In vitro partial reprogramming of hepatocytes

Abstract

Provided herein are methods for facilitating the expansion, reprogramming, and maturation of hepatocytes. The methods include contacting hepatocytes with one or more reprogramming factors to rejuvenate the cells and enhance proliferation.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO. 51540-048WO3

[0003] IN VITRO REPROGRAMMING OF HEPATOCYTES

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates generally to methods of culturing, expanding, and maturing hepatocytes.

[0006] BACKGROUND OF THE INVENTION

[0007] Liver disease (e.g., hepatic disease) is any disease that negatively affects the normal, healthy performance of the liver. The resulting disturbance of liver function causes illness. For example, impaired liver function can result in an accumulation of toxins (e.g., nitrogenous waste compounds) in the blood. These toxins may travel to the brain and affect the nervous system. The CDC reports that 4.5 million Americans have been diagnosed with liver disease. While organ replacement therapy can rescue impaired native liver function, the demand far exceeds availability. A feasible alternative is to implant a population of hepatocytes with therapeutic potential into the subject. However, hepatocytes, such as primary human hepatocytes (PHHs), are in short supply. Therefore, improved methods for culturing and expanding hepatocytes, such as PHH, are needed. Previous studies have identified compositions and methods for use in expanding hepatocytes. However, following initial expansion, continued hepatocyte expansion can slow or deteriorate over time. Accordingly, new methods are needed to produce robust expansion of hepatocytes.

[0008] SUMMARY OF THE INVENTION

[0009] In one aspect, provided herein is a method for producing expanded human hepatocytes. The method includes culturing hepatocytes in contact with an extracellular matrix in the presence of an expansion medium; and contacting the hepatocytes with a polynucleotide (e.g., RNA or DNA) encoding one or more reprogramming factors to produce expanded human hepatocytes.

[0010] In another aspect, featured is a method for culturing human hepatocytes by contacting expanded hepatocytes with a polynucleotide (e.g., RNA or DNA) encoding one or more reprogramming factors to produce expanded human hepatocytes, wherein the expanded hepatocytes were previously cultured in contact with an ECM in the presence of an expansion medium.

[0011] In some embodiments, the polynucleotide is RNA. In other embodiments, the polynucleotide is DNA.

[0012] In some embodiments, the one or more reprogramming factors include one or more transcription factors. For example, the one or more transcription factors may include, Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, Glisl , or a combination thereof. In some embodiments, the one or more transcription factors include Oct3 / 4, Sox2, Klf4, cMyc, Lin28A, and Nanog. In some embodiments, the one or more transcription factors include Oct3 / 4, Sox2, and Nanog.

[0013] In some embodiments, each reprogramming factor is encoded by an RNA.

[0014] In some embodiments, the one or more reprogramming factors are encoded by mRNA, and the contacting step includes transfecting (e.g., via lipid-based transfection) the expanded human hepatocytes with the mRNA. In some embodiments, each reprogramming factor is encoded by an mRNA. In some embodiments, the mRNA includes a modified base (e.g., 5’-methylcytidine or N1-methylpseudouridine). PATENT

[0015] ATTORNEY DOCKET NO. 51540-048WO3

[0016] The mRNA may include multiple modified bases (e.g., 5’-methylcytidine, N1-methylpseudouridine, or a combination thereof).

[0017] In some embodiments, the contacting step includes lipid-based transfection.

[0018] In some embodiments, the polynucleotide (e.g., RNA or DNA) encoding the reprogramming factor is delivered to the hepatocytes via a viral vector (e.g., a lentivirus or retrovirus). In some embodiments, the viral vector is an RNA viral vector. In some embodiments, the viral vector is a DNA viral vector.

[0019] In some embodiments, the reprogramming factor causes expression of one or more pluripotency- associated genes. In some embodiments, the reprogramming factor causes an epigenetic modification in the hepatocytes. In some embodiments, the epigenetic modification results in a change in expression pattern of one or more genes in the hepatocytes. In some embodiments, the reprogramming factor causes a chromatin modification in the hepatocytes.

[0020] In some embodiments, the human hepatocytes are primary human hepatocytes or expanded human hepatocytes.

[0021] In some embodiments, the hepatocytes are genetically engineered hepatocytes. In some embodiments, the genetically engineered hepatocytes are hypoimmunogenic hepatocytes.

[0022] In some embodiments, the culturing step includes expanding plated cells (step PO) prior to contacting the hepatocytes with the polynucleotide (e.g., RNA or DNA).

[0023] In some embodiments, the culturing step includes expanding plated cells (step PO) prior to contacting the hepatocytes with the polynucleotide (e.g., RNA or DNA). In some embodiments, cells are plated and cultured in the presence of Chroman 1 , Emricasan, Polyamine, and Trans-integrated stress response inhibitor (trans-ISRIB) (CEPT) cocktail.

[0024] In some embodiments, the PO step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 ,

[0025] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the PO step has a duration of 13 days.

[0026] In some embodiments, the polynucleotide is RNA.

[0027] In some embodiments, the contacting step includes a first passage of expanded cells (step P1). In some embodiments, the P1 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0028] In some embodiments, the method further includes a second passage of expanded cells (step P2). In some embodiments, the P2 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2,

[0029] 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 PATENT

[0030] ATTORNEY DOCKET NO. 51540-048WO3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0031] In some embodiments, the method further includes a third passage of expanded cells (step P3). In some embodiments, the P3 step has a duration of 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0032] In some embodiments, the method further includes a fourth, fifth, sixth, seventh, eighth, ninth, or tenth passage of expanded cells (step P4-P10).

[0033] In some embodiments, cells are plated and cultured in the presence of CEPT cocktail, e.g., during any one of P1 -P10.

[0034] In some embodiments, the polynucleotide is DNA. In some embodiments, the DNA includes an inducible promoter and one or more open reading frames of the one or more reprogramming factors, wherein the inducible promoter is operably linked to the one or more open reading frames of the one or more reprogramming factors.

[0035] In some embodiments, the contacting step includes a first passage of expanded cells (step P1). In some embodiments, the P1 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light inducible promoter) on day 1 for a total of 1 day. In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light inducible promoter) daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0036] In some embodiments, the method further includes a second passage of expanded cells (step P2). In some embodiments, the P2 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light inducible promoter) on day 1 for a total of 1 day. In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light PATENT

[0037] ATTORNEY DOCKET NO. 51540-048WO3 inducible promoter) daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0038] In some embodiments, the method further includes a third passage of expanded cells (step P3). In some embodiments, the P3 step has a duration of 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light inducible promoter) on day 1 for a total of 1 day. In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and (a) contacting the hepatocytes with an agent that activates the inducible promoter (e.g., a tetracycline responsive element or a cumate switch) or (b) exposing the hepatocytes to a condition that activates the inducible promoter (e.g., a heat shock promoter or light inducible promoter) daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0039] In some embodiments, the method further includes a fourth, fifth, sixth, seventh, eighth, ninth, or tenth passage of expanded cells (step P4-P10).

[0040] In some embodiments, cells are plated and cultured in the presence of CEPT cocktail, e.g., during any one of P1 -P10.

[0041] In some embodiments, the method further includes contacting the expanded human hepatocytes with a maturation medium, e.g., to mature the expanded hepatocytes in two-dimensional planar culture.

[0042] In some embodiments, the method further includes contacting the expanded human hepatocytes with a maturation medium, e.g., to mature the expanded hepatocytes in three-dimensional aggregates. In some embodiments, the method further includes contacting the expanded human hepatocytes with a maturation medium in the presence of a different cell type to mature the expanded hepatocytes in three- dimensional heterocellular aggregates.

[0043] In some embodiments, the method further includes the maturation step begins immediately following hepatocyte reprogramming. In some embodiments, the maturation step does not begin immediately following hepatocyte reprogramming. In some embodiments, the maturation step has a duration of 1 to 14 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days).

[0044] In some embodiments, prior to the contacting step, the hepatocytes are cryopreserved and thawed.

[0045] In some embodiments, the hepatocytes are engineered to express or knock-out one or more genes.

[0046] In some embodiments, the expanded hepatocytes exhibit enhanced proliferation compared to the initial hepatocytes.

[0047] Also featured is a population of expanded hepatocytes produced by a method of any of the above embodiments.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 is a schematic drawing showing the expansion, partial reprogramming, and maturation of hepatocytes. PATENT

[0050] ATTORNEY DOCKET NO. 51540-048WO3

[0051] FIG. 2 is a schematic drawing showing the timing of an expansion and partial reprogramming protocol with transfection occurring for three days during each passage; Ed = expansion day; Td = transfection day.

[0052] FIG. 3A is a set of phase contract images at 4X magnification during passage P1 (step P1) of untransfected control hepatocytes (top row), transfection control (second row), and hepatocytes expanded for seven days (Ed7) and transfected with Oct3 / 4, Sox2, Klf4, cMyc, Lin28A, and GFP (OSKML-G) for three days (Td3); Ed = expansion day; Td = transfection day.

[0053] FIG. 3B is a set of phase contract images from FIG. 3A at 10X magnification.

[0054] FIG. 4A is a set of phase contract images at 4X magnification following a second passage (P2) of untransfected control hepatocytes (top row), transfection control (second row), and hepatocytes expanded for nine days (Ed9) and transfected with OSKMLG for three days (Td3); Ed = expansion day; Td = transfection day.

[0055] FIG. 4B is a set of phase contract images from FIG. 4A at 10X magnification.

[0056] FIG. 5 is a set of phase contract images at 4X magnification (top row) and 10X magnification (bottom row) following a third passage (P3) of hepatocytes expanded for ten days (Ed10) and transfected with OSKMLG for three days (Td3); Ed = expansion day; Td = transfection day. Untransfected and Transfection control groups could not be passaged after P2 as there were very few cells due to deterioration of expansion / proliferation. Therefore, there are no untransfected and transfection control groups in Passage 3.

[0057] FIGS. 6A and 6B are a set of bar graphs showing fold expansion with reprogramming factors OSKML-G wherein OSKML are reprogramming factors, and G is GFP for transfection tracking purposes. FIG. 6A shows fold expansion relative to initial plating following P1 , P2, and P3. Bars are quantified in the table underneath the graph. FIG. 6B shows cumulative fold expansion for partially reprogrammed cells as compared to untransfected control expansion; P1_Trans Ctrl_3d_Ed7 = Passage 1 , Transfection Control, 3 days of transfection, Expansion day7; P1_OSKMLG_3d_Ed7 = Passage

[0058] 1 ,Oct4,Sox2,KLF4,C_MYC,LIN28A,GFP, 3days of transfection, Expansion day7; P2_Untrans Ctrl_Ed9 = Passage 2, Untransfected Control, Expansion day9; P2_Trans Ctrl_Ed9 = Passage 2, Transfection Control, 3days of transfection, Expansion day9; P2_OSKMLG _3d_Ed9 (P1 -3dPR) = Passage 2,Oct4,Sox2,KLF4,C_MYC,LIN28A,GFP, 3days of transfection, Expansion day 9, Passaged from Passage 1 eHH which was 3 day partially reprogrammed; P3_OSKMLG_3d_Ed10 (P1 , 3dPR, P2-3dPR) = Passage 3, Oct4, Sox2, KLF4, cmYC, LIN28A, GFP, 3 days of transfection, Expansion day 10, Passaged from eHH, which were partially reprogrammed for 3 days each in passage 1 and passage 2.

[0059] FIGS. 7A-7F are a set of bar graphs showing expression of hepatic / hepatic progenitor markers or pluripotent cell markers. FIGS. 7A-7C show hepatic / hepatic progenitor markers HNF4a (FIG. 7A), EPCAM (FIG. 7B), and AFP (FIG. 7C) following partial reprogramming; P0_d13_eHH (ev3, fresh) = Passage 0, day13 expanded cells in expand media version 3, fresh expansion. FIGS. 7D-7F is a set of bar graphs showing expression of pluripotent cell markers Nanog (FIG. 7D), ZFP42 (FIG. 7E), and GDF3 (FIG. 7F).

[0060] FIG. 8 is a set of bar graphs showing hepatocyte secretion of albumin (top panel) and A1 AT (bottom panel). PATENT

[0061] ATTORNEY DOCKET NO. 51540-048WO3

[0062] FIG. 9 is a schematic drawing showing the timing of an expansion and partial reprogramming protocol with transfection occurring for four days of each passage; Ed = expansion day; Td = transfection day.

[0063] FIG. 10 is a set of phase contract images at 4X (left panel) and 10X (right panel) magnification during passage P1 (step P1) of untransfected control hepatocytes (top row), and transfection using different sets of reprogramming factors OSKML (second row), OSKMLN (third row), OSN (fourth row), and three different concentrations of Nanog only (rows 5-7). Low, medium, and high concentrations of Nanog tested were 110.6 ng, 500 ng, and 1000 ng Nanog mRNA per well of 6-well plate, respectively.

[0064] FIG. 11 is a bar graph showing fold expansion relative to initial plating from the cells shown in FIG. 10.

[0065] FIG. 12 is a set of phase contract images at 4X (left panel) and 10X (right panel) magnification during passage P2 (step P2) of untransfected control hepatocytes (top row), and transfection using different sets of reprogramming factors OSKML (second row), OSKMLN (third row), OSN (fourth row), and three different concentrations of Nanog only (rows 5-7).

[0066] FIG. 13 is a bar graph showing fold expansion relative to initial plating from the cells shown in FIG. 12.

[0067] FIGS. 14A and 14B are a set of phase contract images at 4X (FIG. 14A) and 10X (FIG. 14B) magnification during passage P3 (step P3) of untransfected control hepatocytes (top row), and transfection using different sets of reprogramming factors OSKML (second row), OSKMLN (third row), OSN (fourth row).

[0068] FIG. 15 is a bar graph showing fold expansion relative to initial plating from the cells shown in FIGS. 14A and 14B.

[0069] FIG. 16 is a bar graph showing cumulative fold expansion for partially reprogrammed cells as compared to untrasfected control expansion for each set of reprogramming factors following each passage (P1 -P3).

[0070] FIG. 17 is a set of fluorescent micrographs at 20X magnification showing immunostaining of partially reprogrammed cells for HOECHST and HNF4A in partially reprogrammed expanded human hepatocytes (pr-eHH) following partial reprogramming with OSKML after P1 and P2. The percentages of HNF4a positive cells indicated are an average from n=3 images per group.

[0071] Definitions

[0072] As used herein, the terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a component” optionally includes a combination of two or more such components, and the like.

[0073] As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10% in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0074] As used herein, the terms “basal cell culture medium” and “basal medium” refer to a nutrient-rich solution that is used for cultivating cells (e.g., human cells; e.g., hepatocytes) in vitro by maintaining cell survival and function and promoting cell growth. A basal cell culture medium contains amino acids (e.g., PATENT

[0075] ATTORNEY DOCKET NO. 51540-048WO3 glycine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or valine), glucose, inorganic salts (e.g., calcium chloride, potassium chloride, sodium chloride, sodium phosphate, sodium bicarbonate, magnesium sulfate, ferric nitrate, manganous chloride, zinc sulfate, sodium selenite, and / or cupric sulfate), and vitamins or synthetic forms thereof (e.g., ascorbic acid, folic acid, inositol, niacinamide, riboflavin, thiamine, vitamin B5, vitamin B6). A basal cell culture medium may include additional reagents or additives such as stable forms of L-glutamine, an additional buffering agent (e.g., N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)), sodium pyruvate, an antioxidant (e.g., glutathione), a protein such as a recombinant protein (e.g., albumin, transferrin, and / or insulin), a pH indicator (e.g., phenol red), and / or an antibiotic solution (e.g., penicillin and / or streptomycin or gentamicin). Examples of commercially available basal cell culture media are Dulbecco’s Modified Eagle Medium / Ham’s Nutrient Mixture F-12 (DMEM / F-12), Advanced DMEM / F-12, LONZA™ HCM™, William’s E, HepatoZYME-SFM, Takara CELLARTIS® POWER™ Primary HEP, and LIFENET HEALTH® Human Hepatocyte media.

[0076] As used herein, the terms “comprise,” “comprising,” “comprises,” and “comprised of” are synonymous with “include,” “including,” “includes,” or “contain,” “containing,” “contains,” and are inclusive or open-ended terms that specify the presence of what follows, e.g., a component, and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0077] As used herein, the term “culturing step” refers to the process of expanding and passaging cells. This process encompasses the phase of cell culture in which the number of cells increases by cell division. When the cells have reached, e.g., 80-90% confluence, they may be passaged and seeded onto additional cell culture surfaces. For example, one passage of PHH may require at least 3 days to reach confluence. Cells can be continuously passaged and cultured for 120 days. Alternatively, cells can be passaged until they become transformed or lose hepatic phenotype.

[0078] As used herein, the terms “decrease,” “reduce,” and related variations refer to a lesser amount or a lesser degree. In some embodiments, the terms “decrease” or “reduce” can mean a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by 10% or more (e.g., by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) as compared to a reference level.

[0079] As used herein, the terms “expand,” “expands,” “expanding,” and “expansion” refer to an increase in the number of what follows, e.g., a population of hepatocytes (e.g., PHHs). An “expansion step” refers to a phase of cell culture in which the number of hepatocytes increases by cell division. As used herein in the context of a plurality of agents that together or collectively “expand” a population of hepatocytes, describes instances in which each agent, individually, may or may not achieve the indicated function, but when the agents are combined, the indicated expansion is achieved.

[0080] As used herein, the term “express” refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. In the context of a gene that encodes a protein product, the terms “gene expression” and the like are used interchangeably PATENT

[0081] ATTORNEY DOCKET NO. 51540-048WO3 with the terms “protein expression” and the like. Expression of a gene or protein of interest in a patient can manifest, for example, by detecting: an increase in the quantity or concentration of mRNA encoding corresponding protein (as assessed, e.g., using RNA detection procedures described herein or known in the art, such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA seq techniques), an increase in the quantity or concentration of the corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), among others), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay described herein or known in the art) in a sample obtained from the patient. As used herein, a cell is considered to “express” a gene or protein of interest if one or more, or all, of the above events can be detected in the cell or in a medium in which the cell resides. For example, a gene or protein of interest is considered to be “expressed” by a cell or population of cells if one can detect (i) production of a corresponding RNA transcript, such as an mRNA template, by the cell or population of cells (e.g., using RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5’ cap formation, and / or 3’ end processing, such as using RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using protein detection procedures described herein); and / or (iv) post-translational modification of the protein product (e.g., using protein detection procedures described herein).

