiR-Tubulin) (Cytoskeleton Inc., CYSC006) and imaged over 5 days.
  • NucBlue Hoechst 33342
  • SiR-Actin and SiR-Tubulin Cytoskeleton Inc., CYSC006
  • Reads were aligned to human genome assembly hg38 and quantified using the quasi-mapper Salmon (v1.8.0). Gene-expression analysis was performed using the R Bioconductor package DESeq2 (v1.36.0). The read count matrix was normalized by size factors, and a variance stabilizing transformation (VST) was applied to the normalized expression data. The data was visualized using clusterProfiler (v4.4.2) and pheatmap (v1.0.12) packages.
  • VST variance stabilizing transformation
  • the ACSS2 (Abcam, ab133543) and ALDH6A1 (Abcam, ab12618) antibody was crosslinked to Protein A Dynabeads (Invitrogen, 10002D).
  • the ChIP assay was then carried out on extracts from organoids as described above.
  • DNA was eluted with elution buffer supplemented with 10 mM DTT.
  • the eluate was then diluted in 2 volumes of wash buffer supplemented with 1x Protease Inhibitor Cocktail and 1 mM DTT.
  • the 2nd ChIP assay was then carried out as described above.
  • DeepTools (v3.5.1) was used to generate BigWig files which were visualized using IGV (v2.13.0). Peaks were identified using MACS2 (v2.2.7.1) and annotated with ChIPseeker (v1.32.0) to generate BED and BEDgraph files for visualization with IGV.
  • DiffBind (v3.6.1) was used to call statistically significant differential peaks after normalization and differential regions were selected based on DESeq2 method FDR531 corrected q-value of 0.05. Heatmap and profile plots were generated with EnrichedHeatmap (v1.26.0). The functional analyses of GO term and KEGG pathway were performed using clusterProfiler.
  • snRNA-seq De novo motif analysis was then carried out on centered 100 bp regions from the peak summits using MEME Suite (v5.4.1). snRNA-seq and analysis [0383] For snRNA-seq, 25-30 mg samples were pulverized with liquid nitrogen and nuclei were prepared using Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich). The nuclei were filtered through a 10 ⁇ m filter, sorted, and counted before the library was generated using the Chromium 3′ v3 GEM Kit (10x Genomics, CG000183RevC).
  • the dataset was then analyzed using Seurat v4.2.0 in RStudio v4.1.1. Quality control was then carried out by using filtering parameters where nuclei with features less than 200 and greater than 4000 or more than 0.5 percentage mitochondrial genes were discarded. In the end, 45,223 parenchymal nuclei were isolated out from a total of 120,195 nuclei. The dataset was then normalized, and top 2000 highly variable genes were selected using the ‘VST’ method. The dataset was then scaled, and principal component analysis (PCA) was run for dimensional reduction. Elbow plots and JackStraw plots were then used to determine the number of PCs to be used. The nuclei were then clustered using Louvain algorithm and KNN.
  • PCA principal component analysis
  • Integrated dataset of published human liver organoids and primary liver reference [0384] For integration 8 different protocol-based human PSC-derived liver organoid and 4 primary adult and fetal datasets were collected according to the descriptions in the original publications 28,34-38. Briefly, available data (either raw FASTQ files, count matrices, H5AD, or Cell Ranger outputs such as “filtered_feature_bc_matrix” files) was obtained for each organoid from databases such as GEO, ArrayExpress, and the Human Cell Atlas (HCA). For FASTQ files, we used Cell Ranger to align and quantify the sequencing reads with the same parameters described in the original publication, generating UMI count data. Subsequent data processing was performed in Seurat using default settings.
  • Metadata was curated for all organoid data, including cell barcodes, sample names, cell type annotations, and cell cycle phase.
  • the public organoid data with was normalized and combined with the mZ-HLO data.
  • the top 3,000 variable genes from the primary liver data was identified and applied these to the organoid dataset.
  • Cell type annotations were based on the original publication and assigned into hepatocytes, hepatoblasts, endothelial cells, cholangiocytes, macrophage, mesenchyme, and stellate cells, which were added as new metadata.
  • Seurat RPCA integration the organoid data comprising 29,526 cells and the primary liver data comprising 8,656 cells was integrated.
  • the same configurations were used to integrate the mZ-HLO dataset. After integration, Louvain clustering and re-annotated cell types was performed based on the expression of known marker genes.
  • the integrated organoid data was used as the query and the primary liver data as the reference.
  • the miloR and scrabbitr R packages was used to compute neighborhood graphs, compare neighborhoods based on similar features, and map neighborhood comparison defined by k-NN graph using UMAP embeddings for primary adult and fetal liver dataset.
  • the neighborhood correlations were computed using 3000 highly variable genes that were found in the highly variable genes in either adult or fetal primary liver compared as reference.
  • the transcriptional similarity graph was computed using 30 dimensional nearest neighbors and UMAP embeddings of cells, while other parameters were implemented as default.
  • Adenovirus mediated gene silencing of p300 in vivo The BLOCK-iT adenoviral RNA interference expression system (Invitrogen, Carlsbad, California) was used to construct adenoviral short hairpin RNA (shRNA) for p300 and scrambled shRNA as previously described 62. Rat pups aged ⁇ P0-P1 were then placed on a sterile heating pad, sanitized using isopropyl alcohol and iodine tincture to clean the skin surface.
