Methods for producing mature hepatocytes

Abstract

The present invention provides methods for producing mature hepatocytes by increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes, and compositions thereof.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 185,735, entitled "METHODS OF GENERATING MATURE HEPATOCYTES," filed May 7, 2021, the entire contents of which are expressly incorporated herein by reference.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to methods and compositions for producing mature hepatocytes. [Background technology]

[0003] background Hepatocytes metabolize drugs and also control the elimination of xenobiotics from the body (Gebhardt et al. 2003, Drug Metab Rev 35, 145-213 (Non-Patent Document 1); and Hewitt et al. 2007, Drug Metab Rev 39, 159-234 (Non-Patent Document 2)). Hepatocytes have important functions in detoxifying drugs, xenobiotics, and endogenous substrates, and therefore are used in drug toxicity screening and development programs. However, when cultured in vitro, primary human hepatocytes quickly lose their function. Furthermore, the drug metabolism capacity of primary human hepatocytes shows significant differences between individuals (Byers et al. 2007, Drug Metab Lett 1, 91-95 (Non-Patent Document 3)).

[0004] Hepatocytes provide a novel platform for testing drugs as well as a potential new therapeutic approach for patients with liver disease. Liver transplantation offers an effective treatment for end-stage liver disease, but the scarcity of living donor organs limits the patient population that can be treated with hepatocytes (Kawasaki et al. 1998, Ann Surg 227, 269-274; and Miro et al. 2006, J Hepatol 44, 5140-145). Hepatocyte transplantation and bioartificial liver devices developed with hepatocytes are life-saving alternative treatments for patients with specific types of liver disease. Given the important functional role of hepatocytes and the fact that the ability to metabolize certain drugs may vary between individuals, there is a need for a means to obtain mature and functional hepatocytes.

[0005] Due to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood, the generation of reproducible and efficient mature hepatocytes has been a challenge to date.Almost all approaches attempt to repeat the key stages of liver development in differentiation culture, including induction of definitive endoderm, specification of the endoderm to hepatic fate, and production of hepatic precursors.Although these early steps are somewhat well documented, the conditions that promote hepatocyte maturation are not fully understood.In addition, populations generated using different protocols differ greatly in their maturation state and represent immature hepatocytes.

[0006] Thus, there is a need in the art for simple and effective methods for generating mature hepatocytes. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gebhardt et al. 2003, Drug Metab Rev 35, 145-213 [Non-Patent Document 2] Hewitt et al. 2007, Drug Metab Rev 39, 159-234 [Non-Patent Document 3] Byers et al. 2007, Drug Metab Lett 1, 91-95 [Non-Patent Document 4] Kawasaki et al. 1998, Ann Surg 227, 269-274 [Non-Patent Document 5] Miro et al. 2006, J Hepatol 44, 5140-145 Summary of the Invention

[0008] overview The present invention addresses the above-mentioned need in the art by providing an efficient and effective method for generating mature hepatocytes by increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes. In one aspect, the present invention provides a novel and effective method for generating mature hepatocytes by increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes.

[0009] The methods of the present invention are both simple, efficient and effective, and result in the production of mature hepatocytes that can be used for a variety of applications disclosed herein, such as, for example, treating liver disease.

[0010] In one aspect, the present invention provides a method for producing mature hepatocytes, the method comprising: increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes, thereby producing mature hepatocytes.

[0011] In some embodiments, the transcription factor is NFIX.

[0012] In some embodiments, the transcription factor is NFIC.

[0013] In some embodiments, the transcription factors are NFIX and NFIC.

[0014] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0015] In some embodiments, the method further comprises increasing expression of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 in the immature hepatocyte.

[0016] In some embodiments, the method further comprises culturing the immature hepatocytes in a culture medium comprising dexamethasone, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or a combination thereof. In some embodiments, the culturing is carried out for at least 2, 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, the concentration of 8-Br-cAMP is at least 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 nM, or 1 mM. In some embodiments, the concentration of dexamethasone is at least 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, or 100 nM.

[0017] In some embodiments, increasing the expression of at least one transcription factor in the immature hepatocyte comprises contacting the immature hepatocyte with at least one transcription factor.

[0018] In some embodiments, the immature hepatocyte comprises an expression vector comprising a nucleic acid encoding at least one transcription factor. In some embodiments, the expression vector is a viral vector. In some embodiments, the expression vector is a non-viral vector. In some embodiments, the expression vector is an inducible expression vector. In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid encoding at least one transcription factor. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is an artificial promoter. In some embodiments, the promoter is an inducible promoter.

[0019] In some embodiments, increasing the expression of at least one transcription factor in the immature hepatocytes comprises transducing the immature hepatocytes with a viral vector encoding the at least one transcription factor.

[0020] In some embodiments, increasing the expression of at least one transcription factor in the immature hepatocytes comprises transfecting the immature hepatocytes with an expression vector encoding the at least one transcription factor.

[0021] In some embodiments, the immature hepatocytes are cultured for at least 2, 3, 4, or 5 days prior to increasing expression of the at least one transcription factor.

[0022] In some embodiments, the immature hepatocytes are cultured for at least 2, 3, 4, 5, 6, 7, 8, or 9 days after increasing expression of the at least one transcription factor.

[0023] In some embodiments, increased expression of NFIX comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold compared to the endogenous expression level of NFIX in immature hepatocytes.

[0024] In some embodiments, increased expression of NFIC includes an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIC in immature hepatocytes.

[0025] In some embodiments, mature hepatocytes exhibit increased expression of albumin (ALB), cytochrome P450 enzyme 1A2 (CYP1A2), cytochrome P450 enzyme 3A4 (CYP3A4), tyrosine aminotransferase (TAT), and / or UDP-glucuronosyltransferase 1A-1 (UGT1A1) compared to immature hepatocytes. In some embodiments, increased expression of CYP1A2 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of CYP3A4 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of TAT comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of UGT1A1 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes.

[0026] In some embodiments, mature hepatocytes exhibit decreased expression of alpha-fetoprotein (AFP) compared to immature hepatocytes. In some embodiments, decreased expression of AFP comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, or 4-fold decrease compared to immature hepatocytes.

[0027] In some embodiments, mature hepatocytes show increased secretion of albumin (ALB), decreased secretion of AFP, and / or increased activity of CYP1A2 compared to immature hepatocytes. In some embodiments, increased secretion of ALB comprises at least 5%, 10%, 15%, 20%, or 25% increase compared to immature hepatocytes. In some embodiments, decreased secretion of AFP comprises at least 5%, 10%, 20%, 40%, or 60% decrease compared to immature hepatocytes. In some embodiments, increased activity of CYP1A2 comprises at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold increase compared to immature hepatocytes.

[0028] In some embodiments, increasing the expression of at least one transcription factor shifts at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the transcriptome of an immature hepatocyte to that of a mature hepatocyte.

[0029] In some embodiments, the immature hepatocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0030] In some embodiments, increasing expression of at least one transcription factor in the immature hepatocyte comprises using a gene switch construct encoding at least one transcription factor. In some embodiments, the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct.

[0031] In some embodiments, the expression vector further comprises a self-cleaving sequence.

[0032] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.

[0033] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2 through SEQ ID NO:6.

[0034] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40.

[0035] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41 to SEQ ID NO:45.

[0036] In another aspect, the present invention provides a method for producing mature hepatocytes from pluripotent stem cells, the method comprising: (a) differentiating pluripotent stem cells into immature hepatocytes, the pluripotent stem cells comprising an expression vector comprising a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC), and (b) increasing expression of the at least one transcription factor from the expression vector in the immature hepatocytes, thereby producing mature hepatocytes.

[0037] In some embodiments, the pluripotent stem cells are embryonic stem cells.

[0038] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0039] In some embodiments, the immature liver cells comprise hepatoblasts.

[0040] In some embodiments, the immature liver cells comprise hepatic stem cells.

[0041] In some embodiments, the transcription factor is NFIX.

[0042] In some embodiments, the transcription factor is NFIC.

[0043] In some embodiments, the transcription factors are NFIX and NFIC.

[0044] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0045] In some embodiments, the method further comprises increasing expression of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 in the immature hepatocyte.

[0046] In some embodiments, the method further comprises culturing the immature hepatocytes in a culture medium comprising dexamethasone, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or a combination thereof. In some embodiments, the culturing is carried out for at least 2, 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, the concentration of 8-Br-cAMP is at least 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 nM, or 1 mM. In some embodiments, the concentration of dexamethasone is at least 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, or 100 nM.

[0047] In some embodiments, the immature hepatocytes comprise an expression vector comprising a nucleic acid encoding at least one transcription factor.

[0048] In some embodiments, the expression vector is a viral vector.

[0049] In some embodiments, the expression vector is a non-viral vector.

[0050] In some embodiments, the expression vector is an inducible expression vector.

[0051] In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid encoding at least one transcription factor. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is an artificial promoter. In some embodiments, the promoter is an inducible promoter.

[0052] In some embodiments, increasing the expression of at least one transcription factor in immature hepatocytes comprises inducing the expression of at least one transcription factor in immature hepatocytes.In some embodiments, inducing the expression of at least one transcription factor in immature hepatocytes comprises using a gene switch construct that encodes at least one transcription factor.In some embodiments, the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct.

[0053] In some embodiments, the expression vector further comprises a self-cleaving sequence.

[0054] In some embodiments, the pluripotent stem cells are transduced with a viral vector encoding at least one transcription factor.

[0055] In some embodiments, the pluripotent stem cells are transfected with an expression vector encoding at least one transcription factor.

[0056] In some embodiments, step (a) of the method comprises culturing pluripotent stem cells in a first differentiation medium comprising activin A, a second differentiation medium comprising at least one of BMP4 and FGF2, and a third differentiation medium comprising HGF, thereby generating immature hepatocytes. In some embodiments, the first differentiation medium, the second differentiation medium, and the third differentiation medium are each cultured for at least 5 days.

[0057] In some embodiments, the immature hepatocytes are cultured for at least 2, 3, 4, or 5 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured in a culture medium that includes hepatocyte growth factor (HGF).

[0058] In some embodiments, the immature hepatocytes are cultured for at least 2, 3, 4, 5, 6, 7, 8, or 9 days after increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured in a culture medium that includes Oncostatin M (OSM).

[0059] In some embodiments, increased expression of NFIX comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIX in immature hepatocytes.

[0060] In some embodiments, increased expression of NFIC includes an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIC in immature hepatocytes.

[0061] In some embodiments, mature hepatocytes exhibit increased expression of albumin (ALB), cytochrome P450 enzyme 1A2 (CYP1A2), cytochrome P450 enzyme 3A4 (CYP3A4), tyrosine aminotransferase (TAT), and / or UDP-glucuronosyltransferase 1A-1 (UGT1A1) compared to immature hepatocytes. In some embodiments, increased expression of CYP1A2 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of CYP3A4 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of TAT comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of UGT1A1 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes.

[0062] In some embodiments, mature hepatocytes exhibit decreased expression of alpha-fetoprotein (AFP) compared to immature hepatocytes. In some embodiments, decreased expression of AFP comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, or 4-fold decrease compared to immature hepatocytes.

[0063] In some embodiments, mature hepatocytes show increased secretion of albumin (ALB), decreased secretion of AFP, and / or increased activity of CYP1A2 compared to immature hepatocytes. In some embodiments, increased secretion of ALB comprises at least 5%, 10%, 15%, 20%, or 25% increase compared to immature hepatocytes. In some embodiments, decreased secretion of AFP comprises at least 5%, 10%, 20%, 40%, or 60% decrease compared to immature hepatocytes. In some embodiments, increased activity of CYP1A2 comprises at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold increase compared to immature hepatocytes.

[0064] In some embodiments, increasing the expression of at least one transcription factor shifts at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the transcriptome of an immature hepatocyte to that of a mature hepatocyte.

[0065] In another aspect, the present invention provides a composition comprising a mature hepatocyte population produced by any one or more of the methods disclosed herein.

[0066] In another aspect, the present invention provides a pharmaceutical composition comprising a mature hepatocyte population produced by any one or more of the methods disclosed herein and a pharma- ceutically acceptable carrier.

[0067] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.

[0068] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2 through SEQ ID NO:6.

[0069] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40.

[0070] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41 to SEQ ID NO:45.

[0071] In another aspect, the present invention provides a composition comprising a hepatocyte population, the hepatocyte population comprising an increased expression level of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) compared to the endogenous expression level of the transcription factor in the hepatocyte population.

[0072] In some embodiments, the transcription factor is NFIX.

[0073] In some embodiments, the transcription factor is NFIC.

[0074] In some embodiments, the transcription factors are NFIX and NFIC.

[0075] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0076] In some embodiments, the hepatocytes further comprise an increased expression level of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 compared to the endogenous expression level of the one or more transcription factors in the hepatocyte population.

[0077] In some embodiments, the increased expression comprises exogenous expression of at least one transcription factor.

[0078] In some embodiments, the hepatic cells comprise an expression vector comprising a nucleic acid encoding at least one transcription factor.

[0079] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a herpes simplex virus vector, a Sendai virus vector, and a retrovirus vector.

[0080] In some embodiments, the expression vector is a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises naked nucleic acid, liposome, dendrimer, nanoparticle, lipid-polymer system, solid lipid nanoparticle, and / or liposome-protamine / DNA lipoplex (LPD).

[0081] In some embodiments, the expression vector is an inducible expression vector.

[0082] In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid encoding at least one transcription factor. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is an artificial promoter. In some embodiments, the promoter is an inducible promoter.

[0083] In some embodiments, the expression vector comprises a gene switch construct encoding at least one transcription factor, hi some embodiments, the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct.

[0084] In some embodiments, the expression vector further comprises a self-cleaving sequence, hi some embodiments, the self-cleaving sequence is selected from the group consisting of T2A, P2A, E2A, and F2A.

[0085] In some embodiments, increased expression of NFIX comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIX in the hepatic cell population.

[0086] In some embodiments, increased expression of NFIC includes an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIC in the hepatocyte population.

[0087] In some embodiments, the hepatocyte cell population is an immature hepatocyte cell population.

[0088] In some embodiments, the hepatocyte population is a mature hepatocyte population.

[0089] In some embodiments, the composition further comprises cells other than hepatocytes.

[0090] In some embodiments, the hepatic cell population is in the form of an organoid.

[0091] In some embodiments, the hepatocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0092] In some embodiments, the population of hepatocytes comprises at least 10 6 Contains hepatocytes.

[0093] In another aspect, the present invention provides a pharmaceutical composition comprising a hepatocyte population of any one or more compositions described herein and a pharma- ceutically acceptable carrier.

[0094] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.

[0095] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2 through SEQ ID NO:6.

[0096] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40.

[0097] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41 to SEQ ID NO:45.

[0098] In another aspect, the present invention provides a composition comprising a pluripotent stem cell population comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC).

[0099] In some embodiments, the transcription factor is NFIX.

[0100] In some embodiments, the transcription factor is NFIC.

[0101] In some embodiments, the transcription factors are NFIX and NFIC.

[0102] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0103] In some embodiments, the pluripotent stem cells further comprise an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1.

[0104] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a herpes simplex virus vector, a Sendai virus vector, and a retrovirus vector.

[0105] In some embodiments, the expression vector is a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises naked nucleic acid, liposome, dendrimer, nanoparticle, lipid-polymer system, solid lipid nanoparticle, and / or liposome-protamine / DNA lipoplex (LPD).

[0106] In some embodiments, the expression vector is an inducible expression vector.

[0107] In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid encoding at least one transcription factor. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is an artificial promoter. In some embodiments, the promoter is an inducible promoter.

[0108] In some embodiments, the expression vector comprises a gene switch construct encoding at least one transcription factor. In some embodiments, the gene switch construct is a transcriptional gene switch construct. In some embodiments, the gene switch construct is a post-transcriptional gene switch construct.

[0109] In some embodiments, the expression vector further comprises a self-cleaving sequence, hi some embodiments, the self-cleaving sequence is selected from the group consisting of T2A, P2A, E2A, and F2A.

[0110] In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0111] In some embodiments, the pluripotent stem cell population comprises at least 10 6 The cell comprises pluripotent stem cells.

[0112] In another aspect, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a composition or pharmaceutical composition of the present disclosure, thereby treating the disease in the subject.

[0113] In some embodiments, the disease is selected from the group consisting of: fulminant hepatic failure due to any cause, viral hepatitis, drug-induced liver injury, cirrhosis, inherited liver dysfunction (e.g., Wilson's disease, Gilbert's syndrome, or alpha-1-antitrypsin deficiency), hepatobiliary cancer, autoimmune liver disease (e.g., autoimmune chronic hepatitis or primary biliary cirrhosis), urea cycle disorders, factor VII deficiency, glycogen storage disease type 1, childhood Refsum's disease, phenylketonuria. , severe childhood oxalosis, cirrhosis, liver damage, acute liver failure, hepatobiliary carcinoma, hepatocellular carcinoma, hereditary cholestasis (PFIC and Alagille syndrome), hereditary hemochromatosis, tyrosinemia type 1, argininosuccinic aciduria (ASL), Crigler-Najjar syndrome, familial amyloidotic polyneuropathy, atypical hemolytic uremic syndrome-1, primary hyperoxaluria type 1, maple syrup urine disease (MSUD), acute intermittent porphyria, coagulation disorders, GSD type Ia (in metabolic control), homozygous familial hypercholesterolemia, organic acidurias, as well as any other condition causing liver dysfunction.

[0114] In another aspect, the present invention provides a kit comprising a composition or pharmaceutical composition described herein.

[0115] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.

[0116] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2 through SEQ ID NO:6.

[0117] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40.

[0118] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41 to SEQ ID NO:45.

[0119] In another aspect, the present invention provides a kit comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC).

[0120] In some embodiments, the transcription factor is NFIX.

[0121] In some embodiments, the transcription factor is NFIC.

[0122] In some embodiments, the transcription factors are NFIX and NFIC.

[0123] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0124] In some embodiments, the kit further comprises an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1.

[0125] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.

[0126] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2 through SEQ ID NO:6.

[0127] In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40.

[0128] In some embodiments, the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41 to SEQ ID NO:45.

[0129] The present invention is further explained by the following detailed description and drawings. [Brief description of the drawings]

[0130] [Figure 1] FIG. 1 shows a schematic diagram for selecting transcription factors (TFs) of the present invention. [Diagram 2]FIG. 2A shows a schematic of the usability versus physiological compatibility of cancer cell lines (HepG2, HuH7, and HepaRG), stem cell-derived hepatocytes (stem cell / iPSC-Heps), and primary human hepatocytes (PHH). FIG. 2B shows the principal component analysis of the cells shown in FIG. 2A. PHH-AQL, PHH-TLY, and PHH-NES are adult hepatocytes. PHH-BVI are stillborn hepatocytes. "Fetal" refers to primary human fetal hepatocytes. HuH7 cells cluster with hepatocytes differentiated from GMP1 iPSCs that were not further treated with Br-cAMP and dexamethasone ("GMP1 control"), and hepatocytes differentiated from GMP1 iPSCs that were further treated with Br-cAMP and dexamethasone for 5 days ("GMPDex"), so the HuH7 cells are used to construct a HuH7 cell line for screening transcription factors. Figure 2C shows a schematic diagram of the construction of the HuH7 cell line (HuH7-Tet-On3G) used to screen the transcription factors of the present invention. Figure 2D shows that the HuH7-Tet-On3G cell line is responsive to doxycycline induction. [Diagram 3] Figure 3 is a panel of bar graphs showing the expression of mature hepatocyte markers CYP1A2 (Figure 3A) and CYP3A4 (Figure 3B) upon increasing expression of various transcription factors in HuH7-Tet-On3G cells. Transduction with transcription factors was performed at a multiplicity of infection (MOI) of 10. Arrows indicate transcription factors that upregulated the expression levels of CYP1A2 and CYP3A4. NFIC transcript variants 1 and 3 (NFIC-1+3) refer to a mixture of NFIC transcript variant 1 (NFIC-1) (NCBI Reference Sequence No.: NM_001245002) and NFIC transcript variant 3 (NFIC-3) (NCBI Reference Sequence No.: NM_001245004), which are alternative splice variants of the transcription factor NFIC, respectively, and were used for transduction at an MOI of 5 for each of NFIC transcript variant 1 (NFIC-1) and NFIC transcript variant 3 (NFIC-3). [Figure 4] Figure 4A is a schematic diagram of NFIC alternative splice variants NFIC transcript variant 1 (NFIC-1); and NFIC transcript variant 3 (NFIC-3). Figure 4B is a panel of bar graphs showing increased expression of mature hepatocyte markers CYP1A2 and CYP3A4 upon increased expression of NFIC alternative splice variants NFIC transcript variant 1 (NFIC-1), NFIC transcript variant 3 (NFIC-3), and their combination (NFIC transcript variants 1 and 3 (NFIC-1+3)) in HuH7-Tet-On3G cells. HuH7-Tet-On3G cells were transduced with lentiviral particles of NFIC transcript variants 1 and 3 (NFIC-1+3), lentiviral particles of NFIC transcript variant 1 (NFIC-1), and lentiviral particles of NFIC transcript variant 3 (NFIC-3) at an MOI of 5. [Diagram 5] FIG. 5 is a panel of bar graphs showing that culturing HuH7-Tet-On3G cells in culture medium containing dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) further increased expression of mature hepatocyte markers CYP1A2 (FIG. 5A), TAT (FIG. 5B), and UGT1A1 (FIG. 5C) upon increasing expression of NFIC transcript variant 1 (NFIC-1). [Figure 6] FIG. 6 is a panel of bar graphs showing expression of the immature hepatocyte marker AFP (FIG. 6A), and mature hepatocyte markers CYP1A2 (FIG. 6B), TAT (FIG. 6C), and CYP3A4 (FIG. 6D) upon increasing expression of various transcription factors in HuH7-Tet-On3G cells. Cells were transduced with NFIC transcription variant 1 (NFIC-1) (MOI of 10) and with individual lentiviruses encoding various transcription factors at an MOI of 10. After transduction, cells were cultured in culture medium containing 1 mM 8-Br-cAMP and 100 nM dexamethasone. [Figure 7]Figure 7A shows a schematic diagram of the four-stage stepwise differentiation of induced pluripotent stem cells (iPSCs) into hepatocyte-like cells. Transduction was performed on day 15 of differentiation toward hepatocyte-like cells with MOI of 5 for Tet-On3G and MOI of 3 for each transcription factor (TF). Cells were then cultured for 5 days in culture medium in the absence or presence of 1 mM 8-Br-cAMP and 100 nM dexamethasone. Figure 7B is a panel of bar graphs showing increased expression of mature hepatocyte markers CYP1A2 and TAT upon increasing expression of NFIC transcription variant 1 (NFIC-1), NFIX, and their combination in iPSC-derived immature hepatocytes. [Figure 8] FIG. 8A shows a schematic diagram of the four-stage stepwise differentiation of induced pluripotent stem cells (iPSCs) into hepatocyte-like cells. Transduction was performed on day 15 of differentiation toward hepatocyte-like cells with MOI of 5 for Tet-On3G and MOI of 3 for each transcription factor (TF). Cells were then cultured in culture medium in the absence or presence of 1 mM 8-Br-cAMP and 100 nM dexamethasone and harvested on days 20 and 24 of cell culture. FIG. 8B is a panel of bar graphs showing the decreased expression of immature hepatocyte marker AFP and increased expression of mature hepatocyte marker CYP1A2 upon increasing expression of NFIC transcription variant 1 (NFIC-1), NFIX, and their combination in iPSC-derived immature hepatocytes. [Figure 9] Figure 9A is a graph showing that when expression of NFIC transcription variant 1 (NFIC-1), NFIX, and a combination thereof is increased in iPSC-derived immature hepatocytes, 30-34% of the transcriptome of iPSC-derived immature hepatocytes is shifted to that of mature hepatocytes. Figure 9B is a graph showing an enlarged view of bracket 1 of the graph in Figure 9A. Figure 9C is a list of samples shown in Figures 9A-9B. [Figure 10]FIG. 10 is a panel of bar graphs showing the results of functional assays to identify CYP1A2 activity (FIG. 10A), albumin (ALB) secretion (FIG. 10B), alpha-fetoprotein (AFP) secretion (FIG. 10C), and urea secretion (FIG. 10D) upon increasing expression of NFIC transcription variant 1 (NFIC-1), NFIX, and their combination in iPSC-derived immature hepatocytes. Transduction was performed on day 15 of differentiation with an MOI of 5 for Tet-On3G and an MOI of 3 for each transcription factor. Cells were then cultured in culture medium in the absence or presence of 1 mM 8-Br-cAMP and 100 nM dexamethasone. Functional assays were performed on days 20 (20d) and 24 (24d) of cell culture. [Figure 11] Figure 11A shows the transcription factors used in the combination experiment, and Figure 11B is a panel of bar graphs showing the expression of mature hepatocyte markers CYP1A2 and CYP3A4 upon increasing expression of various transcription factors in HuH7-Tet-On3G cells. [Figure 12] FIG. 12 shows a time course analysis of the expression of mature hepatocyte markers ALB (FIG. 12A), CYP3A4 (FIG. 12B), and UGT1A1 (FIG. 12C) after forced expression of NFIC transcription variant 1 (NFIC-1); NFIX; and their combination in iPSC-derived immature hepatocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0131] Detailed Description The present invention provides an efficient and effective method for producing mature hepatocytes, which comprises: increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes, thereby producing mature hepatocytes. Compositions produced by these methods are also provided by the present invention, as are methods of using the compositions.