[0082] As used herein, the term “hypoimmunogenic cell” or “hypoimmune cell” refers to a cell that gives rise to a reduced immunological rejection response when transferred into an allogeneic host, e.g., compared to a wild-type or immunogenic cell of the same cell type. In some embodiments, hypoimmunogenic cells do not give rise to an immune response. Thus, “hypoimmunogenic” may refer to a significantly reduced or eliminated immune response when compared to the immune response of a cell prior to immuno-engineering. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof. In other embodiments, a hypoimmunogenic cell has reduced expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell expresses one or more of cluster of differentiation cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof. In some embodiments, a hypoimmunogenic cell overexpresses one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7- H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, PATENT

[0083] ATTORNEY DOCKET NO. 51540-048WO3

[0084] IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and expresses (e.g., overexpresses) one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7- H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof.

[0085] The terms “expression level” or “level of expression” in general are used interchangeably and generally refer to the amount of a marker in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and / or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a posttranslational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).

[0086] The terms “increase,” “enhance,” and related variations are all used herein to mean an elevated amount or elevated degree. In some embodiments, the terms “increase” or “enhance,” can mean an increase of 10% or more (e.g., by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%) as compared to a reference level. In some embodiments, the terms “increase” or “enhance” can refer to an increase of about 2-fold, or about 3-fold, about 4-fold, about 5-fold, about 10-fold, or more, as compared to a reference level.

[0087] As used herein, the term “inhibitor” refers to any compound, natural or synthetic, which can reduce the activity of a target protein or signaling pathway. An inhibitor may attenuate or prevent the activity of a target protein either directly or indirectly. Direct inhibition can be obtained, for instance, by binding to a protein and reducing or eliminating interaction of the protein and an endogenous molecule, such as an enzyme, a substrate, or other binding partner, thereby diminishing the activity of the protein. For instance, an inhibitor may bind an enzyme active site and sterically preclude binding of an endogenous substrate at this location, thus decreasing the enzymatic activity of the protein. Alternatively, indirect inhibition can be obtained, for instance, by binding to a protein that promotes the activity of a target protein by inducing a conformational change or catalyzing a chemical modification of the target protein. For instance, indirect inhibition of a target protein may be achieved by binding and inactivating a kinase that catalyzes the phosphorylation of, and thus activates, the target protein.

[0088] As used herein, the terms “biomarker” or “marker” are used interchangeably herein to refer to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which can be detected by standard methods in the art (or methods disclosed herein) in a sample (e.g., a cell sample, a cell population sample, a culture medium sample, or a tissue sample). Expression of such PATENT

[0089] ATTORNEY DOCKET NO. 51540-048WO3 a marker may be determined to be higher or lower in a population of hepatocytes (e.g., PHHs) that have been expanded and / or matured using the disclosed compositions and / or according to the disclosed methods, as compared to a population of hepatocytes that were not expanded or matured with said compositions or methods.

[0090] As used herein, the term “maturing” or any variation thereof refers to the process of developing cells into a population that is distinct from a progenitor expanded population. For example, a population of hepatocytes that has undergone a maturing step as disclosed herein has a mature hepatocyte phenotype, which may be evaluated by gene expression analysis (e.g., an upregulation of transcripts associated with mature hepatocytes and downregulation of transcripts associated with progenitors or cholangiocytes) and / or hepatocyte functional assays (e.g., urea secretion, CYP3A4 activity).

[0091] As used herein, the term “maturation supplement” refers to a reagent or additive that may be included in a basal cell culture medium for promoting maturation of a cell or a population of cells (e.g., hepatocytes).

[0092] As used herein, the terms “one or more” or “a combination thereof,” in reference to one or more member(s) of a group, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, e.g., >3, >4, >5, >6, or >7, etc., of said members, and up to all said members.

[0093] “Primary cells,” and “primary cultures” are used interchangeably herein to refer to cells and cell cultures that have been harvested from human livers and cryogenically stored immediately without being cultured in vitro for any amount of time. Cells can be harvested from an individual by any convenient method such as biopsy or isolation from whole donated livers. An appropriate solution can be used for dispersion or suspension of the harvested cells. The cells can be used immediately, or they can be stored, frozen, for long periods of time, and then later thawed and reused.

[0094] As used herein, the terms “primary human hepatocytes” and “PHHs” refer to major parenchymal cells in the liver. Specifically, these cells are of human origin and have the capacity to replicate and increase cell number in response to liver injury. It is known in the art that such cells express one or more gene selected from hepatocyte nuclear factor 4-alpha (HNFA), albumin (ALB), and a member of the cytochrome P450 (CYP) gene family (e.g., CYP1 , CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11 , CYP17, CYP19, CYP20, CYP21 , CYP24, CYP26, CYP27, CYP39, CYP46, CYP51 , or a combination thereof). It is also known that such cells do not express the alpha-fetoprotein (AFP) gene. It is additionally known in the art that such cells express biomarkers such as HNF4a, albumin, A1 AT, and transferrin, and also secrete urea.

[0095] As used herein, the term “reprogramming factor” refers to a protein that causes expression of one or more pluripotency-associated genes. In some embodiments, the reprogramming factor gives rise to pluripotency and / or differentiation potential of a cell (e.g., a hepatocyte). The reprogramming factor may rejuvenate hepatocytes (e.g., expanded hepatocytes) while preserving hepatic commitment. The reprogramming factor may be a transcription factor. The reprogramming factor may cause an epigenetic or chromatin modification that results in a change in expression pattern of one or more genes in the cell. Exemplary reprogramming factors are Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf 1 , Klf2, Klf4, Klf5, cMyc, N- myc, L-myc, Nanog, Lin28, and Glisl . PATENT

[0096] ATTORNEY DOCKET NO. 51540-048WO3

[0097] As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. A subject may be any animal (e.g., a mammal). A subject may be in need of treatment and / or may receive treatment by administration of a population of hepatocytes (e.g., a population of hepatocytes prepared by the methods disclosed herein). In preferred embodiments, the subject is a human.

[0098] As used herein, the term “serum replacement component” refers to a reagent that may be added to cell culture medium that does not include serum (i.e., serum-free medium). A serum replacement component may be a specific formulation that includes, e.g., amino acids, antioxidants, transferrin or transferrin substitutes, insulin or insulin substitutes, and lipids.

[0099] As used herein, the term “xeno-free” refers to cell culture components or reagents that do not contain any unrefined or raw materials (e.g., a serum or tissue extract) that originate from a species that is different from the cell in culture. For example, a xeno-free cell culture medium for the cultivation of human cells would does not contain serum or tissue extracts from any non-human animals but may contain serum or tissue extracts that originate from a human (e.g., human platelet lysate). Xeno-free cell culture reagents may include recombinant proteins.

[0100] DETAILED DESCRIPTION

[0101] The present disclosure provides methods that can be used for the expansion and maturation of hepatocytes, which can be used for therapeutic purposes. In vitro expansion of hepatocytes typically employs a starting batch of hepatocytes, which are then expanded in defined culture media in either two- dimensional or three-dimensional cell cultures. As cell count expands and growth media is depleted, the hepatocytes often exhibit a reduction in proliferation potential, thus reducing the potential yield of total hepatocytes. The present disclosure is based upon the discovery that supplementing the growth media with one or more reprogramming factors during expansion partially reprograms the cells and rejuvenates the cell state to promote cell proliferation without altering cellular identity.

[0102] The methods described herein for producing expanded human hepatocytes include culturing hepatocytes (e.g., human hepatocytes, e.g., primary human hepatocytes or expanded hepatocytes) in contact with an extracellular matrix in the presence of an expansion medium and contacting the hepatocytes with RNA encoding one or more reprogramming factors to produce expanded human hepatocytes with enhanced proliferation potential.

[0103] Reprogramming of hepatocytes

[0104] Reprogramming factors are proteins (e.g., transcription factors) that cause expression of one or more pluripotency-associated genes of a cell (e.g., a hepatocyte). The reprogramming factor may rejuvenate hepatocytes to increase their proliferation potential while preserving hepatic commitment, such that the decision to commit to a hepatocyte lineage is not reversed. In some embodiments, the reprogramming factor causes expression of one or more pluripotency-associated genes and / or causes an epigenetic modification in the hepatocytes, thereby resulting in a change in expression pattern of one or more genes in the hepatocytes. In some embodiments, the reprogramming factor causes a chromatin PATENT

[0105] ATTORNEY DOCKET NO. 51540-048WO3 modification in the hepatocytes. Exemplary reprogramming factors are Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, and Glisl .

[0106] The methods described herein include expressing, e.g., transiently expressing, one or more reprogramming factors in the hepatocytes to produce expanded human hepatocytes with enhanced proliferation potential. In some embodiments, the method includes contacting the hepatocytes with RNA encoding one or more reprogramming factors to produce expanded human hepatocytes with enhanced proliferation potential. The RNA may be, for example, an mRNA encoding the one or more reprogramming factors. In other embodiments, the RNA may be a viral vector (e.g., a lentivirus or retrovirus) encoding the one or more reprogramming factors. The method may include transfecting the hepatocytes with the RNA (e.g., mRNA, e.g., via lipid-based transfection).

[0107] In some embodiments, the culturing step includes expanding plated cells (step PO) prior to contacting the hepatocytes with the RNA. In some embodiments, cells are plated and cultured in the presence of Chroman 1 , Emricasan, Polyamine, and Trans-integrated stress response inhibitor (trans- ISRIB) (CEPT) cocktail.

[0108] In some embodiments, the PO step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 ,

[0109] 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the PO step has a duration of 13 days.

[0110] In some embodiments, the contacting step includes a first passage of expanded cells (step P1 ). In some embodiments, the P1 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0111] In some embodiments, the method further includes a second passage of expanded cells (step P2). In some embodiments, the P2 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2,

[0112] 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0113] In some embodiments, the method further includes a third passage of expanded cells (step P3). In some embodiments, the P3 step has a duration of 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day. In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the RNA encoding the one or more PATENT

[0114] ATTORNEY DOCKET NO. 51540-048WO3 reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

[0115] In some embodiments, the method further includes a fourth, fifth, sixth, seventh, eighth, ninth, or tenth passage of expanded cells (step P4-P10).

[0116] In some embodiments, cells are plated and cultured in the presence of CEPT cocktail, e.g., during any one of steps P1 -P10.

[0117] In some embodiments, the method includes transiently expressing one or more reprogramming factors in the hepatocytes by contacting the hepatocytes with a DNA (e.g., a plasmid) that includes an open reading frame (ORF) of a reprogramming factor for protein expression of the reprogramming factor.

[0118] In some embodiments, the method includes expressing one or more reprogramming factors in the hepatocytes by gene editing the hepatocytes. The hepatocytes may be gene edited using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology or other gene editing technologies known in the art, such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs) to transiently express or overexpress one or more of Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, and Glisl . For example, a hepatocyte may be engineered to transiently express or overexpress one or more of Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf 1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, and Glisl , via transduction with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector) or an adeno-associated viral (AAV) vector)).

[0119] The expression of or more of any of the reprogramming factors disclosed herein, e.g., Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, Glisl , or any combination thereof, may be controlled by any suitable control element(s), e.g., promoters or enhancers, including constitutively active promoters (e.g., a cytomegalovirus (CMV), a CMV early enhancer element, a first exon and a first intron of chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene (CAG), chicken beta actin (CBA), CBA hybrid (CBh) , human elongation factor-1 alpha (EF1a), EF1a short (EFS), spleen focus-forming virus (SFFV), ubiquitin C (UBC), murine stem cell virus (MSCV), simian virus 40 (SV40), phosphoglycerate kinase (PGK), minimal cytomegalovirus promoter (minCMV), minimal herpes simplex virus thymidine kinase (minTK), a U6 promoter, or y-Box (yb) promoter) and / or inducible promoters such as those described herein (e.g., a tetracycline-controlled promoter, doxycycline- inducible promoter, heat shock promoter, light inducible promoter, or other responsive promoter). Other constitutively active promoters and inducible promoters are known in the art.

[0120] The expression of a reprogramming factor (e.g., Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf 1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, and / or Glisl) may be temporally controlled by one or more suitable control elements such as a promoter or enhancer that is operably linked to the ORF of the reprogramming factor, thereby controlling expression of the reprogramming factor. In some embodiments, the expression of the reprogramming factor is controlled by an inducible promoter such that when the hepatocytes are contacted with an agent (e.g., tetracycline, doxycycline, a cumate solution, or the like) that activates the inducible promoter or placed under a condition for a period of time sufficient to activate the inducible promoter (e.g., exposed to light (e.g., blue light at a wavelength of 450 nm) or exposed to increased temperature (e.g., temperatures of 39°C to 42°C), the hepatocytes express the one or more reprogramming factors. In some embodiments, the inducible promoter is a tetracycline- or doxycycline- PATENT

[0121] ATTORNEY DOCKET NO. 51540-048WO3 controlled promoter (e.g., a promoter linked to a tetracycline responsive element), e.g., for transient protein expression of the reprogramming factor in the hepatocytes and adding tetracycline or doxycycline to the cell culture medium induces expression of the reprogramming factor in the hepatocytes. Inducible promoters for temporal gene or protein expression are described in the art such as, e.g., in Das et al. Curr Gene Ther. 16(3) :156-167 (2016), and Kallunki et al. Cells. 8(8):796 (2019), each of which is hereby incorporated by reference as they pertain to inducible expression systems.

[0122] In some embodiments, the culturing step includes expanding plated cells (step P0) prior to (a) contacting the hepatocytes with the agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter. In some embodiments, cells are plated and cultured in the presence of CEPT cocktail.

[0123] In some embodiments, the P0 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 ,

[0124] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P0 step has a duration of 13 days.

[0125] In some embodiments, the method includes a first passage of expanded cells (step P1). In some embodiments, the P1 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the agent that activates the inducible promoter on day 1 for a total of 1 day and optionally replacing the culture medium on day 2. In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the agent that activates the inducible promoter daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days and optionally replacing the culture medium. In other embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and exposing the hepatocytes to a condition that activates the inducible promoter (e.g., heat or light) for a period of time that activates the inducible promoter on day 1 . In some embodiments, the P1 step includes culturing the hepatocytes on day 0 of P1 and exposing the hepatocytes to a condition that activates the inducible promoter for a period of time daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days. In some embodiments, the period of time for which the hepatocytes are exposed to the condition that activates the inducible promoter is 0.5 to 5 hours (e.g., 0.5 to 2 hours, 1 to 3 hours, 1 to 5 hours, 2 to 4 hours, or 3 to 5 hours).

[0126] In some embodiments, the method further includes a second passage of expanded cells (step P2). In some embodiments, the P2 step has a duration of 1 day to 20 days, e.g., 5 to 20 days (e.g., 1 , 2,

[0127] 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the agent that activates the inducible promoter on day 1 for a total of 1 day and optionally replacing the culture medium on day 2. In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the agent that activates the inducible promoter daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days and optionally replacing the culture medium.

[0128] In other embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and exposing the hepatocytes to a condition that activates the inducible promoter (e.g., heat or light) for a period of time PATENT

[0129] ATTORNEY DOCKET NO. 51540-048WO3 that activates the inducible promoter on day 1 . In some embodiments, the P2 step includes culturing the hepatocytes on day 0 of P2 and exposing the hepatocytes to a condition that activates the inducible promoter for a period of time daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days. In some embodiments, the period of time for which the hepatocytes are exposed to the condition that activates the inducible promoter is 0.5 to 5 hours (e.g., 0.5 to 2 hours, 1 to 3 hours, 1 to 5 hours, 2 to 4 hours, or 3 to 5 hours).

[0130] In some embodiments, the method further includes a third passage of expanded cells (step P3). In some embodiments, the P3 step has a duration of 5 to 20 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the agent that activates the inducible promoter on day 1 for a total of 1 day and optionally replacing the culture medium on day 2. In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the agent that activates the inducible promoter daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days and optionally replacing the culture medium. In other embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and exposing the hepatocytes to a condition that activates the inducible promoter (e.g., heat or light) for a period of time that activates the inducible promoter on day 1 . In some embodiments, the P3 step includes culturing the hepatocytes on day 0 of P3 and exposing the hepatocytes to a condition that activates the inducible promoter for a period of time daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days. In some embodiments, the period of time for which the hepatocytes are exposed to the condition that activates the inducible promoter is 0.5 to 5 hours (e.g., 0.5 to 2 hours, 1 to 3 hours, 1 to 5 hours, 2 to 4 hours, or 3 to 5 hours).

[0131] In some embodiments, the method further includes a fourth, fifth, sixth, seventh, eighth, ninth, or tenth passage of expanded cells (step P4-P10).

[0132] In some embodiments, cells are plated and cultured in the presence of CEPT cocktail, e.g., during any one of steps P1 -P10.

[0133] Polynucleotide Encoding Reprogramming Factors

[0134] The invention features a polynucleotide (e.g., an exogenous polynucleotide) encoding one or more reprogramming factors, such as an RNA encoding one or more reprogramming factors. The polynucleotide may be an RNA, such as a messenger RNA (mRNA) or circular RNA, encoding the reprogramming factor. In some embodiments, the RNA (e.g., mRNA) includes an ORF encoding the polypeptide. In some embodiments, the mRNA further includes a 5' UTR, 3' UTR, a poly(A) tail, and / or a 5' cap or 5’ cap analog. In some embodiments, the mRNA includes a chemical modification. In some embodiments, the mRNA is chemically modified with N1-methylpseudouridine (m1qj). In some embodiments, each U in the sequence is a 1 N1-methylpseudouridine (m1qj).