  • the rat pups were then returned to the mother by rubbing them with the nesting material to prevent pup rejection.
  • the pups were sacrificed, and the livers were harvested at age P5, as most pups died at P7, to be fixed in 4% PFA and stained. Isolation of freshly isolated PHH for benchmarking [0386]
  • a fresh healthy human transplant rejected liver was harvested and cut into 1 g pieces. The liver pieces were chopped into a fine paste like consistency and submerged in Liver Digest Medium (Gibco, 17703034) for 15 min at 37 °C to isolate single cells 44.
  • the cells were then passed through a 100 ⁇ m strainer on ice and centrifuged at 50 ⁇ g for 3 min at 4 °C.
  • the H40 fraction was isolated by passing the cells through a 40 ⁇ m strainer on ice again, while the H20 fraction was isolated by passing the cells through a 20 ⁇ m strainer on ice.
  • the isolated cells were immediately used for gene expression profiling using RT-qPCR and epigenetic profiling by ChIP.
  • Organoid transplantation [0387] The HLOs and mZ-HLOs were harvested right after co-culture on Day 23 and dissociated into chunks by repeated pipetting, washed with PBS and resuspended with HCM containing 2% FBS and CEPT cocktail to increase viability.
  • the RRG rats were fully anesthetized, and an exploratory laparotomy was performed via midline incision followed by bowel evisceration to expose the portal triad, including the portal vein.
  • the bile duct was ligated using nylon suture proximally and distally.
  • the HLOs and mZ-HLOs (5 ⁇ 10 6 cells) were then transplanted orthotopically at the base of the liver in close proximity to the portal vein using TISSEEL fibrin glue (Baxter), for stabilization.
  • TISSEEL fibrin glue Baxter
  • the HLOs were injected through the portal vein or inferior vena cava using a 32G 1 inch needle in a 200 ⁇ L infusion.
  • Bleeding was controlled by application of a bulldog clamp distal to the site of injection. The incision was then closed in two layers with 5-0 vicryl coated surgical sutures (Ethicon) and GLUture (Zoetis), and Buprenorphine (0.1 mg/kg) was administered as an analgesic. The animal was maintained on ad libitum dox chow until the day of harvest. Blood was collected regularly by the retro-orbital method as needed before the liver was harvested on Day 30. For antegrade and retrograde intravenous transplantation, the RRG rats were fully anesthetized, and an exploratory laparotomy was performed for transplantation through two different routes: portal vein (antegrade) or IVC (retrograde).
  • portal vein antegrade
  • IVC retrograde
  • a 32G 1 inch needle was used to inject 3 ⁇ 10 3 organoids (roughly 5 ⁇ 10 5 cells) in a 200- ⁇ L infusion into the portal vein.
  • Bleeding was controlled by application of a bulldog clamp distal to the site of injection. This also assisted with preferential flow into the liver.
  • Excessive blood loss was controlled by application of a SURGICEL SNoW Absorbable Hemostat (Ethicon). The incision was then closed in two layers with 5-0 vicryl-coated surgical sutures (Ethicon) and GLUture (Zoetis) and buprenorphine (0.1 mg/kg) was administered as an analgesic.
  • Table 2 List of antibodies used for immunostaining (IC), and ChIP-seq (ChIP) in 1110 organoid experiment Antibody Host Source Catalog # Dilution Method EP300 ChIP Rabbit abcam ab14984 5 ug ChIP C C C C C C C C A Anti-Rabbit Alexa Donkey Invitrogen A-21206 1:500 IF Fluor 488 Gene Gene name TaqMan probe (Catalog #) Table 5: List of Custom primers used for ChIP-PCR Orientation Sequence Reverse ALDH6A1 CCTTACGCGGATGTTGAGGT (SEQ ID NO: 4) Orientation Sequence Forward ACSS2 GGAGGTTCTGTGAAGGAAGAAT (SEQ ID NO: 5) Q
  • Protein expression assays [0389] Albumin secretion was measured by collecting 200 ⁇ l of the supernatant from the HLOs cultured in HCM and stored at ⁇ 80°C until use. The supernatant was assayed with Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc) according to the manufacturer’s instructions. For murine GULO expression assay, the organoids were dissociated and washed with PBS.
  • the supernatant and serum were assayed with Bilirubin Assay Kit (Total and Direct, Colorimetric) (Abcam, ab235627) and Bilirubin Assay Kit (Sigma- Aldrich, MAK126) according to the manufacturer’s instructions.
  • Cellular antioxidant levels were measured by harvesting the HLOs, washing in PBS, and plating them into a 96 well assay plate. The levels were then quantitated using Cellular Antioxidant Assay Kit (ab242300) according to the manufacturer’s instructions.
  • the nitrogen related metabolite assays were carried out by harvesting the HLOs, washing in PBS, and plating them into a 96 well assay plate.
  • the glutathione, ammonia, urea, glutamine, glucose and triglyceride levels were then assayed by using the corresponding glutathione, ammonia, urea, glutamine, glucose and triglyceride assay kits (Abcam ab65322, ab83360, ab83362, ab197011, ab65333, and ab65336) according to the manufacturer’s instructions.