[0132] In one aspect, the present invention provides a method for producing mature hepatocytes from pluripotent stem cells, such as human embryonic stem (hES) cells, embryo-derived cells, and induced pluripotent stem cells (iPS cells). The method of the present invention is efficient and effective, and results in the production of mature hepatocytes that can be used for various applications disclosed herein, such as treating liver disease.

[0133] The following detailed description discloses how to make and use the invention.

[0134] In order to facilitate a more readily understood understanding of the present invention, certain terms are first defined. It should also be noted that whenever a value or range of values ​​is given for a parameter, it is intended that values ​​and ranges intermediate to the given values ​​are also part of the present invention.

[0135] In the following description, certain values, materials, and configurations are described for the purpose of explanation in order to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention can be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, references in this specification to phrases such as "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described with respect to the embodiment is included in at least one embodiment of the present invention. Although phrases such as "one embodiment" appear in various places in this specification, they do not necessarily all refer to the same embodiment.

[0136] definition Unless otherwise specified, each of the following terms has the meaning set forth in this section.

[0137] The indefinite articles "a" and "an" refer to at least one of the following noun and are used interchangeably with the terms "at least one" and "one or more."

[0138] The conjunctions "or" and "and / or" are used interchangeably as non-exclusive disjunctions.

[0139] The term "hepatocyte" as used herein refers to hepatocyte parenchymal cells.Hepatocytes constitute the majority of the cytoplasmic mass of the liver and are involved in: protein synthesis and accumulation; carbohydrate metabolism; cholesterol, bile salts, and phospholipid synthesis; and detoxification, modification, and excretion of exogenous and endogenous substances.Hepatocytes include immature hepatocytes that exhibit some, but not all, of the characteristics of mature hepatocytes, as well as mature and fully functional hepatocytes that have all the characteristics of hepatocytes as determined by morphology, marker expression, and in vitro and in vivo functional assays.

[0140] The term "primary hepatocyte" as used herein refers to hepatocytes that are directly collected from living tissue, such as living liver tissue. In some embodiments, the functionality of primary hepatocytes can be shown by, for example, albumin production, urea production, and various metabolic enzyme activities, and can have the characteristics of mature hepatocytes. In some embodiments, primary hepatocytes are primary human hepatocytes ("PHH").

[0141] The term "immature hepatocyte" as used herein refers to hepatocytes or hepatic progenitor cells that need to undergo maturation to acquire the characteristics and / or functionality of mature hepatocytes. In some embodiments, immature hepatocytes are hepatocyte-like cells that exhibit some, but not all, of the characteristics of mature hepatocytes. In some embodiments, immature hepatocytes do not express detectable levels of one or more of the following: albumin (ALB), cytochrome P450 enzyme 3A4 (CYP3A4), cytochrome P450 enzyme 1A2 (CYP1A2), tyrosine aminotransferase (TAT), and UDP-glucuronosyltransferase 1A-1 (UGT1A1). In some embodiments, immature hepatocytes express detectable levels of alpha-fetoprotein (AFP). In some embodiments, immature hepatocytes show reduced secretion of albumin (ALB), increased secretion of AFP, and / or reduced activity of CYP1A2 compared to mature hepatocytes or primary hepatocytes. In some embodiments, the immature hepatic cells include hepatic stem cells and / or hepatic progenitor cells.

[0142] The term "hepatic precursor", "hepatic progenitor cell", "hepatoblast" or "hepatoblast cell" as used herein refers to cells that have the ability to differentiate into hepatocytes or bile duct cells. In some embodiments, hepatic progenitor cells are defined by expressing at least one liver-related marker, such as Hex, HNF4, alpha-fetoprotein (AFP), cytokeratin 19 (CK18), cytokeratin 19 (CK19), hepatocyte nuclear factor 6 (HNF6), and albumin (ALB). In some embodiments, hepatic progenitor cells have reduced expression levels of stem cell genes, such as Nanog, Oct4, and ckit.

[0143] The term "hepatic stem cell" as used herein refers to the cell that can self-renew and differentiate into hepatocytes and bile duct cells in vivo or in vitro.In one embodiment, hepatic stem cell expresses leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and / or epithelial cell adhesion molecule (EpCAM).

[0144] "Mature hepatocytes", as used herein, refer to hepatocytes that (i) contain a gene expression profile that is more similar to that of primary hepatocytes or known mature hepatocytes than to that of immature hepatocytes, and / or (ii) exhibit one or more characteristics of mature hepatocytes. Non-limiting examples of cell markers useful for distinguishing mature hepatocytes include: albumin, asialoglycoprotein receptor, alpha 1-antitrypsin, alpha-fetoprotein, apoE, arginase I, apoAI, apoAII, apoB, apoCIII, apoCII, aldolase B, alcohol dehydrogenase 1, catalase, CYP3A4, glucokinase, glucose-6-phosphatase, insulin growth factor 1 and 2, IGF-1 receptor, insulin receptor, leptin, liver-specific organic anion transporter, and the like. These include LST-1, L-type fatty acid binding protein, phenylalanine hydroxylase, transferrin, retinol binding protein, erythropoietin (EPO), albumin, α1-antitrypsin, asialoglycoprotein receptor, cytokeratin 8 (CK8), cytokeratin 18 (CK18), CYP3A4, fumarylacetoacetate hydrolase (FAH), glucose-6-phosphate, tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and tryptophan 2,3-dioxygenase.

[0145] In some embodiments, mature hepatocytes show increased expression of albumin (ALB), cytochrome P450 enzyme 1A2 (CYP1A2), cytochrome P450 enzyme 3A4 (CYP3A4), tyrosine aminotransferase (TAT), and / or UDP-glucuronosyltransferase 1A-1 (UGT1A1) compared to immature hepatocytes. In some embodiments, mature hepatocytes show decreased expression of alpha-fetoprotein (AFP) compared to immature hepatocytes.

[0146] In some embodiments, mature hepatocytes exhibit increased secretion of albumin (ALB), decreased secretion of AFP, and / or increased activity of CYP1A2 compared to immature hepatocytes.

[0147] In some embodiments, mature hepatocytes comprise increased expression of at least one, two, three, four, five, six, seven, eight, nine, ten, or more genes or proteins selected from the group consisting of ALB, CPS1, G6P, TDO, CYP2C9, CYP2D6, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, NAT2, TAT, ASGPR-1, and UGT1A1, compared to a population of cells comprising immature hepatocytes.

[0148] In yet another embodiment, the mature hepatocytes exhibit a global gene expression profile indicative of hepatocyte maturation. The global gene expression profile may be compared to the profile for primary hepatocytes or known mature hepatocytes, and may be obtained by any method known in the art, such as by transcriptome analysis or microarray analysis.

[0149] In one embodiment, one or more characteristics of mature hepatocytes include, but are not limited to, epithelial morphology, polarization, polyploidization, gene expression, CYP activity, transferase activity, transporter activity, bile acid synthesis, glycogen accumulation, serum protein synthesis, cholesterol metabolism, lipid uptake, urea metabolism, clotting factors, engraftment and repopulation, liver function recovery, and tumorigenicity.See, for example, Chen et al. Gastroenterology 2018;154:1258-1272, the entirety of which is incorporated herein by reference.

[0150] The term "increased expression" as used herein refers to the increase in the level and / or activity of the nucleic acid, such as RNA or DNA, encoding the transcription factor disclosed herein and / or the level and / or activity of the transcription factor disclosed herein compared to the endogenous nucleic acid level and / or protein level for the transcription factor.In some embodiments, increasing the expression of at least one transcription factor comprises contacting a cell (e.g., immature hepatocyte, hepatic progenitor cell, or pluripotent stem cell, such as embryonic stem cell or induced pluripotent stem cell) with at least one transcription factor.In some embodiments, increasing the expression of at least one transcription factor comprises transducing a cell (e.g., immature hepatocyte, hepatic progenitor cell, or pluripotent stem cell, such as embryonic stem cell or induced pluripotent stem cell) with a viral vector encoding at least one transcription factor. In some embodiments, increasing the expression of at least one transcription factor comprises transfecting a cell (e.g., an immature hepatocyte, a hepatic progenitor cell, or a pluripotent stem cell, e.g., an embryonic stem cell or an induced pluripotent stem cell) with an expression vector encoding the at least one transcription factor.

[0151] In some embodiments, the increased expression of at least one transcription factor comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold compared to the endogenous expression level of the at least one transcription factor in a cell (e.g., an immature hepatocyte, a hepatic progenitor cell, or a pluripotent stem cell, such as an embryonic stem cell or an induced pluripotent stem cell). In some embodiments, the increased expression of at least one transcription factor comprises an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or 1000% compared to the endogenous expression level of the at least one transcription factor in a cell (e.g., an immature hepatocyte, a hepatic progenitor cell, or a pluripotent stem cell, e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0152] The term "endogenous" as used herein refers to the native form of a nucleic acid, polynucleotide, oligonucleotide, DNA, RNA, gene, peptide, or polypeptide in its natural location within a cell or in its natural location within the genome of a cell.

[0153] The term "maturation" as used herein refers to the process by which cells, such as immature hepatocytes, become more specialized and / or more functional, for example, the process required to become similar to their functional and / or phenotypic state in vivo, or similar to the functional and / or phenotypic state of known mature hepatocytes or primary hepatocytes.In one embodiment, the process by which immature hepatocytes become mature hepatocytes is referred to as maturation.

[0154] As used herein, the term "pluripotent stem cell", "PS cell" or "PSC" includes embryonic stem cell, induced pluripotent stem cell and embryo-derived pluripotent stem cell, regardless of the method by which the pluripotent stem cell is generated. Pluripotent stem cell is functionally defined as a stem cell that: (a) can induce teratoma when transplanted into immune-deficient (SCID) mice; (b) can differentiate into cell types of all three germ layers (e.g., can differentiate into cell types of ectoderm, mesoderm and endoderm); (c) expresses one or more markers of embryonic stem cell (e.g., expresses OCT4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, NANOG, TRA-1-60, TRA-1-81, SOX2, REX1, etc.); and d) can self-renew. The term "pluripotency" refers to the ability of a cell to form all cell lineages of the body or somatic cells (i.e., the embryonic body). For example, embryonic stem cells and induced pluripotent stem cells are types of pluripotent stem cells that can form cells derived from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential that spans between incomplete or partial pluripotent cells that cannot give rise to a complete organism, and more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism. Exemplary pluripotent stem cells can be generated, for example, by using methods known in the art. Exemplary pluripotent stem cells include, but are not limited to, embryonic stem cells derived from the inner cell mass of a blastocyst stage embryo; embryonic stem cells derived from one or more blastomeres of a cleavage or morula stage embryo (optionally without destroying the remaining part of the embryo); induced pluripotent stem cells produced by reprogramming somatic cells to a pluripotent state; and pluripotent cells produced from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF). Such embryonic stem cells can be produced from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgeny.

[0155] In one embodiment, pluripotent stem cells may be genetically engineered or modified, for example, to increase life span, potency, homing, prevent or reduce immune responses, or deliver desirable factors in cells obtained from such pluripotent cells (e.g., hepatocytes). For example, pluripotent stem cells, and thus differentiated cells resulting therefrom, may be engineered or modified to lack or have reduced expression of the following genes: β2 microglobulin, HLA-A, HLA-B, HLA-C, TAP1, TAP2, tapasin, CTIIA, RFX5, TRAC, and / or TRAB. As described in WO2012145384 and WO2013158292, which are incorporated herein by reference in their entirety, in some embodiments, the cells, such as pluripotent stem cells and differentiated cells resulting therefrom, such as hepatocytes, comprise a disruption of the β2 microglobulin (B2M) gene by genetic engineering. In some embodiments, the cells further comprise a polynucleotide capable of encoding a single-chain fusion human leukocyte antigen (HLA) class I protein, wherein the protein comprises at least a portion of the B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of the HLA-1 alpha chain. In some embodiments, the HLA-1 alpha chain is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the cells comprise a disruption of the human leukocyte antigen (HLA) class II associated gene by genetic engineering. In some embodiments, the HLA class II associated gene is selected from: regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), class II transactivator (CIITA), HLA-DPA (alpha chain), HLA-DPB (beta chain), HLA-DQA, HLA-DQB, HLA-DRA, HLA-DRB, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB.In some embodiments, the cells comprise one or more polynucleotides encoding a single chain fusion HLA class II protein or an HLA class II protein.

[0156] Pluripotent stem cells and the differentiated cells derived therefrom may be engineered or modified to increase the expression of a gene.In one embodiment, pluripotent stem cells may be engineered to express one or more of the transcription factors of the present invention or to increase the expression of one or more of the transcription factors.There are various techniques for engineering cells to change the expression of one or more genes (or proteins), including: using viral vectors, such as AAV vectors, using zinc finger nucleases (ZFNs), using transcription activator-like effector nucleases (TALENs), and using CRISPR / Cas-based methods to manipulate genomes; and using transcription and translation inhibitors, such as antisense and RNA interference, which can be achieved using stably integrated vectors and episomal vectors.

[0157] The term "embryo" or "embryonic" refers to a developing mass of cells that has not been implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained within an embryo. The term also includes blastomeres obtained as early as the two-cell stage or aggregated after collection.

[0158] The term "embryonic-derived cells" (EDCs), as used herein, refers broadly to morula-derived or blastocyst-derived cells, including cells of the inner cell mass, embryonic shield, or epiblast; or other pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm, and cells derived therefrom. "EDCs" also include aggregated single blastomeres or blastomeres and cell clusters from the embryo from various stages of development, but exclude human embryonic stem cells that have been subcultured as cell lines.

[0159] The term "embryonic stem cell", "ES cell" or "ESC" as used herein broadly refers to cells isolated from the inner cell mass of blastocyst or morula and cells that are serially subcultured as cell lines. The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remaining parts of the embryo (see, for example, Chung et al. Cell Stem Cell. 2008 Feb 7;2(2):1 13-7; US Patent Publication No. 20060206953; US Patent Publication No. 2008 / 0057041, each of which is incorporated herein by reference in its entirety). ES cells may be derived from fertilization of eggs with sperm or DNA, nuclear transfer, parthenogenesis, or any means that produces ES cells with homozygosity in the HLA region. ES cells may also refer to cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, produced by the fusion of sperm and egg, nuclear transfer, parthenogenesis, or by reprogramming chromatin and subsequent integration of the reprogrammed chromatin into the cell membrane to produce cells. In one embodiment, the embryonic stem cells may be human embryonic stem cells (or "hES cells"). In one embodiment, the human embryonic stem cells are not derived from embryos that are more than 14 days old since fertilization. In another embodiment, the human embryonic stem cells are not derived from embryos that have developed in vivo. In another embodiment, the human embryonic stem cells are derived from preimplantation embryos produced by in vitro fertilization.

[0160] "Induced pluripotent stem cells" or "iPS cells", as used herein, generally refer to pluripotent stem cells obtained by reprogramming somatic cells to a less differentiated state. iPS cells can be generated by expressing or inducing the expression of a combination of factors ("reprogramming factors") in somatic cells, such as, for example, OCT4 (sometimes referred to as OCT 3 / 4), SOX2, MYC (e.g., c-MYC or any MYC variant), NANOG, LIN28, and KLF4. In one embodiment, the reprogramming factors include OCT4, SOX2, c-MYC, and KLF4. In another embodiment, the reprogramming factors include OCT4, SOX2, NANOG, and LIN28. In an embodiment, at least two reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In another embodiment, at least three reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In another embodiment, at least four reprogramming factors are expressed in somatic cells, and somatic cells are successfully reprogrammed.In another embodiment, at least five reprogramming factors are expressed in somatic cells, and somatic cells are successfully reprogrammed.In yet another embodiment, at least six reprogramming factors are expressed in somatic cells, such as OCT4, SOX2, c-MYC, NANOG, LIN28, and KLF4.In another embodiment, additional reprogramming factors are identified, and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0161] iPS cells may be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. Somatic cells may include, but are not limited to, fibroblasts, keratinocytes, adipocytes, muscle cells, organ and tissue cells, and various blood cells, including but not limited to hematopoietic cells (e.g., hematopoietic stem cells). In one embodiment, the somatic cells are fibroblasts, such as skin fibroblasts, synovial fibroblasts, or lung fibroblasts, or the somatic cells are non-fibroblast somatic cells.

[0162] iPS cells can be obtained from cell banks. Alternatively, iPS cells can be newly generated by methods known in the art. iPS cells can be specifically generated using materials from a specific patient, or can be specifically generated using materials from a donor that serve the purpose of generating tissue-matched cells. In one embodiment, iPS cells can be the cells of a universal donor that are substantially free of immunogenicity.

[0163] Induced pluripotent stem cells can be produced by expressing one or more reprogramming factors in somatic cells or by inducing the expression of said reprogramming factors.Reprogramming factors can be expressed in somatic cells by infection with viral vectors, such as retroviral vectors, or reprogramming factors can be expressed in somatic cells by other gene editing techniques, such as CRISPR, TALEN, zinc finger nuclease (ZFN).Reprogramming factors can also be expressed in somatic cells by non-integrating vectors, such as episomal plasmids, or by RNA, such as synthetic mRNA, or reprogramming factors can be expressed in somatic cells via RNA viruses, such as Sendai virus.When using non-integrating vectors to express reprogramming factors, they can be expressed in cells by electroporation, transfection, or transformation with said vectors in somatic cells. For example, in mouse cells, four factors (OCT3 / 4, SOX2, c-MYC, and KLF4) can be expressed using integrating viral vectors to reprogram somatic cells, and in human cells, four factors (OCT3 / 4, SOX2, NANOG, and LIN28) can be expressed using integrating viral vectors to reprogram somatic cells.

[0164] Expression of the reprogramming factors may be induced by contacting the somatic cells with at least one agent, such as an organic small molecule agent, that induces expression of the reprogramming factors.

[0165] Somatic cells may also be reprogrammed using combinatorial approaches, in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules).

[0166] Once reprogramming factors are expressed or induced in cells, the cells can be cultured. Cells with ES characteristics gradually emerge in the culture dish. The cells can be selected and subcultured, for example, based on ES cell morphology or based on the expression of selectable or detectable markers. The cells can be cultured to generate a culture of cells that resemble ES cells.

[0167] To confirm the pluripotency of iPS cells, the cells can be tested in one or more assays for pluripotency.For example, the cells can be tested for the expression of ES cell markers; the cells can be evaluated for the ability to generate teratomas when transplanted into SCID mice; the cells can be evaluated for the ability to differentiate to produce the cell types of all three germ layers.

[0168] iPS cells can be derived from any species. The above-mentioned iPS cells have been successfully produced using mouse cells and human cells. In addition, iPS cells have also been successfully produced using embryonic tissue, fetal tissue, neonatal tissue, and adult tissue. Therefore, it is possible to easily produce iPS cells using donor cells from any species. Therefore, iPS cells can be produced from any species, including but not limited to: human, non-human primate, rodent (mouse, rat), ungulate (cattle, sheep, etc.), dog (domestic dog and wild canine), cat (domestic cat and wild cat, for example, lion, tiger, cheetah, etc.), rabbit, hamster, goat, elephant, panda (including giant panda), pig, raccoon, horse, zebra, marine mammal (dolphin, whale, etc.), etc.

[0169] The term "contacting" (e.g., contacting a cell, e.g., an immature hepatocyte, a hepatic progenitor cell, or a pluripotent stem cell, e.g., an embryonic stem cell or an induced pluripotent stem cell, with a transcription factor according to the present invention) is intended to include any manner of introducing a transcription factor into a cell and / or any manner of incubating the transcription factor and the cell together in vitro (e.g., adding a transcription factor to the cell in culture). In some embodiments, the term "contacting" is not intended to include in vivo exposure of a cell to a transcription factor disclosed herein, which may occur naturally in a subject. The step of contacting a cell with a transcription factor disclosed herein can be carried out in any suitable manner. The cells can be treated in adherent or suspension culture, and the transcription factors can be added substantially simultaneously (e.g., together as a cocktail), or the transcription factors can be added sequentially (e.g., within an hour, within a day, or longer after adding the first transcription factor). It is understood that the cells contacted with the transcription factors disclosed herein can also be contacted simultaneously or subsequently with another agent to stabilize the cells or to further differentiate the cells, such as, for example, a growth factor or other agent or environment that induces differentiation. In one embodiment, contacting the cells with the transcription factors includes transducing the cells with a vector comprising a nucleic acid encoding the transcription factor or transfecting the cells with an expression vector comprising a nucleic acid encoding the transcription factor, and contacting the cells with the transcription factors can include culturing the cells under conditions known in the art, for example, for culturing pluripotent or differentiated cells, for example, as further described in the Examples.