[0135] In some embodiments, each reprogramming factor is encoded by an RNA. In some embodiments, a plurality of RNAs are delivered to a cell, e.g., each mRNA encoding a reprogramming factor. In some embodiments, the RNA encodes a plurality of reprogramming factors. PATENT

[0136] ATTORNEY DOCKET NO. 51540-048WO3

[0137] In some embodiments, the RNA (e.g., mRNA) includes at least one synthetic modification. Suitable mRNA modifications are described, e.g., in Gao et al. Acta Biomater. 1 ;131 :1 -15, 2021 , which is hereby incorporated by reference in its entirety.

[0138] In some embodiments, the at least one synthetic modification is a 5’ cap analog. In some embodiments, the 5’ cap analog is an m7GpppG, anti-reverse cap analog (ARCA), two-headed cap, S cap, or 2S cap.

[0139] In some embodiments, the at least one synthetic modification is a tail modification. In some embodiments, the tail modification is a ribose-modified adenosine, 8-azaadenosine, cordycepin, or a fluorescent modification.

[0140] In some embodiments, the at least one synthetic modification is a modified nucleobase. In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1qj), 2-thiouridine (S2U), 5- methylcytidine (m5C), N6-methyladenosine (m6A), 2’-O-methyluridine (Um), 2’-O-methylcytidine (Cm), 2’- O-methyladenosine (Am), 2’-0-methylguanosine (Gm), 5’-methoxyuridine, 5-methylcytosine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2- propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4- thiouracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8- haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-bromouracil, 5- trifluoromethyluracil, 5-bromocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 2- fluoroadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or 3- deazaadenine.

[0141] In some embodiments, the at least one synthetic modification is a modified sugar. In some embodiments, the modified sugar is a 2’-Ome or 2’-0-fluoro ribose modification. The modified sugar may be a bicyclic sugar, a 2’-0-methoxyethyl (2MOE) modified sugar, a 2’-0-methoxy (2-OMe) modified sugar, a 2’-methoxy modified sugar, a 2’-O-alkyl modified sugar, a constrained ethyl (cEt) modified sugar, a locked sugar, and an unlocked sugar.

[0142] In some embodiments, the mRNA has a plurality of synthetic modifications, e.g., one or more cap, tail, nucleobase, and ribose modifications.

[0143] In some embodiments, the polynucleotide encoding the one or more reprogramming factors provided to a hepatocyte is DNA, such that the hepatocyte expresses the one or more reprogramming factors (e.g., by way of transcription of the DNA and subsequent translation of transcribed RNA) following contacting the hepatocyte with DNA (e.g., exogenous DNA). In some embodiments, the polynucleotide is an episomal vector within the hepatocyte. Episomal vectors (e.g., episomal plasmids) are polynucleotides that are not part of the genome of the cell and replicate independent of the eukaryotic chromosome. However, episomal vectors may still replicate together with the rest of the genome and subsequently associated with metaphase chromosomes during mitosis. Advantageously, episomes do not degrade.

[0144] In other embodiments, the polynucleotide may be integrated into a genome of the hepatocyte. By integrating the polynucleotide into the genome, the cells can stably express the one or more reprogramming factors encoded by the one or more ORFs without risk of losing, for example, a standard plasmid encoding the polypeptide that may easily degrade over time or get lost during cell division. In some embodiments, the polynucleotide is integrated into a safe harbor locus of the genome of the PATENT

[0145] ATTORNEY DOCKET NO. 51540-048WO3 hepatocyte. Such integration can be achieved by transducing the cell with a viral vector, such as a lentiviral vector.

[0146] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a CMV, a CMV early enhancer element, a first exon and a first intron of chicken beta-actin gene, and a splice acceptor of the rabbit beta-globin gene (CAG), CBA, CBh, EF1 a, EFS, SFFV, UBC, MSCV, SV40, PGK, minCMV, minTK, a U6 promoter, or yb promoter.

[0147] In some embodiments, the inducible promoter is a chemically inducible promoter, such as an alcohol inducible promoter, a steroid regulated promoter, a tetracycline responsive element, a cymene repressor (CymR), a cumate switch (SparQ), a lac operon (Lac) promoter (pLAC), an arabinose inducible promoter (e.g., pBAD), an Aspergillus nidulans alcA promoter, or a LexA promoter.

[0148] In some embodiments, the inducible promoter is a temperature inducible promoter, such as a Hsp70- or Hsp90-derived promoter.

[0149] In some embodiments, the inducible promoter is a light inducible promoter, such as a lightsensing protein-derived promoter (e.g., YFI or FixK2 promoter).

[0150] In some embodiments, the promoter is a physiologically responsive promoter, such as a glucoseresponsive promoter. The physiologically responsive promoter may be, for example, L-pyruvate kinase (LPK) promoter, glucose transporter-2 (GLUT2) promoter, carbohydrate response element binding protein (ChREBP) promoter, insulin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, glucose- 6-phosphatase (G6Pase) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, activating transcription factor 6 (ATF6) promoter, FAS (TNFRSF6 / CD95 / APO-1) promoter, or insulin-like growth factor binding protein 1 (IGFBP-1) promoter.

[0151] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive element, cymene repressor (CymR), cumate switch (SparQ), or lac operon (Lac) promoter.

[0152] Viral Vectors

[0153] The RNA encoding the one or more reprogramming factors may be a viral vector (e.g., an RNA viral vector) encoding a reprogramming factor or a plurality of reprogramming factors. In some embodiments, the RNA viral vector is a replicon RNA or self-amplifying RNA.

[0154] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector), parvovirus, coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type PATENT

[0155] ATTORNEY DOCKET NO. 51540-048WO3 viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (US 5,801 ,030), the teachings of which are incorporated herein by reference.

[0156] Retroviral Vectors

[0157] The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that may be used in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene encoding the polypeptide or RNA. An overview of optimization strategies for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

[0158] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the agent of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, encapsidation, and expression, in which the sequences to be expressed are inserted.

[0159] A LV used in the methods and compositions described herein may include one or more of a 5'- Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 -alpha promoter and 3'-self inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE. The lentiviral vector may further include a pHR' backbone, which may include for example as provided below.

[0160] The Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) may be used to express the DNA molecules and / or transduce cells. A LV used in the methods and compositions described herein may a 5'-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 - alpha promoter and 3'-self inactivating L TR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.

[0161] Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may PATENT

[0162] ATTORNEY DOCKET NO. 51540-048WO3 include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.

[0163] In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. The vector used in the methods and compositions described herein may include multiple promoters that permit expression more than one polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in Klump et al., Gene Ther.; 8:811 (2001 ), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004), the disclosures of which are incorporated herein by reference as they pertain to protein cleavage sites that allow expression of more than one polypeptide. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein.

[0164] The viral vectors (e.g., retroviral vectors, e.g., lentiviral vectors) may include a promoter operably coupled to the transgene encoding the polypeptide or the polynucleotide encoding the RNA to control expression. The promoter may be a ubiquitous promoter. Alternatively, the promoter may be a tissue specific promoter. The promoter may be an inducible promoter.

[0165] Expansion of Hepatocytes

[0166] The methods described herein include expansion of hepatocytes. The expansion may occur e.g., prior to reprogramming, during reprogramming, and / or after reprogramming. In some embodiments of the compositions and methods described herein, the hepatocytes are primary human hepatocytes (PHHs). In some embodiments of the compositions and methods described herein, the hepatocytes are expanded human hepatocytes (eHHs). In some embodiments of the compositions and methods described herein, the hepatocytes are genetically engineered hepatocytes, e.g., hypoimmunogenic hepatocytes. In some embodiments, the reprogramming occurs with cryopreserved hepatocytes that have been thawed. In some embodiments, the hepatocytes are expanded and then reprogrammed. In some embodiments, the hepatocytes are expanded, reprogrammed, and further expanded. In some embodiments, the hepatocytes are PHHs that are obtained from mature tissue (e.g., mature liver tissue). A PHH or a population of PHHs may be obtained by any suitable method. In some embodiments, cells are isolated by PATENT

[0167] ATTORNEY DOCKET NO. 51540-048WO3 collagenase digestion, for example, as described in Dorell et al. Hepatology. 48:1282-91 , 2008, which is hereby incorporated by reference. In some embodiments, collagenase digestion is performed on a tissue biopsy. In some embodiments, collagenase and accutase digestion are used to obtain the PHHs.

[0168] PHHs are present in the liver. In some embodiments, the method includes culturing a fragment of tissue which includes liver epithelium. In some embodiments, the PHHs are isolated from a tissue fragment. For example, in the context of liver, the tissue fragment may include a portal vein, a liver biliary duct, or biliary duct tissue. Liver PHHs can be isolated from normal liver tissues using FACS-based sorting to exclude EpCAM+progenitor cells such that the population of PHHs after processing does not include a detectable amount of EpCAM+progenitor cells. In some embodiments, PHHs isolated from normal liver tissue may contain EpCAM+ progenitor cells such that the level of EpCAM+ progenitor cells is less than 10% (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%).

[0169] In some embodiments, a single cell is isolated and cultured in a cell culture medium. In other embodiments, a population of cells is isolated and cultured a cell culture medium.

[0170] In some embodiments, the hepatocytes are genetically engineered hepatocytes, e.g., hypoimmunogenic hepatocytes. In certain embodiments, genetically engineered hepatocytes exhibit reduced expansion ability. Thus, the methods described herein allow for improved expansion ability of genetically engineered hepatocytes, e.g., hypoimmunogenic hepatocytes. In some embodiments, the hypoimmunogenic cells lack expression of one or more of the following endogenous genes: beta-2- microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof.

[0171] In some embodiments, the hypoimmunogenic cells include one or more alterations that inactivate the endogenous B2M, HLA-A, HLA-B, HLA-C, CIITA, or PVR gene.

[0172] In some embodiments, the hypoimmunogenic cells express one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3- dioxygenase 1 (IDO1), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), transforming growth factor beta-1 (TGFB1), carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof.

[0173] In some embodiments, expression of the one or more genes is controlled by one or more inducible promoters or constitutive promoters.

[0174] In some embodiments, the hypoimmunogenic cells overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof relative to a wild-type hepatocyte as the hypoimmunogenic cell. PATENT

[0175] ATTORNEY DOCKET NO. 51540-048WO3

[0176] In some embodiments, the cells may be cultured after isolation for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. In some embodiments, the cells may be cultured after isolation for at least about 5 days. In some embodiments, the cells may be cultured after isolation for at least 10 days. In some embodiments, the cells may be cultured after isolation for at least 15 days. In some embodiments, the cells may be cultured after isolation for at least 20 days. In some embodiments, the cells may be cultured after isolation for at least 25 days. In some embodiments, the cells may be cultured after isolation for at least about 30 days.

[0177] The length of time the cells are cultured may depend on the recovery of the cells after isolation. For instance, the cells may be cultured until they reach about 80%, about 85%, or about 90% confluence in a cell culture vessel (e.g., a microwell plate, a flask, or a Petri dish). In certain aspects, the cells are expanded in culture longer to improve the homogeneity of the cell phenotype in the cell population or to stabilize the cell state of expanded cells.

[0178] In some embodiments, provided is a method for obtaining a population of expanded hepatocytes including culturing hepatocytes in an expansion medium using the method as described herein.

[0179] In some embodiments, following isolation and subsequent culturing, the cells (e.g., hepatocytes, e.g., PHHs) are prepared for storage and are frozen for later use. In other embodiments, following isolation and subsequent culturing, the cells (e.g., hepatocytes, e.g., PHHs) are expanded into a larger population via methods of cell cultivation described herein.

[0180] In some embodiments, the method includes culturing the hepatocytes or obtaining the population of expanded hepatocytes from a single cell. Advantageously, this allows a homogenous population of cells to form. In some embodiments, the method includes culturing the cells in an expansion medium for a duration of 7 to 50 days (e.g., 7 to 50 days, 7 to 45 days, 7 to 40 days, 7 to 35 days, or 7 to 30 days; e.g., 7, 8, 9, 10, 1 1 , 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, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 days).

[0181] In some embodiments, the culturing step includes expanding plated cells (step P0) and a first passage of expanded cells (step P1).

[0182] In some embodiments, step P0 has a duration of 5 to 20 days (e.g., 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, step P0 has a duration of longer than 20 days (e.g., about 25 days). In some embodiments step P0 has a duration of about 10 to about 16 days. In some embodiments, step P0 has a duration of about 14 days.

[0183] In some embodiments, step P1 has a duration of 5 to 20 days (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days). In some embodiments, step P1 has a duration of longer than 20 days (e.g., about 25 days). In some embodiments, step P1 has a duration of about 10 to about 16 days. In some embodiments, step P1 has a duration of about 14 days.

[0184] In some embodiments, step P0 and / or step P1 includes seeding the hepatocytes at a density of 200 viable cells / cm2to 14,000 viable cells / cm2, e.g., 200 to 1 ,000 viable cells / cm2(e.g., about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1 ,000 viable cells / cm2), about 1 ,000 viable cells / cm2to about 10,000 viable cells / cm2(e.g., about 1 ,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, or about 10,000 viable cells / cm2), or 10,000 viable cells / cm2to 14,000 viable cells / cm2(e.g., about 10,000, about 11 ,000, about 12,000, about 13,000, or about 14,000 viable cells / cm2). In some embodiments, step P0 includes PATENT

[0185] ATTORNEY DOCKET NO. 51540-048WO3 seeding the hepatocytes at a density of about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, or about 800 viable cells / cm2. In some embodiments, step P1 includes seeding the hepatocytes at a density of about 900, about 925, about 1 ,000, about 1 ,025, about 1 ,050, about 1 ,075, or about 1 ,1 10 viable cells / cm2.

[0186] In some embodiments, the population of hepatocytes (e.g., PHHs) expand by at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90- fold, at least 100-fold, at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, or at least 150-fold during step P0. For example, upon seeding hepatocytes at a density of 200 to 1 ,000 viable cells / cm2during the initial passage (i.e., step P0), the cell yield following expansion of hepatocytes during step P0 may be about 4 x 103viable cells / cm2to about 1 .5 x 105viable cells / cm2. In preferred embodiments, the expanded hepatocytes (e.g. PHH) are further passaged (i.e., step P1) to further expand the population of hepatocytes. The hepatocytes may be passaged after reaching about 80% (e.g., 75%, 80%, 85%, 90%, or 95%) confluence during step P0 of the cell culturing method. In some embodiments, the population of hepatocytes (e.g., PHHs) expand by about 1 ,000-fold, about 1 ,100-fold, about 1 ,200-fold, about 1 ,300-fold, about 1 ,400-fold, about 1 ,500-fold, about 1 ,600-fold, about 1 ,700-fold, about 1 ,800-fold, about 1 ,900-fold, about 2,000-fold, about 2,100-fold, about 2,200-fold, about 2,300-fold, about 2,400-fold, about 2,500-fold, about 2,600-fold, about 2,700-fold, about 2,800-fold, about 2,900-fold, about 3,000-fold, about 3,100-fold, about 3,200-fold, about 3,300-fold, about 3,400-fold, about 3,500-fold, about 3,600-fold, about 3,700-fold, about 3,800-fold, about 3,900-fold, about 4,000-fold, about 4,100-fold, about 4,200-fold, about 4,300-fold, about 4,400-fold, about 4,500-fold, about 4,600-fold, about 4,700-fold, about 4,800-fold, about 4,900-fold, about 5,000-fold, about 5,100-fold, about 5,200-fold, about 5,300-fold, about 5,400-fold, about 5,500-fold, about 5,600-fold, about 5,700-fold, about 5,800-fold, about 5,900-fold, about 6,000-fold, about 6,100-fold, about 6,200-fold, about 6,300-fold, about 6,400-fold, about 6,500-fold, about 6,600-fold, about 6,700-fold, about 6,800-fold, about 6,900-fold, or about 7,000-foldcumulatively in step P0 and step P1 .

[0187] Achieving high cell yields of hepatocytes following expansion (e.g., expansion of PHH) is critical for therapeutic applications in which hepatocytes may be administered to, engrafted in, or implanted in a subject in need thereof (e.g., a subject that has liver dysfunction or liver failure or a subject that is at risk of liver dysfunction or liver failure) since the availability of PHH for transplantation is limited and therefore poses a significant problem. A mouse transplant, for example, requires a minimum of 1 x 105hepatocytes for successful engraftment. Approximately 1 x 107hepatocytes may be required for a successful graft in a human subject.

[0188] In some embodiments, the hepatocytes (e.g., PHHs) are cultured in the presence of 5% carbon dioxide. In some embodiments, the hepatocytes (e.g., PHHs) are cultured at a temperature of 37°C. In some embodiments, PHH are cultured in hypoxic conditions, in which atmospheric oxygen level is below 20.9%.

[0189] In some embodiments, the cells will expand at a rate of more than two (e.g., 3, 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) population doublings a week.

[0190] In some embodiments, the method includes replacing the culture medium one or more times during a method of culturing. For example, the culture medium may be replaced with fresh medium during PATENT

[0191] ATTORNEY DOCKET NO. 51540-048WO3 culturing because one or more components of the culture medium (e.g., one or more components added to the culture medium) degrade or are metabolized by the cells during culturing. In some embodiments, the culture medium is replaced about every 16 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 60 hours, or about every 72 hours. In some embodiments, the culture medium is replaced every 16 to 24 hours, every 24 to 48 hours, every 24 to 60 hours, or every 24 to 72 hours. In some embodiments, the culture medium is replaced every 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , or 72 hours.

[0192] The expansion medium may induce or promote the survival and / or proliferation of cells for at least 7 days of culture (e.g., at least 7, at least 10, at least 15, at least 20, at least 30, at least 50, or at least 100). In some embodiments, the population of expanded hepatocytes includes 70% or more viable cells (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). Cellular viability may be assessed using any viability determination method known in the art such as, e.g., Hoechst staining, trypan blue staining, propidium iodide staining and measuring by any suitable quantitative method including microscopy (e.g., immunofluorescent microscopy) or flow cytometry. Proliferation can be evaluated using techniques known in the art, such as bromo-2’-deoxyuridine (BrdU) staining, 5-ethynyl-2’-deoxyuridine (Edu) staining, Ki67 staining, or calculating cell growth via a growth curve.