  • Metabolic activity assays [0391] CYP3A4 and CYP1A2 assays were performed by harvesting the HLOs, washing in PBS, plating them into a 96 well assay plate, and treating them with rifampicin and omeprazole respectively for 24 hrs.
  • Dual Luciferase Assay System (amsbio, 60683-2) for Notch1 assay was then used to measure the Firefly luciferase activity and compared to Renilla luciferase activity to normalize the transfection efficiency.
  • the luciferase assay indicates Notch activity using a CSL (CBF1/RBPJK) luciferase reporter vector, Notch pathway responsive reporter.
  • Notch1 is cleaved by gamma secretase and NICD is released into the nucleus which is detected by the luciferase reporter as active Notch signaling.
  • the nitrogen metabolism related enzyme assays were carried out by harvesting the HLOs, washing in PBS, and plating them into a 96 well assay plate.
  • the GS activity and GST activity levels were then assayed by using the Glutamine Synthetase Activity and Glutathione S Transferase Activity, Lipase Activity, and Glucokinase Activity Assay Kit (abcam ab284572, ab65325, ab102524, ab273303) according to the manufacturer’s instructions.
  • the apoptosis assay was carried out by lysing the HLOs and assaying the lysate with a Caspase-3 Assay Kit (Colorimetric) (ab39401) according to the manufacturer’s instructions.
  • rat serum was assayed with AST and ALT Activity Assay Kit (Sigma-Aldrich, MAK055 and MAK052) and quantified by a BioTek® Synergy H1 plate reader.
  • Zonal toxicity assay [0392] The HLOs were induced with 3-MC (50 ⁇ M) for alcohol degradation and drug conjugation metabolism 24 hours prior to the toxicity assay. After induction a toxic dose of the zone 1 toxin allyl alcohol (200 ⁇ M) was supplemented for 2 hr at 37 °C. On the other hand, a toxic dose of the zone 3 toxin acetaminophen (10 mM) was supplemented for 4 hr at 37 °C on different batches.
  • the toxins were incubated for 1 hr at 37 °C. Subsequently, organoids were supplied with fresh media and the organoids were fixed after 24 hr in 4% PFA and stained. The cultures were then tested for Caspase 3 activity using the cellular lysate collected from the organoid culture. Separately, the cultures were also tested for viability using CellTiter-Glo Luminescent Cell Viability Assay. Plasticity assay [0393] The HLOs were treated with Dox starting at Day 17 and some organoids were fixed in 4% PFA before Dox withdrawal on Day 20.
  • RNA-seq, ChIP-seq and snRNA-seq data reported in this paper have been deposited to NCBI Gene Expression Omnibus (GEO) with the following accession number: GSE222654. Publicly available datasets were downloaded from the following sources: Camp et al. (GSE96981), Guan et al. (GSE154883), Harrison et al.
  • Glutaminase2 Glutaminase2
  • GS Glutamate Synthetase
  • mGULO murine Gulo
  • Presumptive mGULO was paralleled by the mCherry expression. In ascorbate-deprived conditions, the iPSCs lacking mGulo transgene failed to differentiate into HLO properly, whereas the mGULO containing iPSCs generated healthy viable HLOs without ascorbate (Fig.1B and Fig.2B). [0398] The expression of dox-induced mGULO contributed to higher cellular antioxidant concentration and lower ROS levels when compared to exogenous ascorbate supplements in medium (Fig.1C-E).
  • ELISA assay showed that the Z1-HLOs synthesized much higher levels of albumin compared to control HLOs and primary human hepatocytes (PHH), a characteristic prominent in periportal hepatocytes (Fig. 1G). More importantly, the Z1-HLOs expressed CPS1 and ACSS2 proteins, as verified by immunofluorescence and compared to control HLOs (Fig. 1H). Overall, these data demonstrated that functional mGULO induction with elevated intracellular ascorbate primed differentiation into CPS1+ periportal hepatocyte in Z1-HLOs.
  • EXAMPLE 3 Extracellular bilirubin specifies GLUL+ in Z3-HLOs [0400]
  • a separate batch of HLOs expressing constitutive GFP was treated with bilirubin at around Day 20, and 1 mg/L was found to be the concentration that enabled the greatest cellular survival in the HLOs (Fig.2A and Fig.3A-B).
  • Morphological analysis of the organoids revealed that they were more compact resulting in a smaller irregular lumen (Fig. 2C and Fig.3C-D).
  • the bilirubin-treated human liver organoid expressed more zone 3 genes, such as ALDH6A1 (Aldehyde dehydrogenase 6A1), OATP2 (Organic anion transporter polypeptide 2), and GHR (Growth hormone receptor), hereafter defined as Z3-HLOs (Fig. 3E). Additionally, the Z3-HLOs expressed zone 3-specific ALDH1A2 (Aldehyde dehydrogenase 1A2), GLUL, HIF1A, and SREBF1 (Sterol regulatory element-binding transcription factor 1), which were higher when compared to control HLOs and similar to PHH expression (Fig.2C).
  • ALDH6A1 Aldehyde dehydrogenase 6A1
  • OATP2 Organic anion transporter polypeptide 2
  • GHR Greenth hormone receptor
  • pan-hepatocyte marker genes such as A1AT, HNF4A, and CEBPA
  • Z3-HLOs expressed more pericentral specific genes, such as GHR, BCHE (Butyrylcholinesterase), and RCAN1 (Regulator of calcineurin 1)
  • Z1-HLOs expressed ACSS2, SLBP (Stem-loop binding protein), and RND3; however, they lacked expression of widespread markers, such as ARG1 and interzonal markers, such as AKR1C1 (Aldo-keto reductase 1C1) and APOM (Apolipoprotein M) (Fig.3H).