[0170] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as hepatocytes. Differentiated cells are cells that are in a more specialized position in cell lineage. For example, hES cells can be differentiated into a variety of more differentiated cell types, including hepatocytes. In some embodiments, cell differentiation is performed in vitro, and this excludes in vivo differentiation.

[0171] As used herein, the term "cultured" or "culturing" refers to placing cells in a medium that contains, among other things, the nutrients necessary to sustain the life of the cultured cells, and also contains any specific additives. If the medium in which the cells are maintained contains a specific substance, such cells are cultured "in the presence" of such a specific substance. Culturing can be carried out in any vessel or device that allows the cells to remain exposed to the medium, including, but not limited to, Petri dishes, culture dishes, blood collection bags, roller bottles, flasks, test tubes, microtiter wells, hollow fiber cartridges, or any other device known in the art.

[0172] As used herein, the term "subculturing" or "passaging" refers to transferring some or all of the cells from a previous culture into fresh growth medium and / or plating the cells into a new culture dish and further culturing. Subculturing may be performed in a culture, for example, to extend the life span, enrich for a desired cell population, and / or to increase the number of cells. For example, the term includes transferring, culturing, or plating some or all of the cells into a new culture vessel at a lower cell density that allows for cell growth.

[0173] As used herein, "administration", "administer" and variants thereof refer to the introduction of a composition or agent into a subject, and include simultaneous or sequential introduction of the composition or agent. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also includes in vitro and ex vivo treatments. Administration includes self-administration and administration by another. Administration can be performed by any suitable route. A suitable route of administration is one that allows the composition or agent to perform its intended function. For example, if a suitable route is an intravenous route, the composition is administered by introducing the composition or agent into the subject's vein.

[0174] As used herein, the terms "subject", "individual", "host" and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in certain embodiments, the subject is a human.

[0175] As used herein, the terms "therapeutic amount", "therapeutically effective amount", "effective amount" or "pharmaceutical effective amount" of an active agent (e.g., hepatocytes) are used interchangeably to refer to an amount that is sufficient to provide the intended benefit from treatment. However, dosage levels are based on a variety of factors, including: type of injury; age, weight, sex, medical condition of the patient; severity of the condition; route of administration; expected cell engraftment; long-term survival rate; and / or the particular active agent utilized. Thus, dosage regimens can vary widely, but can be determined by a physician using standard methods as a matter of routine. In addition, the terms "therapeutic amount", "therapeutically effective amount" and "pharmaceutical effective amount" include prophylactic or preventative amounts of the compositions of the present invention described. In the prophylactic or preventative use of the present invention described, the pharmaceutical composition or medicament is administered to a patient susceptible to or at risk of a disease, disorder, or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition; complications thereof; and intermediate pathological phenotypes that appear during the progression of the disease, disorder, or condition. In general, it is preferred to use the maximum dose, which is the maximum safe amount according to any medical judgment. The terms "dose" and "administration" are used interchangeably herein.

[0176] As used herein, the term "therapeutic effect" refers to the outcome of a treatment that is deemed desirable and beneficial. Therapeutic effect can include the direct or indirect arrest, reduction, or elimination of disease symptoms. Therapeutic effect can also include the direct or indirect arrest, reduction, or elimination of the progression of disease symptoms.

[0177] For therapeutic agents (e.g., hepatocytes) described herein, the therapeutically effective amount may be initially determined from preliminary in vitro tests and / or animal models. The therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjustment of the dose to achieve maximum efficacy based on the above-mentioned methods and other known methods is within the ability of one skilled in the art.

[0178] The principles of pharmacokinetics provide a basis for modifying dosing regimens to obtain a desired degree of therapeutic efficacy while minimizing unacceptable adverse effects. In situations where the plasma concentration of an agent is measurable and can be related to a therapeutic window, an additional guide for modifying dosage is available.

[0179] As used herein, the terms "treat," "treating," and / or "treatment" include arresting, substantially inhibiting, slowing, or reversing the progression of a condition, substantially ameliorating the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition (e.g., a pathological condition), and obtaining a beneficial or desired clinical outcome. Treating further refers to achieving one or more of the following: (a) reducing the severity of the abnormality; (b) limiting the progression of symptoms characteristic of the abnormality being treated; (c) limiting the worsening of symptoms characteristic of the abnormality being treated; (d) limiting the recurrence of the abnormality in patients who previously had the abnormality; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the abnormality.

[0180] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, the following: prevention of the occurrence of a disease, disorder, or condition in a subject who may be susceptible to the disease, disorder, or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment); alleviation of the symptoms of the disease, disorder, or condition; reducing the severity of the disease, disorder, or condition; stabilization (i.e., not worsening) of the disease, disorder, or condition; prevention of the spread of the disease, disorder, or condition; delaying or slowing the progression of the disease, disorder, or condition; amelioration or temporary palliation of the disease, disorder, or condition; and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0181] I. The Methods of the Invention The present invention is based on the discovery of a method, which comprises increasing the expression of at least one transcription factor selected from the group consisting of NFIC and NFIX to enhance hepatocyte maturation, thereby allowing the generation of mature and functional hepatocytes. The method of the present invention is efficient and effective, and results in the generation of mature hepatocytes, e.g., from pluripotent stem cells, which can be used for various applications disclosed herein, e.g., for the treatment of liver diseases.

[0182] In some embodiments, increased expression of NFIX comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold compared to the endogenous expression level of NFIX in immature hepatocytes.

[0183] In some embodiments, increased expression of NFIC includes an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIC in immature hepatocytes.

[0184] In some embodiments, the method further comprises culturing the immature hepatocytes in a culture medium comprising dexamethasone, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or a combination thereof.

[0185] In some embodiments, the immature hepatocytes comprise an expression vector comprising a nucleic acid encoding at least one transcription factor.

[0186] In some embodiments, increasing the expression of at least one transcription factor in the immature hepatocytes comprises inducing expression of the at least one transcription factor in the immature hepatocytes.

[0187] In some embodiments, the immature hepatocyte is derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. Any method for differentiating pluripotent cells into immature hepatocytes can be used. For example, the immature hepatocyte can be obtained by differentiating pluripotent stem cells as described herein.

[0188] In some embodiments, the pluripotent stem cells may be engineered to contain an expression vector that includes a nucleic acid encoding at least one transcription factor. In some embodiments, the expression vector includes a promoter, such as an endogenous promoter, an artificial promoter, or an inducible promoter, that is operably linked to the nucleic acid encoding at least one transcription factor.

[0189] Cells for generating hepatocytes In one embodiment of the present invention, methods and compositions are disclosed for generating mature hepatocytes by increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes.In some embodiments, mature hepatocytes and immature hepatocytes are derived from pluripotent stem cells, such as embryonic stem cells, induced pluripotent stem cells, fetal stem cells, and / or adult stem cells.In further embodiments, mature hepatocytes and immature hepatocytes can be derived from somatic cells.

[0190] A. Stem cells In the developing embryo, stem cells are capable of differentiating into all of the specialized embryonic tissues. In the adult organism, stem and progenitor cells act as the body's repair system, replenishing specialized cells, but also maintaining the normal turnover of regenerative organs, such as blood, skin, or intestinal tissue.

[0191] Pluripotent stem cells, such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can be expanded in vitro for long periods while retaining the potential to differentiate into all cell types of the body, including immature hepatocytes. Thus, these cells may have the potential to provide an unlimited source of functional patient-specific hepatocytes for both drug development and transplantation therapy. Differentiation of pluripotent stem cells into hepatocytes in vitro may involve the addition of various growth factors at different stages of differentiation, and this may require about 15-20 days of differentiation (see, for example, Figures 5A and 6A). One of the challenges in differentiating pluripotent stem cells into hepatocytes in vitro is that the hepatocytes appear to be functionally more similar to fetal hepatocytes, such as immature hepatocytes, and have not yet shown the full functional spectrum of mature hepatocytes, such as primary human hepatocytes (PHHs). Pluripotent stem cells with unlimited proliferation capacity, such as human ESCs / iPSCs, offer advantages over somatic cells as a starting cell population for differentiation into hepatocytes.

[0192] Pluripotent stem cells, such as embryonic stem (ES) cells or iPS cells, can be the starting material for the disclosed method. In any of the embodiments herein, the pluripotent stem cells can be human pluripotent stem cells (hPSCs). Pluripotent stem cells (PSCs) can be cultured in any manner known in the art, such as in the presence or absence of feeder cells. In addition, PSCs generated using any method can be used as starting material for generating hepatocytes. For example, hES cells can be derived from a blastocyst stage embryo, which is the product of in vitro fertilization of an egg and a sperm. Alternatively, hES cells can be derived from one or more blastomeres isolated from an early cleavage stage embryo, optionally without destroying the remaining part of the embryo. In yet another embodiment, hES cells can be generated using nuclear transfer. In a further embodiment, iPSCs can be used. Previously cryopreserved PSCs can be used as starting material. In another embodiment, never cryopreserved PSCs can be used.

[0193] In one aspect of the present invention, PSCs are plated on extracellular matrix under feeder or feeder-free conditions. In one embodiment, PSCs can be cultured on extracellular matrix, including but not limited to laminin, fibronectin, vitronectin, matrigel, CellStart, collagen, or gelatin. In some embodiments, the extracellular matrix is ​​laminin with or without e-cadherin. In some embodiments, the laminin can be selected from the group including laminin 521, laminin 511, or iMatrix 511. In some embodiments, the feeder cells are human feeder cells, such as human dermal fibroblasts (HDFs). In other embodiments, the feeder cells are mouse embryonic fibroblasts (MEFs).

[0194] In some embodiments, the medium used in culturing PSCs can be selected from any medium suitable for culturing PSCs.In some embodiments, any medium that can support the culture of PSCs can be used.For example, those skilled in the art can select from commercially available medium or proprietary medium.

[0195] The medium supporting pluripotency may be any such medium known in the art. In some embodiments, the medium supporting pluripotency is Nutrisem™. In some embodiments, the medium supporting pluripotency is TeSR™. In some embodiments, the medium supporting pluripotency is StemFit™. In other embodiments, the medium supporting pluripotency is Knockout™ DMEM (Gibco), which may be supplemented with Knockout™ Serum Replacement (Gibco), LIF, bFGF, or any other factor. Each of these exemplary media is known in the art and commercially available. In further embodiments, the medium supporting pluripotency may be supplemented with bFGF or any other factor. In one embodiment, bFGF may be added at a low concentration (e.g., 4 ng / mL). In another embodiment, bFGF may be added at a higher concentration (e.g., 100 ng / mL), which may prime PSCs to differentiate.

[0196] The concentration of PSCs used in the production method of the present invention is not particularly limited. For example, when a 10 cm culture dish is used, 1 x 10 4 ~1 x 10 8 Cells are preferably 5 x 10 per dish. 4 ~5 x 10 6 cells, more preferably 1 x 10 cells per culture dish. 5 ~1 x 10 7 cells are used.

[0197] In some embodiments, the PSCs are present at about 1,000-100,000 cells / cm. 2In some embodiments, the PSCs are plated at a cell density of about 5,000-100,000 cells / cm. 2 , about 5000~50,000 cells / cm 2 or approximately 5,000-15,000 cells / cm 2 In other embodiments, the PSCs are plated at a cell density of about 10,000 cells / cm. 2 The cells are plated at a density of 100 μg / ml.

[0198] In some embodiments, the pluripotency-supporting medium, such as StemFit™ or other similar medium, is replaced with a differentiation medium to differentiate the cells into immature hepatocytes. In some embodiments, the medium exchange from the pluripotency-supporting medium to the differentiation medium may be performed at various time points during the cell culture of the PSCs, and the exchange may vary depending on the initial plating density of the PSCs. In some embodiments, the medium exchange may be performed after culturing the PSCs in the pluripotency medium for 3 to 14 days. In some embodiments, the medium exchange may be performed on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, or 14th day.

[0199] In some embodiments, stem cells useful in the methods described herein include, but are not limited to, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, chondrocyte progenitor cells, epidermal stem cells, gastrointestinal stem cells, neural stem cells, hepatic stem cells, adipose-derived mesenchymal stem cells, pancreatic progenitor cells, hair follicle stem cells, endothelial progenitor cells, and smooth muscle progenitor cells.

[0200] In some embodiments, the stem cells used for the methods described herein are isolated from umbilical cord, placenta, amniotic fluid, chorionic villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, gastrointestinal tract, umbilical cord blood, blood vessels, skeletal muscle, skin, liver, and menstrual blood.

[0201] Detailed procedures for isolating human stem cells from various sources are described in "Current Protocols in Stem Cell Biology" (2007), which is incorporated herein by reference in its entirety. Methods for isolating and culturing stem cells from various sources are also described in U.S. Patent Nos. 5,486,359, 6,991,897, 7,015,037, 7,422,736, 7,410,798, 7,410,773, and 7,399,632, each of which is incorporated herein by reference in its entirety.

[0202] B. Somatic cells In certain aspects of the invention, methods of transdifferentiation, i.e., directly converting one somatic cell type into another, may also be provided, for example, to give rise to hepatocytes from other somatic cells. Transdifferentiation may involve using genes or gene products of transcription factors that induce hepatocyte differentiation, to increase the expression levels of such genes in somatic cells to generate hepatocytes.

[0203] However, human somatic cells, especially those from living donors, may be in limited supply. To provide an unlimited source of starting cells for differentiation into hepatocytes, somatic cells may be immortalized by the introduction of immortalization genes or proteins, such as hTERT and / or other oncogenes. Cell immortalization may be reversible (e.g., using a removable expression cassette) or inducible (e.g., using an inducible promoter).

[0204] Somatic cells, in certain aspects of the invention, may be primary cells (non-immortalized cells), such as cells freshly isolated from an animal, or may be derived from a cell line (immortalized cells). The cells may be maintained in cell culture after isolation from a subject. In certain embodiments, the cells are passaged one or more times (e.g., 2-5 times, 5-10 times, 10-20 times, 20-50 times, 50-100 times, or more) before being used in the methods of the invention. In some embodiments, the cells are passaged no more than once, no more than two, no more than five, no more than 10, no more than 20, or no more than 50 times before being used in the methods of the invention.

[0205] The somatic cells used or described herein may be natural somatic cells or engineered somatic cells, i.e. genetically altered somatic cells. The somatic cells of the present invention are typically mammalian cells, such as human cells, primate cells, or mouse cells. These cells may be obtained by well-known methods and may be obtained from any organ or any tissue that contains living somatic cells, such as blood, bone marrow, skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs.

[0206] Mammalian somatic cells useful in the present invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac myocytes, and other muscle cells.

[0207] The methods described herein can be used to program one or more types of somatic cells, such as colonies or populations of somatic cells, into hepatocytes.In some embodiments, the cell population of the present invention is substantially homogeneous in that at least 90% of cells exhibit phenotypes or characteristics of interest.In some embodiments, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95% or more of cells exhibit phenotypes or characteristics of interest.In some embodiments of the present invention, somatic cells have the ability to divide, i.e., somatic cells are not postmitotic cells.

[0208] Somatic cells may be partially or fully differentiated. As described herein, both partially and fully differentiated somatic cells can be differentiated to produce hepatocytes.

[0209] Transcription Factors for Use in the Methods of the Invention Mature hepatocytes can be produced by increasing the expression of at least one transcription factor as described herein in immature hepatocytes.Any transcription factor that is important for promoting hepatocyte differentiation, hepatocyte maturation, or hepatocyte function can be used, for example, at least one transcription factor selected from the transcription factors listed in Table 1.All of the isoforms and variants of the transcription factors listed in Table 1 can be included in the present invention.Non-limiting examples of the accession numbers of some isoforms or variants of the transcription factors of the present invention are listed in Table 1.

[0210] Table 1. Transcription factors for generating mature hepatocytes TIFF2024518409000001.tif198151

[0211] In some embodiments, the at least one transcription factor is selected from the group consisting of NFIX, NFIC, RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1.

[0212] In some embodiments, the transcription factor is nuclear factor IX (NFIX). As used herein, "NFIX" refers to its well-known gene and protein. NFIX is also known as nuclear factor IX, nuclear factor 1 type X, NF1-X, or NF-I / X. The protein encoded by the NFIX gene is a palindromic sequence in viral and cellular promoters and in the origin of replication of adenovirus type 2. NFIX is a transcription factor that binds to TIFF2024518409000002.tif4128. NFIX protein can activate transcription and replication by itself. The sequence of human NFIX mRNA transcript can be found in the National Center for Biotechnology Information (NCBI) RefSeq accession number NM_002501.4 (SEQ ID NO: 1). Further examples of NFIX mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0213] Exemplary sequences for NFIX include the nucleotide sequence of SEQ ID NO: 1, or the amino acid sequence encoded thereby. In some embodiments, NFIX comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

[0214] In some embodiments, the method of the present invention is directed to increasing the expression of NFIX by at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 0.1-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 0.2-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 0.5-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 1-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 2-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 5-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 10-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 20-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 50-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 100-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 200-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, increased expression of NFIX comprises at least a 500-fold increase as compared to the endogenous expression level of NFIX in immature hepatocytes.In some embodiments, the increased expression of NFIX comprises at least a 1,000-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes. In some embodiments, the increased expression of NFIX comprises at least a 10,000-fold increase compared to the endogenous expression level of NFIX in immature hepatocytes.

[0215] In some embodiments, the transcription factor is nuclear factor IC (NFIC). As used herein, "NFIC" refers to its well-known gene and protein. The term NFIC includes alternative splice variants or transcript variants (e.g., NFIC transcript variants 1-5) and protein isoforms. NFIC is also known as nuclear factor IC, CTF, nuclear factor 1 type C, NF1-C, or NF-I / C. The proteins encoded by the NFIC gene belong to the CTF / NF-I family. They are dimeric DNA-binding proteins and function as transcription factors in cells and as replication factors for adenovirus DNA replication. The NFIC protein is a palindromic sequence present in viral and cellular promoters, as well as in the origin of replication of adenovirus type 2. The NFIC protein recognizes and binds to TIFF2024518409000003.tif4128. The NFIC protein can activate transcription and replication independently. The NFIC gene encodes an alternative splice variant. In some embodiments, the NFIC is NFIC transcript variant 1. The sequence of the mRNA transcript of human NFIC transcript variant 1 can be found in NCBI RefSeq Accession No. NM_001245002 (SEQ ID NO: 2). In some embodiments, the NFIC is NFIC transcript variant 2. The sequence of the mRNA transcript of human NFIC transcript variant 2 can be found in NCBI RefSeq Accession No. NM_205843 (SEQ ID NO: 3). In some embodiments, the NFIC is NFIC transcript variant 3. The sequence of the mRNA transcript of human NFIC transcript variant 3 can be found at NCBI RefSeq Accession No. NM_001245004 (SEQ ID NO: 4). In some embodiments, the NFIC is NFIC transcript variant 4. The sequence of the mRNA transcript of human NFIC transcript variant 4 can be found at NCBI RefSeq Accession No. NM_001245005 (SEQ ID NO: 5). In some embodiments, the NFIC is NFIC transcript variant 5. The sequence of the mRNA transcript of human NFIC transcript variant 5 can be found at NCBI RefSeq Accession No. NM_005597 (SEQ ID NO: 6). In some embodiments, the NIFIC is any combination of NFIC transcript variants 1-5. In some embodiments, the NFIC is NFIC transcript variant 1 and NFIC transcript variant 3. Further examples of NFIC mRNA sequences are readily available using public databases such as GenBank, UniProt, and OMIM.

[0216] Exemplary sequences of NFIC transcript variant 1 include the nucleotide sequence of SEQ ID NO: 2, or the amino acid sequence encoded thereby. In some embodiments, NFIC transcript variant 1 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 2. In another embodiment, NIFC transcript variant 1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2.

[0217] Exemplary sequences of NFIC transcript variant 2 include the nucleotide sequence of SEQ ID NO: 3, or the amino acid sequence encoded thereby. In some embodiments, NFIC transcript variant 2 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 3. In one embodiment, NFIC transcript variant 2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.

[0218] Exemplary sequences of NFIC transcript variant 3 include the nucleotide sequence of, or the amino acid sequence encoded thereby, SEQ ID NO: 4. In some embodiments, NFIC transcript variant 3 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4. In one embodiment, NFIC transcript variant 3 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4.

[0219] Exemplary sequences of NFIC transcript variant 4 include the nucleotide sequence of SEQ ID NO: 5, or the amino acid sequence encoded thereby. In some embodiments, NFIC transcript variant 4 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5. In one embodiment, NFIC transcript variant 4 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5.

[0220] Exemplary sequences of NFIC transcript variant 5 include the nucleotide sequence of SEQ ID NO: 6, or the amino acid sequence encoded thereby. In some embodiments, NFIC transcript variant 5 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6. In one embodiment, NFIC transcript variant 5 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6.

[0221] In some embodiments, the method of the present invention is directed to increase the expression of NFIC by at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 0.1-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 0.2-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 0.5-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 1-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 2-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 5-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 10-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 20-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 50-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 100-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 200-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, increased expression of NFIC comprises at least a 500-fold increase as compared to the endogenous expression level of NFIC in immature hepatocytes.In some embodiments, the increased expression of NFIC comprises at least a 1,000-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes. In some embodiments, the increased expression of NFIC comprises at least a 10,000-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes.

[0222] In some embodiments, the transcription factor is RORC. The sequence of the mRNA transcript of human RORC can be found in NCBI RefSeq Accession No. NM_005060.3 (SEQ ID NO: 7). Exemplary sequences of RORC include the nucleotide sequence of SEQ ID NO: 7, or the amino acid sequence encoded thereby. In some embodiments, RORC includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. In one embodiment, the RORC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7.

[0223] In some embodiments, the transcription factor is NR0B2. The sequence of the mRNA transcript of human NR0B2 can be found in NCBI RefSeq Accession No. NM_021969.2 (SEQ ID NO: 8). Exemplary sequences of NR0B2 include the nucleotide sequence of SEQ ID NO: 8, or the amino acid sequence encoded thereby. In some embodiments, NR0B2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8. In one embodiment, NR0B2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:8.