[0193] In some embodiments, following the culturing step, the expression profile of the hepatocytes (e.g., PHHs) includes expression of Ki67 (e.g., protein or mRNA transcript expression levels) by about 15% or more hepatocytes in the population (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more). In some embodiments, following the culturing step, the expression profile of the PHH includes expression of Ki67 by at most 15% (e.g., at most 10%, 5%, 4%, 3%, 2%, or 1%) of the PHH.

[0194] Following culturing, the method may further include obtaining and / or isolating one or more cultured hepatocytes. For example, following cultivation of a population of hepatocytes (e.g., PHHs), it may be useful to remove one or more hepatocytes cultured in the cell culture medium (e.g., an expansion medium or a maturation medium described herein) for use in subsequent applications. For example, it may be useful to isolate a single hepatocyte following hepatocyte expansion in an expansion medium. Alternatively, it may be useful to obtain a population of hepatocytes following expansion in an expansion medium.

[0195] In some embodiments, following hepatocyte expansion, the expanded hepatocyte population may undergo maturation in the presence of a maturation medium including a basal medium for human cells to which is added one or more hepatocyte maturation supplements. In some embodiments, the maturation step immediately follows hepatocyte expansion. In some embodiments, the maturation step does not immediately follow hepatocyte expansion.

[0196] The cells produced by the methods described herein can be used immediately. Alternatively, the cells can be frozen in liquid nitrogen and stored for long periods of time for thawing and culturing later. For example, cells can be frozen in a culture medium that includes a cryoprotective agent, such as a medium that includes 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or one or more other agents known in the art. PATENT

[0197] ATTORNEY DOCKET NO. 51540-048WO3

[0198] In some embodiments, following culturing in an expansion medium, the expanded hepatocytes (e.g., PHHs) are prepared for cryopreservation. For example, a population of expanded hepatocytes may be prepared for cryopreservation by dissociating expanded population of hepatocyte cultures and mixing them with a freezing medium that includes a cryoprotective agent such as Recovery cell culture freezing medium (Gibco) or CRYOSTOR® (Biolife Solutions) and freezing following standard procedures. One or more aliquots of expanded hepatocytes may be cryopreserved for at least a week, at least a month, at least a year, or longer. One or more aliquots of frozen cells may be recovered after cryopreservation by thawing the frozen expanded hepatocytes, embedding the thawed hepatocytes in an extracellular matrix (ECM) (e.g., a laminin, a collagen, a vitronectin, a fibronectin, or a combination thereof) and culturing the expanded hepatocytes in an expansion medium of the invention.

[0199] In some embodiments, the culture medium may be supplemented with a rho kinase (ROCK) inhibitor, such as Y-27632 (CAS No. 129830038-2), following thawing. In some embodiments, the culture medium may be supplemented with about 10 pM Y-27632 after thawing. In some embodiments, the culture medium is supplemented with Y-27632 for the first 1 , 2, 3, 4, 5 or less days after thawing, preferably for the first 3 or 4 days. In some embodiments, Y-27632 is not present in the culture medium after the first 3, 4, 5, 6 or more days, preferably after the first 3 or 4 days. This thawing method may be used for expansion of hepatocytes. In some embodiments, a ROCK inhibitor such as Y-27632 is not added to the culture medium after thawing.

[0200] An expansion medium of the disclosure is composed of a basal cell culture medium to which one or more proteins and / or supplements are added. The expansion medium may be used for cultivating hepatocytes (e.g., PHHs) as described herein. Components of an expansion medium are described below.

[0201] Basal Cell Culture Medium

[0202] The expansion medium may include a basal cell culture medium such as a commercially available basal cell culture medium that is suitable for the in vitro cultivation of mammalian (e.g., human) cells. In some embodiments, the basal cell culture medium is Advanced DMEM / F-12 medium. In some embodiments, the basal cell culture medium is Takara CELLARTIS® POWER™ Primary HEP medium. In some embodiments, the basal cell culture medium is Lonza HCM™ cell culture medium. In In some embodiments, the basal cell culture medium is William’s E medium. In some embodiments, the basal cell culture medium is LIFENET HEALTH® Human Hepatocyte Media.

[0203] In some embodiments, the expansion medium includes a basal cell culture medium that includes amino acids glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L- threonine, L-tryptophan, L-tyrosine, and L-valine; vitamins ascorbic acid, biotin, choline chloride, D- calcium pantothenate, ergocalciferol, folic acid, menadione sodium bisulfate, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin A, vitamin B12, alpha-tocopherol, i-inositol; inorganic salts calcium chloride, cupric sulfate, ferric nitrate, magnesium sulfate, manganese chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, zinc sulfate; D- glucose; glutathione; methyl linoleate; phenol red; and sodium pyruvate. PATENT

[0204] ATTORNEY DOCKET NO. 51540-048WO3

[0205] In some embodiments, the expansion medium includes a basal cell culture medium that includes amino acids glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L- threonine, L-tryptophan, L-tyrosine, and L-valine; vitamins ascorbic acid, biotin, choline chloride, D- calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol; inorganic salts calcium chloride, cupric sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, and zinc sulfate; proteins albumin (e.g., a bovine serum albumin that is enriched with lipids such as cholesterol and fatty acids (e.g., GIBCO™ AlbuMAX™ II Lipid-Rich BSA)), transferrin, and insulin; D-glucose; glutathione; ammonium metavanadate; manganous chloride; sodium selenite; ethanolamine; hypoxanthine; linoleic acid; lipoic acid; putrescine; phenol red; and sodium pyruvate.

[0206] Growth Factors and Signaling Pathway Agonists

[0207] The expansion medium may include one or more different types of growth factors, such as a fibroblast growth factors (FGFs), an epidermal growth factor (EGF), a hepatocyte growth factor (HGF), or a combination thereof. In some embodiments, an FGF, an EGF, and an HGF are added to the basal cell culture medium. In some embodiments, the expansion medium includes an FGF or a fragment thereof (e.g., a fragment that retains binding to a fibroblast growth factor receptor (FGFR)). In some embodiments, the expansion medium includes an EGF or a fragment thereof (e.g., a fragment that retains binding to an epidermal growth factor receptor (EGFR)).

[0208] FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGFs stimulate cells by interacting with cell surface FGFR. Four closely related receptors (i.e., FGFR1 , FGFR2, FGFR3, and FGFR4) have been identified. Most FGFs bind more than one receptor (Ornitz J. Biol. Chem. 273(9) :5349-57, 1998). However, FGF10 and FGF7 are unique among FGFs in that they interact only with a specific isoform of FGFR2, designated FGFR2b, which is expressed exclusively by epithelial cells (Igarashi, J. Biol. Chem. 273(21 ):13230-5, 1998).

[0209] In some embodiments, the FGF is a human FGF. In some embodiments, the FGF is a recombinant FGF. In some embodiments, the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF includes only one FGF. In some embodiments, the FGF is FGF10. In some embodiments, the FGF includes FGF10 and one or more FGFs (e.g., 1 , 2, 3, or more than 3 FGFs). In preferred embodiments, the FGF does not include FGF7, which is also known in the art as keratinocyte growth factor (KGF). In some embodiments, the expansion medium includes one or more FGFs at a total concentration of 5 ng / mL to 500 ng / mL (e.g., 5 to 50 ng / mL, 25 to 100 ng / mL, 50 to 150 ng / mL, 100 to 200 ng / mL, 200 to 300 ng / mL, 300 to 400 ng / mL, or 400 to 500 ng / mL; e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 ng / mL). PATENT

[0210] ATTORNEY DOCKET NO. 51540-048WO3

[0211] EGF is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. In some embodiments, any suitable EGF may be used. In some embodiments, the EGF includes a human EGF. In some embodiments, the EGF includes a recombinant EGF (e.g., a recombinant human EGF).

[0212] In some embodiments, the expansion medium includes EGF at a concentration of 5 ng / mL to 500 ng / mL (e.g., 5 to 50 ng / mL, 25 to 100 ng / mL, 50 to 150 ng / mL, 100 to 200 ng / mL, 200 to 300 ng / mL, 300 to 400 ng / mL, or 400 to 500 ng / mL; e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 ng / mL). In some embodiments, EGF is substituted with an alternative compound that activates EGFR. For example, an insulin-like growth factor (IGF) may be substituted for EGF.

[0213] In some embodiments, the expansion medium includes an HGF. Hepatocyte growth factor / scatter factor (HGF / SF) is a morphogenic factor that regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogenic c-Met receptor. In some embodiments, the HGF is human HGF. In some embodiments, the HGF is a recombinant HGF. In some embodiments, the expansion medium includes HGF at a concentration of 1 ng / mL to 200 ng / mL (e.g., 1 to 20 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, 50 to 75 ng / mL, 60 to 80 ng / mL, 80 to 100 ng / mL, 100 to 125 ng / mL, 125 to 150 ng / mL, 150 to 175 ng / mL, or 175 to 200 ng / mL; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9,

[0214] 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL). In some embodiments, HGF is substituted with a compound that activates the HGF receptor, such as oligopeptide N-hexanoid-Tyr-lle-(6)aminohexanoic amide, which is also known in the art as dihexa (CAS NO. 1401708-83-5).

[0215] In some embodiments, the expansion medium further includes a transforming growth factor (TGF), a polypeptide growth factor. The TGF may be TGFa, TGFpl , TGFp2, TGFp3, or a combination thereof. In some embodiments, the TGF is a human TGF. In some embodiments, the TGF is a recombinant TGF. In some embodiments, the expansion medium includes TGF at a concentration of 2 ng / mL to 50 ng / mL (e.g., 2 to 10 ng / mL, 5 to 20 ng / mL, or 20 to 50 ng / mL; e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,

[0216] 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 ng / mL). In preferred embodiments, the expansion medium does not include TGFa.

[0217] In some embodiments, during culturing of hepatocytes (e.g., PHHs), the one or more growth factors (e.g., an EGF, an FGF, an HGF, a TGF, or a combination thereof) is added to the culture medium when required, for example, daily or every other day. The one or more growth factors may be added singularly or in combination. In some embodiments, the one or more growth factors are added upon replacement of the cell culture medium (e.g., the expansion medium). In some embodiments, the one or more growth factors are added directly to the cell culture medium prior to adding the cell culture medium to the cells.

[0218] In some embodiments, the expansion medium includes a signal pathway agonist such as a Wnt signaling pathway agonist. The Wnt signaling pathway is an evolutionarily conserved developmental signaling pathway that is initiated upon activation of the cell-surface Wnt receptor complex, which includes a Frizzled receptor, a low density lipoprotein receptor-related protein (LRP), and a leucine-rich repeat-containing G-protein-coupled receptor (LGR). The cell-surface Wnt receptor complex is usually activated by an extracellular signaling molecule, such as a member of the Wnt family, which then results PATENT

[0219] ATTORNEY DOCKET NO. 51540-048WO3 in the activation of Disheveled family proteins, which inhibit a complex of proteins that includes axin, GSK-3, and APC to degrade intracellular p-catenin. The resulting enriched nuclear p-catenin enhances transcription by TCF / LEF family transcription factors. A Wnt agonist is an agent that activates TCF / LEF- mediated transcription in a cell. Wnt agonists are therefore selected from Wnt agonists that bind and activate the Wnt receptor complex including any and all of the Wnt family proteins, such as an inhibitor of intracellular p-catenin degradation, including a GSK inhibitor (such as the small molecule GSK3 inhibitor CHIR99021 (CAS No. 252917-06-9) and activators of TCF / LEF.

[0220] The Wnt agonist in the expansion medium may be any agonist able to stimulate the Wnt pathway via the LGR5 cell surface receptor, e.g., an LGR5 agonist. Known LGR5 agonists include an R-spondin protein and anti-LGR5 antibodies, including protein and antibody fragments that retain binding to LGR5. Exemplary LGR5 agonists are described in WO 2012 / 140274 and De Lau, W. et al. Nature.

[0221] 476(7360) :293-7, 2011 , each of which is hereby incorporated by reference. In preferred embodiments, an LGR5 agonist in the expansion medium is R-spondin. In some embodiments, the expansion medium includes an R-spondin, wherein the R-spondin includes R-spondin 1 , R-spondin 2, R-spondin 3, R- spondin 4, or a combination thereof. In some embodiments, the R-spondin is a human R-spondin. In some embodiments, the R-spondin is a recombinant R-spondin (e.g., a recombinant human R-spondin; e.g., a recombinant human R-spondin 1 or a recombinant human R-spondin 3). In some embodiments, one or more R-spondin proteins are added to the expansion medium at a concentration of 10 ng / mL to 1 pg / mL (e.g., 10 to 100 ng / mL, 50 to 200 ng / mL, 150 to 300 ng / mL, 200 to 500 ng / mL, 500 to 600 ng / mL, 600 to 700 ng / mL, 700 to 800 ng / mL, 800 to 900 ng / mL, or 900 to 1 pg / mL; e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1 ,000 ng / mL).

[0222] In some embodiments, a Wnt agonist is a secreted glycoprotein selected from the following: Wnt- l / lnt-1 , Wnt-2 / lrp (InM-related protein), Wnt-2b / 13, Wnt-3 / lnt-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al., 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D systems), Wnt-7b, Wnt- 8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnMI, and Wnt-16. In preferred embodiments, the expansion medium does not include Wnt-3a. An overview of human Wnt proteins is provided in “THE WNT FAMILY OF SECRETED PROTEINS,” R&D Systems Catalog, 2004.

[0223] The Wnt agonist is preferably added to the expansion medium in an amount effective to stimulate Wnt activity in a cell. Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example by TOPFLASH and FOPFLASH Tcf luciferase reporter constructs, such as those described in Korinek et al., Science. 275:1784-1787, 1997, which is hereby incorporated by reference. In some embodiments, a Wnt agonist is added to the expansion medium at a concentration of 10 ng / mL to 1 pg / mL (e.g., 10 to 100 ng / mL, 50 to 200 ng / mL, 150 to 300 ng / mL, 200 to 500 ng / mL, 500 to 600 ng / mL, 600 to 700 ng / mL, 700 to 800 ng / mL, 800 to 900 ng / mL, or 900 to 1 pg / mL; e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1 ,000 ng / mL). PATENT

[0224] ATTORNEY DOCKET NO. 51540-048WO3

[0225] In some embodiments, during culturing of hepatocytes (e.g., PHHs), the one or more Wnt agonist such as one or more R-spondin proteins may be added to the culture medium when required, for example, daily or every other day. In some embodiments, the one or more Wnt agonists are added upon replacement of the cell culture medium (e.g., the expansion medium). In some embodiments, the Wnt agonist may be added to the culture medium prior to adding the cell culture medium to the cells.

[0226] Transforming Growth Factor-Beta Inhibitor

[0227] The expansion medium described herein may include one or more transforming growth factorbeta (TGFp) inhibitors. A TGFp inhibitor may be any agent (e.g., a small molecule, a protein, or an inhibitory nucleic acid) that can attenuate or prevent the transcription of one or more genes that are transcribed due to the activity of a SMAD transcription co-activator protein. A TGFp inhibitor may disrupt the signal transduction cascade that leads to SMAD-induced gene transcription at one or more points within the pathway. For instance, a TGFp inhibitor may disrupt or prevent TGFp or a TGFp superfamily ligand, such as activin, inhibin, nodal, lefty, bone morphogenetic protein (BMP), growth and differentiation factor (GDF), or mullerian inhibitory factor (MIF), from binding to its endogenous receptor, thus inhibiting the phosphorylation and activation of the receptor-associated SMAD proteins.

[0228] The presence of a TGFp inhibitor in the expansion media is advantageous because it reduces the likelihood that the hepatocytes (e.g., PHHs) differentiate by reducing or inhibiting the activity of the TGFp signaling pathway, thereby preventing development of the mesenchymal phenotype. TGFp signaling is involved in many cellular functions, including cell growth, cell fate, and apoptosis. Signaling is generally initiated by the binding of a TGFp superfamily ligand to a type II receptor, which subsequently recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates SMAD proteins, which act as transcription factors in the nucleus and regulate target gene expression.

[0229] The TGFp superfamily ligands include bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGFp isoforms TGFpl , TGFp2, and TGFp3. In general, SMAD2 and SMAD3 are phosphorylated by activin receptor-like kinases (ALK) ALK4, ALK5, and ALK7 in the TGFp / activin pathway. In contrast, SMAD1 , SMAD5 and SMAD8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. Although there is some crossover between pathways, in the context of this disclosure, a “TGFp inhibitor” or an “inhibitor of TGFp Signaling” is preferably an inhibitor of the TGFp pathway which acts via SMAD2 and SMAD3. Therefore, in some embodiments the TGFp inhibitor is not a BMP inhibitor such that, for example, the TGFp inhibitor is not Noggin.

[0230] Thus, the TGFp inhibitor may be any agent that reduces the activity of the TGFp signaling pathway. Many targets and inhibition strategies thereof of the TGFp signaling pathway are known in the art. For example, TGFp signaling may be dampened by any one or more of the following: inhibition of TGFp expression by a small-interfering RNA strategy; inhibition of furin (a TGFp activating protease); inhibition of the pathway by physiological inhibitors; neutralization of one or more TGFp isoforms with a monoclonal antibody; inhibition with small-molecule inhibitors of TGFp receptor kinase 1 (also known as ALK5), or other TGFp-related receptor kinases; inhibition of SMAD2 and SMAD3 signaling, e.g., by overexpression of their physiological inhibitor SMAD7 or by using thioredoxin as a SMAD anchor to inhibit SMADactivation (Fuchs, O. Inhibition of TGF-Signaling for the Treatment of Tumor Metastasis and PATENT

[0231] ATTORNEY DOCKET NO. 51540-048WO3

[0232] Fibrotic Diseases. Current Signal Transduction Therapy, Volume 6, Number 1 , January 2011 , pp. 29- 43(15)).