  • bilirubin-induced Notch and Ezrin signaling activated cytoskeletal rearrangement to induce fusion.
  • These fused organoids expanded canalicular connectivity as was observed in a fluorescently labelled bile acid analogue transport assay (Fig.4H). Quantification of self-assembly efficiency indicated the preferential fusion efficiency in the cell line-derived HLOs as follows: Z3- Z3 HLO (60%), Z3-Z1 HLO (35%), and Z1-Z1 HLO (5%) (Fig.4I). [0406]
  • the self-assembled dual organoids generated from the bilirubin-treated Z3-HLOs and the dox-treated Z1-HLOs were then further characterized (Fig.2F).
  • Zone 1, Zone 2, Zone 3, and pan-liver markers e.g., ACSS2, ALDH6A1, AKR1C1, and HNF4A
  • Fig.2G pan-liver markers
  • the mZ-HLOs also expressed several pan hepatocyte markers: ALB, HNF1A, A1AT, CEBPB (CCAAT/enhancer-binding protein beta), PROX1, HNF4A, and TUBA1A (Tubulin alpha-1A) (Fig. 2H). They maintained a tubular structure, as indicated by the basal marker CTNNB1, and a continuous lumen, as indicated by the ZO-1 (Zonula occludens 1) apical marker (Fig.5C).
  • Immunofluorescence also confirmed protein expression of three distinct regions within the newly generated mZ-HLOs: ARG1 (Arginase 1)-positive region, TERT-positive region, and AHR (Aryl hydrocarbon receptor)-positive region, consistent with periportal, interzonal and pericentral zonal marker expression (Fig.5C), indicating the emergence of multi-zonal properties.
  • Other zone-specific liver markers such as apical MRP2 (Multidrug resistance-associated protein 2) and nuclear SLBP indicate the variable hepatic characters, while only a tiny proportion of the cells were found to express the cholangiocyte marker CK7 (Keratin-7) (Fig.5C).
  • the mCherry+ zone 1 side expressed the zone 1 markers TET1 and GLS (Glutaminase), while the GFP+ zone 3 side exhibited expression of the zone 3 markers ALDH6A1, GHR, and AR (Androgen receptor) (Fig. 2H).
  • These stains were consistant with the spatial patterning of TUBA1A, CK7, SLBP, GLS, ALDH6A1, ARG1, TERT, AHR, and MRP2 in human neonatal liver tissue (Fig.5D).
  • Hepatoblasts are the most immature population in mZ-HLO and enriched for fetal markers such as AFP, and other growth mitogenic markers such as IGF2 (Insulin-like growth factor 2), and MAP2K2 (mitogen-activated protein kinase 2), which regulate the growth and differentiation of the cells.
  • Interzonal hepatocytes are known for expressing glutathione and DNA repair enzymes and as such express TERT and GSS.
  • periportal hepatocytes expressed GLS2, CPS1, OTC, ACSS2 and ARG1, while the pericentral hepatocyte population expressed GLUL, CYP2E1, HIF1A, ALDH1A2, ALDH6A1, and AR (Fig. 6C-E and Fig. 7B).
  • TAT, HAMP, and CYP3A4 are localized in the periportal, interzonal and pericentral hepatocyte populations as cross-referenced by primary liver spatial transcriptomic dataset.
  • Fig. 7A and Fig. 8A-C Cholangiocyte marker KRT7 was expressed at a minimal level in the hepatoblast population only (Fig.
  • the periportal cells were involved in gluconeogenesis, lipid and glutamine catabolism, ROS and oxygen response, and was enriched for Notch signaling. On the other hand, xenobiotic and pigment metabolism, glutamine biosynthesis, and Wnt signaling were more enriched in the pericentral population (Fig. 6D). Additionally, the mZ253 HLO dataset was integrated with multiple previously published adult and fetal hepatocyte snRNAseq datasets (Andrews, T. S. et al. Single- Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatology Communications 6, 821-840 (2022); Aizarani, N.
  • RNA velocity and pseudotime analysis which is based on the kinetics of the splicing rate of mRNA and the expression of each gene (Fig.7A and Fig.9A-B). Both methods predicted that the zonal hepatocytes originated from hepatoblasts through the interzonal hepatocytes population (Fig. 7A and Fig. 9C).
  • GSEA Gene Set Enrichment Analysis
  • EP300 regulation executes zonal transcription [0414]
  • EP300 E1A-associated protein p300
  • EP300 is a histone acetyltransferase that acetylates enhancer regions and activates transcription leading to hepatoblast differentiation.
  • EP300 marks poised and active enhancers and activates expression of zonal genes.
  • EP300 ChIPseq analysis revealed that the presently described mZ-HLOs had increased binding of EP300 at putative enhancer sites (7875 binding sites) as compared to controls and singly treated HLOs (2852, 4891 and 5219 binding sites) (Fig.10A).