[0224] In some embodiments, the transcription factor is ESR1. The sequence of the mRNA transcript of human ESR1 can be found in NCBI RefSeq Accession No. NM_001291230.1 (SEQ ID NO: 9). Exemplary sequences of ESR1 include the nucleotide sequence of SEQ ID NO: 9 or the amino acid sequence encoded thereby. In some embodiments, ESR1 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 9. In one embodiment, ESR1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:9.

[0225] In some embodiments, the transcription factor is THRSP. The sequence of the mRNA transcript of human THRSP can be found in NCBI RefSeq Accession No. NM_003251.3 (SEQ ID NO: 10). Exemplary sequences of THRSP include the nucleotide sequence of SEQ ID NO: 10, or the amino acid sequence encoded thereby. In some embodiments, THRSP includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10. In one embodiment, THRSP comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:10.

[0226] In some embodiments, the transcription factor is TBX15. The sequence of the mRNA transcript of human TBX15 can be found in NCBI RefSeq Accession No. NM_152380 (SEQ ID NO: 11). Exemplary sequences of TBX15 include the nucleotide sequence of SEQ ID NO: 11 or the amino acid sequence encoded thereby. In some embodiments, TBX15 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 11. In one embodiment, TBX15 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:11.

[0227] In some embodiments, the transcription factor is HLF. The sequence of the mRNA transcript of human HLF can be found in NCBI RefSeq accession number NM_002126.4 (SEQ ID NO: 12). Exemplary sequences of HLF include the nucleotide sequence of SEQ ID NO: 12 or the amino acid sequence encoded thereby. In some embodiments, HLF includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the HLF comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:12.

[0228] In some embodiments, the transcription factor is ATOH8. The sequence of the mRNA transcript of human ATOH8 can be found in NCBI RefSeq Accession No. NM_032827.7 (SEQ ID NO: 13). Exemplary sequences of ATOH8 include the nucleotide sequence of SEQ ID NO: 13, or the amino acid sequence encoded thereby. In some embodiments, ATOH8 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 13. In one embodiment, ATOH8 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:13.

[0229] In some embodiments, the transcription factor is NR1I2. The sequence of the mRNA transcript of human NR1I2 can be found in NCBI RefSeq Accession No. NM_003889.3 (SEQ ID NO: 14). Exemplary sequences of NR1I2 include the nucleotide sequence of SEQ ID NO: 14, or the amino acid sequence encoded thereby. In some embodiments, NR1I2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 14. In one embodiment, NR1I2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:14.

[0230] In some embodiments, the transcription factor is CUX2. The sequence of the mRNA transcript of human CUX2 can be found at NCBI RefSeq Accession No. NM_015267.3 (SEQ ID NO: 15). Exemplary sequences of CUX2 include the nucleotide sequence of SEQ ID NO: 15, or the amino acid sequence encoded thereby. In some embodiments, CUX2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 15. In one embodiment, CUX2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:15.

[0231] In some embodiments, the transcription factor is ZNF662. The sequence of the mRNA transcript of human ZNF662 can be found in NCBI RefSeq Accession No. NM_001134656.1 (SEQ ID NO: 16). Exemplary sequences of ZNF662 include the nucleotide sequence of SEQ ID NO: 16 or the amino acid sequence encoded thereby. In some embodiments, ZNF662 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 16. In one embodiment, ZNF662 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:16.

[0232] In some embodiments, the transcription factor is TSHZ2. The sequence of the mRNA transcript of human TSHZ2 can be found in NCBI RefSeq Accession No. NM_173485.5 (SEQ ID NO: 17). Exemplary sequences of TSHZ2 include the nucleotide sequence of SEQ ID NO: 17 or the amino acid sequence encoded thereby. In some embodiments, TSHZ2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 17. In one embodiment, TSHZ2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:17.

[0233] In some embodiments, the transcription factor is ATF5. The sequence of the mRNA transcript of human ATF5 can be found in NCBI RefSeq accession number NM_001193646.1 (SEQ ID NO: 18). Exemplary sequences of ATF5 include the nucleotide sequence of SEQ ID NO: 18 or the amino acid sequence encoded thereby. In some embodiments, ATF5 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 18. In one embodiment, ATF5 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:18.

[0234] In some embodiments, the transcription factor is NFIA. The sequence of the mRNA transcript of human NFIA can be found in NCBI RefSeq Accession No. NM_001134673.3 (SEQ ID NO: 19). Exemplary sequences of NFIA include the nucleotide sequence of SEQ ID NO: 19, or the amino acid sequence encoded thereby. In some embodiments, NFIA includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 19. In one embodiment, NFIA comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:19.

[0235] In some embodiments, the transcription factor is NFIB. ​​The sequence of the mRNA transcript of human NFIB can be found in NCBI RefSeq Accession No. NM_005596.3 (SEQ ID NO: 20). Exemplary sequences of NFIB include the nucleotide sequence of SEQ ID NO: 20, or the amino acid sequence encoded thereby. In some embodiments, NFIB includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 20. In one embodiment, NFIB comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:20.

[0236] In some embodiments, the transcription factor is NPAS2. The sequence of the mRNA transcript of human NPAS2 can be found in NCBI RefSeq Accession No. XM_005263953.2 (SEQ ID NO: 21). Exemplary sequences of NPAS2 include the nucleotide sequence of SEQ ID NO: 21 or the amino acid sequence encoded thereby. In some embodiments, NPAS2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 21. In one embodiment, NPAS2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21.

[0237] In some embodiments, the transcription factor is FOS. The sequence of the mRNA transcript of human FOS can be found in NCBI RefSeq Accession No. NM_005252.3 (SEQ ID NO: 22). Exemplary sequences of FOS include the nucleotide sequence of SEQ ID NO: 22 or the amino acid sequence encoded thereby. In some embodiments, FOS includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 22. In one embodiment, the FOS comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:22.

[0238] In some embodiments, the transcription factor is ONECUT2. The sequence of the mRNA transcript of human ONECUT2 can be found in NCBI RefSeq Accession No. NM_004852.2 (SEQ ID NO: 23). Exemplary sequences of ONECUT2 include the nucleotide sequence of SEQ ID NO: 23 or the amino acid sequence encoded thereby. In some embodiments, ONECUT2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 23. In one embodiment, ONECUT2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:23.

[0239] In some embodiments, the transcription factor is PROX1. The sequence of the mRNA transcript of human PROX1 can be found in NCBI RefSeq Accession No. NM_001270616.2 (PROX1 transcript variant 1; SEQ ID NO: 24), or NM_002763.5 (PROX1 transcript variant 2; SEQ ID NO: 39). Exemplary sequences of PROX1 include the nucleotide sequence of SEQ ID NO: 24, or the amino acid sequence encoded thereby. In some embodiments, PROX1 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 24. In one embodiment, PROX1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 24. Exemplary sequences of PROX1 include the nucleotide sequence of SEQ ID NO: 39, or the amino acid sequence encoded thereby. In some embodiments, PROX1 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 39.In one embodiment, PROX1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:39.

[0240] In some embodiments, the transcription factor is NR1H4. The sequence of the mRNA transcript of human NR1H4 can be found in NCBI RefSeq Accession No. NM_001206979.1 (SEQ ID NO: 25). Exemplary sequences of NR1H4 include the nucleotide sequence of SEQ ID NO: 25 or the amino acid sequence encoded thereby. In some embodiments, NR1H4 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 25. In one embodiment, NR1H4 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:25.

[0241] In some embodiments, the transcription factor is MLXIPL. The sequence of the mRNA transcript of human MLXIPL can be found in NCBI RefSeq Accession No. NM_032951.2 (SEQ ID NO: 26). Exemplary sequences of MLXIPL include the nucleotide sequence of SEQ ID NO: 26, or the amino acid sequence encoded thereby. In some embodiments, MLXIPL includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 26. In one embodiment, MLXIPL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:26.

[0242] In some embodiments, the transcription factor is ETV1. The sequence of the mRNA transcript of human ETV1 can be found at NCBI RefSeq Accession No. NM_001163147 (SEQ ID NO: 27). Exemplary sequences of ETV1 include the nucleotide sequence of SEQ ID NO: 27, or the amino acid sequence encoded thereby. In some embodiments, ETV1 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 27. In one embodiment, ETV1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:27.

[0243] In some embodiments, the transcription factor is AR. The sequence of the mRNA transcript of human AR can be found in NCBI RefSeq accession number NM_000044.3 (SEQ ID NO: 28). Exemplary sequences of AR include the nucleotide sequence of SEQ ID NO: 28, or the amino acid sequence encoded thereby. In some embodiments, AR includes the nucleotide sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 28. In one embodiment, the AR comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:28.

[0244] In some embodiments, the transcription factor is CEBPB. The sequence of the mRNA transcript of human CEBPB can be found in NCBI RefSeq Accession No. NM_005194.3 (SEQ ID NO: 29). Exemplary sequences of CEBPB include the nucleotide sequence of SEQ ID NO: 29 or the amino acid sequence encoded thereby. In some embodiments, CEBPB includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 29. In one embodiment, CEBPB comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:29.

[0245] In some embodiments, the transcription factor is NR1D1. The sequence of the mRNA transcript of human NR1D1 can be found in NCBI RefSeq Accession No. NM_021724.4 (SEQ ID NO: 30). Exemplary sequences of NR1D1 include the nucleotide sequence of SEQ ID NO: 30, or the amino acid sequence encoded thereby. In some embodiments, NR1D1 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 30. In one embodiment, NR1D1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:30.

[0246] In some embodiments, the transcription factor is HEY2. The sequence of the mRNA transcript of human HEY2 can be found at NCBI RefSeq Accession No. NM_012259.2 (SEQ ID NO: 31). Exemplary sequences of HEY2 include the nucleotide sequence of SEQ ID NO: 31, or the amino acid sequence encoded thereby. In some embodiments, HEY2 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 31. In one embodiment, HEY2 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:31.

[0247] In some embodiments, the transcription factor is ARID3C. The sequence of the mRNA transcript of human ARID3C can be found in NCBI RefSeq accession number NM_001017363.1 (SEQ ID NO: 32). Exemplary sequences of ARID3C include the nucleotide sequence of SEQ ID NO: 32 or the amino acid sequence encoded thereby. In some embodiments, ARID3C includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 32. In one embodiment, ARID3C comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:32.

[0248] In some embodiments, the transcription factor is KLF9. The sequence of the mRNA transcript of human KLF9 can be found at NCBI RefSeq Accession No. NM_001206.2 (SEQ ID NO: 33). Exemplary sequences of KLF9 include the nucleotide sequence of SEQ ID NO: 33, or the amino acid sequence encoded thereby. In some embodiments, KLF9 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 33. In one embodiment, KLF9 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:33.

[0249] In some embodiments, the transcription factor is DMRTA1. The sequence of the mRNA transcript of human DMRTA1 can be found in NCBI RefSeq Accession No. NM_022160.2 (SEQ ID NO: 34). Exemplary sequences of DMRTA1 include the nucleotide sequence of SEQ ID NO: 34, or the amino acid sequence encoded thereby. In some embodiments, DMRTA1 includes a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 34. In one embodiment, DMRTA1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:34.

[0250] Increased expression of transcription factors The vector for delivering the nucleic acid encoding the transcription factor of the present invention can be constructed to express the transcription factor in the cell of the present invention, for example, the cell is an immature hepatocyte, a hepatic progenitor cell, or a pluripotent stem cell, for example, an embryonic stem cell or an induced pluripotent stem cell.In some embodiments, the nucleic acid is DNA.In some embodiments, the nucleic acid is RNA.In some embodiments, the nucleic acid is modified DNA.In some embodiments, the nucleic acid is modified RNA.

[0251] In addition, protein delivery compositions or methods may also be used to effect expression of transcription factors in the methods of the invention.

[0252] A. Nucleic Acid Delivery Systems Those skilled in the art will be fully equipped to construct vectors by standard recombinant techniques (see, for example, Sambrook et al. 2001; Ausubel et al. 1996; Maniatis et al. 1988; and Ausubel et al. 1994; each of which is incorporated herein by reference in its entirety). Vectors comprising the nucleic acid encoding at least one transcription factor of the present disclosure include, but are not limited to, viral vectors, non-viral vectors, and / or inducible expression vectors.

[0253] Vectors can also include other components or functional elements that further alter gene delivery and / or gene expression or provide beneficial properties to target cells, including, for example, components that affect cell binding or targeting (including components that mediate cell-type or tissue-specific binding), components that affect cellular uptake of vector nucleic acid, components that affect cellular localization of the polynucleotide once uptake (e.g., agents that mediate nuclear localization), and components that affect expression of the polynucleotide.

[0254] Such components may also include markers, such as detectable and / or selectable markers that can be used to detect or select cells that take up and express the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., the use of certain viral vectors that have components or functional elements that mediate binding and uptake), or the vector may be modified to provide such functional elements. A wide variety of such vectors are known and widely available in the art. When a vector is maintained in a host cell, it may either be stably replicated by the cell during mitosis as an autonomous structure, be integrated into the genome of the host cell, or be maintained in the nucleus or cytoplasm of the host cell.

[0255] 1. Viral Vectors In some aspects of the present disclosure, a viral vector that encodes at least one transcription factor of the present invention can be provided.A viral vector is a type of expression construct that utilizes viral sequence to introduce nucleic acid and optionally protein into cells.A non-limiting example of the viral vector that can be used to deliver the nucleic acid of some aspects of the present invention is described below.

[0256] In some embodiments, the viral vector is a non-integrating viral vector.The exemplary non-integrating viral vector of the present disclosure is selected from the group consisting of: adeno-associated viral (AAV) vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, etc.; adenovirus (Ad) vector, such as Ad7, Ad4, Ad2, Ad5, etc., including their replication-competent, replication-incompetent and gutless forms; Simian Virus 40 (SV-40) vector, bovine papilloma virus vector, Epstein-Barr virus vector, herpes virus vector, vaccinia virus vector, Harvey murine sarcoma virus vector, mouse mammary tumor virus vector, or Rous sarcoma virus vector.

[0257] In some embodiments, the viral vector is an integrating viral vector, such as, for example, a retroviral vector. Retroviruses are promising gene delivery vectors due to their ability to integrate genes into the host genome, transport large amounts of foreign genetic material, infect a wide range of organisms and cell types, and be packaged in specific cell lines.

[0258] In some embodiments, the integrating viral vector is derived from a retroviral vector (e.g., Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), a lentiviral vector (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), or a vector derived therefrom.

[0259] Recombinant vectors can also infect non-dividing cells and can be used in the methods of the present invention to deliver genes and express nucleic acid sequences both in vivo and ex vivo. For example, recombinant lentiviruses capable of infecting non-dividing cells are described in U.S. Patent No. 5,994,136, the entire contents of which are incorporated herein by reference, and suitable host cells (i.e., virus-producing cells, not hepatocytes of the present disclosure) are transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat.

[0260] 2. Episomal and other non-viral vectors The use of plasmid-based or liposome-based extrachromosomal vectors (i.e., episomal vectors) may also be provided in certain aspects of the present invention. Such episomal vectors may include, for example, oriP-based vectors and / or vectors encoding derivatives of EBNA-1. These vectors may allow large DNA fragments to be introduced into cells and maintained extrachromosomally, replicating the fragments once per cell cycle, and efficiently distributing the fragments to daughter cells while not eliciting a substantial immune response.

[0261] Other extrachromosomal vectors include other vectors based on lymphotropic herpesviruses. Exemplary lymphotropic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); Herpesvirus saimiri (HS), and Marek's disease virus (MDV). Also contemplated are other sources of episome-based vectors, such as yeast ARS, adenovirus, SV40, or BPV.

[0262] In some embodiments, the vector is a non-viral vector.In some embodiments, the non-viral vector is selected from the group consisting of plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA) and double-stranded mRNA (dsRNA).

[0263] In some embodiments, the non-viral vector comprises naked nucleic acid, liposomes, dendrimers, nanoparticles, lipid-polymer systems, solid lipid nanoparticles, and / or liposome-protamine / DNA lipoplexes (LPD).

[0264] In some embodiments, the non-viral vector comprises mRNA. In some embodiments, the mRNA may be delivered as modified naked mRNA, for example in sucrose-citrate buffer or saline. In other embodiments, the non-viral vector comprises mRNA complexed with a transfection reagent, such as Lipofectamine 2000, jetPEI, RNAiMAX, and / or Invivofectamine. Amine-containing materials are also commonly used as non-viral vectors to protect mRNA from degradation by nucleases and to shield its negative charge. One of the most developed methods for delivering mRNA is its combination with lipid nanoparticles (LNPs). LNP formulations typically consist of: (1) lipids or polymeric materials that are ionizable or cationic, carrying tertiary or quaternary amines to encapsulate polyanionic mRNA; (2) zwitterionic lipids that resemble lipids in cell membranes (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]); (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) polyethylene glycol (PEG) lipids to hydrate the nanoparticles, improve colloidal stability, and reduce protein absorption. Exemplary non-viral vectors containing mRNA are described in Kowalksi et al. 2019, Mol Ther.; 27(4): 710-728, which is incorporated herein by reference in its entirety.

[0265] 3. Transposon-based systems In certain embodiments, the introduction of the nucleic acid may use a transposon-transposase system, which may be the well-known Sleeping Beauty or Frog Prince transposon-transposase system (for a description of the latter, see, for example, EP1507865), or the piggyBac system, which is a TTAA-specific transposon.

[0266] Transposons are DNA sequences that can move around to different locations in the genome of a cell, a process called transposition. In the process, transposons can cause mutations and change the amount of DNA in the genome. There are various mobile genetic elements, and they can be classified based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first transcribing themselves into RNA, then reverse transcribing it into DNA with reverse transcriptase, and then inserting it into another location in the genome. Class II mobile genetic elements move directly from one location to another in the genome using a transposase that "cuts" and pastes itself.

[0267] 4. Homologous Recombination Homologous recombination (HR) is a targeted genome modification technique that has been a standard method for genome engineering in mammalian cells since the mid-1980s. Meganucleases, or homing endonucleases, such as I-SceI, have been used to increase the efficiency of HR. Both natural meganucleases and engineered meganucleases with modified targeting specificity have been utilized to increase the efficiency of HR. Another route to increase the efficiency of HR has been the engineering of chimeric endonucleases with programmable DNA specificity domains. Zinc finger nucleases (ZFNs) are an example of such chimeric molecules, in which a zinc finger DNA binding domain is fused to the catalytic domain of a type IIS restriction endonuclease, such as FokI. Another class of such specificity molecules includes the DNA binding domain of a transcription activator-like effector (TALE) fused to the catalytic domain of a type IIS restriction endonuclease, such as FokI. Another class of such molecules that facilitate targeted genome modification includes the CRISPR / Cas system, which is described, for example, in Ran et al. 2013; Nature Protocols 8:2281-2308, which is incorporated herein by reference in its entirety.

[0268] B. Regulatory Elements The eukaryotic expression cassette contained in the vector preferably comprises (in a 5' to 3' orientation) a eukaryotic transcription promoter operably linked to the protein coding sequence, splice signals including intervening sequences, and transcription termination / polyadenylation sequences.

[0269] 1. Promoter / Enhancer A "promoter" is a control sequence, which is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. It can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. The terms "operably located," "operatively linked," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct and functional location and / or orientation with respect to the nucleic acid sequence whose transcription initiation and / or expression it controls.

[0270] Promoters generally contain sequences that function to define the start site for RNA synthesis. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located 30-110 positions upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and enhances expression of the encoded RNA.

[0271] Spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased up to 50 apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either in concert or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refers to cis-acting regulatory sequences involved in activating transcription of a nucleic acid sequence.

[0272] With respect to the compositions disclosed herein, in addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™ (see U.S. Patent Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference in its entirety).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in organelles other than the nucleus, such as mitochondria, chloroplasts, etc., may be similarly utilized.

[0273] The promoter utilized may be one that is constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high level expression of the introduced DNA segment, such as those advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be artificial or endogenous.

[0274] In some embodiments, the promoter is an inducible promoter. The term "inducible promoter" is known in the art and refers to a promoter that is active only in response to a stimulus. An inducible promoter selectively expresses a nucleic acid molecule in response to an endogenous or exogenous stimulus, such as the presence of a chemical compound (chemical inducer), or in response to an environmental, hormonal, chemical, and / or developmental signal. Inducible promoters include, for example, promoters that are induced or regulated by light, heat, stress (e.g., salt or osmotic stress), plant hormones, wounding, or chemicals, such as ethanol, abscisic acid (ABA), jasmonic acid, salicylic acid, or safener. In some embodiments, the inducible promoter is an EF1a promoter. In some embodiments, the inducible promoter is a PGK promoter.

[0275] In addition, any combination of promoters / enhancers (e.g., as per the Eukaryotic Promoter Data Base, EPDB, available on the World Wide Web at "epd.isb-sib.ch / ") can also be used to drive expression. Non-limiting examples of promoters include the following: constitutive EF1α promoter; early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter, and the like; eukaryotic promoters, such as β-actin promoter, GADPH promoter, metallothionein promoter, and the like; and tethered response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and minimal TATA box proximal response element promoter (tre), and the like.

[0276] Several enhancer sequences for liver-specific genes have been described.For example, PCT publication WO2009130208 describes several liver-specific regulatory enhancer sequences, and the entirety of the document is incorporated herein by reference.PCT publication WO95 / 011308 describes a gene therapy vector that comprises a hepatocyte-specific control region (HCR) enhancer linked to a promoter and a transgene, and the entirety of the document is incorporated herein by reference.PCT publication WO01 / 098482 teaches the combination of a specific ApoE enhancer sequence or its truncated version with a liver promoter, and the entirety of the document is incorporated herein by reference.

[0277] 2. Initiation signals, internal ribosome binding sites, and self-cleavage sequences Specific initiation signals may also be used to efficiently translate coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including the ATG initiation codon. Those skilled in the art will be able to easily determine such necessity and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be improved by including appropriate transcriptional enhancer elements.

[0278] In some embodiments of the present invention, an internal ribosome entry site (IRES) element is used to create multiple genes, or polycistronic messengers. The IRES element can avoid the ribosome scanning model of 5' methylated cap-dependent translation and start translation at a site within the sequence. The IRES element can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together and separated by IRES, thereby creating polycistronic messengers. The IRES element allows ribosome access to each open reading frame for efficient translation. Multiple genes can be efficiently expressed using one promoter / enhancer to transcribe one messenger (see U.S. Patent Nos. 5,925,565 and 5,935,819; each of which is incorporated herein by reference in its entirety).

[0279] In some embodiments, a self-cleaving sequence can be used to co-express genes. The term "self-cleaving sequence" as used herein refers to a sequence that links open reading frames to form a single cistron and induces ribosome skipping during translation. Two coding sequences linked by a self-cleaving sequence are translated into two separate peptides by ribosome skipping. For example, the self-cleaving sequence 2A can be used in the construct provided in the present disclosure to cause the linked expression or co-expression of multiple genes. Exemplary self-cleaving sequences include, but are not limited to, T2A, P2A, E2A, and F2A listed in Table 2.