[0233] Various methods for determining whether a substance is a TGFp inhibitor are known in the art. For example, a cellular assay may be used in which cells are stably transfected with a reporter construct including the human PAI-1 promoter or SMAD binding sites, driving a luciferase reporter gene. Inhibition of luciferase activity relative to control groups can be used as a measure of compound activity (De Gouville et al., Br J Pharmacol. 145(2) :166-177, 2005).

[0234] A TGFp inhibitor that is added to the cell culture medium (e.g., expansion medium) may be a protein, a peptide, a small molecule, a small interfering RNA, an antisense oligonucleotide, an aptamer, or an antibody. The inhibitor may be naturally occurring or synthetic. In some embodiments, the TGFp inhibitor is an inhibitor of ALK5. For example, the TGFp inhibitor may bind to and directly inhibit ALK5. In some embodiments, the TGFp inhibitor is A83-01 (CAS No. 909910-43-6), a small molecule that inhibits Smad signaling by inhibiting ALK4, ALK5, and ALK7.

[0235] In some embodiments, the expansion medium includes about 500 nM to about 5 pM A83-01 (e.g., 500 nM to 1 pM, 1 to 2 pM, 2 to 3 pM, 3 to 4 pM, or 4 to 5 pM; e.g., about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1 .1 pM, about 1 .2 pM, about 1 .3 pM, about 1 .4 pM, about 1 .5 pM, about 1 .6 pM, about 1 .7 pM, about 1 .8 pM, about 1 .9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, or about 5 pM).

[0236] In some embodiments, one TGFp inhibitor is present in the expansion medium. In some embodiments, more than one (e.g., two, three, four, or more) TGFp inhibitors are present in the expansion medium.

[0237] Serum Replacement Component or Serum

[0238] An expansion medium described herein may include a serum replacement component or a serum.

[0239] In some embodiments, the expansion medium includes a serum replacement component (i.e., a component that may substitute fetal bovine serum as an additive in a cell culture medium). In some embodiments, the expansion medium includes a serum replacement component at a volumetric concentration of about 0.1% to about 20% (e.g., about 0.1% to about 1%, about 0.1% to about 5%, about 0.1% to about 10%, about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%; e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%).

[0240] Any suitable serum replacement component may be used. In some embodiments, the serum replacement component is KNOCKOUT™ Serum Replacement (KOSR), human platelet lysate, human serum, insulin transferrin selenium (ITS), Trace Elements A (i.e., a cell culture medium additive that includes copper, zinc, iron, and selenium), or Trace Elements B (i.e., a cell culture medium additive that PATENT

[0241] ATTORNEY DOCKET NO. 51540-048WO3 includes ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride, and hydrochloric acid). KOSR is a serum-free eukaryotic cell culture medium supplement that includes or is obtained by combining albumin or an albumin supplement and one or more ingredients selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L- phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, |- Mn2+, F-, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+(see, e.g., U.S. Publication No. US20020076747, the disclosure of which is hereby incorporated by reference in its entirety).

[0242] In some embodiments, the serum replacement component includes KOSR. In some embodiments, the expansion medium includes KOSR at a volumetric concentration of about 0.1 % to about 20% (e.g., about 0.1 % to about 1 %, about 0.1 % to about 5%, about 0.1 % to about 10%, about 1 % to about 5%, about 1 % to about 10%, about 1 % to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%; e.g., about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%).

[0243] In some embodiments, during culturing of hepatocytes (e.g., PHHs), the serum replacement component or serum may be added to the culture medium when required, for example, daily or every other day. In some embodiments, the serum replacement component or serum is added upon replacement of the cell culture medium (e.g., the expansion medium). In some embodiments, the serum replacement component or serum may be added to the culture medium prior to adding the cell culture medium to the cells.

[0244] In some embodiments, the concentration of the serum replacement component is changed depending on the cell density or confluence of the hepatocytes during expansion.

[0245] In some embodiments, the serum replacement component is present in the cell culture medium (e.g., the expansion medium) at a volumetric concentration of 1 % upon seeding the hepatocytes in step P0. In some embodiments, the serum replacement component is present in the cell culture medium (e.g, the expansion medium) at a volumetric concentration of 5% upon seeding the hepatocytes in step P0.

[0246] In some embodiments, the volumetric concentration of the serum replacement component is raised to 5% in the cell culture medium (e.g., the expansion medium) when the cell density reaches about 15% to about 30% confluency (e.g., about 15%, about 20%, about 25%, or about 30%) at step P0. In some embodiments, the volumetric concentration of the serum replacement component is raised to 5% in the cell culture medium (e.g., the expansion medium) at day 3 to day 7 (e.g., day 3, day 4, day 5, day 6, or day 7) of step P0.

[0247] In some embodiments, the volumetric concentration of the serum replacement component is raised to 10% when the cell density reaches about 40% to about 60% confluency (e.g., about 40%, about 45%, about 50%, about 55%, or about 60%) of step P0. In some embodiments, the volumetric concentration of the serum replacement component is raised to 10% at day 7 to day 13 (e.g., day 7, day 8, day 9, day 10, day 1 1 , day 12, or day 13) of step P0. PATENT

[0248] ATTORNEY DOCKET NO. 51540-048WO3

[0249] In some embodiments, the serum replacement component is present in the cell culture medium (e.g., the expansion medium) at a volumetric concentration of 1 % upon the passage of the hepatocytes in step P1 . In some embodiments, the serum replacement component is present in the cell culture medium (e.g., the expansion medium) at a volumetric concentration of 5% upon the passage of hepatocytes in step P1 .

[0250] In some embodiments, the volumetric concentration of the serum replacement component is raised to 5% in the cell culture medium (e.g., the expansion medium) when the cell density reaches about 15% to about 30% confluency (e.g., about 15%, about 20%, about 25%, or about 30%) at step P1 . In some embodiments, the volumetric concentration of the serum replacement component is raised to 5% in the cell culture medium (e.g., the expansion medium) at day 3 to day 7 (e.g., day 3, day 4, day 5, day 6, or day 7) of step P1 .

[0251] In some embodiments, the volumetric concentration of the serum replacement component is raised to 10% when the cell density reaches about 40% to about 60% confluency (e.g., about 40%, about 45%, about 50%, about 55%, or about 60%) of step P1 . In some embodiments, the volumetric concentration of the serum replacement component is raised to 10% between day 5 to day 13 (e.g., day 5, day 6, day 7, day 8, day 9, day 10, day 1 1 , day 12, or day 13) of step P1 .

[0252] In some embodiments, the expansion medium includes a serum. In some embodiments, the serum includes fetal bovine serum. In some embodiments, the expansion medium includes a serum at a volumetric concentration of about 0.1 % to about 20% (e.g., about 0.1 % to about 1 %, about 0.1 % to about 5%, about 0.1 % to about 10%, about 1 % to about 5%, about 1 % to about 10%, about 1 % to about 20%, about 5% to about 10%, or about 10% to about 20%; e.g., about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%). In preferred embodiments, the expansion medium does not include a serum.

[0253] Additional Medium Components

[0254] An expansion medium described herein optionally includes one or more additional agents or supplements, such as an antioxidant, a cell survival agent, an amino acid supplement, a buffering agent, an antibiotic, or any combination thereof.

[0255] In some embodiments, the expansion medium includes an antioxidant. In some embodiments, the antioxidant is added to the expansion medium at a concentration of 100 pM to 10 mM (e.g., 100 pM to 500 pM, 500 pM to 1 mM, 1 mM to 2 mM, 2 mM to 3 mM, 3 mM to 4 mM, 4 mM to 5 mM, 5 mM to 6 mM, 6 mM to 7 mM, 7 mM to 8 mM, 8 mM to 9 mM, or 9 mM to 10 mM; e.g., 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 mM, 1 .25 mM, 1 .5 mM, 1 .75 mM, 2 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3 mM, 3.25 mM, 3.5 mM, 3.75 mM, 4 mM, 4.25 mM, 4.5 mM, 4.75 mM, 5 mM, 5.25 mM, 5.5 mM, 5.75 mM, 6 mM, 6.25 mM, 6.5 mM, 6.75 mM, 7 mM, 7.25 mM, 7.5 mM, 7.75 mM, 7 mM, 7.25 mM, 7.5 mM, 7.75 mM, 8 mM, 8.25 mM, 8.5 mM, 8.75 mM, 9 mM, 9.25 mM, 9.5 mM, 9.75 mM, or 10 mM). PATENT

[0256] ATTORNEY DOCKET NO. 51540-048WO3

[0257] In some embodiments, the antioxidant includes N-acetyl cysteine. In some embodiments, the antioxidant includes nicotinamide. In some embodiments, the antioxidant includes both N-acetyl cysteine and nicotinamide.

[0258] In some embodiments, the expansion medium includes 100 pM to 5 mM N-acetylcysteine (e.g., 100 pM to 500 pM, 500 pM to 1 mM, 1 mM to 2 mM, 2 mM to 3 mM, 3 mM to 4 mM, or 4 mM to 5 mM; e.g., 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 mM, 1 .25 mM, 1 .5 mM, 1 .75 mM, 2 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3 mM, 3.25 mM, 3.5 mM, 3.75 mM, 4 mM, 4.25 mM, 4.5 mM, 4.75 mM, or 5 mM).

[0259] In some embodiments, the expansion medium includes nicotinamide at a concentration of 1 mM to 250 mM (e.g., 1 mM to 5 mM, 1 to 10 mM, 1 to 25 mM, 5 mM to 25 mM, 5 mM to 50 mM, 5 mM to 100 mM, 10 mM to 25 mM, 10 mM to 50 mM, 10 mM to 100 mM, 50 mM to 100 mM, 50 mM to 200 mM, 100 mM to 200 mM, 100 mM to 250 mM, 150 mM to 200 mM, 150 to 250 mM, or 200 mM to 250 mM; e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, or 250 mM).

[0260] In some embodiments, the expansion medium includes agents or additives that promote cell survival of the cell culture. In some embodiments, a cell survival agent is a supplement such as a supplement for a serum-free cell culture medium (i.e. , a cell culture medium that does not contain animal serum, e.g., fetal bovine serum). In some embodiments, the supplement includes a B27 supplement. In some embodiments, the supplement includes an N2 supplement. In some embodiments, the supplement includes both a B27 supplement and an N2 supplement.

[0261] In some embodiments, the B27 does not contain vitamin A. The B27 supplement may be used to formulate a cell culture medium that includes biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin. The B27 supplement without vitamin A was shown to work particularly well in an expansion medium for liver cells (e.g., hepatocytes; e.g., PHHs). The B27 supplement may be purchased as a liquid 50X concentrate that includes biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin. Of these ingredients, at least linolenic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists. B27 supplement may be added to a culture medium as a concentrate or diluted before addition to a culture medium. It may be used at a 1X final concentration or at other concentrations. Use of B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin into a culture medium of the invention. It is also envisaged that some or all of these components may be added separately to the expansion medium instead of using the B27 supplement. Thus, the expansion medium may include some or all of these components.

[0262] In some embodiments, the expansion medium includes a B27 supplement at a concentration of about 0.1X to about 100X (e.g., about 0.1X to about 90X, about 0.5X to about 80X, about 1 X to about PATENT

[0263] ATTORNEY DOCKET NO. 51540-048WO3

[0264] 70X, about 5X to about 60X, and about 10X to about 50X; e.g., about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 15X, about 20X, about 25X, about 30X, about 35X, about 40X, about 45X, about 50X, about 60X, about 70X, about 80X, about 90X, or about 100X).

[0265] In some embodiments, the expansion medium includes an N2 supplement. N2 supplement may be purchased as a 100X liquid concentrate, containing human transferrin, bovine insulin, progesterone, putrescine, and sodium selenite. N2 supplement may be added to a culture medium as a concentrate or diluted before addition to a culture medium. It may be used at a 1X final concentration or at other concentrations. Use of N2 supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine, and sodium selenite into a culture medium of the invention. It is also envisaged that some or all of these components may be added separately to the expansion medium instead of using the N2 supplement. Thus, in some embodiments, the expansion medium may include some or all of these components.

[0266] In some embodiments, the expansion medium includes an N2 supplement at a concentration of about 0.1X to about 100X (e.g., about 0.1X to about 90X, about 0.5X to about 80X, about 1 X to about 70X, about 5X to about 60X, and about 10X to about 50X; e.g., about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 15X, about 20X, about 25X, about 30X, about 35X, about 40X, about 45X, about 50X, about 60X, about 70X, about 80X, about 90X, or about 100X).

[0267] In some embodiments, the expansion medium includes an amino acid supplement. In some embodiments, the amino acid supplement is a non-essential amino acid (NEAA) supplement. In some embodiments, the NEAA supplement includes glycine, L-alanine, L-asparagine, L-aspartic acid, L- glutamic acid, L-proline, L-serine, or any combination thereof. In some embodiments, the NEAA supplement includes glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L- serine. In some embodiments, the expansion medium includes a non-essential amino acid supplement containing 1 pM to 100 mM, e.g., 1 pM to 10 pM (e.g., 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM), 10 pM to 100 pM ( e.g., 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM), 100 pM to 1 mm (e.g., 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, or 1 mm), 1 mM to 10 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM), or 10 mM to 100 mM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM) of each of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and L-serine. In some embodiments, the expansion medium includes 100 pM of each of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, the NEAA supplement is purchased as a solution including glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine at a 100X concentration. In some embodiments, the expansion medium includes an NEAA supplement solution at a final concentration of about 0.1X to about 10X (e.g., about 0.1 X to about 1X, about 0.1X to about 5X, about 1 X to about 5X, about 1 X to about 10X, or about 5X to about 10X; e.g., about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, PATENT

[0268] ATTORNEY DOCKET NO. 51540-048WO3 about 0.7X, about 0.8X, about 0.9X, about 1X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, or about 10X).

[0269] In some embodiments, the expansion medium includes L-glutamine or an L-glutamine substitute or derivative thereof. In some embodiments, the L-glutamine is an L-glutamine supplement, e.g., L-alanyl- L-glutamine dipeptide, e.g., 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCI (e.g., GLUTAMAX™). In some embodiments, the expansion medium includes GLUTAMAX™ at a volumetric concentration of about 0.1% to about 20% (e.g., about 0.1% to about 1%, about 0.1% to about 5%, about 0.1% to about 10%, about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%; e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9%, about 10%, about 1 1%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%). In some embodiments, L-glutamine or an L-glutamine substitute or derivative thereof is provided as a solution at a 100X concentration and is added at a concentration of about 0.1X to about 10X (e.g., about 0.1 X to about 1 X, about 0.1 X to about 5X, about 1 X to about 5X, about 1 X to about 10X, or about 5X to about 10X; e.g., about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, or about 10X).

[0270] In some embodiments, an expansion medium includes a buffering agent. A buffering agent may be any buffering agent suitable for mammalian cell culture. In some embodiments, the expansion medium includes a buffering agent at a concentration of 1 mM to 100 mM (e.g., 1 to 5 mM, 1 to 10 mM, 1 to 20 mM, 1 to 50 mM, 10 to 50 mM, 10 to 100 mM, 20 to 100 mM, or 50 to 100 mM; e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM).

[0271] In some embodiments, the buffering agent is HEPES. In some embodiments, the expansion medium includes 1 mM to 50 mM HEPES (e.g., 1 to 5 mM, 1 to 10 mM, 1 to 20 mM, 1 to 50 mM, 10 to 20 mM, 10 to 50 mM, or 20 to 50 mM; e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM).

[0272] In some embodiments, the expansion medium includes an antibiotic. In some embodiments, the expansion medium does not include an antibiotic. In some embodiments, the antibiotic includes penicillin, streptomycin, or both penicillin and streptomycin. In some embodiments, the expansion medium includes about 0.1% or more (e.g., about 0.5%, about 1%, or about 5%) of a solution of penicillin and streptomycin. In some embodiments, the expansion medium includes from 0.1% to 10%, e.g., 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) or 1 % to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) a solution of penicillin and streptomycin.

[0273] In some embodiments, during culturing of hepatocytes (e.g., PHHs), any one or more of the foregoing supplements and agents (e.g., an antioxidant, a cell survival agent, an amino acid supplement a buffering agent, and / or an antibiotic) may be added to the culture medium when required, for example, daily or every other day. In some embodiments, the one or more supplements and agents are added upon replacement of the cell culture medium (e.g., the expansion medium). In some embodiments, the PATENT

[0274] ATTORNEY DOCKET NO. 51540-048WO3 one or more supplements and agents may be added to the culture medium prior to adding the cell culture medium to the cells.

[0275] Culture Surface

[0276] In some examples of the methods described herein, the hepatocytes (e.g., PHHs) are cultured on a two-dimensional surface (e.g., a two-dimensional surface of a cell culture vessel). The cell culture surface can be made of any material suitable for culturing mammalian cells. For example, the surface may be a biocompatible and easily sterilized material such as plastic or other artificial polymer material. In some embodiments, the cell culture surface may contain plastic or glass. In some embodiments, the cells are grown in one plane.

[0277] Any number of materials can be used to form the cell culture surface including polyamides, polyesters, polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate, polytetrafluoroethylene (PTFE), nitrocellulose, cotton, polyglyolic acid (PGA), cellulose, dextran, gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, and silicon substrates (e.g., fused silica, polysilicon, or single silicon crystals), and the like. Metals such as gold, silver, or titanium films may also be used.

[0278] In some embodiments, the surface may be modified to promote cellular adhesion (e.g., coated with a material that promotes engagement with a cell surface, e.g., via engagement of an extracellular cell adhesion protein or receptor). For example, a glass surface may be treated with a protein or a peptide to promote cell adhesion to the cell culture surface. In some embodiments, a single protein is adhered to the surface. In some embodiments, two or more proteins are adhered to the surface. In some embodiments, the surface is coated with an extracellular matrix (ECM) (e.g., an ECM described herein) to facilitate cell adhesion.