  • Pan liver markers, such as HNF4A and CTNNB1 ( ⁇ -catenin) had EP300 peaks upstream of the TSS (Transcription Start Site) in all the samples similar to the PHH dataset in Smith et al. (Fig. 10B-C).
  • zone-specific genes such as ACSS2 (zone 1), ALDH6A1 (zone 3), and HPR (Haptoglobin-related protein) (zone 2)
  • ACSS2 zone 1
  • ALDH6A1 zone 3
  • HPR Haptoglobin-related protein
  • zone 2 zone 2
  • SLBP zone 1
  • GHR zone 3
  • AKR1C1 zone 2
  • Fig.10D-F GSEA analysis of the peaks revealed enrichment for mixed zonal processes in the mZ-HLOs while the Z1- and Z3-HLOs had zone specific biological process enrichment (Fig. 10G-H and Fig. 11D).
  • RNAseq dataset was then integrated with the ChIPseq dataset to look at the regulation of upregulated RNA in each condition (Fig. 11F).
  • the Z3-HLOs exhibited HIF1A, TBX3 (T-box transcription factor TBX3), and AHR as the top motifs co-occupied by EP300, while Z1-HLOs revealed TET1, NRF1 (Nuclear respiratory factor 1), and TFEB (Transcription factor EB) as the top motifs (Fig.11F).
  • the p300 shRNA mediated EP300 silencing resulted in impaired liver development where the hepatocytes underwent over proliferation and under differentiation causing portal and central vein ambiguity, i.e., zonal impairment.
  • PROX1 nuclear specificity or expression was lost in some hepatic cells, while for ARG1 and GLUL the zone-specific expression was altered to spotty non-zone-specific expression (Fig. 12A-B).
  • the Z1- and Z3-HLO gene expression profile was compared with size fractionated freshly isolated primary human hepatocytes.
  • the Z1-HLOs had similar expression of ACSS2, ALB, ASL, CPS1, and OTC when compared to ⁇ 20 ⁇ m periportal hepatocytes (H20) (Fig.12C).
  • the Z3-HLOs expressed ALDH1A2, ALDH6A1, GLUL, HIF1A, and SREBF1 similar to the ⁇ 40 ⁇ m pericentral hepatocytes (H40) (Fig.12D).
  • the mZ-HLOs were found to express urea cycle genes, such as CPS1, OTC, and ARG1, while expressing detoxification genes, such as GSTA2 (Glutathione S-transferase A2), ALDH1A2, and GLUL as well in response to 10mM NH4CL when compared to PHH and control HLOs (Fig. 13A-C).
  • BSO buthionine sulphoximine
  • the mZ-HLOs therefore metabolized the highest amount of ammonia, while BSO inhibits ammonia removal (Fig. 13E).
  • the Z1-HLO exhibited zone 3 features as evident in switching of CPS1 and TET1 expression to GLUL and NR3C1 expression at Day 25 following doxycycline withdrawal and persistent bilirubin treatment starting at Day 20 when the zone1 features started to appear (Fig.15E).
  • This switch was also observed in reduced expression of ACSS2 and increased expression of ALDH1A2 following doxycycline withdrawal and bilirubin treatment (Fig. 15F).
  • EXAMPLE 8 Transplantating of mZ-HLOs after bile duct ligation rats [0424] To determine multi-zonal functionality in vivo, post-transplant mZ-HLO metabolic performance was evaluated in ammonium and bilirubin removal relative to a singular zonal HLO system. In choosing a model, bile duct ligation (BDL) was adopted because BDL in rats exhibits hyperammonemia and hyperbilirubinemia, leading to the progression of total hepatic dysfunction. The ligation results in large accumulation of bilirubin and ammonia in the serum that accelerates liver injury.
  • BDL bile duct ligation
  • mZ-HLOs exhibited dual zonal functionality
  • fibrin glue as a scaffold in bile duct ligated Il2rg-deficient, Rag1-deficient rats, which were observed for 30 days (Fig.16A).
  • transplanted rats exhibited approximately 350 ng/ml peak human albumin in their serum at Day 20, thus indicating functional engraftment (Fig. 16C).
  • transplanted mZ- HLOs were observed to invade into the hepatic parenchyma and retained their structure as indicated by the TUBA1A and ASGR1 stain (Fig.17A-B).
  • TUBA1A and ASGR1 stain Fig.17A-B
  • These partially integrated mZ-HLOs retained their mCherry and GFP expression which also expressed ARG1 and GLUL (Fig. 17C).
  • the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
  • An embodiment for producing a multi-zonal liver organoid comprising: co-culturing one or more first human liver organoid (HLO) with one or more second HLO, wherein the second HLO is a bilirubin-treated HLO; and contacting the co-cultured first HLO and the bilirubin-treated second HLO with bilirubin for a period of time to provide a liver organoid with at least one phenotypically distinct hepatocyte subpopulation.
  • HLO human liver organoid
  • second HLO is a bilirubin-treated HLO
  • contacting the co-cultured first HLO and the bilirubin-treated second HLO with bilirubin for a period of time to provide a liver organoid with at least one phenotypically distinct hepatocyte subpopulation.
  • the first HLO comprises an ascorbate-treated HLO.