[0280] Table 2: Exemplary 2A sequences TIFF2024518409000004.tif26156

[0281] In some embodiments, T2A comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35, or a nucleic acid encoding such an amino acid sequence.

[0282] In some embodiments, P2A comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:36, or a nucleic acid encoding such an amino acid sequence.

[0283] In some embodiments, E2A comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:37, or a nucleic acid encoding such an amino acid sequence.

[0284] In some embodiments, F2A comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 38, or a nucleic acid encoding such an amino acid sequence.

[0285] 3. Origin of replication For the purpose of propagating the vector in a host cell, the vector may contain one or more origins of replication (often called "ori"), which are specific nucleic acid sequences at which replication is initiated, such as the oriP of EBV described above, or a nucleic acid sequence corresponding to an engineered oriP with a similar or enhanced function in programming. Alternatively, the origins of replication of other viruses that replicate extrachromosomally, or autonomously replicating sequences (ARS), described above, can be used.

[0286] 4. Selectable and Screenable Markers In some embodiments of the present invention, cells containing the nucleic acid construct of the present invention can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker can confer identifiable changes to cells, allowing cells containing the expression vector to be easily identified. Generally, a selection marker confers a property that allows selection. A positive selection marker is one whose presence allows its selection, while a negative selection marker is one whose presence prevents its selection. One example of a positive selection marker is a drug resistance marker.

[0287] Generally, the inclusion of a drug selection marker facilitates cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, histidinol, etc. are useful selection markers. In addition to markers that confer a phenotype that allows transformants to be identified based on the implementation of certain conditions, other types of markers are also contemplated, including screenable markers whose basis is colorimetric, such as GFP.

[0288] Alternatively, a screenable enzyme may be utilized, such as a negative selection marker. In some embodiments, the negative selection marker comprises one or more suicide genes, which upon administration of a prodrug, convert the gene product into a compound that kills the host cell. Exemplary suicide genes for the present disclosure include, but are not limited to, inducible caspase 9 (or caspase 3 or 7), CD20, CD52, EGFRt, thymidine kinase, cytosine deaminase, HER1, and any combination thereof. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, and thymidine phosphorylase (TP).

[0289] Those skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis.The marker used will not have a significant impact, as long as it can be expressed simultaneously with the nucleic acid encoding the gene product.Further examples of selection markers and screenable markers are well known to those skilled in the art.One feature of the present invention includes the use of selection markers and screenable markers to select hepatocytes after transcription factors have caused desired changes in hepatocytes.

[0290] In some embodiments of the present invention, cells containing the nucleic acid construct of the present invention can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker can confer identifiable changes to cells, allowing cells containing the expression vector to be easily identified. Generally, a selection marker confers a property that allows selection. A positive selection marker is one whose presence allows its selection, while a negative selection marker is one whose presence prevents its selection. One example of a positive selection marker is a drug resistance marker.

[0291] Generally, the inclusion of a drug selection marker facilitates cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, histidinol, etc. are useful selection markers. In addition to markers that confer a phenotype that allows transformants to be identified based on the implementation of certain conditions, other types of markers are also contemplated, including screenable markers whose basis is colorimetric, such as GFP.

[0292] C. Nucleic Acid Delivery In certain embodiments, increasing expression of at least one transcription factor in an immature hepatocyte comprises contacting the cell, such as a pluripotent stem cell, an immature hepatocyte, or a hepatic progenitor cell, with at least one transcription factor. In some embodiments, the cell, such as a pluripotent stem cell, an immature hepatocyte, or a hepatic progenitor cell, comprises an expression vector comprising a nucleic acid encoding at least one transcription factor.

[0293] To introduce a nucleic acid, such as DNA, RNA, modified DNA, or modified RNA, into a cell of the invention, such as a pluripotent stem cell, an immature hepatic cell, or a hepatic progenitor cell, any suitable method for delivering a nucleic acid for transformation of a cell may be used, as described herein or as known to one of skill in the art. Such methods include, for example, but are not limited to, direct delivery of DNA, such as by ex vivo transfection (Wilson et al. 1989; Nabel et al. 1989; each of which is incorporated herein by reference in its entirety); microinjection (Harland and Weintraub, No. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated by reference in its entirety), including injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated by reference in its entirety); by electroporation (U.S. Pat. No. 5,384,253; Tur-Kaspa et al. 1986; Potter et al. 1984, each of which is incorporated by reference in its entirety); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990; each of which is incorporated herein by reference in its entirety); using DEAE-dextran followed by polyethylene glycol; by direct sonoloading (Fechheimer et al., 1987; each of which is incorporated herein by reference in its entirety); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991; each of which is incorporated herein by reference in its entirety), and by receptor-mediated transfection (Wu and Wu, 1987;Wu and Wu, 1988; each of which is incorporated herein by reference in its entirety); by microprojectile bombardment (PCT applications WO 94 / 09699 and WO 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880; each of which is incorporated herein by reference in its entirety); by stirring with silicon carbide fibers (Kaeppler et al. 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference in its entirety); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference in its entirety); by desiccation / inhibition mediated DNA uptake (Potrykus et al. 1985, each of which is incorporated herein by reference in its entirety), and any combination of such methods. By applying such techniques, organelles, cells, tissues, or organisms can be stably or transiently transformed;

[0294] In some embodiments of the invention, the nucleic acid may be encapsulated in a lipid complex, such as a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and encapsulate water and dissolved solutes between the lipid bilayers. Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated. The amount of liposomes used may vary depending on the nature of the liposomes and cells used, for example, about 5 to about 20 μg of vector DNA may be contemplated for 1 to 10 million cells.

[0295] In some embodiments of the invention, nucleic acids are introduced into organelles, cells, tissues, or organisms by electroporation. Electroporation involves exposing a suspension of cells and DNA to a high voltage discharge. Recipient cells can be mechanically damaged to make them more susceptible to transformation. Again, the amount of vector used can vary depending on the nature of the cells used, for example, about 5 to about 20 μg of vector DNA for 1 to 10 million cells can be contemplated.

[0296] In another embodiment of the invention, the nucleic acid is introduced into the cell using calcium phosphate precipitation.

[0297] In another embodiment, the nucleic acid is delivered to the cell by using DEAE-dextran followed by polyethylene glycol.

[0298] A further aspect of the invention involves introducing nucleic acids by direct sonication.

[0299] Microprojectile bombardment techniques can also be used to introduce nucleic acid into at least one organelle, cell, tissue, or organism (U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT application WO 94 / 09699; each of which is incorporated herein by reference). This method utilizes the ability to accelerate DNA-coated microprojectiles to high speeds, which allows them to penetrate cell membranes and enter cells without killing the cells (Klein et al. 1987; each of which is incorporated herein by reference in its entirety). A wide variety of microprojectile bombardment techniques are known in the art and are suitable for use in the methods of the present invention.

[0300] D. Genetic Switch In some embodiments, cells of the present disclosure, such as pluripotent stem cells or immature hepatocytes, are engineered to contain gene switch constructs encoding transcription factors of the present invention. Gene switch constructs provide the basic building blocks for constructing complex genetic circuits, which convert cells into cell-based machines useful for biomedical applications. Ligand-responsive gene switch constructs are cellular sensors that can process certain signals and generate a response in the form of a gene product. Incorporation of these into complex genetic circuits results in sophisticated circuit topologies reminiscent of electronics, which can provide engineered cells with the ability to remember events, start and stop protein production, and perform complex information processing tasks (see Auslander et al. 2016; Cold Spring Harb Perspect Biol.; 8(7): a023895; which is incorporated herein by reference in its entirety). Based on the gene switch construct design strategy, cells of the present disclosure, such as pluripotent stem cells or immature hepatocytes, can be engineered to contain gene switch constructs encoding transcription factors of the present disclosure along with various synthetic systems that sense various ligand inputs, which in turn mediate expression of gene switch constructs encoding transcription factors of the present disclosure.

[0301] 1. Transcriptional Gene Switches In some embodiments, the gene switch construct is a transcriptional gene switch construct. In some embodiments, the transcriptional gene switch construct comprises the use of a prokaryotic regulator protein fused to a transcriptional regulator protein, which binds to a DNA operator sequence that controls expression of the gene switch construct in a ligand-responsive manner. In some embodiments, the transcriptional gene switch construct comprises the use of a combination of a prokaryotic regulator protein and a ligand- or light-induced dimerization system (DS), which allows for signal-dependent recruitment of the transcriptional regulator protein. In some embodiments, the transcriptional gene switch construct comprises the use of a G protein-coupled receptor (GPCR) located on the surface of a eukaryotic cell, which senses an extracellular signal and initiates signal transduction through a signal transduction pathway to control expression of the gene switch construct. In some embodiments, the transcriptional gene switch construct comprises the use of an engineered diguanylate cyclase (DGCL), which synthesizes the second messenger cyclic di-GMP in a red light-responsive manner to initiate downstream signal transduction pathways and cause transcriptional activation of the gene switch construct. In some embodiments, the transcriptional gene switch construct comprises any of the synthetic systems described in Auslander et al. 2016, which is incorporated by reference in its entirety.

[0302] 2. Post-transcriptional Gene Switches In some embodiments, the gene switch construct is a post-transcriptional gene switch construct. In some embodiments, the post-transcriptional gene switch construct comprises the use of an aptazyme fused to a primary microRNA (pri-miRNA) molecule, which allows for ligand-responsive control of pri-miRNA processing and post-transcriptional control of target genes. In some embodiments, the post-transcriptional gene switch construct comprises the use of a protein-responsive aptazyme incorporated into a messenger RNA (mRNA), which regulates the stability of the messenger RNA in response to the presence or absence of a protein ligand. In some embodiments, the post-transcriptional gene switch construct comprises the use of a protein-binding aptamer, which is incorporated into a short hairpin RNA (shRNA) and inhibits shRNA processing, and allows for protein-controlled expression of the gene switch construct. In some embodiments, the post-transcriptional gene switch construct comprises the use of a protein-binding aptamer incorporated into the 5' untranslated region (UTR) of an mRNA, which controls the initiation of translation in a protein-dependent manner. In some embodiments, the post-transcriptional gene switch construct comprises the use of incorporation of a protein-binding aptamer near the splice site, which allows for regulation of alternative splicing in response to the protein. In some embodiments, the post-transcriptional gene switch construct comprises the use of an ATetR-binding aptamer combined with a theophylline-responsive aptamer, which allows for theophylline-dependent folding of the TetR-binding aptamer. Upon binding to its close aptamer, the TetR protein loses its ability to bind to the DNA operator and affects gene expression at the transcriptional level.

[0303] Integrases can also act as functional regulators of genetic switches, activating coding sequences or promoter switches designed to operate in eukaryotic cells. Integrases are accurate in their site recognition and recombination process and do not have cytotoxicity. In some embodiments, the genetic switch construct comprises the use of a genetic switch controlled by serine integrase, as described in Gomide et al. 2020, Commun Biol.;3(1):255, which is incorporated herein by reference in its entirety.

[0304] E. Protein Delivery In some embodiments, the cells of the present disclosure, such as immature hepatocytes, may be contacted with a transcription factor that comprises a sufficient amount of polypeptide to generate mature hepatocytes.Protein transduction has been used as a method to enhance the delivery of macromolecules to cells.Protein transduction domains may be used to directly transduce transcription factor polypeptides or functional fragments thereof into cells.

[0305] A "protein transduction domain" or "PTD" is an amino acid sequence capable of crossing biological membranes, particularly cell membranes. When linked to a heterologous polypeptide, the PTD can enhance the translocation of the heterologous polypeptide across the biological membrane. The PTD is typically covalently linked (e.g., by a peptide bond) to a heterologous DNA-binding domain. For example, the PTD and the heterologous DNA-binding domain may be encoded by a single nucleic acid, e.g., in a common open reading frame or in one or more exons of a common gene. Exemplary PTDs may contain 10-30 amino acids and may form an amphipathic helix. Many PTDs are basic in nature. For example, a basic PTD may contain at least 4, 5, 6, or 8 basic residues (e.g., arginine or lysine). The PTD may be capable of enhancing the transfer of a polypeptide into a cell that lacks a cell wall or into a cell of a particular species, such as a mammalian cell, such as a human, monkey, mouse, bovine, equine, feline, or ovine cell.

[0306] The PTD can be linked to the artificial transcription factor, for example, by using a flexible linker. The flexible linker can include one or more glycine residues that allow free rotation. For example, the PTD can be at least 10, 20, or 50 amino acids away from the DNA-binding domain of the transcription factor. The PTD can be located N-terminal or C-terminal to the DNA-binding domain. Being located N-terminal or C-terminal to a particular domain does not require being adjacent to the particular domain. For example, a PTD that is N-terminal to a DNA-binding domain can be separated from the DNA-binding domain by a spacer and / or other types of domain. The PTD can be chemically synthesized and then chemically conjugated to a separately prepared DNA-binding domain, with or without a linker peptide. The artificial transcription factor can also include multiple PTDs, for example, multiple separate PTDs or at least two copies of one type of PTD.

[0307] Several proteins and short peptides have the ability to transduce or cross biological membranes independent of classical receptor- or endocytosis-mediated pathways. Examples of these proteins include the TAT protein of HIV-1, the DNA-binding protein VP22 of Herpes Simplex Virus Type 1 (HSV-1), and the Antennapedia (Antp) homeotic transcription factor of Drosophila. Short protein transduction domains (PTDs) from these proteins can be fused with other macromolecules, peptides, or proteins to successfully transport them into cells. Sequence alignments of the transduction domains from these proteins show a high content of basic amino acids (Lys and Arg), which may facilitate the interaction of these regions with negatively charged lipids in the membrane. Secondary structure analysis shows no structural congruence between all three domains.

[0308] The advantage of using these transduction domain fusions is that protein entry is rapid and concentration dependent, and appears to occur even with difficult cell types. PTDs are further described in U.S. Patent Application Publication Nos. 2003 / 0082561; 2002 / 0102265; and 2003 / 0040038, each of which is incorporated herein by reference in its entirety.

[0309] In addition to PTDs, it is possible to use endocytosis signals. Such signals include amino acid sequences that are specifically recognized by cell receptors or other surface proteins. Interaction between the endocytosis signal and the cell leads to internalization of the artificial transcription factor that contains the endocytosis signal. Some PTDs can also function by interacting with cell receptors or other surface proteins.

[0310] cell culture Generally, the cells of the present invention are cultured in a culture medium, which is a nutrient-rich buffered solution capable of sustaining the growth of the cells.

[0311] The hepatocytes of the present invention can be produced by culturing pluripotent stem cells or other cells, such as immature hepatocytes, in a medium under conditions that increase the intracellular levels of the transcription factors described herein sufficiently to promote the production of mature hepatocytes. The medium may also contain one or more agents that effect differentiation into hepatocytes, such as various growth factors. These agents may either help induce cells to commit to a more mature phenotype - or selectively promote the survival of mature cells - or have a combination of both of these effects.

[0312] Agents exemplified in the present disclosure that effect differentiation into hepatocytes can include soluble growth factors (peptide hormones, cytokines, ligand-receptor complexes, and other compounds) capable of promoting proliferation of cells of the hepatocyte lineage. Non-limiting examples of such agents include, but are not limited to, epidermal growth factor (EGF), insulin, TGF-α, TGF-β, fibroblast growth factor (FGF), heparin, hepatocyte growth factor (HGF), oncostatin M (OSM), IL-1, IL-6, insulin-like growth factors I and II (IGF-I, IGF-2), heparin-binding growth factor 1 (HBGF-1), Wnt family member 3A (WNT3A), A83, CHIR, and glucagon. Those skilled in the art will already understand that oncostatin M is structurally related to leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and ciliary neurotrophic factor (CNTF).

[0313] In some embodiments, the methods of the present invention include increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes, and culturing the immature hepatocytes in a culture medium containing dexamethasone, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or a combination thereof.

[0314] In some embodiments, the step of culturing immature hepatocytes in a culture medium comprising dexamethasone, 8-Br-cAMP, or a combination thereof is performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 days. In some embodiments, the step of culturing immature hepatocytes in a culture medium comprising dexamethasone, 8-Br-cAMP, or a combination thereof is performed for at least 1-3 days. In some embodiments, the step of culturing immature hepatocytes in a culture medium comprising dexamethasone, 8-Br-cAMP, or a combination thereof is performed for at least 2-5 days. In some embodiments, the step of culturing immature hepatocytes in a culture medium comprising dexamethasone, 8-Br-cAMP, or a combination thereof is performed for at least 3-7 days. In some embodiments, the step of culturing immature hepatocytes in a culture medium comprising dexamethasone, 8-Br-cAMP, or a combination thereof is performed for at least 5-9 days.

[0315] In some embodiments, the concentration of 8-Br-cAMP is at least 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 nM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, the concentration of 8-Br-cAMP is about 0.1-0.5 mM, 0.2-0.7 mM, 0.3-0.9 mM, 0.5-1 mM, 1-5 mM, 5-10 mM, or 10-50 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 0.1 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 0.2 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 0.5 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 1 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 5 mM. In some embodiments, the concentration of 8-Br-cAMP is at least 10 mM.

[0316] In some embodiments, the concentration of dexamethasone is at least 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 200 nM, 300 nM, 500 nM, 1 mM, 5 mM, or 10 mM. In some embodiments, the concentration of dexamethasone is about 5-10 nM, 20-50 nM, 30-90 nM, 50-100 nM, 200-500 nM, 1-3 mM, 2-5 mM, or 5-10 mM. In some embodiments, the concentration of dexamethasone is at least 5 nM. In some embodiments, the concentration of dexamethasone is at least 10 nM. In some embodiments, the concentration of dexamethasone is at least 20 nM. In some embodiments, the concentration of dexamethasone is at least 50 nM. In some embodiments, the concentration of dexamethasone is at least 100 nM. In some embodiments, the concentration of dexamethasone is at least 200 nM. In some embodiments, the concentration of dexamethasone is at least 500 nM. In some embodiments, the concentration of dexamethasone is at least 1 mM. In some embodiments, the concentration of dexamethasone is at least 5 mM. In some embodiments, the concentration of dexamethasone is at least 10 mM.

[0317] In some embodiments, immature hepatocytes are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before increasing the expression of at least one transcription factor disclosed herein.In some embodiments, immature hepatocytes are cultured for at least 2 days before increasing the expression of at least one transcription factor.In some embodiments, immature hepatocytes are cultured for at least 5 days before increasing the expression of at least one transcription factor.In some embodiments, immature hepatocytes are cultured for at least 10 days before increasing the expression of at least one transcription factor.

[0318] In some embodiments, the immature hepatocytes are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after increasing the expression of at least one transcription factor disclosed herein.In some embodiments, the immature hepatocytes are cultured for at least 2 days after increasing the expression of at least one transcription factor.In some embodiments, the immature hepatocytes are cultured for at least 5 days after increasing the expression of at least one transcription factor.In some embodiments, the immature hepatocytes are cultured for at least 10 days after increasing the expression of at least one transcription factor.

[0319] In some embodiments, the immature hepatocytes are derived from pluripotent stem cells. Suitable culture media for isolating, expanding and differentiating pluripotent stem cells into immature hepatocytes by the methods described herein include, but are not limited to, high glucose Dulbecco's Modified Eagle Medium (DMEM), DMEM / F-15, Leibovitz L-15, RPMI 1640, Iscove's Modified Dulbecco's Medium (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically defined media include minimal essential medium, such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco), to which the addition of human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine, and mitogens is also suitable. As used herein, mitogen refers to an agent that stimulates cell division of cells. The agent may be a chemical that acts on cells to initiate cell division and initiate mitosis, typically some form of protein. In one embodiment, serum-free medium (US Patent Application Serial No. 08 / 464,599 and PCT Publication WO 96 / 39487; each of which is incorporated herein by reference in its entirety) as well as complete medium (US Patent No. 5,486,359; each of which is incorporated herein by reference in its entirety) are intended to be used with the methods described herein. In some embodiments, the culture medium is supplemented with 10% fetal bovine serum (FBS), human autologous serum, human AB serum, or platelet-rich plasma supplemented with heparin (2 U / ml). To maintain the pH of the culture fluid, the cell culture may be maintained under a CO2 atmosphere, for example, a 5%-12% CO2 atmosphere, the cell culture may be incubated at 37 degrees in a humid atmosphere, and the cell culture may be subcultured to maintain less than 85% confluence.

[0320] Pluripotent stem cells to be differentiated into immature hepatocytes may be cultured in a medium that can maintain pluripotency. The induced pluripotent stem (iPS) cells produced in one aspect of the present invention can be cultured using a variety of media and techniques developed for culturing primate pluripotent stem cells, more specifically embryonic stem cells (US Patent Application Publication No. 20070238170 and US Patent Application Publication No. 20030211603, each of which is incorporated herein by reference in its entirety). For example, like human embryonic stem (hES) cells, iPS cells can be maintained in 80% DMEM (Gibco, No. 10829-018 or No. 11965-092), 20% non-heat-inactivated chemically defined fetal bovine serum (FBS), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Alternatively, ES cells can be maintained in serum-free medium made of 80% Knock-Out DMEM (Gibco, No. 10829-018), 20% serum replacement (Gibco, No. 10828-028), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol.

[0321] In some embodiments, the method of culturing pluripotent stem cells and inducing the formation of immature hepatocytes comprises culturing the pluripotent stem cells in a first differentiation medium comprising activin A, a second differentiation medium comprising at least one of BMP4 and FGF2, and a third differentiation medium comprising HGF, thereby producing immature hepatocytes.

[0322] In some embodiments, the first differentiation medium, the second differentiation medium, and the third differentiation medium are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, respectively. In some embodiments, the first differentiation medium is cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the second differentiation medium is cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the third differentiation medium is cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0323] In some embodiments, the immature hepatocytes are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before increasing the expression of at least one transcription factor disclosed herein. In some embodiments, the immature hepatocytes are cultured for at least 2 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 5 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 10 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured in a culture medium containing hepatocyte growth factor (HGF) before increasing the expression of at least one transcription factor.

[0324] In some embodiments, the immature hepatocytes are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after increasing the expression of at least one transcription factor disclosed herein. In some embodiments, the immature hepatocytes are cultured for at least 2 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 5 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 10 days before increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured in a culture medium containing hepatocyte growth factor (HGF) before increasing the expression of at least one transcription factor.

[0325] In some embodiments, the immature hepatocytes are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after increasing the expression of at least one transcription factor disclosed herein. In some embodiments, the immature hepatocytes are cultured for at least 2 days after increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 5 days after increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured for at least 10 days after increasing the expression of at least one transcription factor. In some embodiments, the immature hepatocytes are cultured in a culture medium containing Oncostatin M (OSM) after increasing the expression of at least one transcription factor.