[0279] In some embodiments, the hepatocytes (e.g., PHHs) are cultured on a two-dimensional surface, wherein the two-dimensional surface has a surface area of 9.5 cm2to 10,000 cm2(e.g., 9.5 to 500 cm2, 500 to 1 ,000 cm2, or 1 ,000 cm2to 10,000 cm2; e.g., 9.5 cm2, 100 cm2, 200 cm2, 300 cm2, 400 cm2, 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, 1 ,000 cm2, 2,000 cm2, 3,000 cm2, 4,000 cm2, 5,000 cm2, 6,000 cm2, 7,000 cm2, 8,000 cm2, 9,000 cm2, or 10,000 cm2). In some embodiments the PHH are cultured on a two-dimensional surface, wherein the two-dimensional surface has a surface area of about 600 cm2to about 6,000 cm2(e.g., about 600 cm2, about 650 cm2, about 700 cm2, about 750 cm2, about 800 cm2, about 850 cm2, about 900 cm2, about 950 cm2, about 1 ,000 cm2, about 1 ,500 cm2, about 2,000 cm2, about 2,500 cm2, about 3,000 cm2, about 3,500 cm2, about 4,000 cm2, about 4,500 cm2, about 5,000 cm2, about 5,500 cm2, or about 6,000 cm2).

[0280] Extracellular Matrix

[0281] As described herein, the methods for culturing hepatocytes (e.g., PHHs) may include culturing the one or more hepatocytes in contact with an extracellular matrix (ECM). In some embodiments, the hepatocytes contact the ECM through physical, mechanism, or chemical means, or any combination thereof. Any suitable ECM may be used. Isolated hepatocytes (e.g., PHHs) are preferably cultured in a microenvironment that mimics, at least in part, a cellular niche in which the hepatocytes naturally reside. A cellular niche is determined in part by the hepatocytes (e.g., PHHs) and surrounding cells, and the ECM PATENT

[0282] ATTORNEY DOCKET NO. 51540-048WO3 that is produced by the cells in said niche. This cellular niche may be mimicked by culturing the one or more hepatocytes (e.g., PHHs) in the presence of biomaterials, such as an ECM that provides key regulatory signals that control hepatocyte fate.

[0283] In some embodiments, the cell culture surface is coated with an ECM. In some embodiments, the hepatocytes (e.g., PHHs) adhere to the ECM.

[0284] ECM includes a variety of polysaccharides, proteoglycans, water, elastin, and glycoproteins, wherein the glycoproteins may include a laminin, a collagen, an entactin (nidogen), a vitronectin, and / or a fibronectin. ECM components are secreted by connective tissue cells. Different types of ECM are known, including different compositions including different types of glycoproteins and / or different combinations of glycoproteins.

[0285] An ECM may be a one glycoprotein or a particular isoform thereof, or a purified or partially purified fraction that is enriched for ECM species based on their molecular weight (e.g., high molecular weight or low molecular weight species). An ECM may include a mixture of components, such as a mixture of glycoproteins. An ECM may include a laminin (e.g., laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-311 , laminin-321 , laminin-411 , laminin-421 , laminin-511 , laminin-521 , and / or laminin-213), a collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XIII, and / or type XXIV collagen), a vitronectin (e.g., type I, type II, and / or type III vitronectin), a fibronectin (e.g., type I, type II, and / or type III fibronectin), or any combination thereof.

[0286] An ECM can be provided by culturing ECM-producing cells, such as for example fibroblasts, in a receptacle, prior to the removal of these cells and the addition of isolated tissue fragments (e.g., tissue fragments that have been isolated from a liver) or isolated hepatocytes (e.g., PHHs). Examples of ECM- producing cells are chondrocytes that primarily produce collagen and proteoglycans; fibroblasts that primarily produce type IV collagen, laminin, interstitial procollagens, and fibronectin; and colonic myofibroblasts that primarily produce type I, type III, and type V collagens, chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C.

[0287] Alternatively, an ECM can be commercially provided. Examples of commercially available ECMs are ECM proteins (Invitrogen). A synthetic ECM material may be used. Mixtures of ECM materials may be used, if desired. In some embodiments, the ECM does not include a hydrogel (e.g., MATRIGEL™). In some embodiments, the ECM includes a hydrogel (e.g., MATRIGEL™). In some embodiments, the hydrogel is MATRIGEL™.

[0288] In some embodiments, the ECM includes a xeno-free substrate. In some embodiments, the ECM includes one or more recombinant proteins (e.g., recombinant glycoproteins).

[0289] In some embodiments, the ECM includes a native human-derived liver ECM that has been extracted from human liver tissue such as Huma HepatoMatrix Coat (Humabiologics, Inc.).

[0290] In some embodiments, the ECM includes collagen. In some embodiments, the collagen is type I collagen or type IV collagen. In some embodiments, the collagen is type I collagen. In some embodiments, the collagen is type IV collagen.

[0291] In some embodiments, the ECM includes laminin. In some embodiments, the laminin is laminin- 111 , laminin-211 , laminin-221 , laminin-332, laminin-41 1 , laminin-421 , laminin-511 , or laminin-521 . In PATENT

[0292] ATTORNEY DOCKET NO. 51540-048WO3 some embodiments, the laminin is laminin-1 1 1 . In some embodiments, the laminin is laminin-51 1 . In some embodiments, the laminin is laminin-521 .

[0293] In some embodiments, the ECM is applied to a surface (e.g., the surface of a cell culture vessel). In some embodiments, the ECM is applied to the surface at a density of about 0.050 pg / cm2to about 0.150 pg / cm2(e.g., about 0.050, about 0.055, about 0.060, about 0.065, about 0.070, about 0.075, about 0.080, about 0.085, about 0.090, about 0.095, about 0.100, about 0.105, about 0.1 10, about 0.1 15, about 0.120, about 0.125, about 0.130, about 0.135, about 0.140, about 0.145, or about 0.150 pg / cm2). In some embodiments, the ECM is applied to the surface at a density of about 0.150 pg / cm2to about 1 pg / cm2(e.g., about 0.150 pg / cm2to about 0.300 pg / cm2, about 0.150 pg / cm2to about 0.500 pg / cm2, about 0.200 pg / cm2to about 0.600 pg / cm2, or about 0.600 pg / cm2to about 1 pg / cm2; e.g., about 0.160, about 0.170, about 0.180, about 0.190, about 0.200, about 0.210, about 0.220, about 0.230, about 0.240, about 0.250, about 0.300, about 0.350, about 0.400, about 0.450, about 0.500, about 0.550, about 0.600, about 0.650, about 0.700, about 0.750, about 0.800, about 0.850, about 0.900, about 0.950, or about 1 pg / cm2).

[0294] In some embodiments, the culture medium is placed on top of the ECM.

[0295] In some embodiments the culture medium of the invention is in contact with an ECM or a three- dimensional matrix that mimics the ECM by its interaction with the cellular membrane proteins, such as, e.g., integrins.

[0296] Hypoxic Culture Conditions

[0297] The culture methods described herein may include culturing hepatocytes (e.g., PHHs) under hypoxic conditions or in the presence of a hypoxic mimetic. Hypoxic conditions, as described herein, include any condition where oxygen is present in concentrations below normal oxygen concentrations (normoxic conditions). Hypoxic mimetics mimic hypoxia by inducing the accumulation of hypoxia-inducible factor one alpha (HIF1a), which is a protein subunit of a transcription factor that responds to decreases in available oxygen.

[0298] In some embodiments, culturing includes culturing the cells under hypoxic conditions. Hypoxic conditions may include, e.g., an oxygen level of less than 20%. In some embodiments, the culturing under hypoxic conditions includes culturing the cells at an oxygen level of between 1 % to 19% (e.g., between 1 % and 10%, e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the culturing under hypoxic conditions includes culturing the cells at an oxygen level of between 1 % to 19%. In some embodiments, the culturing includes culturing the cells at an oxygen level of between 1 % to 10%. In some embodiments, the culturing includes culturing the cells at an oxygen level of 5%. In some embodiments, the culturing includes culturing the cells under normoxic conditions.

[0299] In some embodiments, culturing includes culturing the cells in the presence of a hypoxic mimetic (e.g., a HIF-1 a stabilizer or a PHD inhibitor). Exemplary hypoxia mimetics are an iron chelator (e.g., deferoxamine (DFO), compound A, deferasirox, and 2,2'-dipyridyl (DP)), an ion competitor (e.g., cobalt chloride (C0CI2) or a divalent metal ion such as Ni2+, Mn2+, Co2+, or Zn2+), and a 2 oxoglutarate (2OG) analog (e.g., dihydroxybenzoic acid (DHB), N-oxalylglycine, dimethyloxalylglycine (DMOG)), a prolyl hydroxylase domain (PHD( inhibitor (e.g., FG-4497, GSK360A, TM6008, or folic acid), or a HIF-1 a stabilizer (e.g., miR-335, isoflurane, N-acetylcysteine, MG-132, BSc21 18, and tilorone). PATENT

[0300] ATTORNEY DOCKET NO. 51540-048WO3

[0301] Hypoxia mimetics are also described, e.g., in Davis et al. Front. Cell Dev. Biol. 6:175, 2018, which is hereby incorporated by reference. In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of cobalt chloride. In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of DFO. In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of DMOG. In some embodiments, the culturing includes culturing the cells in the absence of a hypoxic mimetic.

[0302] Hepatocyte Maturation

[0303] Methods of the disclosure may include maturing a population of hepatocytes (e.g., PHHs), such as a population of expanded hepatocytes. In some embodiments, maturation of a population of hepatocytes (e.g., PHHs) occurs immediately after hepatocyte expansion (e.g., a method of hepatocyte expansion described herein). In some embodiments, maturation of a population of hepatocytes (e.g., PHHs) does not occur immediately after hepatocyte expansion. In some embodiments, a population of expanded hepatocytes have been cryopreserved prior to maturation.

[0304] In some embodiments, the maturation step has a duration of 3 to 14 days (e.g., 3 to 7 days, 3 to 10 days, 5 to 10 days, 7 to 10 days, 7 to 14 days, or 10 to 14 days; e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days). In some embodiments, the maturation step is 7 days.

[0305] In some embodiments, the population of hepatocytes are matured in a two-dimensional environment, such as on a surface of a culture vessel. In some embodiments, the population of hepatocytes are matured in a three-dimensional environment, such as in a suspension culture, in which the hepatocytes are cultivated via dispersion (e.g., by shaking or agitation) in a maturation medium. In some embodiments, the specific components that are included in a maturation medium depend on the culture format (i.e., a two-dimensional or three-dimensional culture).

[0306] In some embodiments, the population of hepatocytes are matured in vivo, such as following administration of hepatocytes (e.g., expanded hepatocytes) into a subject (e.g., a human subject).

[0307] Maturation Medium

[0308] A maturation medium of the disclosure is composed of a basal cell culture medium to which one or more maturation supplements are added. The maturation medium may be used for cultivating hepatocytes (e.g., expanded hepatocytes; e.g., PHHs) in vitro. Components of a maturation medium are described below.

[0309] Basal Cell Medium

[0310] The basal cell culture medium of the maturation medium may be a commercially available basal cell culture medium that is suitable for the in vitro cultivation of mammalian (e.g., human) cells. In some embodiments, the basal cell culture medium is Advanced DMEM / F-12 medium. In some embodiments, the basal cell culture medium is Takara CELLARTIS® POWER™ Primary HEP medium. In some embodiments, the basal cell culture medium is Lonza HCM™ cell culture medium. In In some embodiments, the basal cell culture medium is William’s E medium. In some embodiments, the basal cell culture medium is LIFENET HEALTH® Human Hepatocyte Media. PATENT

[0311] ATTORNEY DOCKET NO. 51540-048WO3

[0312] In some embodiments, the expansion medium includes a basal cell culture medium that includes amino acids glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L- threonine, L-tryptophan, L-tyrosine, and L-valine; vitamins ascorbic acid, biotin, choline chloride, D- calcium pantothenate, ergocalciferol, folic acid, menadione sodium bisulfate, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin A, vitamin B12, alpha-tocopherol, i-inositol; inorganic salts calcium chloride, cupric sulfate, ferric nitrate, magnesium sulfate, manganese chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, zinc sulfate; D- glucose; glutathione; methyl linoleate; phenol red; and sodium pyruvate.

[0313] In some embodiments, the expansion medium includes a basal cell culture medium that includes amino acids glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L- threonine, L-tryptophan, L-tyrosine, and L-valine; vitamins ascorbic acid, biotin, choline chloride, D- calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol; inorganic salts calcium chloride, cupric sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, and zinc sulfate; proteins albumin (e.g., a bovine serum albumin that is enriched with lipids such as cholesterol and fatty acids (e.g., GIBCO™ AlbuMAX™ II Lipid-Rich BSA)), transferrin, and insulin; D-glucose; glutathione; ammonium metavanadate; manganous chloride; sodium selenite; ethanolamine; hypoxanthine; linoleic acid; lipoic acid; putrescine; phenol red; and sodium pyruvate.

[0314] Maturation Supplements

[0315] In some embodiments, the one or more maturation supplements includes a buffering agent that is suitable for mammalian cell culture. In some embodiments, the maturation medium includes a buffering agent at a concentration of 1 mM to 100 mM (e.g., 1 to 5 mM, 1 to 10 mM, 1 to 20 mM, 1 to 50 mM, 10 to 50 mM, 10 to 100 mM, 20 to 100 mM, or 50 to 100 mM; e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM). In some embodiments, the buffering agent is HEPES.

[0316] In some embodiments, the maturation medium includes L-glutamine or an L-glutamine substitute or derivative thereof. In some embodiments, the L-glutamine is an L-glutamine supplement, e.g., L-alanyl- L-glutamine dipeptide, e.g., 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCI (e.g., GLUTAMAX™). In some embodiments, the maturation medium includes GLUTAMAX™ at a volumetric concentration of about 0.1% to about 20% (e.g., about 0.1% to about 1%, about 0.1% to about 5%, about 0.1% to about 10%, about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%; e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9%, about 10%, about 1 1%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%). PATENT

[0317] ATTORNEY DOCKET NO. 51540-048WO3

[0318] In some embodiments, L-glutamine or an L-glutamine substitute or derivative thereof is provided as a solution at a 100X concentration and is added at a concentration of about 0.1X to about 10X (e.g., about 0.1 X to about 1 X, about 0.1 X to about 5X, about 1 X to about 5X, about 1 X to about 10X, or about 5X to about 10X; e.g., about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, or about 10X).

[0319] In some embodiments, the maturation medium includes a Notch inhibitor. A Notch inhibitor may be any agent that inhibits or decreases signaling activity of the Notch signaling pathway, e.g., by inhibiting or decreasing activity of a Notch receptor (e.g., NOTCH1 , NOTCH2, NOTCH3, and / or NOTCH4). A Notch inhibitor may reduce cell surface expression of one or more Notch receptors or reduce the expression of Notch Intracellular Domain (NICD) induced by the transmembrane ligand Delta-like 4 (DLL4). A Notch inhibitor may be an antibody that targets one or more Notch receptors. A Notch inhibitor may be a small molecule inhibitor.

[0320] In some embodiments, a Notch inhibitor is a gamma secretase inhibitor, which inhibits proteolytic cleavage of a Notch receptor intracellular domain. Gamma secretase inhibitors are known in the art and are described elsewhere, such as Golde et al., Biochim Biphys Acta. 1828(12) :2898-907, 2013, which is hereby incorporated by reference. In some embodiments, the Notch inhibitor includes gamma secretase inhibitor Compound E (CAS No. 209986-17-4). In some embodiments, the Notch inhibitor includes Gamma Secretase Inhibitor XX (CAS No. 209985-56-5). In some embodiments, the Notch inhibitor includes both Compound E and Gamma Secretase Inhibitor XX.

[0321] In some embodiments, the maturation medium includes an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the maturation medium includes an EGFR inhibitor at a concentration of 200 nM to 20 pM (e.g., 200 nM to 1 pM, 500 nM to 5 pM, 1 pM to 5 pM, 1 pM to 10 pM, 5 pM to 10 pM, or 10 pM to 20 pM; e.g., 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 pM, 1 .5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, or 20 pM). In some embodiments, the EGFR inhibitor is erlotinib hydrochloride.

[0322] In some embodiments, the maturation medium includes oncostatin M. In some embodiments, the maturation medium includes oncostatin M at a concentration of 1 to 200 ng / mL (e.g., 1 to 20 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, 50 to 75 ng / mL, 60 to 80 ng / mL, 80 to 100 ng / mL, 100 to 125 ng / mL, 125 to 150 ng / mL, 150 to 175 ng / mL, or 175 to 200 ng / mL; e.g., 1 , 2, 3, 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, 30, 31 , 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL).

[0323] In some embodiments, the maturation medium includes an antioxidant. In some embodiments, the maturation medium includes the antioxidant at a concentration of 0.1 mM to 10 mM (e.g., 0.1 mM to 1 mM, 1 mM to 5 mM, or 5 mM to 10 mM; e.g., 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM). In some embodiments, the antioxidant includes vitamin C.

[0324] In some embodiments, the maturation medium includes a glucocorticoid. In some embodiments, the glucocorticoid includes dexamethasone, hydrocortisone, prednisone, triamcinolone, methylprednisolone, or a combination thereof. In some embodiments, the maturation medium includes PATENT

[0325] ATTORNEY DOCKET NO. 51540-048WO3 two or more glucocorticoids (e.g., two, three, four, or more). In some embodiments, the maturation medium includes a glucocorticoid at a concentration of 1 gM to 1 mM (e.g., 1 gM to 5 gM, 1 gM to 10 gM, 1 gM to 50 gM, 1 gM to 100 gM, 100 gM to 200 gM, 100 gM to 500 gM, 100 gM to 1 mM, or 500 gM to 1 mM; e.g., 1 gM, 2 gM, 3 gM, 4 gM, 5 gM, 6 gM, 7 gM, 8 gM, 9 gM, 10 gM, 1 1 gM, 12 gM, 13 gM, 14 gM, 15 gM, 16 gM, 17 gM, 18 gM, 19 gM, 20 gM, 30 gM, 40 gM, 50 gM, 60 gM, 70 gM, 80 gM, 90 gM, 100 gM, 150 gM, 200 gM, 250 gM, 300 gM, 350 gM, 400 gM, 450 gM, 500 gM, 550 gM, 600 gM, 650 gM, 700 gM, 750 gM, 800 gM, 850 gM, 900 gM, or 1 mM). In some embodiments, the maturation medium includes dexamethasone. In some embodiments, the maturation medium includes hydrocortisone. In some embodiments, the maturation medium includes both dexamethasone and hydrocortisone.