  • Embodiment 1 or 2 wherein the first HLO is a functional L-gulonolactone oxidase (GULO)- expressing HLO. 4. Any of embodiments 1-4, wherein the first HLO is treated with doxyclcine. 5. Any of embodiments 1-5, wherein the first HLO and/or the bilirubin-treated second HLO comprises an immature HLO. 6. Any of embodiments 1-5, wherein the first HLO comprises an ascorbate-enriched progenitor cell population, and wherein the bilirubin-treated second HLO comprises a bilirubin-enriched progenitor cell population. 7.
  • each phenotypically distinct hepatocyte subpopulation comprises a zone 1 (Z1) or zone 1-like (Z1-like) hepatocyte subpopulation, a zone 2 (Z2) or zone 2-like (Z2-like) hepatocyte subpopulation, or a zone 3 (Z3) or zone 3-like (Z3-like) hepatocyte subpopulation.
  • Z1-like hepatocyte subpopulation comprises a zone 1 (Z1) or zone 1-like (Z1-like) hepatocyte subpopulation, a zone 2 (Z2) or zone 2-like (Z2-like) hepatocyte subpopulation, or a zone 3 (Z3) or zone 3-like (Z3-like) hepatocyte subpopulation.
  • any of embodiments 8-10 wherein the period of time sufficient to develop two or more phenotypically distinct hepatocyte subpopulations, to self-assemble into multizonal HLOs, and/or to fuse into interconnected dual organoids is equal to or at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. 12. Any of embodiments 1-11, wherein first HLO comprises a Z1 or Z1-like, Z2 or Z2-like, or Z3 or Z3-like hepatocyte subpopulation, and/or wherein the bilirubin-treated HLO comprises a Z1 or Z1-like, Z2 or Z2-like, or Z3 or Z3-like hepatocyte subpopulation. 13.
  • the multi-zonal liver organoid comprises two or more hepatocyte subpopulations.
  • the two or more hepatocyte subpopulations comprise a Z1 or Z1- like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte subpopulation; or a Z1 or Z1-like hepatocyte subpopulation and a Z2 or Z2-like hepatocyte subpopulation; or a Z2 or Z2-like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte subpopulation.
  • the two or more hepatocyte subpopulations comprise a Z1 or Z1- like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte subpopulation; or a Z1 or Z1-like hepatocyte subpopulation and a Z2 or Z2-like hepatocyte subpopulation; or a Z2 or Z2-like hepatocyte subpopulation and a Z3 or Z3-like hepatocyte sub
  • the multi-zonal liver organoid comprises three or more hepatocyte subpopulations.
  • Embodiment 16 wherein the three or more hepatocyte subpopulations comprise a Z1 or Z1- like hepatocyte subpopulation, a Z2 or Z2-like hepatocyte subpopulation, and a Z3 or Z3-like hepatocyte subpopulation. 18.
  • any of embodiments 1-17, wherein the liver organoid comprises a tubular structure with a single lumen. 19. Any of embodiments 1-18, wherein the liver organoid does not contain hematopoietic tissue and/or acquired immune cells. 20. Any of embodiments 1-19, wherein, during the co-culturing, the concentration of bilirubin is maintained continuously. 21. Any of embodiments 1-20, wherein, during the co-culturing, the concentration of bilirubin is refreshed through addition of exogenous bilirubin during every media change. 22. Any of embodiments 1-21, wherein, during the co-culturing, the concentration of bilirubin is maintained continuously at a level less than or equal to about 5 mg/L. 23.
  • the bilirubin concentration during the co-culturing step is maintained continuously at about 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L, 2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, or 3 mg/L; preferably at about 1 mg/L.
  • any of embodiments 1-25 wherein the first HLO and the bilirubin-treated second HLO are seeded for co-culturing at a density of greater than about 1 ⁇ 10 4 cells/well, greater than about 0.5 ⁇ 10 5 cells/well, greater than about 1 ⁇ 10 5 cells/well, greater than about 2 ⁇ 10 5 cells/well, greater than about 3 ⁇ 10 5 cells/well, greater than about 4 ⁇ 10 5 cells/well, greater than about 5 ⁇ 10 5 cells/well, or higher.
  • 27 Any of embodiments 1-26, wherein the first HLO and the bilirubin-treated second HLO are seeded for co-culturing at a density of greater than about 50-5000 organoids per well; preferably about 500-2000 organoids per well.
  • the functional GULO-expressing HLO is produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of a heterologous expression system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells under conditions to induce expression from the heterologous expression system, to provide the functional GULO-expressing HLO. 30.
  • any of embodiments 1-29, wherein the functional GULO-expressing HLO is produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of an inducible system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with an induction agent, to provide the functional GULO-expressing HLO. 31.
  • the functional GULO-expressing HLO is produced by: genetically modifying a progenitor cell population with a functional GULO protein and/or a gene or mRNA, or both, that encodes for the functional GULO protein, wherein the functional GULO protein and/or a gene or mRNA, or both, are under the control of an tetracycline inducible system; culturing the genetically modified progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with doxycycline, to provide the functional GULO- expressing HLO. 32.
  • the functional GULO protein is a Rodentia GULO, preferably a murine GULO (mGULO).
  • the culturing the posterior foregut cells under conditions to induce expression from the heterologous expression system occurs on or about day 17 of culture of the progenitor cell population. 35.
  • any of embodiments 29-34, wherein the functional GULO-expressing HLO is engineered with the gene that encodes for the functional GULO protein using CRISPR.