[0326] For the purpose of generating immature hepatocytes derived from pluripotent stem cells, in some embodiments, a monolayer of pluripotent cells is harvested and cultured, for example, at least 2 x 10 5 cells / cm 2The cells are plated at a density of 100-1500 μg / ml. Stage 1 of the differentiation process is initiated by culturing the pluripotent stem cells in a culture medium containing one or more of activin A, BMP4, FGF-2, or B27 for at least 1, 2, or 3 days. This is followed by culturing the cells in a culture medium containing one or more of activin A and B27 for at least 1, 2, or 3 days. Stage 2 of the differentiation process involves culturing the cells resulting from stage 1 in a culture medium containing one or more of BMP4, FGF-2, or B27 for at least 1, 2, 3, 4, or 5 days. Stage 3 is initiated by culturing the cells resulting from stage 2 in a culture medium containing one or more of HGF or B27 (e.g., supplemented with insulin) for at least 1, 2, 3, 4, or 5 days. The final stage 4 involves culturing the cells resulting from stage 3 in culture medium containing one or more of Oncostatin-M or SingleQuots (without EGF) for at least 1, 2, 3, 4, or 5 days.

[0327] In some embodiments, pluripotent stem cell-derived hepatocytes are derived from culture dishes using a stage 4, 20 day protocol as previously described in Mallanna et al. 2013 (Curr Protoc Stem Cell Biol.; 26:1G.4.1-1G.4.13; which is incorporated herein by reference in its entirety).

[0328] Characteristics of hepatocytes Cells can be characterized according to a number of phenotypic and / or functional criteria, including, but not limited to, detection or quantification of expressed cellular markers, enzymatic activity, and characterization of morphological traits and intracellular signaling.

[0329] Hepatocytes, such as mature hepatocytes embodied in some aspects of the present invention, have morphological features characteristic of native hepatocytes, such as primary hepatocytes from an organ donor. Such features are readily understood by those skilled in the art and include any or all of the following: polygonal cell shape, binuclear phenotype, presence of rough endoplasmic reticulum for secretory protein synthesis, presence of Golgi-endoplasmic reticulum-lysosome complex for sorting intracellular proteins, presence of peroxisomes and glycogen granules, relatively abundant mitochondria, and the ability to form intracellular tight junctions leading to the formation of bile canalicular spaces. Some of these features present in a cell are consistent with those of cells that are members of the hepatocyte lineage.

[0330] The mature hepatocytes of the present invention can also be characterized by whether they express phenotypic markers characteristic of cells of the hepatocyte lineage. Non-limiting examples of cell markers useful for distinguishing mature hepatocytes include: albumin, asialoglycoprotein receptor, alpha 1-antitrypsin, alpha-fetoprotein, apoE, arginase I, apoAI, apoAII, apoB, apoCIII, apoCII, aldolase B, alcohol dehydrogenase 1, catalase, CYP3A4, glucokinase, glucose-6-phosphatase, insulin growth factor 1 and 2, IGF-1 receptor, insulin receptor, leptin, liver-specific organic anion transporter, and the like. These include LST-1, L-type fatty acid binding protein, phenylalanine hydroxylase, transferrin, retinol binding protein, erythropoietin (EPO), albumin, α1-antitrypsin, asialoglycoprotein receptor, cytokeratin 8 (CK8), cytokeratin 18 (CK18), CYP3A4, fumarylacetoacetate hydrolase (FAH), glucose-6-phosphate, tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and tryptophan 2,3-dioxygenase.

[0331] The mature hepatocyte may also exhibit a global gene expression profile indicative of hepatocyte maturation. The global gene expression profile may be compared to the profile for primary hepatocytes or known mature hepatocytes, and may be obtained by any method known in the art, such as by transcriptome analysis or microarray analysis, or as described in the Examples. In some embodiments, increasing the expression of at least one transcription factor shifts at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the transcriptome of the immature hepatocyte to that of the mature hepatocyte. In some embodiments, increasing the expression of at least one transcription factor shifts at least 1% of the transcriptome of the immature hepatocyte to that of the mature hepatocyte. In some embodiments, increasing the expression of at least one transcription factor shifts at least 5% of the transcriptome of the immature hepatocyte to that of the mature hepatocyte. In some embodiments, increasing the expression of at least one transcription factor shifts at least 10% of the transcriptome of the immature hepatocyte to that of the mature hepatocyte. In some embodiments, increasing the expression of at least one transcription factor shifts at least 20% of the transcriptome of immature hepatocytes to the transcriptome of mature hepatocytes.In some embodiments, increasing the expression of at least one transcription factor shifts at least 30% of the transcriptome of immature hepatocytes to the transcriptome of mature hepatocytes.In some embodiments, increasing the expression of at least one transcription factor shifts at least 40% of the transcriptome of immature hepatocytes to the transcriptome of mature hepatocytes.In some embodiments, increasing the expression of at least one transcription factor shifts at least 50% of the transcriptome of immature hepatocytes to the transcriptome of mature hepatocytes.

[0332] An assessment of the expression levels of such markers in mature hepatocytes can be determined in comparison to other cells, such as immature hepatocytes, etc. Positive controls for markers of mature hepatocytes include adult hepatocytes of the species of interest, such as primary human hepatocytes (PHH).

[0333] The tissue-specific (e.g., hepatocyte-specific) protein and oligosaccharide determinants listed in this disclosure can be detected using any suitable immunological technique, such as flow immunocytochemical analysis for cell surface markers, immunohistochemical analysis for intracellular or cell surface markers (e.g., immunohistochemical analysis of fixed cells or tissue sections), Western blot analysis for cell extracts, and enzyme-linked immunoassays for cell extracts or products secreted into the medium. Expression of an antigen by a cell is said to be "detectable by an antibody" if a significant detectable amount of antibody binds to the antigen in a standard immunocytochemical or flow cytometric assay, optionally after fixation of the cells and optionally using a labeled secondary antibody or other conjugate that amplifies the label (e.g., biotin-avidin conjugates).

[0334] Expression of tissue-specific (e.g., mature hepatocyte-specific) markers can also be detected at the mRNA level by Northern blot analysis, by dot blot hybridization analysis, or by real-time polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods (US Pat. No. 5,843,780). Sequence data for certain markers listed in this disclosure can be obtained from public databases, such as GenBank. Expression at the mRNA level is said to be "detectable" by one of the assays described in this disclosure if the assay performed on a cell sample according to standard procedures in a typical control experiment results in clearly identifiable hybridization or amplification products within a standard time window. Unless otherwise required, the expression of a particular marker is indicated if its corresponding mRNA is detectable by RT-PCR. Expression of a tissue-specific marker, detected at the protein or mRNA level, is considered positive when the level is at least twice as high, and preferably more than 10-fold or 50-fold higher, than that of control cells, such as undifferentiated pluripotent stem cells, fibroblasts, or other unrelated cell types.

[0335] Mature hepatocytes can also be characterized by whether they show enzyme activity characteristic of mature hepatocytes.For example, the assay for glucose-6-phosphatase activity is described in Bublitz (1991); Yasmineh et al. (1992); and Ockerman (1968); each of which is incorporated herein by reference in its entirety.The assay for alkaline phosphatase (ALP) and 5-nucleotidase (5'-Nase) in hepatocytes is described in Shiojiri (1981); each of which is incorporated herein by reference in its entirety.

[0336] In other embodiments, the mature hepatocytes of the present invention are assayed for activities indicative of xenobiotic detoxification. Cytochrome p450 is a key catalytic component in the monooxygenase system. Cytochrome p450 constitutes a family of heme proteins that perform the oxidative metabolism of xenobiotics (administered drugs) and many endogenous compounds. Different cytochromes exhibit unique and overlapping substrate specificities. The majority of biotransformation capacity is attributed to cytochromes called 1A2, 2A6, 2B6, 3A4, 2C9-2C11, 2D6, and 2E1 (Gomes-Lechon et al. 1997); the entirety of which is incorporated herein by reference.

[0337] Numerous assays for measuring the detoxification of xenobiotics by cytochrome p450 enzyme activity are known in the art. Detoxification by CYP3A4 is demonstrated using the P450-Glo™ CYP3A4 DMSO tolerance assay (luciferin-PPXE) and the P450-Glo™ CYP3A4 cell-based / biochemical assay (luciferin-PFBE) (Promega Inc., No. V8911 and No. V8901). Detoxification by CYP1A1 and / or CYP1B1 is demonstrated using the P450-Glo™ assay (luciferin-CEE) (Promega Inc., No. V8762). Detoxification by CYP1A2 and / or CYP4A is demonstrated using the P450-Glo™ Assay (Luciferin-ME) (Promega Inc., No. V8772) and detoxification by CYP2C9 is demonstrated using the P450-Glo™ CYP2C9 Assay (Luciferin-H) (Promega Inc., No. V8791).

[0338] In another aspect, the biological function of the mature hepatocytes of the present invention is evaluated, for example, by analyzing glycogen accumulation. Glycogen accumulation is characterized by assaying the functional staining of glycogen granules with periodic acid Schiff (PAS). Cells are first oxidized with periodic acid. The oxidation process results in the formation of aldehyde groups by cleavage of carbon-carbon bonds. For oxidation to occur, a free hydroxyl group must be present. Oxidation is complete when the aldehyde stage is reached. The aldehyde group is detected by Schiff's reagent. A colorless, unstable dialdehyde compound is formed, which is then converted to a colored end product by reduction of the quinoid chromophore group (Thompson, 1966; Sheehan and Hrapchak, 1987; each of which is incorporated herein by reference in its entirety). PAS staining can be performed according to the protocols described on the World Wide Web at jhu.edu / ~iic / PDF jrotocols / LM / Glycogen Staining pdf and at library.med.utah.edu / WebPath / HISTHTML / MANUALS / PAS.PDF, with some modifications for in vitro culture of hepatocyte-like cells. The skilled artisan should be able to make the appropriate modifications.

[0339] In another aspect, the mature hepatocytes of the present invention are characterized for the production of urea, which can be assayed colorimetrically using a kit from Sigma Diagnostic (Miyoshi et al. 1998, which is incorporated herein by reference in its entirety) and is based on the biochemical reaction of urease reduction to urea and ammonia, followed by the reaction of 2-oxoglutarate to produce glutamate and NAD.

[0340] In another aspect, bile secretion is analyzed. Bile secretion can be determined by fluorescein diacetate time course assay. Briefly, cell monolayer culture, for example, mature hepatocyte monolayer culture, is rinsed three times with phosphate buffered saline (PBS) and incubated at 37 degrees for 35 minutes in serum-free hepatocyte growth medium supplemented with doxycycline and fluorescein diacetate (20 μg / ml) (Sigma-Aldrich). Cells are washed three times with PBS and fluorescence imaging is performed. Fluorescein diacetate is a non-fluorescent precursor of fluorescein. Images are evaluated to determine that the compound is taken up by hepatocyte-like cells and metabolized to fluorescein. In some embodiments, the compound is secreted into the intercellular space of the cell monolayer. Alternatively, bile secretion is determined by the sodium fluorescein method described in Gebhart and Wang (1982), which is incorporated herein by reference in its entirety.

[0341] In yet another aspect, lipid synthesis is analyzed. Lipid synthesis in mature hepatocytes can be determined by Oil Red O staining, Oil Red O (Solvent Red 27, Sudan Red 5B, CI 26125, C26H24N4O) is a lysochrome (lipid-soluble) diazo dye that is used to stain neutral triglycerides and lipids on frozen sections and to stain some lipoproteins on paraffin sections. It has the appearance of a red powder and has a maximum absorption of 518 nm (359 nm). Oil Red O is one of the dyes used in Sudan staining, and similar dyes include Sudan III, Sudan IV, and Sudan Black B. Staining should be performed on fresh and / or formalin-fixed samples. Hepatocyte-like cells are cultured on microscope slides, rinsed three times in PBS, the slides air-dried at room temperature for 30-60 minutes, fixed in ice-cold 10% formalin for 5-10 minutes, and then quickly rinsed with three changes of distilled water. The slides are then placed in anhydrous propylene glycol for 2-5 minutes to prevent water transfer into the Oil Red O, and stained in pre-warmed Oil Red O solution for 8 minutes in a 600° oven. The slides are then placed in 85% propylene glycol solution for 2-5 minutes and rinsed with two changes of distilled water. Oil Red O staining can also be performed by one of skill in the art following the protocol described at "library.med.utah.edu / WebPath / HISTHTML / MANUALS / OILRED.PDF" with some modifications for in vitro culture of hepatocyte-like cells.

[0342] In yet another aspect, mature hepatocytes are assayed for glycogen synthesis. Glycogen assays are well known to those skilled in the art, for example, Passonneau and Lauderdale (1974). Alternatively, commercially available glycogen assays can be used, such as, for example, BioVision, Inc. catalog number K646-100.

[0343] Mature hepatocytes can also be evaluated by their ability to accumulate glycogen. One suitable assay uses Periodic Acid Schiff (PAS) staining, which does not react with monosaccharides and disaccharides, but stains long chain polymers such as glycogen and dextran. The PAS reaction provides a quantitative estimate of complex carbohydrates, as well as soluble and membrane-bound carbohydrate compounds. Kirkeby et al. (1992) describe a quantitative PAS assay for carbohydrate compounds and detergents. van der Laarse et al. (1992) describe a microdensitometric histochemical assay for glycogen using the PAS reaction. Evidence of accumulating glycogen is determined when the cells are PAS positive at least two-fold and preferably more than ten-fold higher than the levels of control cells, such as fibroblasts, and the cells can also be characterized by karyotyping by standard methods.

[0344] Assays are also available for enzymes involved in the conjugation, metabolism, or detoxification of small molecule drugs. For example, mature hepatocytes can be characterized by their ability to conjugate bilirubin, bile acids, and small molecule drugs for excretion via the urinary or biliary tract. The cells are contacted with the appropriate substrate, incubated for an appropriate period, and then the medium is analyzed (by GCMS or other suitable techniques) to determine whether conjugated products are formed. Drug metabolizing enzyme activities include deethylation, dealkylation, hydroxylation, demethylation, oxidation, glucuronidation, sulfate conjugation, glutathione conjugation, and N-acetyltransferase activity (A. Guillouzo, "In vitro Methods in Pharmaceutical Research", pp 411-431, Academic Press, 1997; the entirety of which is incorporated herein by reference). Assays include peenacetin deethylation, procainamide N-acetylation, paracetamol sulfate conjugation, and paracetamol glucuronidation (Chesne et al. 1988; incorporated herein by reference in its entirety).

[0345] A further feature of certain cell populations, such as mature hepatocyte populations, of the present invention is that under appropriate conditions, the population is susceptible to pathogens with primate hepatocyte tropism. Such pathogens include Hepatitis A, B, C, and Delta, Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), tuberculosis, and malaria. For example, hepatitis B infectivity can be determined by combining cultured mature hepatocytes with a source of infectious hepatitis B particles, such as serum from a human HBV carrier. The hepatocytes can then be tested for synthesis of viral core antigen (HBcAg) by immunohistochemical analysis or RT-PCR.

[0346] In yet another aspect, mature hepatocytes can be evaluated for their ability to engraft in a subject and / or show long-term survival in a subject. In one embodiment, to determine whether hepatocytes survive and maintain their phenotype in vivo, hepatocytes are administered to an animal (e.g., SCID mouse) at a site that is easy to further observe, such as under the kidney capsule, in the spleen, or in the liver lobule. Tissues are harvested after a few days to weeks or longer, and the presence and phenotype of the administered cells are evaluated, for example, by immunohistochemical analysis or ELISA using human-specific antibodies, or by RT-PCR analysis. Markers suitable for evaluating gene expression at the mRNA or protein level are provided in the present disclosure. Effects on liver function can also be determined by evaluating markers expressed in liver tissue, such as cytochrome p450 activity, and by evaluating blood indicators, such as alkaline phosphatase activity, bilirubin conjugation, and prothrombin time.

[0347] Assays for determining the ability of mature hepatocytes to engraft in a subject and / or exhibit long-term survival in a subject are described, for example, in U.S. Pat. No. 9,260,722; and U.S. Patent Application Publication No. 2020 / 0216823; each of which is incorporated by reference in its entirety herein.

[0348] In some embodiments, mature hepatocytes are engrafted into the target tissue of the subject.In some embodiments, mature hepatocytes comprise a mature hepatocyte population, wherein at least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% mature hepatocytes are engrafted into the target tissue of the subject.In some embodiments, the target tissue is the liver.

[0349] Those skilled in the art will readily understand that while primary hepatocyte cultures isolated from adult or fetal liver tissues are typically contaminated with other cell types, one advantage of mature hepatocytes is that they will essentially be free of such other cell types. The mature hepatocytes provided by certain aspects of the present invention may have some of the characteristics of the cell stage that they are intended to exhibit. The more of these characteristics present in a particular cell, the more likely it is that the cell will be characterized as a cell of hepatocyte lineage. More cells having at least 2, 3, 5, 7, or 9 of these characteristics are more preferred. For a particular cell population that may be present in a culture vessel or preparation for administration, it is often advantageous for the expression of these characteristics to be uniform among cells. In this context, more populations in which at least about 10%, 20%, 30%, 40%, 60%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells have the desired characteristics are more preferred.

[0350] Other desirable features of the hepatocytes provided in certain aspects of the invention are their ability to serve as target cells in drug screening assays and to reconstitute liver function both in vivo and as part of an extracorporeal device, which are further described in the following sections.

[0351] II. Cells and Compositions of the Invention A further aspect of the present invention provides a composition comprising a hepatocyte population, for example, the population is produced by any of the methods described herein.In some embodiments, the composition comprises an enriched, purified or isolated hepatocyte population, for example, the population is produced by any of the methods described herein.The enriched, purified or isolated hepatocyte population can be a single cell suspension, aggregates, chimeric aggregates, and / or structures, where the structures include branched structures and / or cysts.

[0352] In some embodiments, the hepatic cell population comprises an increased expression level of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) compared to the endogenous expression level of the transcription factor in the hepatic cell population.

[0353] In some embodiments, the increased expression of NFIX comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 0.1-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 0.2-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 0.5-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 1-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 2-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 5-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 10-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 20-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 50-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 100-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 200-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, increased expression of NFIX comprises at least a 500-fold increase as compared to the endogenous expression level of NFIX in the hepatic cell population.In some embodiments, the increased expression of NFIX comprises at least a 1,000-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population. In some embodiments, the increased expression of NFIX comprises at least a 10,000-fold increase compared to the endogenous expression level of NFIX in the hepatocyte population.

[0354] In some embodiments, the increased expression of NFIC comprises at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least 0.1-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least 0.2-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least 0.5-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least 1-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 2-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 5-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 10-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 20-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 50-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 100-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 200-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, increased expression of NFIC comprises at least a 500-fold increase as compared to the endogenous expression level of NFIC in the hepatic cell population.In some embodiments, the increased expression of NFIC comprises at least a 1,000-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population. In some embodiments, the increased expression of NFIC comprises at least a 10,000-fold increase compared to the endogenous expression level of NFIC in the hepatocyte population.

[0355] In some embodiments, the hepatocyte population further comprises an increase in the expression level of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 compared to the endogenous expression level of the one or more transcription factors in the hepatocyte population.In some embodiments, the one or more transcription factors are RORC.In some embodiments, the one or more transcription factors are NR0B2.In some embodiments, the one or more transcription factors are ESR1.In some embodiments, the one or more transcription factors are THRSP.In some embodiments, the one or more transcription factors are TBX15. In some embodiments, the one or more transcription factors are HLF. In some embodiments, the one or more transcription factors are ATOH8. In some embodiments, the one or more transcription factors are NR1I2. In some embodiments, the one or more transcription factors are CUX2. In some embodiments, the one or more transcription factors are ZNF662. In some embodiments, the one or more transcription factors are TSHZ2. In some embodiments, the one or more transcription factors are ATF5. In some embodiments, the one or more transcription factors are NFIA. In some embodiments, the one or more transcription factors are NFIB. ​​In some embodiments, the one or more transcription factors are NPAS2. In some embodiments, the one or more transcription factors are FOS. In some embodiments, the one or more transcription factors are ONECUT2. In some embodiments, the one or more transcription factors are PROX1. In some embodiments, the one or more transcription factors are NR1H4. In some embodiments, the one or more transcription factors are MLXIPL. In some embodiments, the one or more transcription factors is ETV1.In some embodiments, the one or more transcription factors are AR. In some embodiments, the one or more transcription factors are CEBPB. In some embodiments, the one or more transcription factors are NR1D1. In some embodiments, the one or more transcription factors are HEY2. In some embodiments, the one or more transcription factors are ARID3C. In some embodiments, the one or more transcription factors are KLF9. In some embodiments, the one or more transcription factors are DMRTA1.

[0356] In some embodiments, the hepatocyte population is an immature hepatocyte population. In some embodiments, the hepatocyte population is a mature hepatocyte population. In some embodiments, the hepatocyte population comprises both mature and immature hepatocytes.

[0357] In some embodiments, mature hepatocytes exhibit increased expression of albumin (ALB), cytochrome P450 enzyme 1A2 (CYP1A2), cytochrome P450 enzyme 3A4 (CYP3A4), tyrosine aminotransferase (TAT), and / or UDP-glucuronosyltransferase 1A-1 (UGT1A1) compared to immature hepatocytes.

[0358] In some embodiments, the increased expression of CYP1A2 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of CYP3A4 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, the increased expression of TAT comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. In some embodiments, increased expression of UGT1A1 comprises at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes.

[0359] In some embodiments, mature hepatocytes exhibit decreased expression of alpha-fetoprotein (AFP) compared to immature hepatocytes. In some embodiments, decreased expression of AFP comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, or 4-fold decrease compared to immature hepatocytes.

[0360] In some embodiments, mature hepatocytes show increased albumin secretion, decreased AFP secretion, and / or increased CYP1A2 activity compared to immature hepatocytes. In some embodiments, increased ALB secretion comprises at least 5%, 10%, 15%, 20%, or 25% increase compared to immature hepatocytes. In some embodiments, decreased AFP secretion comprises at least 5%, 10%, 20%, 40%, or 60% decrease compared to immature hepatocytes. In some embodiments, increased CYP1A2 activity comprises at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold increase compared to immature hepatocytes.

[0361] In some embodiments, the composition of the hepatocyte population comprises about 1 x 106 pcs ~ approx. 1 x 10 12 In some embodiments, the composition of the hepatocyte population comprises at least 1 x 10 hepatocytes. 5 pcs, 1x10 6 pcs, 1x10 7 pcs, 1x10 8 pcs, 1x10 9 pcs, 1x10 10 pcs, 1x10 11 Pieces, or 1 x 10 12 Contains hepatocytes.