[0326] In some embodiments, the maturation medium includes a pregnane X receptor (PXR) activator. PXR is also known in the art as the steroid and xenobiotic sensing nuclear receptor (SXR) and is a ligand-activated nuclear receptor. PXR may be activated by a hydrophobic ligand such as a hydrophobic vitamin, a bile acid (e.g., a bile acid described herein), or a combination thereof. In some embodiments, the PXR activator is a hydrophobic vitamin such as vitamin A, vitamin D, vitamin E, or vitamin K. In some embodiments, the PXR activator is vitamin K. The maturation medium may include a PXR activator at a concentration of 1 to 100 gM (e.g., 1 to 5 gM, 1 to 10 gM, 1 to 20 gM, 5 to 20 gM, 10 to 50 gM, or 50 to 100 gM; e.g., 1 gM, 2 gM, 3 gM, 4 gM, 5 gM, 6 gM, 7 gM, 8 gM, 9 gM, 10 gM, 1 1 gM, 12 gM, 13 gM, 14 gM, 15 gM, 16 gM, 17 gM, 18 gM, 19 gM, 20 gM, 25 gM, 30 gM, 35 gM, 40 gM, 45 gM, 50 gM, 55 gM, 60 gM, 65 gM, 70 gM, 75 gM, 80 gM, 85 gM, 90 gM, 95 gM, or 100 gM).

[0327] In some embodiments, the maturation medium includes a bile acid. In some embodiments, the bile acid is a secondary bile acid. In some embodiments, the bile acid includes lithocholic acid, urso deoxycholic acid, or a combination thereof. In some embodiments, the maturation medium includes a bile acid at a concentration of 1 to 100 gM (e.g., 1 to 5 gM, 1 to 10 gM, 1 to 20 gM, 5 to 20 gM, 10 to 50 gM, or 50 to 100 gM; e.g., 1 gM, 2 gM, 3 gM, 4 gM, 5 gM, 6 gM, 7 gM, 8 gM, 9 gM, 10 gM, 1 1 gM, 12 gM, 13 gM, 14 gM, 15 gM, 16 gM, 17 gM, 18 gM, 19 gM, 20 gM, 25 gM, 30 gM, 35 gM, 40 gM, 45 gM, 50 gM, 55 gM, 60 gM, 65 gM, 70 gM, 75 gM, 80 gM, 85 gM, 90 gM, 95 gM, or 100 gM).

[0328] In some embodiments, the maturation medium includes a cholesterol.

[0329] In some embodiments, the maturation medium includes a cAMP or a cAMP analog. In some embodiments, the cAMP analog includes 8-bromo cAMP. In some embodiments, the cAMP analog includes forskolin. In some embodiments, the cAMP analog includes both 8-bromo cAMP and forskolin. In some embodiments, the maturation medium includes cAMP or a cAMP analog at a concentration of 1 to 100 gM (e.g., 1 to 5 gM, 1 to 10 gM, 1 to 20 gM, 5 to 20 gM, 10 to 50 gM, or 50 to 100 gM; e.g., 1 gM, 2 gM, 3 gM, 4 gM, 5 gM, 6 gM, 7 gM, 8 gM, 9 gM, 10 gM, 1 1 gM, 12 gM, 13 gM, 14 gM, 15 gM, 16 gM, 17 gM, 18 gM, 19 gM, 20 gM, 25 gM, 30 gM, 35 gM, 40 gM, 45 gM, 50 gM, 55 gM, 60 gM, 65 gM, 70 gM, 75 gM, 80 gM, 85 gM, 90 gM, 95 gM, or 100 gM). In some embodiments, the maturation medium includes cAMP or a cAMP analog at a concentration of 0.1 to 10 mM (e.g., 0.1 to 1 mM, 0.1 to 5 mM, 1 to 5 mM, 1 to 10 mM, 1 to 20 mM, 1 to 50 mM, 10 to 50 mM, 10 to 100 mM, 20 to 100 mM, or 50 to 100 mM; e.g., 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM). PATENT

[0330] ATTORNEY DOCKET NO. 51540-048WO3

[0331] In some embodiments, the maturation medium includes a thyroid hormone. In some embodiments, the thyroid hormone is triiodothyronine (T3). In some embodiments, the maturation medium includes a thyroid hormone at a concentration of 1 to 30 |_iM (e.g., 1 to 5 pM, 1 to 10 |_iM, 10 to 20 |_lM, or 10 to 30 |lM; e.g., 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 |_iM).

[0332] In some embodiments, the maturation medium includes a peptide hormone. In some embodiments, the peptide hormone includes glucagon or glucagon-like peptide-1 (GLP-1 ). In some embodiments, the peptide hormone includes glucagon. In some embodiments, the peptide hormone includes GLP-1 . In some embodiments, the maturation medium includes the peptide hormone at a concentration of 10 nM to 1 pM (e.g., 10 nM to 50 nM, 10 nM to 100 nM, 50 nM to 200 nM, 50 nM to 500 nM, 100 nM to 500 nM, 100 nM to 1 pM, or 500 nM to 1 pM; e.g., 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 1 10 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 525 nM, 550 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 850 nM, 875 nM, 900 nM, 925 nM, 950 nM, 975 nM, or 1 pM).

[0333] In some embodiments, the maturation medium includes an amino acid supplement. In some embodiments, the amino acid supplement is an NEAA supplement or an essential amino acid (EAA) supplement. In some embodiments, the maturation medium includes both an NEAA supplement and an EAA supplement. In some embodiments, the NEAA supplement includes glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or any combination thereof. In some embodiments, the NEAA supplement includes glycine, L-alanine, L-asparagine, L-aspartic acid, L- glutamic acid, L-proline, and L-serine. In some embodiments, the EAA supplement includes L-histidine, L- isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, or any combination thereof. In some embodiments, the EAA supplement includes L-histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine. In some embodiments, the amino acid supplement is provided as a solution at a 50X or 100X stock concentration. In some embodiments, the maturation medium includes the amino acid supplement at a final concentration of about 0.1 X to about 100X (e.g., about 0.1 X to about 90X, about 0.5X to about 80X, about 1 X to about 70X, about 5X to about 60X, and about 10X to about 50X; e.g., about 0.1 X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1 X, about 1 .5X, about 2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 15X, about 20X, about 25X, about 30X, about 35X, about 40X, about 45X, about 50X, about 60X, about 70X, about 80X, about 90X, or about 100X).

[0334] In some embodiments, the maturation medium includes an antibiotic. In some embodiments, the maturation medium does not include an antibiotic. In some embodiments, the antibiotic includes penicillin, streptomycin, or both penicillin and streptomycin. In some embodiments, the maturation medium includes about 0.1 % or more (e.g., about 0.5%, about 1 %, or about 5%) of a solution of penicillin and streptomycin. In some embodiments, the maturation medium includes from 0.1 % to 10%, e.g., 0.1 % to 1 % (e.g., 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1 %) or 1 % to 10% (e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) a solution of penicillin and streptomycin. PATENT

[0335] ATTORNEY DOCKET NO. 51540-048WO3

[0336] In some embodiments, the maturation medium includes additional additives. In some embodiments, the maturation medium includes a ROCK inhibitor, such as Y-27632 (CAS No. 129830038- 2). In some embodiments, the maturation medium includes a nuclease (e.g., a DNase or an RNase). In some embodiments, the maturation medium includes a DNase. In some embodiments, the maturation medium includes a surfactant such as a non-ionic detergent to reduce shear forces applied to cells in a suspension culture. In some embodiments, the non-ionic detergent includes Pluronic F68, Pluronic F127, Poloxamer 188, Poloxamer 407, polysorbate 20, polysorbate 80, or sorbitan monolaurate. In some embodiments, the non-ionic detergent includes Pluronic F68.

[0337] Serum Replacement Component

[0338] The maturation medium may include a serum replacement component, in which any suitable serum replacement component may be used. In some embodiments, the serum replacement component is KOSR, human platelet lysate, human serum, ITS, Trace Elements A (i.e., a cell culture medium additive that includes copper, zinc, iron, and selenium), or Trace Elements B (i.e., a cell culture medium additive that includes ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride, and hydrochloric acid). In some embodiments, the serum replacement component includes a combination of two or more (e.g., two, three, or four) of the foregoing serum replacement components. In some embodiments, the maturation medium includes KOSR and ITS.

[0339] Cellular Signatures and Biomarkers

[0340] Following expansion and / or maturation of a population of hepatocytes (e.g., PHHs), the hepatocytes may be evaluated for expression of biomarkers that confirm hepatocyte cellular identity and function. Such biomarkers include proteins that are secreted into the bloodstream, such as coagulation factors, surface-expressed biomarkers and receptors, and liver enzymes, including urea cycle enzymes. Exemplary biomarkers that are expressed following expansion and / or maturation (e.g., via the expansion and maturation methods described herein) are discussed below.

[0341] Albumin is a globular protein that is produced by the liver and secreted into the bloodstream, in which it stabilizes osmotic pressure in the vasculature and carries endogenous ligands to various tissues. Albumin is a biomarker of both hepatoblasts (i.e., hepatic progenitor cells) and terminally differentiated hepatocytes. In some embodiments, after culturing, the population of hepatocytes secrete albumin. In general, expanded hepatocytes (e.g., PHHs) may secrete lower levels of albumin as compared to overnight plated control PHH. In some embodiments, after maturation, the mature hepatocytes secrete albumin.

[0342] In some embodiments, the expanded and / or matured hepatocytes (e.g., PHHs) secrete 1 to 50 pg / million cells / day of albumin (e.g., 1 to 5 pg / million cells / day, 1 to 10 pg / million cells / day, 5 to 25 pg / million cells / day, 10 to 25 pg / million cells / day, 25 to 50 pg / million cells / day, or more than 50 pg / million cells / day; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, or more than 50 pg / million cells / day). In some embodiments, at least 70% of hepatocytes in a population of expanded and / or matured hepatocytes secrete albumin. In some embodiments, at least 75%, at least 80%, at least 85%, or at least 90% of hepatocytes in a population of expanded and / or matured hepatocytes secrete albumin. PATENT

[0343] ATTORNEY DOCKET NO. 51540-048WO3

[0344] In some embodiments, the expanded and / or matured hepatocytes (e.g., PHHs) secrete coagulation factor IX. In some embodiments, the expanded and / or matured hepatocytes secrete 1 to 50 ng / mil lion cells / day of factor IX (e.g., 1 to 5 ng / million cells / day, 1 to 10 ng / m illion cells / day, 5 to 25 ng / million cells / day, 10 to 25 ng / million cells / day, 25 to 50 ng / million cells / day, or more than 50 ng / million cells / day; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, or more than 50 ng / million cells / day). In some embodiments, at least 70% of hepatocytes in a population of expanded and / or matured hepatocytes secrete factor IX. In some embodiments, at least 75%, at least 80%, at least 85%, or at least 90% of hepatocytes in a population of expanded and / or matured hepatocytes secrete factor IX.

[0345] In some embodiments, the expanded and / or matured hepatocytes express one or more other biomarkers that confirms hepatocyte identity. In some embodiments, the expanded and / or matured hepatocytes express hepatocyte biomarkers including HNFa, NR1 I2, SERPINA1 , alpha-1 antitrypsin, ASGR1 , or a combination thereof.

[0346] In some embodiments, the expanded and / or matured hepatocytes express one or more biomarkers associated with liver metabolic function. In some embodiments, the expanded and / or matured hepatocytes secrete urea or express one or more biomarkers associated with the urea cycle, such as a gene encoding an enzyme in the urea cycle. The expanded and / or matured hepatocytes may express one or more urea cycle biomarkers including CYP protein family (e.g., CYP3A4, CYP1 A2, or both), ARG1 , ASL, ASS1 , CPS1 , NAGS, OTC, AQP9, SLC24A13, SLC25A15, or a combination thereof. In some embodiments, the expanded and / or matured hepatocytes express one or more glutamine synthesis biomarkers including GLS2, GLUL, GPX1 , UGT 1 A1 , or a combination thereof. In some embodiments, at least 75%, at least 80%, at least 85%, or at least 90% of hepatocytes in a population of expanded and / or matured hepatocytes express one or more urea cycle biomarkers.

[0347] In some embodiments, the expanded and / or matured hepatocytes express one or more biomarkers associated with cell polarity, such as apical and basolateral polarity membrane proteins. In some embodiments, the expanded and / or matured hepatocytes express ABCG2, ABCC2, ABCC3, ABCB11 , SCARB1 , ALC10A1 , or a combination thereof.

[0348] In some embodiments, the expanded and / or matured hepatocytes do not express hepatic progenitor or cholangiocyte biomarkers. In some embodiments, the expanded and / or matured hepatocytes do not have detectable expression of hepatic progenitor or cholangiocyte biomarkers including AFP, CYP3A7, EPCAM, LGR5, KRT7, KRT19, AQP1 , or a combination thereof. In some embodiments, a population of expanded hepatocytes (e.g., PHHs) have increased expression of hepatic progenitor or cholangiocyte markers such as AFP, CYP3A7, EPCAM, LGR5, KRT7, KRT 19, AQP1 , or a combination thereof as compared to a population of PHH that were not expanded. In some embodiments, a population of expanded PHH require a maturation step to downregulate or reduce the expression of hepatic progenitor or cholangiocyte biomarkers. In some embodiments, less than 10% (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%) of hepatocytes in a population of expanded and / or matured hepatocytes express a hepatic progenitor or cholangiocyte biomarker.

[0349] The presence and / or expression level of cell biomarkers (e.g., biomarkers of hepatocyte identity or function described herein) may be detected or measured by any suitable methodology known in the art PATENT

[0350] ATTORNEY DOCKET NO. 51540-048WO3 for evaluating the expression of nucleic acids such as RNA (e.g., mRNA transcripts), proteins (e.g., intracellularly expressed proteins, surface-expressed proteins, or secreted proteins), or metabolic products or byproducts (e.g., urea). In some embodiments, expression level of a protein (e.g., an enzyme) includes detecting or measuring its activity (e.g., by product yield and / or by a fluorescent or luminescent reporter assay).

[0351] Exemplary methods of detecting or measuring the relative expression of one or more transcription factors or biomarkers are flow cytometry, fluorescence activated cell sorting (FACS), massively parallel DNA sequencing (e.g., next-generation sequencing), RNA-sequencing (RNA-seq) (e.g., bulk RNA-seq or single-cell RNA-seq), real-time reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), RT-qPCR, Northern blot analysis, mass spectrometry and proteomic modalities, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunofluorescence or immunodetection methods, in situ hybridization (e.g., fluorescence in situ hybridization (FISH))among other detection methods known in the art.

[0352] In some embodiments, the presence and / or expression level of a biomarker in a first sample (e.g., a cell such as an expanded hepatocyte or a matured hepatocyte, a tissue such as a portion of a liver, or a sample of culture medium such as culture medium that has been in contact with a population of hepatocytes) is compared to the expression level of the biomarker in a second sample. In some embodiments, the second sample is a reference sample such as, a reference cell or population of cells (e.g., PHHs cultured in the absence of a culture medium such as a culture medium described herein), a reference tissue, a control sample, a control cell, or a control tissue.

[0353] Hepatocyte Aggregates

[0354] The expanded and / or matured hepatocytes (e.g., PHHs) may be aggregated. Aggregates described herein include a population of hepatocytes. In some embodiments, the hepatocytes are admixed under conditions which cause the cell population to form aggregates. In some embodiments, the hepatocytes are admixed using tissue fabrication techniques. In some embodiments, the hepatocytes are cultured by hanging drop, microwell molding, non-adhesive surfaces, spheroid suspension culture using a spinner flask, vertical wheel bioreactor, horizontal wheel bioreactor, or a microfluidic spheroid system. Additional methods include those using acoustical waves and using positively-charged surfaces on a plate. In some embodiments, the hepatocytes (e.g., PHHs) are admixed in the presence of stromal cells (e.g., fibroblasts, e.g., normal human dermal fibroblasts (NHDF)). In some embodiments, the hepatocytes (e.g., PHHs) are admixed in the presence of NHDF. In some embodiments, the hepatocytes (e.g., PHHs) are admixed in the absence of stromal cells (e.g., NHDF).

[0355] In other aspects, the compositions provided herein can contain additional components, growth factors, ligands, cytokines, drugs, and the like. In some embodiments, the cell mixture can include molecules which elicit additional microenvironmental cues such as small molecules or growth factors which stimulate or enhance proliferation and expansion of a cell population.

[0356] In certain embodiments, the aggregates disclosed herein include one or more adherence materials to facilitate maintenance of the desired phenotype of the grafted cells in vivo. The material may include an antibody, a protein or peptide, a nucleic acid, an aptamer (e.g., an RNA or DNA aptamer), a PATENT

[0357] ATTORNEY DOCKET NO. 51540-048WO3 carbohydrate, a proteoglycan, or a matrix. The type of adherence materials (e.g., ECM materials, sugars, proteoglycans, etc.) will be determined, in part, by the cell type (e.g., PHH) to be cultured.

[0358] In some embodiments, organizing cells and material into spatial arrangements, such as aggregates, can be accomplished by physically constraining the placement of cells / material by the use of wells or grooves, or injecting cells into microfluidic channels or oriented void spaces / pores. In certain embodiments, the cells can be organized by physically positioning cells with electric fields, magnetic tweezers, optical tweezers, ultrasound waves, pressure waves, or micromanipulators.

[0359] The cells produced by the culturing methods described herein can be used immediately in the making of an aggregate. Alternatively, the cells can be frozen in liquid nitrogen and stored for long periods of time for thawing and later making an aggregate. For example, the cells can be frozen in a culture medium that includes a cryoprotective agent, such as a medium that includes 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or one or more other agents known in the art.