  • Any of embodiments 29-35, wherein the gene or mRNA, or both, that encodes for the functional GULO protein is introduced to the functional GULO-expressing HLO by transfection.
  • Any of embodiments 1-36, wherein the bilirubin-treated HLO is produced by: culturing a progenitor cell population to form posterior foregut cells; and culturing the posterior foregut cells with bilirubin, to provide the bilirubin-treated HLO. 38.
  • the multi-zonal liver organoid has: a) expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; b) expression of one or more Z2-associated genes and/or expresses one or more Z2-associated proteins; c) expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins; and/or d) expression of one or more pan-hepatocyte-associated genes and/or expresses one or more pan-hepatocyte-associated proteins.
  • any of embodiments 1-38, wherein the multi-zonal liver organoid has expression of one or more Z1-associated genes and/or expresses one or more Z1-associated proteins; and expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins. 40.
  • Embodiment 40 wherein the Z3-associated genes and/or Z3-associated proteins function in xenobiotic metabolism, WNT signaling, glycolysis, and/or lipogenesis. 42.
  • Embodiment 40 or 41 wherein the Z1-associated genes and/or Z1-associated proteins function in gluconeogenesis, lipid catabolism, glutamine catabolism, and/or reactive oxygen species (ROS) metabolism.
  • ROI reactive oxygen species
  • the Z2-associated genes and/or Z2-associated proteins function in DNA repair, amino acid metabolism, and/or cell growth.
  • the first HLO has elevated expression of one or more Z1- associated genes and/or expresses one or more Z1-associated proteins; and/or wherein the bilirubin-treated second HLO has elevated expression of one or more Z3-associated genes and/or expresses one or more Z3-associated proteins. 45.
  • the one or more Z1-associated genes are selected from Fumarylacetoacetase (FAH), 4-Hydroxyphenylpyruvate dioxygenase (HPD), Stearoyl-CoA desaturase (SCD), Acyl- coenzyme A synthetase 2 (ACSS2), Argininosuccinate lyase (ASL), Carbamoyl phosphate synthetase I (CPS1), Ornithine transcarbamylase (OTC), Stem-loop binding protein (SLBP), Glutaminase (GLS), and Rho family GTPase 3 (RND3) genes; the one or more Z1-associated proteins are selected from CPS1 and ACSS2; the one or more Z2-associated genes are selected from Glutathione synthetase (GSS), Telomerase reverse transcriptase (TERT), and Aldo-keto reductase family 1
  • FH Fumarylacetoace
  • ROS reactive oxygen species
  • Embodiment 46 wherein the multi-zonal liver organoid has hepatocyte proliferation, hepatocyte differentiation, urea cycle, ammonia removal, glycolysis, bilirubin removal, lipid catabolism, glutamine catabolism and biosynthesis, reactive oxygen species (ROS) catabolism and/or metabolism, oxygen response, xenobiotic metabolism, DNA repair, pigment metabolism, lipogenesis, gluconeogenesis, glutathione, and glutamine metabolic functionality. 49. Any of embodiments 1-48, wherein the multi-zonal liver organoid is enriched for Notch signaling and/or Wnt signaling. 50.
  • ROS reactive oxygen species
  • the multi-zonal liver organoid comprises hepatocytes and additionally comprises one or more additional cell types selected from cholangiocytes, endothelial cells, macrophages, stellate cells, and mesenchyme cells.
  • the multi-zonal liver organoid comprises hepatocytes and additionally comprises cholangiocytes, endothelial cells, macrophages, stellate cells, and mesenchyme cells. 52.
  • the multi-zonal liver organoid comprises two or more cell types selected from pericentral or pericentral-like (Z3 or Z3-like) hepatocytes, periportal or periportal-like (Z1 or Z1-like) hepatocytes, and interzonal or interzonal-like (Z2 or Z2-like) hepatocytes. 53.
  • Embodiment 52 wherein the multi-zonal liver organoid comprises: a) pericentral or pericentral-like (Z3 or Z3-like) hepatocytes, b) periportal or periportal-like (Z1 or Z1-like) hepatocytes, and c) interzonal or interzonal-like (Z2 or Z2-like) hepatocytes.
  • the multi-zonal liver organoid further comprises hepatoblasts.
  • the hepatoblasts are characterized as expressing fetal markers and/or growth mitogenic markers. 56.
  • Embodiment 55 wherein the fetal markers comprise Alpha-Fetoprotein (AFP) and the growth mitogenic markers comprise Insulin-like growth factor 2 (IGF2) and/or mitogen-activated protein kinase 2 (MAP2K2).
  • AFP Alpha-Fetoprotein
  • IGF2 Insulin-like growth factor 2
  • MA2K2 mitogen-activated protein kinase 2
  • 57 Any of embodiments 1-56, wherein the multi-zonal liver organoid is human.
  • the first HLO and the bilirubin-treated second HLO have been differentiated from pluripotent stem cells, optionally embryonic stem cells and/or induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • DE definitive endoderm cells
  • RA retinoic acid
  • 60 An embodiment including a multi-zonal liver organoid, comprising Z1-like (periportal-like) hepatocytes, and Z3-like (pericentral-like) hepatocytes, produced by the method of any of the preceding embodiments.