[0362] Also provided herein are pharmaceutical compositions and formulations comprising hepatocytes, such as mature or immature hepatocytes, and a pharma- ceutically acceptable carrier.

[0363] In some embodiments, the pharmaceutical composition comprises about 1 x 10 6 pcs ~ approx. 1 x 10 12 In some embodiments, the dose is in the range of about 1 x 10 hepatocytes. 5 pcs, 1x10 6 pcs, 1x10 7 pcs, 1x10 8 pcs, 1x10 9 pcs, 1x10 10 pcs, 1x10 11 Pieces, or 1 x 10 12 In some embodiments, the pharmaceutical composition comprises about 1 x 10 hepatocytes. 6 pcs ~ approx. 1 x 10 12 The doses include those in the range of hepatocytes.

[0364] A further aspect of the invention provides a composition comprising a pluripotent stem cell population comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor of the present disclosure.

[0365] In some embodiments, the transcription factor is NFIX. In some embodiments, the transcription factor is NFIC. In some embodiments, the transcription factor is NFIX and NFIC.

[0366] In some embodiments, the pluripotent stem cell population further comprises an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1. In some embodiments, the one or more transcription factors are RORC. In some embodiments, the one or more transcription factors are NR0B2. In some embodiments, the one or more transcription factors are ESR1. In some embodiments, the one or more transcription factors are THRSP. In some embodiments, the one or more transcription factors are TBX15. In some embodiments, the one or more transcription factors are HLF. In some embodiments, the one or more transcription factors are ATOH8. In some embodiments, the one or more transcription factors are NR1I2. In some embodiments, the one or more transcription factors are CUX2. In some embodiments, the one or more transcription factors are ZNF662. In some embodiments, the one or more transcription factors are TSHZ2. In some embodiments, the one or more transcription factors are ATF5. In some embodiments, the one or more transcription factors are NFIA. In some embodiments, the one or more transcription factors are NFIB. ​​In some embodiments, the one or more transcription factors are NPAS2. In some embodiments, the one or more transcription factors are FOS. In some embodiments, the one or more transcription factors are ONECUT2. In some embodiments, the one or more transcription factors are PROX1. In some embodiments, the one or more transcription factors are NR1H4. In some embodiments, the one or more transcription factors are MLXIPL. In some embodiments, the one or more transcription factors are ETV1. In some embodiments, the one or more transcription factors is AR.In some embodiments, the one or more transcription factors are CEBPB. In some embodiments, the one or more transcription factors are NR1D1. In some embodiments, the one or more transcription factors are HEY2. In some embodiments, the one or more transcription factors are ARID3C. In some embodiments, the one or more transcription factors are KLF9. In some embodiments, the one or more transcription factors are DMRTA1.

[0367] In some embodiments, the composition comprising a pluripotent stem cell population comprises about 1 x 10 6 pcs ~ approx. 1 x 10 12 In some embodiments, the composition comprising a population of pluripotent stem cells comprises at least 1 x 10 5 pcs, 1x10 6 pcs, 1x10 7 pcs, 1x10 8 pcs, 1x10 9 pcs, 1x10 10 pcs, 1x10 11 Pieces, or 1 x 10 12 The cell comprises pluripotent stem cells.

[0368] In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0369] A further aspect of the invention provides a composition comprising a population of immature hepatocytes comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor of the present disclosure.

[0370] In some embodiments, the transcription factor is NFIX. In some embodiments, the transcription factor is NFIC. In some embodiments, the transcription factor is NFIX and NFIC.

[0371] In some embodiments, the immature hepatocyte population further comprises an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1. In some embodiments, the one or more transcription factors are RORC. In some embodiments, the one or more transcription factors are NR0B2. In some embodiments, the one or more transcription factors are ESR1. In some embodiments, the one or more transcription factors are THRSP. In some embodiments, the one or more transcription factors are TBX15. In some embodiments, the one or more transcription factors are HLF. In some embodiments, the one or more transcription factors are ATOH8. In some embodiments, the one or more transcription factors are NR1I2. In some embodiments, the one or more transcription factors are CUX2. In some embodiments, the one or more transcription factors are ZNF662. In some embodiments, the one or more transcription factors are TSHZ2. In some embodiments, the one or more transcription factors are ATF5. In some embodiments, the one or more transcription factors are NFIA. In some embodiments, the one or more transcription factors are NFIB. ​​In some embodiments, the one or more transcription factors are NPAS2. In some embodiments, the one or more transcription factors are FOS. In some embodiments, the one or more transcription factors are ONECUT2. In some embodiments, the one or more transcription factors are PROX1. In some embodiments, the one or more transcription factors are NR1H4. In some embodiments, the one or more transcription factors are MLXIPL. In some embodiments, the one or more transcription factors are ETV1. In some embodiments, the one or more transcription factors is AR.In some embodiments, the one or more transcription factors are CEBPB. In some embodiments, the one or more transcription factors are NR1D1. In some embodiments, the one or more transcription factors are HEY2. In some embodiments, the one or more transcription factors are ARID3C. In some embodiments, the one or more transcription factors are KLF9. In some embodiments, the one or more transcription factors are DMRTA1.

[0372] In some embodiments, the composition comprising an immature hepatocyte population comprises about 1 x 10 6 pcs ~ approx. 1 x 10 12 In some embodiments, the composition comprising a population of immature hepatocytes comprises at least 1 x 10 immature hepatocytes. 5 pcs, 1x10 6 pcs, 1x10 7 pcs, 1x10 8 pcs, 1x10 9 pcs, 1x10 10 pcs, 1x10 11 Pieces, or 1 x 10 12 Contains immature hepatocytes.

[0373] Also provided herein are pharmaceutical compositions and formulations comprising the immature hepatocytes and a pharma- ceutically acceptable carrier.

[0374] In some embodiments, the pharmaceutical composition comprises about 1 x 10 6 pcs ~ approx. 1 x 10 12 In some embodiments, the dose ranges from about 1 x 10 immature hepatocytes. 5 pcs, 1x10 6 pcs, 1x10 7 pcs, 1x10 8 pcs, 1x10 9 pcs, 1x10 10 pcs, 1x10 11 Pieces, or 1 x 10 12 In some embodiments, the pharmaceutical composition comprises about 1 x 10 immature hepatocytes. 6pcs ~ approx. 1 x 10 12 The doses include a range of immature hepatocytes.

[0375] The pharmaceutical compositions and formulations described herein can be prepared in the form of an aqueous solution by mixing the cells of the present disclosure, such as mature hepatocytes, with one or more pharma- ceutically acceptable carriers (see "Remington's Pharmaceutical Sciences" 22nd ed. 2012, which is incorporated herein by reference in its entirety). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecules. polypeptides of small amounts (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).Exemplary pharma- ceutically acceptable carriers herein further include agents that disperse the agent in the intercellular matrix, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Some exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968, each of which is incorporated herein by reference in its entirety. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0376] In some embodiments, the compositions and pharmaceutical compositions comprising hepatocytes comprise a substantially purified hepatocyte population. For example, the hepatocyte composition may comprise less than 25%, less than 20%, less than 15%, 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% of cells other than hepatocytes. In some embodiments, the hepatocyte composition comprises less than 25%, less than 20%, less than 15%, 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% of pluripotent stem cells. In another embodiment, the hepatocyte composition does not have pluripotent stem cells or pluripotent stem cells are undetectable. In some embodiments, a composition comprising a substantially purified hepatocyte population is one in which hepatocytes constitute at least about 75% of the cells in the composition. In other embodiments, a substantially purified hepatocyte population is one in which the hepatocytes constitute at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, or even greater than 99% of the cells in the population. In any of the embodiments, the hepatocytes may be mature hepatocytes.

[0377] In another embodiment, the compositions and pharmaceutical compositions comprising hepatocytes may include cells other than hepatocytes that may be useful for enhancing or complementing the function of hepatocytes, including but not limited to mesenchymal stem cells, endothelial cells, cholangiocytes, stellate cells, and / or Kupffer cells. In other embodiments, the compositions and pharmaceutical compositions comprising hepatocytes include organoids, which are three-dimensional structures of cells that are often capable of self-organization and provide an environment for advanced cell-extracellular matrix and cell-cell interactions in vivo. See, for example, Olgasi et al. International Journal of Molecular Sciences 21:6215 (2020), which is incorporated herein by reference in its entirety. The organoids include hepatocytes and may further include other cells, such as mesenchymal stem cells, endothelial cells, cholangiocytes, stellate cells, and Kupffer cells. In any of the embodiments, the hepatocytes may be mature hepatocytes.

[0378] III. Methods of Use of Hepatocytes The hepatocytes and pharmaceutical compositions produced by the methods described herein may be used for cell-based treatments where hepatocytes are required or where hepatocytes can improve treatment. Methods of using the hepatocytes provided by the present invention to treat various conditions that may benefit from hepatocyte-based therapy are described herein. Individual treatment regimens, routes of administration, and any adjuvant therapy are tailored based on the individual condition, the severity of the condition, and the overall health of the patient. In addition, in some embodiments, administration of hepatocytes may be effective in completely restoring loss of liver function or other symptoms. In other embodiments, administration of hepatocytes may be effective in reducing the severity of symptoms and / or preventing further deterioration of the patient's condition. The present invention contemplates that administration of a composition comprising hepatocytes may be used to treat any of the conditions described herein, including reducing the severity of symptoms in whole or in part.

[0379] The present invention contemplates that hepatocytes generated using any of the methods described herein, including compositions comprising hepatocytes, can be used to treat any of the indications described herein.Furthermore, the present invention contemplates that any of the compositions comprising hepatocytes described herein can be used to treat any of the indications described herein.In another embodiment, the hepatocytes of the present invention can be administered together with other therapeutic cells or other therapeutic agents.Hepatocytes can be administered simultaneously or sequentially as a combined preparation or as separate preparations.

[0380] In one embodiment, the present invention provides a method of treating a disease or disorder selected from the group consisting of: fulminant hepatic failure from any cause, viral hepatitis, drug-induced liver injury, cirrhosis, inherited liver dysfunction (e.g., Wilson's disease, Gilbert's syndrome, or alpha-1-antitrypsin deficiency), hepatobiliary cancer, autoimmune liver disease (e.g., autoimmune chronic hepatitis or primary biliary cirrhosis), urea cycle disorders, factor VII deficiency, glycogen storage disease type 1, childhood Refsum's disease, Phenylketonuria, Severe childhood oxalosis, Cirrhosis, Liver damage, Acute liver failure, Hepatobiliary carcinoma, Hepatocellular carcinoma, Hereditary cholestasis (PFIC and Alagille syndrome), Hereditary hemochromatosis, Tyrosinemia type 1, Argininosuccinic aciduria (ASL), Crigler-Najjar syndrome, Familial amyloidotic polyneuropathy, Atypical hemolytic uremic syndrome-1, Primary hyperoxaluria type 1, Maple syrup urine disease (MSUD), Acute intermittent porphyria, Coagulation disorders, GSD type Ia (in metabolic control), Homozygous familial hypercholesterolemia, Organic acidurias, and any other condition causing liver dysfunction.

[0381] The hepatocytes provided by the method and composition of the present invention can also be used for various applications.These applications include but are not limited to: transplantation or implantation of hepatocytes in vivo; in vitro screening of cytotoxic compounds, carcinogens, mutagens, growth factors / regulators, or pharmaceutical compounds; elucidation of the mechanism of liver disease and infection; study of the mechanism of action of drugs and / or growth factors; diagnosis and monitoring of cancer in patients; gene therapy; and production of biologically active products.In some embodiments, the hepatocytes include mature hepatocytes, immature hepatocytes, or combinations thereof.

[0382] Screening of test compounds The hepatocytes of the present invention can be used to screen factors (e.g., solvents, small molecule drugs, peptides, polynucleotides, etc.) or environmental conditions (e.g., culture conditions or manipulations) that affect the characteristics of the hepatocytes provided herein.

[0383] In some applications, stem cells (differentiated or undifferentiated) are used to screen for factors that enhance the maturation of cells along the hepatocyte lineage or enhance the proliferation and maintenance of such cells under long-term culture. For example, candidate hepatocyte maturation or proliferation factors are tested by adding them to stem cells in various wells and then determining any changes in phenotype that occur against the desired criteria for further culturing and use of the cells.

[0384] Certain screening applications of the present invention relate to the testing of pharmaceutical compounds in drug research, for example, as described in "In vitro Methods in Pharmaceutical Research", Academic Press, 1997, and U.S. Pat. No. 5,030,015; each of which is incorporated herein by reference in its entirety. In certain aspects of the present invention, hepatocytes serve as test cells for standard drug screening and toxicity assays, as previously performed on hepatocyte cell lines or primary hepatocytes in short-term culture. Evaluating the activity of candidate pharmaceutical compounds generally involves: combining hepatocytes provided in certain aspects of the present invention with a candidate compound; determining any changes in cell morphology, marker phenotype, or metabolic activity caused by the compound (compared to untreated cells or compared to cells treated with an inactive compound); and then correlating the compound's action with the observed changes. Screening can be performed either because the compound is designed to have a pharmaceutical effect on hepatocytes, or because compounds designed to have an effect on other sites may have unintended side effects on the liver. Two or more drugs can be tested in combination (by combining them with the cells either simultaneously or sequentially) to detect possible interactions between drugs.

[0385] In some applications, compounds are first screened for hepatotoxic potential (Castell et al. 1997; incorporated herein by reference in its entirety). Cytotoxicity can be initially determined by its effect on cell vitality, cell viability, cell morphology, and enzyme leakage into the culture medium. More detailed analysis is performed to determine whether the compound affects cell function (e.g., gluconeogenesis, ureogenesis, and plasma protein synthesis) without causing toxicity. Lactate dehydrogenase (LDH) is a good marker because the hepatic isoenzyme (type V) is stable under culture conditions and can be reproducibly measured in culture supernatants after 12-24 hours of incubation. Enzyme leakage can also be used, such as mitochondrial glutamic oxaloacetic transaminase and glutamic pyruvate transaminase. Gomez-Lechon et al. (1996), the entire contents of which are incorporated herein by reference, describe a microassay for measuring glycogen, which can be used to measure the effect of pharmaceutical compounds on hepatocyte gluconeogenesis.

[0386] Other current methods for assessing hepatotoxicity include: determining the synthesis and secretion of albumin, cholesterol, and lipoproteins; transport of conjugated bile acids and bilirubin; ureogenesis; cytochrome p450 levels and activity; glutathione levels; release of α-glutathione S-transferase; metabolism of ATP, ADP, and AMP; intracellular K+ and Ca2+ concentrations; release of nuclear matrix proteins or oligonucleosomes; and induction of apoptosis (as indicated by cell rounding, chromatin condensation, and nuclear fragmentation). DNA synthesis can be measured as incorporation of [3H]-thymidine or BrdU. Drug effects on DNA synthesis or structure can be determined by measuring DNA synthesis or repair. Incorporation of [3H]-thymidine or BrdU, especially at unscheduled times in the cell cycle or above the levels required for cell replication, is consistent with drug effects. Unwanted effects can also include abnormal rates of sister chromatid exchanges, as determined by metaphase spreads.

[0387] Liver Treatment and Liver Transplantation The present invention also provides for the use of the hepatocytes described herein to restore certain liver functions in a subject in need thereof, for example resulting from acute, chronic, or hereditary liver dysfunction.

[0388] To determine whether the hepatocytes provided herein are suitable for therapeutic use, the cells can be first tested in a suitable animal model. At one level, the cells are evaluated for their ability to survive and maintain their phenotype in vivo. The hepatocytes provided herein are administered to immunodeficient animals (such as SCID mice, or animals that have been rendered immunodeficient chemically or by irradiation) at sites that are easy to further observe, such as under the kidney capsule, in the spleen, or in the liver lobule. After a period of days to weeks or longer, tissue is harvested and evaluated. This can be carried out by adding detectable labels (such as green fluorescent protein or β-galactosidase) to the administered cells; or by measuring the constitutive markers specific to the administered cells. When the hepatocytes provided herein are tested in rodent models, the presence and phenotype of administered cells can be evaluated by immunohistochemical analysis or ELISA using human specific antibodies, or by RT-PCR analysis using primers and hybridization conditions that cause specific amplification of human polynucleotide sequences.Markers suitable for evaluating gene expression at mRNA level or protein level are provided herein.General descriptions for determining the fate of hepatocyte-like cells in animal models are described, for example, in Grompe et al. (1999); Peeters et al. (1997); and Ohashi et al. (2000); each of which is incorporated herein by reference in its entirety.

[0389] At another level, the hepatocytes provided herein are evaluated for their ability to restore liver function in animals that do not have complete liver function. Braun et al. (2000), the entirety of which is incorporated herein by reference, outlines a toxic liver disease model in transgenic mice for HSV-tk gene. Rhim et al. (1995) and Lieber et al. (1995), each of which is incorporated herein by reference in its entirety, outline a liver disease model by expression of urokinase. Mignon et al. (1998), the entirety of which is incorporated herein by reference, outlines liver disease induced by antibodies against cell surface marker Fas. Overturf et al. (1998), the entirety of which is incorporated herein by reference, develop a model in mice of hereditary tyrosinemia type I by targeted disruption of the Fah gene. By providing a supply of 2-(2-nitro-4-fluoro-methyl-benzoyl)-1,3-cyclohexanedione (NTBC), animals can be rescued from the deficiency, but when NTBC is withdrawn, the animals develop liver disease. Acute liver disease can be modeled by 90% hepatectomy, as described in Kobayashi et al. 2000, which is incorporated herein by reference in its entirety. Acute liver disease can also be modeled by treating animals with hepatotoxins, such as galactosamine, CCl4, or thioacetamide.

[0390] Chronic liver disease, such as cirrhosis, can be modeled by treating animals with sublethal doses of hepatotoxins for a period long enough to induce fibrosis (Rudolph et al. 2000; the entirety of which is incorporated herein by reference). The assessment of the ability of hepatocytes provided herein to reconstitute liver function involves administering the cells to such animals and then determining survival rates over a period of 1-8 weeks or longer while monitoring the animals for progression of disease. The effect on liver function can be determined by assessing markers expressed in liver tissue, cytochrome p450 activity, and blood indicators, such as alkaline phosphatase activity, bilirubin conjugation, and prothrombin time, and by assessing host survival rates. Any improvement in survival rates, disease progression, or maintenance of liver function in light of any of these criteria may be associated with the effectiveness of the therapy and may lead to further optimization.

[0391] Hepatocytes (e.g., mature hepatocytes) provided in certain aspects of the present invention that demonstrate desirable functional characteristics in light of their metabolic enzyme profile or their efficacy in animal models may also be suitable for direct administration to human subjects with liver dysfunction. For hemostasis purposes, cells can be administered at any site that is adequately accessible to the circulation, typically intraperitoneally. For some metabolic and detoxification functions, it is advantageous for the cells to be accessible to the biliary tract. Thus, cells are administered near the liver (e.g., for the treatment of chronic liver disease) or near the spleen (e.g., for the treatment of fulminant hepatic failure). In one method, cells are administered to the hepatic circulation via either the hepatic artery or the portal vein by injecting with an indwelling catheter. The catheter in the portal vein can be manipulated to allow the cells to flow primarily to the spleen or liver, or a combination of both. In another method, cells are administered by placing a bolus in a cavity close to the target organ, typically in an excipient or matrix that holds it in place. In another method, cells are injected directly into the liver lobe or spleen.

[0392] The hepatocytes provided in one aspect of the present invention can be used to treat any subject in need of restoring or supplementing liver function. Human conditions that may be suitable for such treatment include, but are not limited to, fulminant hepatic failure of any cause, viral hepatitis, drug-induced liver injury, liver cirrhosis, inherited liver dysfunction (e.g., Wilson's disease, Gilbert's syndrome, or α1-antitrypsin deficiency), hepatobiliary cancer, autoimmune liver disease (e.g., autoimmune chronic hepatitis or primary biliary cirrhosis), urea cycle disorders, factor VII deficiency, glycogen storage disease type 1, childhood Refsum's disease, and Fibroblast disease. Phenylketonuria, severe childhood oxalosis, cirrhosis, liver damage, acute liver failure, hepatobiliary cancer, hepatocellular carcinoma, hereditary cholestasis (PFIC and Alagille syndrome), hereditary hemochromatosis, tyrosinemia type 1, argininosuccinic aciduria (ASL), Crigler-Najjar syndrome, familial amyloidotic polyneuropathy, atypical hemolytic uremic syndrome-1, primary hyperoxaluria type 1, maple syrup urine disease (MSUD), acute intermittent porphyria, coagulation disorders, GSD type Ia (in metabolic control), homozygous familial hypercholesterolemia, organic acidurias, and any other condition that causes liver dysfunction. For human treatment, the dose is generally about 10 cells / mL. 9 ~10 12 and typically about 5 x 10 cells 9 ~5 x 10 10 This is adjusted with regard to the weight of the subject, the nature and severity of the illness, and the replicative capacity of the administered cells.

[0393] Use of liver support devices The present invention also provides a method of using the hepatocytes disclosed herein, which are encapsulated in or part of a bioartificial liver device.Various forms of encapsulation are described in the art, for example in "Cell Encapsulation Technology and Therapeutics", 1999, which is incorporated herein by reference in its entirety.The hepatocytes provided in one aspect of the present invention may be encapsulated by such methods for either in vitro or in vivo use.

[0394] Bioartificial organs for clinical use are designed to support individuals with impaired liver function - either as part of a long-term therapy or to bridge the gap between fulminant liver failure and liver reconstruction or transplantation. Bioartificial liver devices are described in Macdonald et al., "Cell Encapsulation Technology and Therapeutics", pp. 252-286, and exemplified in U.S. Patent Nos. 5,290,684, 5,624,840, 5,837,234, 5,853,717, and 5,935,849, each of which is incorporated herein by reference in its entirety. Suspension-type bioartificial livers include cells suspended in a flat-plate dialyzer, cells microencapsulated in a suitable substrate, or cells bound to microcarrier beads coated with extracellular matrix. Alternatively, the hepatocytes may be arranged on a solid support in a packed bed, in a multiplate flatbed, on a microchannel screen, or surrounding hollow fiber capillaries. The device has an inlet and an outlet through which the subject's blood passes, and the device may also have a separate set of ports for supplying nutrients to the cells.