[0360] Cell Implants

[0361] As described herein, the methods disclosed herein may include introducing the population of expanded and / or matured hepatocytes (e.g., PHHs) or progeny thereof into a subject. In some embodiments, the population of expanded and / or matured hepatocytes or progeny thereof is introduced into a subject in the form of a hepatocyte aggregate. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient suffering from a liver disease. The population of expanded and / or matured hepatocytes (e.g., PHHs) or progeny thereof may be incorporated into an engineered tissue construct, e.g., for implantation into a subject. The engineered tissue construct may include a biocompatible hydrogel scaffold (e.g., a scaffold that includes fibrin). The biocompatible scaffold may contain an encapsulated population of aggregated hepatocytes.

[0362] In some embodiments, uses of the population of expanded and / or matured hepatocytes (e.g., PHHs) cultured as described herein are likewise provided. For example, in some embodiments, the invention also provides the use of the population of expanded and / or matured hepatocytes of the invention or a lot of frozen hepatocytes derived from said population of expanded hepatocytes in a discovery screen; toxicity assay; gene expression studies including recombinant gene expression; research of mechanisms involved in tissue injury and repair; research of inflammatory and infectious diseases; studies of pathogenetic mechanisms; or studies of mechanisms of cell transformation and etiology of liver disease.

[0363] In some embodiments, the invention also provides cells derived from the population of expanded and / or matured hepatocytes (e.g., PHHs) of the invention for use in medicine. In some embodiments, the invention also provides cells derived from the population of expanded and / or matured hepatocytes of the invention for use in treating a disorder, condition, or disease. In some embodiments, the invention also provides the population of expanded hepatocytes, or cells derived from the population of expanded hepatocytes of the invention, for use in regenerative medicine, for example, wherein the use involves implantation of the population of expanded cells or cells derived from the population of expanded hepatocytes into a subject. In some embodiments, the invention also provides the population of matured hepatocytes (e.g., PHHs), or cells derived from the population of matured hepatocytes of the invention, PATENT

[0364] ATTORNEY DOCKET NO. 51540-048WO3 for use in regenerative medicine, for example, wherein the use involves implantation of the population of matured cells or cells derived from the population of matured hepatocytes into a subject.

[0365] Pharmaceutical Compositions

[0366] The invention also provides a pharmaceutical formulation including one or more population of expanded and / or matured hepatocytes (e.g., PHHs) and a pharmaceutically acceptable diluent and / or excipient.

[0367] The cells or aggregates prepared by one or more of the methods described herein may be formulated into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo. For example, the cells (e.g., expanded and / or matured hepatocytes) or aggregates of the cells described herein may be administered in a suitable diluent, carrier, stabilizer, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nd ed. and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0368] The cells or aggregates described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is hereby incorporated by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0369] In some embodiments, a pharmaceutical composition is prepared for administration by injection, implantation, or engraftment. In some embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or a saline buffer (e.g., phosphate buffered saline). In some embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In some embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents PATENT

[0370] ATTORNEY DOCKET NO. 51540-048WO3 and the like. Certain pharmaceutical formulations for injection may be presented in unit dosage form, e.g., in a vial.

[0371] Kits

[0372] The compositions described herein can be provided in a kit for use in expanding freshly harvested or previously cryopreserved hepatocytes (e.g., PHHs) or for maturing hepatocytes, such as hepatocytes that have been expanded. In some embodiments, the kit can include one or more cell culture medium components or additives as described herein. In some embodiments, the kit further includes a package insert that instructs a user of the kit, such as a laboratory scientist, to perform any one of the methods described herein. In some embodiments, the kit includes cryopreserved hepatocytes (e.g., PHHs) that were previously expanded according to the method described herein. In some embodiments, the kit includes cryopreserved hepatocytes (e.g., PHHs) that were previously matured according to a method described herein. In some embodiments, the kit can include equipment for administering a pharmaceutical composition including hepatocytes (e.g., a pharmaceutical composition including expanded and / or matured hepatocytes).

[0373] EXAMPLES

[0374] The following examples are set forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0375] Example 1. Reprogramming of Hepatocytes

[0376] Primary human hepatocytes were expanded, reprogrammed, and matured as depicted in FIGS. 1, 2, and 9. PO expanded cells were plated at 6400 cells per well of 6-well plate for P1 expansion in 5% KOSR containing Expand 3.0 media containing CEPT (Bio-Techne, 7991) and 200 ng / ml of B18R (Bio- Techne, 8185-BR). After culturing for a day, transfection was initiated in 5% KOSR containing Expand 3.0 media (see, e.g., PCT Pub. No. WO 2023 / 076292) containing CEPT and B18R protein. Reprogramming mRNA was transfected at a molar stoichiometry of 3:1 :1 :1 :1 :1 for the Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog. Specifically, 462 ng of Oct4 (Reprocell, 05-0014), 143 ng of Sox2 (Miltenyi, 130-101 -075), 187 ng of Klf4 (Miltenyi, 130-101 -115), 177 ng of cMyc (Miltenyi, 130-101 -112), 99 ng of Lin28 (Miltenyi, 130-101 - 117), and 133 ng of Nanog (Miltenyi, 130-101 -118) mRNA was used per well of 6-well plate. Molar stoichiometry and quantities of individual mRNAs used per well indicated above was maintained for different mRNA cocktails tested such as OSKMLN, OSKML, and OSN with 1 .2 ug being the maximum total mRNA used per well of 6-well plate. jetMESSENGER® transfection reagent (Polyplus, 101000056) was used at a ratio of 1 .6 pl for every pg of mRNA. Transfection was repeated every day for a total of 3 or 4 transfections as indicated above. After completion of 3 or 4 days of transfection, media exchange was done using 5% KOSR containing Expand 3.0 media containing CEPT and B18R and cultured for next 24- hours. Subsequently, media exchange was done using 10% KOSR containing Expand 3.0 media and kept in culture until they are ready to be passaged with media exchange on alternate days. Partial reprogramming was repeated as indicated above in P2 and P3. During partial reprogramming in each PATENT

[0377] ATTORNEY DOCKET NO. 51540-048WO3 passage, appropriate control conditions such as untransfected control and transfection control involving transfection reagent lacking mRNA cargo were used.

[0378] As shown in FIGS. 3-5, hepatocytes were expanded following partial reprogramming. Untransfected and transfection control groups could not be passaged after P2 as there were very few cells. Therefore, there were no untransfected and transfection control groups in Passages. As shown in FIGS. 6A and 6B, PassageO expanded hepatocytes were partially reprogrammed in passagel , passage2, and passages. OSKML reprogramming factors provided a 24.5-fold, 12.2-fold, and 5.6-fold expansion relative to initial plating in each of P1 , P2, and P3, respectively (FIG. 6A), and a 189,144 cumulative fold expansion (FIG. 6B). Furthermore, hepatic markers were maintained, and endogenous pluripotent markers were not upregulated in partially reprogrammed cells (FIGS. 7A-7F). OSKML partial reprogrammed eHH cells also showed albumin and A1 AT secretion (FIG. 8). These data indicate that partial reprogramming of expanded hepatocytes demonstrated rejuvenation and promoted proliferation. Hepatic lineage markers were maintained, while hepatic progenitor / fetal markers were upregulated. Endogenous pluripotency markers were not upregulated, and partially reprogrammed eHH cells continued to secrete Albumin and A1 AT in all passages.

[0379] A modified reprogramming scheme was implemented as shown in FIG. 9 in which cells were transfected for one additional day during each passage step and tested with different combinations of reprogramming factors (OSKML, OSKMLN, OSN), and three different concentrations of Nanog only. Cells were visualized and quantified during each passage (P1 -P3) as shown in FIGS. 10-15. Following P1 , each of OSKML, OSKMLN, and OSN exhibited at least 13.8-fold expansion relative to initial plating (FIGS. 10 and 11). Following P2, OSKML and OSKMLN showed at least 14.2-fold expansion, while OSN showed 5.0-fold expansion (FIGS. 12 and 13). Following P3, OSKMLN showed 10.3-fold expansion (FIGS. 14A, 14B, and 15). Following P3, OSKMLN showed 174,626 cumulative fold expansion, while OSKML showed 18,318 cumulative fold expansion, and OSN showed 1 ,035 cumulative fold expansion (FIG. 16), indicating that a reduced set of reprogramming factors (OSN) was able to induce expansion. The OSKMLN combination promoted expansion better than OSKML in advance passages (P2, P3). Although OSN seemed to cause some level of eHH expansion in P1 , P2 steps, it did not work in P3 to support eHH expansion. Nanog alone did not support eHH expansion in any passage (P1 -P3). FIG. 17 shows immunostaining of partially reprogrammed cells for HOECHST and HNF4A in partially reprogrammed expanded human hepatocytes (pr-eHH) following partial reprogramming with OSKML after P1 and P2. The percentages of HNF4a positive cells indicated and show that hepatic / hepatic progenitor marker HNF4a was expressed in a vast majority of partially reprogrammed cells.

[0380] Other Embodiments

[0381] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0382] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art PATENT

[0383] ATTORNEY DOCKET NO. 51540-048WO3 to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0384] Other embodiments are within the claims.

Claims

PATENTATTORNEY DOCKET NO. 51540-048WO3CLAIMS1 . A method for producing expanded human hepatocytes, the method comprising:(a) culturing hepatocytes in contact with an extracellular matrix in the presence of an expansion medium; and(b) contacting the hepatocytes with a polynucleotide encoding one or more reprogramming factors to produce expanded human hepatocytes.

2. A method for culturing human hepatocytes, the method comprising contacting expanded hepatocytes with a polynucleotide encoding one or more reprogramming factors to produce expanded human hepatocytes, wherein the expanded hepatocytes were previously cultured in contact with an ECM in the presence of an expansion medium.

3. The method of claim 1 or 2, wherein the polynucleotide is RNA.

4. The method of claim 1 or 2, wherein the polynucleotide is DNA.

5. The method of any one of claims 1 -4, wherein the one or more reprogramming factors comprise one or more transcription factors.

6. The method of claim 5, wherein the one or more transcription factors is Oct3 / 4, Sox2, Sox3, Sox15, Sox18, Klf1 , Klf2, Klf4, Klf5, cMyc, N-myc, L-myc, Nanog, Lin28, Glisl , or a combination thereof.

7. The method of claim 6, wherein the one or more transcription factors comprise Oct3 / 4, Sox2, Klf4, cMyc, Lin28A, and Nanog.

8. The method of claim 7, wherein the one or more transcription factors comprise Oct3 / 4, Sox2, and Nanog.

9. The method of any one of claims 1 -8, wherein each reprogramming factor is encoded by an RNA.

10. The method of any one of claims 1 -3 and 5-9, wherein the one or more reprogramming factors are encoded by mRNA, and the contacting step comprises transfecting the expanded human hepatocytes with the mRNA.1 1 . The method of claim 10, wherein each reprogramming factor is encoded by an mRNA.

12. The method of claim 10 or 1 1 , wherein the mRNA comprises a modified base.

13. The method of claim 12, wherein modified base is 5’-methylcytidine or N1-methylpseudouridine.PATENTATTORNEY DOCKET NO. 51540-048WO314. The method of any one of claims 4-9, wherein the contacting step comprises transfecting the expanded human hepatocytes with DNA, and the DNA transcribes RNA encoding the one or more reprogramming factors.

15. The method of any one of claims 8-14, wherein the contacting step comprises lipid-based transfection.

16. The method of any one of claims 1 -9, wherein the polynucleotide encoding the reprogramming factor is delivered to the hepatocytes via a viral vector.

17. The method of claim 16, wherein the viral vector is an RNA viral vector.

18. The method of claim 16, wherein the viral vector is a DNA viral vector.

19. The method of any one of claims 16-18, wherein the viral vector is a lentivirus or a retrovirus.

20. The method of any one of claims 1 -19, wherein the reprogramming factor causes expression of one or more pluripotency-associated genes.21 . The method of any one of claims 1 -20, wherein the reprogramming factor causes an epigenetic modification in the hepatocytes.

22. The method of claim 21 , wherein the epigenetic modification results in a change in expression pattern of one or more genes in the hepatocytes.

23. The method of any one of claims 1 -22, wherein the reprogramming factor causes a chromatin modification in the hepatocytes.

24. The method of any one of claims 1 -23, wherein the human hepatocytes are primary human hepatocytes or expanded human hepatocytes.

25. The method of any one of claims 1 -24, wherein hepatocytes are genetically engineered hepatocytes.

26. The method of claim 25, wherein the genetically engineered hepatocytes are hypoimmunogenic hepatocytes.

27. The method of any one of claims 1 -26, wherein the culturing step comprises expanding plated cells (step P0) prior to contacting the hepatocytes with the polynucleotide.

28. The method of claim 27, wherein the cells are plated and cultured in the presence of Chroman 1 , Emricasan, Polyamine, and Trans-integrated stress response inhibitor (trans-ISRIB) (CEPT) cocktail.PATENTATTORNEY DOCKET NO. 51540-048WO329. The method of claim 27 or 28, wherein the PO step has a duration of 5 to 20 days.

30. The method of claim 29, wherein the PO step has a duration of 13 days.31 . The method of any one of claims 1 -30, wherein the contacting step comprises a first passage of expanded cells (step P1).

32. The method of claim 31 , wherein the P1 step has a duration of 5 to 20 days.

33. The method of claim 31 or 32, wherein the polynucleotide is RNA.

34. The method of claim 33, wherein the P1 step comprises culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day.

35. The method of 33, wherein the P1 step comprises culturing the hepatocytes on day 0 of P1 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

36. The method of any one of claims 33-35, further comprising a second passage of expanded cells (step P2).

37. The method of claim 36, wherein the P2 step has a duration of 5 to 20 days.

38. The method of claim 36 or 37, wherein the P2 step comprises culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day 1 for a total of 1 day.

39. The method of claim 36 or 37, wherein the P2 step comprises culturing the hepatocytes on day 0 of P2 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

40. The method of any one of claims 36-39, further comprising a third passage of expanded cells (step P3).41 . The method of claim 40, wherein the P3 step has a duration of 5 to 20 days.PATENTATTORNEY DOCKET NO. 51540-048WO342. The method of claim 40 or 41 , wherein the P3 step comprises culturing the hepatocytes on day 0 of P3 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors on day1 for a total of 1 day.

43. The method of claim 40 or 41 , wherein the P3 step comprises culturing the hepatocytes on day 0 ofP3 and contacting the hepatocytes with the RNA encoding the one or more reprogramming factors daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

44. The method of claim 31 or 32, wherein the polynucleotide is DNA.

45. The method of claim 44, wherein the DNA comprises an inducible promoter and one or more open reading frames of the one or more reprogramming factors, wherein the inducible promoter is operably linked to the one or more open reading frames of the one or more reprogramming factors.

46. The method of claim 45, wherein the P1 step comprises culturing the hepatocytes on day 0 of P1 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter on day 1 for a total of 1 day.

47. The method of claim 45, wherein the P1 step comprises culturing the hepatocytes on day 0 of P1 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

48. The method of any one of claims 45-47, further comprising a second passage of expanded cells (step P2).

49. The method of claim 48, wherein the P2 step has a duration of 5 to 20 days.

50. The method of claim 48 or 49, wherein the P2 step comprises culturing the hepatocytes on day 0 of P2 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter on day 1 for a total of 1 day.51 . The method of claim 48 or 49, wherein the P2 step comprises culturing the hepatocytes on day 0 of P2 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter daily from day 1 to day 2 for a total of2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.PATENTATTORNEY DOCKET NO. 51540-048WO352. The method of any one of claims 45-51 , further comprising a third passage of expanded cells (step P3).

53. The method of claim 52, wherein the P3 step has a duration of 5 to 20 days.

54. The method of claim 52 or 53, wherein the P3 step comprises culturing the hepatocytes on day 0 of P3 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter on day 1 for a total of 1 day.

55. The method of claim 52 or 53, wherein the P3 step comprises culturing the hepatocytes on day 0 of P3 and (a) contacting the hepatocytes with an agent that activates the inducible promoter or (b) exposing the hepatocytes to a condition that activates the inducible promoter daily from day 1 to day 2 for a total of 2 days, from day 1 to day 3 for a total of 3 days, or from day 1 to day 4 for a total of 4 days, or from day 1 to day 7 for a total of 7 days.

56. The method of any one of claims 40-55, further comprising a fourth, fifth, sixth, seventh, eighth, ninth, or tenth passage of expanded cells (step P4-P10).

57. The method of any one of claims 31 -56, wherein the cells are plated and cultured in the presence of CEPT cocktail.

58. The method of any one of claims 1 -57, further comprising contacting the expanded human hepatocytes with a maturation medium to mature the expanded hepatocytes in two-dimensional planar culture.

59. The method of any one of claims 1 -58, further comprising contacting the expanded human hepatocytes with a maturation medium to mature the expanded hepatocytes in three-dimensional aggregates.

60. The method of claim 59, further comprising contacting the expanded human hepatocytes with a maturation medium in the presence of a different cell type to mature the expanded hepatocytes in three- dimensional heterocellular aggregates.61 . The method of any one of claims 58-60, wherein the maturation step begins immediately following hepatocyte reprogramming.

62. The method of any one of claims 58-60, wherein the maturation step does not begin immediately following hepatocyte reprogramming.

63. The method of any one of claims 58-62, wherein the maturation step has a duration of 1 to 14 days.PATENTATTORNEY DOCKET NO. 51540-048WO364. The method of any one of claims 1 -63, wherein prior to the contacting step, the hepatocytes are cryopreserved and thawed.

65. The method of any one of claims 1 -64, the hepatocytes are engineered to express or knock-out one or more genes.

66. The method of any one of claims 1 -65, wherein the expanded hepatocytes exhibit enhanced proliferation compared to the initial hepatocytes.

67. A population of expanded hepatocytes produced by the method of any one of claims 1 -66.

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