  • 61 An embodiment including an artificial multi-zonal liver organoid, comprising Z1-like (periportal-like) hepatocytes, and Z3-like (pericentral-like) hepatocytes.
  • 62 Any of embodiments 60 or 61, wherein the hepatocytes self-assemble into the multi-zonal liver organoid, optionally wherein there is an observable and/or measurable boundary between two or more types of hepatocytes.
  • 63
  • any of embodiments 60-62, wherein the multi-zonal liver organoid comprises a tubular structure with a single lumen.
  • the multi-zonal liver organoid of any of claims 60-65 wherein the multi-zonal liver organoid comprises SERPINA1+ hepatocytes, KRT7+ cholangiocytes, PECAM1+ endothelial cells, LYZ+ macrophages, COL1A1+ stellate cells, and CD44+ mesenchyme.
  • GUILO L-gulonolactone oxidase
  • any of embodiments 71 or 72, wherein the three-dimensional artificial multi-zonal liver organoid comprises a tubular structure with a single lumen.
  • Embodiment 75 wherein the hepatocytes self-assemble into the three-dimensional artificial multi-zonal liver organoid.
  • any of embodiments 75 or 76, wherein the three-dimensional artificial multi-zonal liver organoid comprises a tubular structure with a single lumen.
  • 80. Any of embodiments 60-79, further comprising hepatoblasts.
  • composition comprises exogenously provided bilirubin at a concentration of about 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.4 mg/L, 0.5 mg/L, 0.6 mg/L, 0.7 mg/L, 0.8 mg/L, 0.9 mg/L, 1 mg/L, 1.1 mg/L, 1.2 mg/L, 1.3 mg/L, 1.4 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2 mg/L, 2.1 mg/L, 2.2 mg/L, 2.3 mg/L, 2.4 mg/L, 2.5 mg/L, 2.6 mg/L, 2.7 mg/L, 2.8 mg/L, 2.9 mg/L, or 3 mg/L; preferably at about 1 mg/L.
  • any of embodiments 60-85 comprising about 20-40% Z3-like (pericentral-like) cells, about 20-40% Z1-like (periportal-like) cells, about 20-40% hepatoblasts, and about 10-30% Z2-like (interzonal-like) cells.
  • Any of embodiments 60-86 comprising greater than or equal to 10% Z2-like cells, and/or greater than or equal to 20% hepatoblasts.
  • An embodiment including a method of treating a liver-related disease or disorder comprising: transplanting, into a subject having liver dysfunction and/or failure, a multi-zonal liver organoid according to any one of embodiments 60 to 88. 90.
  • Embodiment 89 wherein the multi-zonal liver organoid is according to any one of claims 60- 88. 91.
  • Embodiment 89 or 90 wherein the transplanting comprises: a) ligating a bile duct in a subject; and b) transplanting the multi-zonal liver organoid at base of liver. 92.
  • liver-related disease or disorder comprises one or more types of liver dysfunction and/or failure, hepatitis, viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease, autoimmune liver disease, Wilson’s disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, metabolic dysfunction–associated liver disease (MASLD), MetALD, metabolic dysfunction-associated steatohepatitis (MASH), drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, and/or alcohol- associated liver disease.
  • MASLD metabolic dysfunction–associated liver disease
  • MASH metabolic dysfunction-associated steatohepatitis
  • DILI drug-induced liver injury
  • Embodiment 92 wherein the liver dysfunction and/or failure comprises hyperammonemia and/or hyperbilirubinemia.
  • the metabolic disease comprises nonalcoholic fatty liver disease (NAFLD).
  • NAFLD nonalcoholic fatty liver disease
  • MASH metabolic dysfunction-associated steatohepatitis
  • Embodiment 92, wherein the hepatitis comprises hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and/or autoimmune hepatitis.
  • MASH metabolic dysfunction-associated steatohepatitis
  • 105. An embodiment including a use of the multi-zonal liver organoid according to any one of embodiments 60 to 88, as an in vitro human model system for studying hepatocyte function and developmental divergence; studying liver-related disease; identifying therapeutic targets; and/or identifying therapeutic compounds and/or compositions effective in treating a liver-related disease or disorder.

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Abstract

Sont divulgués des organoïdes hépatiques multi-zonaux dérivés de cellules souches pluripotentes améliorées, ayant deux sous-populations d'hépatocytes ou plus, ainsi que leurs procédés de production, par co-culture d'un premier organoïde hépatique humain avec un second organoïde hépatique humain traité par bilirubine, et la mise en contact des organoïdes hépatiques humains co-cultivés avec de la bilirubine pendant une période de temps suffisante pour que les organoïdes hépatiques humains co-cultivés s'auto-assemblent et fusionnent en organoïdes doubles interconnectés. Le premier organoïde hépatique humain peut être un organoïde hépatique humain enrichi en ascorbate, tel qu'un organoïde hépatique humain exprimant la L-gulonolactone oxidase (GULO) fonctionnelle. Dans des modes de réalisation particuliers, les sous-populations d'hépatocytes peuvent comprendre des sous-populations d'hépatocytes de zone 1 et de zone 3 ou des sous-populations d'hépatocytes de zone 1, 2 et 3. Sont également divulgués des procédés d'étude ou de traitement d'une maladie ou d'un trouble lié au foie à l'aide des organoïdes hépatiques multi-zonaux décrits.
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