[0395] Hepatocytes are prepared according to the methods described herein and then plated onto the device on a suitable substrate, such as a matrix such as Matrigel® or collagen. The efficacy of the device can be assessed by comparing the composition of blood in the import channel with that in the export channel - this is done in terms of metabolites removed from the import flow and newly synthesized proteins in the export flow. This style of device can be used to detoxify fluids, such as blood, which are contacted with hepatocytes provided in certain aspects of the invention under conditions that allow the hepatocytes to remove or modify toxins in the fluid. Detoxification can involve removing or altering at least one ligand, metabolite, or other compound (either natural and synthetic) that is normally processed by the liver. Such compounds include, but are not limited to: bilirubin, bile acids, urea, heme, lipoproteins, carbohydrates, transferrin, hemopexin, asialoglycoprotein, hormones such as insulin and glucagon, and various small molecule drugs. The device can also be used to enrich the efferent fluid with synthesized proteins, such as albumin, acute phase reactants, and unloaded carrier proteins. The device can also be optimized to perform such a variety of functions, thereby restoring as much liver function as needed. In the context of therapeutic care, the device processes blood flow from a patient with hepatocellular failure, and the blood is then returned to the patient.

[0396] The present invention also provides the method of using the hepatocyte disclosed herein as organoid, for example, in combination with other cell types.Organoid can be established from hepatocyte, and can be grown for several months while retaining important morphological, functional and gene expression features.See, for example, Hu et al. 2018, Cell; 175(6):1591-1606; the entirety of which is incorporated herein by reference.

[0397] Furthermore, for purposes of manufacture, sale and use, the hepatocytes of the present invention may be supplied in the form of a cell culture or cell suspension in an isotonic excipient or culture medium, which may optionally be frozen for ease of transport or storage.

[0398] The present invention also includes various reagent systems that include sets or combinations of cells that are constantly persisting in the course of manufacture, sale, and use. The cell sets include any combination of two or more cell populations described in this disclosure, such as mature hepatocytes, their precursors, and their subtypes, combined with undifferentiated stem cells, somatic cell-derived hepatocytes, or other differentiated cell types. The cell populations in the set may have the same genome or genetically modified versions thereof.

[0399] The present invention contemplates that compositions of hepatocytes, such as hepatocytes obtained from human pluripotent stem cells (e.g., human embryonic stem cells or other pluripotent stem cells), may be used to treat any of the aforementioned diseases or conditions. These diseases may be treated with hepatocyte compositions that include hepatocytes of various levels of maturity, and may be treated with hepatocyte compositions enriched for mature hepatocytes.

[0400] IV. Methods of Hepatocyte Administration The hepatocytes of the present invention may be administered by any route of administration appropriate for the disease or disorder being treated. In one embodiment, the hepatocytes of the present invention may be administered topically, systemically, or locally, for example, by injection or as part of a device or implant (e.g., a sustained release implant). For example, the hepatocytes of the present invention may be transplanted into the space in which the hepatocytes should reside, using a surgical procedure, for example, when treating a patient with the following disorders or disorders: fulminant hepatic failure due to any cause, viral hepatitis, drug-induced liver injury, liver cirrhosis, inherited liver dysfunction (e.g., Wilson's disease, Gilbert's syndrome, or alpha 1-antitrypsin deficiency), hepatobiliary cancer, autoimmune liver disease (e.g., autoimmune chronic hepatitis or primary biliary cirrhosis), urea cycle disorders, factor VII deficiency, glycogen storage disorders, and the like. Disease type 1, childhood Refsum's disease, phenylketonuria, severe childhood oxalosis, cirrhosis, liver damage, acute liver failure, hepatobiliary cancer, hepatocellular carcinoma, hereditary cholestasis (PFIC and Alagille syndrome), hereditary hemochromatosis, tyrosinemia type 1, argininosuccinic aciduria (ASL), Crigler-Najjar syndrome, familial amyloidotic polyneuropathy, atypical hemolytic uremic syndrome-1, primary hyperoxaluria type 1, maple syrup urine disease (MSUD), acute intermittent porphyria, coagulation disorders, GSD type Ia (in metabolic control), homozygous familial hypercholesterolemia, organic acidurias, and any other condition that causes liver dysfunction. One skilled in the art would be able to determine the route of administration for the disease or disorder being treated.

[0401] The hepatocytes of the present invention may be delivered by injection as a pharma- ceutically acceptable formulation. The concentration for injection may be in any amount that is effective and non-toxic, depending on the factors described herein. In one embodiment, at least 1 x 10 6 pieces, 2 x 10 6 pieces, 5 x 10 6 pieces, 1 x 10 7 pieces, 1 x 10 8 Pieces, or 1 x 10 10The hepatocytes may be administered to a patient in need thereof.

[0402] Products and systems, such as delivery vehicles, that include the agents of the invention, particularly those formulated as pharmaceutical compositions, are also contemplated as part of the invention, as are kits that include such delivery vehicles and / or systems.

[0403] In some embodiments, the therapeutic methods of the invention include administering the hepatocytes of the invention using an implant or device, hi some embodiments, the device is a bioerodible implant for treating a disease or condition described herein.

[0404] The amount of the composition administered by the methods described herein will also vary depending on factors such as, for example, the mode of administration, the number of liver cells, the age of the patient, and the type and severity of the disease being treated.

[0405] The hepatocytes may be delivered once or multiple times periodically throughout the patient's life. For example, the hepatocytes may be delivered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. When administered by implant or device, the hepatocytes may be administered once or delivered once or multiple times periodically throughout the patient's life, depending on the needs of the particular patient and the particular disorder or condition being treated. Similarly, gradually changing treatment regimens are also contemplated. In some embodiments, the patient is also administered immunosuppressive therapy, either prior to administration of the hepatocytes, simultaneously with administration of the hepatocytes, or after administration of the hepatocytes. Immunosuppressive therapy may be required for the patient's life or for a shorter period of time. Examples of immunosuppressive therapies include, but are not limited to, one or more of the following: antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, tacrolimus (Prograf™), and mycophenolate mofetil (MMF).

[0406] In some embodiments, the hepatocytes of the present invention are formulated with a pharmaceutically acceptable carrier. For example, the hepatocytes may be administered alone or as a component of a pharmaceutical formulation. The hepatocytes may be formulated for administration in any manner convenient for use as a human medicine. In some embodiments, a pharmaceutical composition suitable for parenteral administration may comprise the hepatocytes in combination with one or more of a pharmaceutically acceptable, sterile, isotonic aqueous or non-aqueous solution, dispersion, suspension, or emulsion, or a pharmaceutically acceptable, sterile powder that can be reconstituted immediately prior to use as a sterile injectable solution or dispersion, which may include antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be utilized in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0407] V. Kit In another aspect, the present invention provides an article of manufacture or kit comprising a hepatocyte population of the present disclosure, e.g., a pluripotent stem cell population, an immature hepatocyte population, a mature hepatocyte population, and / or a pharmaceutical composition of the present disclosure.

[0408] In another aspect, the present invention provides an article of manufacture or kit comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC).

[0409] In some embodiments, the transcription factor is NFIX. In some embodiments, the transcription factor is NFIC. In some embodiments, the transcription factor is NFIX and NFIC.

[0410] In some embodiments, the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; and NFIC transcript variant 5. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 3. In some embodiments, the NFIC alternative splice variant is NFIC transcript variant 1 and NFIC transcript variant 3.

[0411] The article of manufacture or kit may further include a package insert containing instructions for use, for example, instructions for use of the hepatocyte population or pharmaceutical composition of the present invention for treating or delaying the progression of any disease disclosed herein. The article of manufacture or kit may further include other materials desirable from a commercial and user perspective, along with instructions for use, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., chemotherapeutic agent). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0412] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. EXAMPLES

[0413] Example 1: Materials and Methods 1. Preparation of Lentivirus : Tet-On 3G viral particles were purchased from Takara Bio (Takara Bio, Catalog No. 0055VCT). pLVX-TRE3G (Takara Bio, Catalog No. 631187) was used as a lentiviral vector to express the gene of interest under the Tet-On inducible promoter, and pLVX-TRE3G-luciferase was used as a positive control. Lentiviral particles were produced using a series of products developed by Takara Bio (www.takarabio.com). Virus packaging was performed using a fourth generation lentiviral packaging system consisting of Lenti-X 293T cells (Takara Bio, Catalog No. 632180) and Lenti-X Packaging Single Shot (Takara Bio, Catalog Nos. 631275 and 631276). The virus concentration and amount were determined using Lenti-X™ Concentration Reagent (Takara Bio, Catalog No. 631231 and 631232) and Lenti-X qRT-PCR Titer Measurement Kit (Takara Bio, Catalog No. 631235), respectively. All work was performed using the manufacturer's recommended protocols. The virus was divided into aliquots and stored at -80 degrees until use.

[0414] Viral titration by GFP limiting dilution : GFP under the EF1a promoter (GeneCopoeia, Cat. No. Lv215) was used as a source of GFP viral particles. Viral particles were produced as described above. To determine the relationship between the multiplicity of infection (MOI) of the virus and the number of copies per microliter, 1.1 x 10 5Cells were plated in 12-well plates. One day after plating, 1.2 mL of GFP lentivirus serial dilutions were used for transduction. For each concentration of GFP, polybrene (6 μg / μl) (Sigma, Cat. No. H9268) and 0.5 mL of virus serial dilutions were added to duplicate wells, and centrifugal infection was performed for 1 h at room temperature, 2000 rpm. The day after transduction, the medium was replaced with 1 mL of fresh medium. 72 h after transduction, the percentage of GFP-positive cells was determined using flow cytometry (Macsquant). Only wells with 1%-20% GFP+ were used for calculation of transforming units.

[0415] Culture conditions for HuH7 cells : The hepatoma cell line, HuH7, was grown in low glucose DMEM (ThermoFisher, Cat. No. 11885-084) medium containing 10% FBS (ThermoFisher, Cat. No. 26140-079). HuH7 cells were passaged twice a week. To dissociate, cells were first washed in PBS - / - (ThermoFisher, Cat. No. 14190-144), followed by the addition of 0.25% trypsin·0.02% EDTA (Sigma, Cat. No. 59428C) and incubation for 4 min at room temperature. Cells were harvested in 9 ml of HuH7 growth medium and centrifuged at 1000 rpm for 5 min. The supernatant was removed and cells were seeded at a passage ratio of 1:4.

[0416] Development of the HuH7-Tet-On3G cell line : The hepatoma cell line HuH7 was transduced with lentiviral particles encoding the Tet-On3G transactivator (Tet-On3G) under the constitutive EF1Eα promoter. Transduction was performed in the presence of 6 μg / μl polybrene for 1 h at room temperature and centrifugation at 2000 rpm. The cell medium was changed the day after transduction. The virus contained a neomycin selectable marker, which allowed for the selection of pools containing lentiviral integration. The optimal concentration of neomycin (G418) (ThermoFisher, Cat. No. 10131027) for selection (1.1 mg / ml) was determined experimentally based on the minimal amount of neomycin inducing cell death after 4 days. For cell line validation, HuH7 cells with Tet-On3G integration (HuH7-Tet-On3G) were transduced with TRE-luciferase control lentiviral particles. The medium was replaced in the presence or absence of doxycycline (1 μg / ml, Sigma, catalogue no. D3072).

[0417] Screening of transcription factors in HuH7-Tet-On3G cell line : A schematic diagram of selecting the transcription factors of the present invention is shown in FIG. 1. Transduction with lentiviral particles encoding candidate transcription factors was performed in HuH7-Tet-On3G cells in the presence of polybrene (6 μg / μl) using centrifugal infection at MOI of 10 for 1 h at room temperature, 2000 rpm. The cell medium was changed the day after transduction. Medium changes in the presence or absence of doxycycline (1 μg / ml) were performed every 2 days for a total of 4-5 days.

[0418] Stem cell culture : Human iPSC cells ("hiPSC-GMP1" or "GMP1 iPSC") were maintained in mTeSR™1 medium (STEMCELL Technologies, Catalog No. 85850) in flasks coated with vitronectin (ThermoFisher, Catalog No. A14700) diluted 1 / 100 in PBS - / -. Cells were maintained in 20% O2 / 5% CO 2 and were subcultured every 3 to 4 days by forming small clumps with EDTA (0.5 mM; ThermoFisher, catalog no. AM9260G).

[0419] Hepatocyte Differentiation Protocol : Pluripotent stem cell-derived hepatocytes were derived from culture dishes using a stage 4, 20-day protocol as previously described in Mallanna et al. 2013 (Curr Protoc Stem Cell Biol.; 26:1G.4.1-1G.4.13; incorporated herein by reference in its entirety). To generate hepatocytes, the monolayer of pluripotent cells was harvested using Accutase (STEMCELL Technologies, Cat. No. 07920) for 7 minutes at 37 degrees and 2 x 10 5 cells / cm 2At a density of 1000 x 1000, the cells were transferred to LN521 (ThermoFisher, Catalog No. A29248) pre-coated plates. Prior to induction, the cells were cultured for 24 hours in mTeSR™1 medium. The basal medium for differentiation included RPMI (ThermoFisher, Catalog No. 22400-089) with 1X penicillin-streptomycin (ThermoFisher, Catalog No. 15140-122) and 1% MEM-NEAA (ThermoFisher, Catalog No. 11140-050). Stage 1 of the differentiation process was initiated by culturing pluripotent stem cells for 2 days in culture medium containing 100 ng / ml Activin A (R&D systems, Cat. No. 338-AC-010), 20 ng / ml BMP4 (R&D, Cat. No. 314BP), and 10 ng / ml FGF-2 (ThermoFisher, Cat. No. PHG0266) in RPMI medium, optionally supplemented with 2% insulin-free B27 (ThermoFisher, Cat. No. A1895601). This was followed by culturing the cells for 3 days in culture medium containing 100 ng / ml Activin A (R&D systems, Cat. No. 338-AC-010) in RPMI medium, optionally supplemented with 2% insulin-free B27. Stage 2 of the differentiation process involved culturing cells derived from stage 1 for 5 days in culture medium containing 20 ng / ml BMP4 (R&D, Cat. No. 314BP), and 10 ng / ml FGF-2 in RPMI medium, optionally supplemented with 2% insulin-free B27 (ThermoFisher, Cat. No. A3582801). Stage 3 was initiated by culturing cells derived from stage 2 for 5 days in culture medium containing 20 ng / ml HGF (Peprotech, Cat. No. 100-39) in RPMI medium, optionally supplemented with 2% insulin-free B27.The final stage 4 involved culturing cells from stage 3 for 5 days in culture medium containing 20 ng / ml Oncostatin-M (R&D systems, catalog number 295-OM-010) in Hepatocyte Culture Medium (Lonza, catalog number CC-3198) and optionally supplemented with SingleQuots (no EGF).

[0420] Transduction of iPSC-derived hepatocytes : Transduction with lentiviral particles encoding transcription factors (using an MOI of 3) and Tet-On3G (using an MOI of 5) lentiviral particles was performed at the end of stage 3 of the differentiation protocol (day 15-16 of cell culture) using centrifugal infection at room temperature and 2000 rpm for 1 h in the presence of polybrene (6 μg / μl). The culture medium was changed the day after transduction. Medium changes during stage 4 were performed daily for the entire 5 or 9 days (i.e. cells were harvested on day 20 or 24 of cell culture) using culture medium containing doxycycline (1 μg / ml).

[0421] Real-time PCR analysis of mature hepatocyte markers : Total RNA from cultured cells was isolated using the RNeasy Micro kit (Qiagen, Cat. No. 74004), and cDNA was generated using the High Capacity RNA-to-cDNA Transcription System (ThermoFisher, Cat. No. 4387406). Real-time quantitative PCR reactions were performed using Taqman probes and Fast advance mix (ThermoFisher, Cat. No. A44360) on a QuantStudio 7 Flex instrument (ThermoFisher). cDNA levels of target genes were analyzed using the comparative Ct method, where Ct is the threshold cycle number normalized to RPL13A.

[0422] compound : 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) (Sigma, Catalog No. B7880) was dissolved in PBS - / - (ThermoFisher, Catalog No. 14190-144) at a concentration of 100 mM (100X) and dexamethasone (sigma, Catalog No. D4902) was dissolved in DMSO at a concentration of 100 μM (1000X).

[0423] Functional assays for CYP1A2 : CYP1A2 activity was measured using a Promega kit (Promega, Cat. No. V8422) according to the manufacturer's recommendations. Primary human hepatocytes (150,000 viable cells) were plated in 250 μl of InVitroGRO CP medium (BioIVT, Cat. No. Z99029) in each well of a collagen I-coated 48-well plate. Cells were maintained in InVitroGRO CP medium for 2 days, with the medium replaced daily. CYP1A2 activity was induced with 100 μM omeprazole in InVitroGRO HI medium (BioIVT, Cat. No. Z99009) for 2 days, with the InVitroGRO HI medium containing omeprazole replaced daily. After 48 hours of incubation with omeprazole, cells were washed twice with InVitroGRO KHB medium (BioIVT, Cat. No. Z99074) without supplements, and 150 μl of fresh KHB medium containing 6 uM luciferin-1A2 and 3 mM salicylamide was added per well. Inhibitor control wells contained 5 μM a-naphthoflavone, a CYP1A2 inhibitor, along with luciferin-1A2 substrate. As background luminescence control, 150 μl of KHB medium containing luciferin-1A2 and 3 mM salicylamide was added to empty wells. 20 μl of the supplied 2M D-cysteine ​​was added to 10 ml of reconstituted luciferin detection reagent. After 60 min incubation at 37°C, 50 μl of the supernatant was transferred to an opalescent assay plate (Costar 3912) and 50 μl of luciferin detection reagent was added. After 20 min incubation at room temperature, luminescence was read using a luminometer. hiPSC-GMP1 was incubated at 2 x 10 5 cells / cm 2The hiPSC-GMP1 cells were seeded in 48-well plates at a density of 100 μM ...

[0424] Secretion of AFP, ALB, and urea into the medium : Primary human hepatocytes (150,000 viable cells) were plated in 250 μl of InVitroGRO CP medium (BioIVT, Cat. No. Z99029) in each well of a collagen I-coated 48-well plate. Cells were maintained in InVitroGRO CP medium for 2 days. hiPSC-GMP1 were differentiated into hepatocyte-like cells as described in detail above. Transduction with Teton, NFIC, and NFIX was performed on day 15 as described in detail above. Supernatants were harvested from primary human hepatocytes on day 2 after plating or from GMP1-derived hepatocyte-like cells on day 20 or 24 of differentiation. These supernatants were used to perform an ELISA assay for human alpha-fetoprotein (AFP) (Abcam, catalog number ab108838), an ELISA assay for human albumin (ALB) (Abcam, catalog number ab108788), or an enzymatic assay to measure urea secretion (Sigma, catalog number MAK006). For each of the aforementioned assays, the work was carried out according to the manufacturer's recommendations. Secretion of AFP, ALB, and urea in the culture medium was normalized to the cell number.

[0425] Example 2: A model system for screening candidate transcription factors Principal component analysis (PCA) was performed on cancer cell lines (HepG2, HuH7, and HepaRG), stem cell-derived hepatocytes (stem cell / iPSC-Heps), and primary human hepatocytes (PHH) (Figures 2A-2B). PHH-AQL, PHH-TLY, and PHH-NES are adult hepatocytes. PHH-BVI are stillborn hepatocytes, and "fetal" refers to primary human fetal hepatocytes. HuH7 cells clustered with hepatocytes differentiated from GMP1 iPSCs that were not further treated with Br-cAMP and dexamethasone ("GMP1 control"), and hepatocytes differentiated from GMP1 iPSCs that were further treated with Br-cAMP and dexamethasone for 5 days ("GMPDex"), so the HuH7 cells were used to construct the HuH7-Tet-On3G cell line for screening the transcription factors of the present invention, as described in Example 1 (Figure 2C). As shown in FIG. 2D, the HuH7-Tet-On3G cell line was responsive to doxycycline induction. The HuH7-Tet-On3G cell line was transduced with lentiviral particles containing a Tet response element (TRE-Luc) upstream of luciferase at MOIs of 0, 5, and 10. After transduction, cells were grown in the presence or absence of 1 μg / ml doxycycline for 48 hours. Luciferase expression relative to the housekeeping gene RPL13A was normalized to uninfected control samples in the absence of doxycycline. This study illustrates an exemplary model system used to screen transcription factor candidates of the present invention.

[0426] Example 3: Increased expression of various transcription factors in immature hepatocytes Screening for transcription factors that enhance hepatocyte maturation was performed in the HuH7-Tet-On3G cell line, which was generated as described in Example 1 above. Screening for transcription factors was performed by measuring increased expression of mature hepatocyte markers CYP1A2 (FIG. 3A) and CYP3A4 (FIG. 3B) after transduction of cells with lentiviral particles containing various transcription factor candidates. Transduction with transcription factors was performed at a multiplicity of infection (MOI) of 10. NFIC transcript variants 1 and 3 (NFIC-1+3) refer to a mixture of alternative splice variants of the transcription factor NFIC, NFIC transcript variant 1 (NFIC-1) (NCBI reference sequence: NM_001245002) and NFIC transcript variant 3 (NFIC-3) (NCBI reference sequence: NM_001245004), which were transduced with MOI of 5 for each of NFIC transcript variant 1 (NFIC-1) and NFIC transcript variant 3 (NFIC-3). After transduction, cells were cultured for 5 days in HuH7 medium containing 1 μg / ml doxycycline. Expression of mature hepatocyte markers was plotted against the housekeeping gene RPL13A and normalized to uninfected cells. Adult primary human hepatocytes (PHH) from lots AQL and TLY were used as positive controls. PHH cells were obtained from BioIVT and mRNA was extracted from frozen vials. Arrows in Figures 3A-3B represent various transcription factors that upregulated the expression levels of mature hepatocyte markers CYP1A2 and CYP3A4.

[0427] Example 4: Increasing expression of the transcription factor NFIC in immature hepatocytes increases expression of mature hepatocyte markers HuH7-Tet-On3G cells, generated as described in Example 1 above, were transduced with lentiviral particles containing the transcription factors NFIC transcript variants 1 and 3 (NFIC-1+3); NFIC transcript variant 1 (NFIC-1); or NFIC transcript variant 3 (NFIC-3) at an MOI of 5. NFIC transcript variants 1 and 3 (NFIC-1+3) refer to a mixture of NFIC transcript variant 1 (NFIC-1) and NFIC transcript variant 3 (NFIC-3), which are alternative splice variants of the transcription factor NFIC, respectively (FIG. 4A). After transduction, cells were cultured for 5 days in culture medium containing 1 μg / ml doxycycline. Expression levels of mature hepatocyte markers CYP1A2 and CYP3A4 were determined relative to the housekeeping gene RPL13A and normalized to non-infected ("NI") cells. The results of this study, as shown in Figure 4B, demonstrate that increasing expression of NFIC in immature hepatocytes increases the expression levels of mature hepatocyte markers and, consequently, enhances the production of mature hepatocytes.

[0428] Example 5: Increasing expression of transcription factor NFIC in immature hepatocytes cultured in the presence of dexamethasone and 8-Br-cAMP increases expression of mature hepatocyte markers HuH7-Tet-On3G cells, generated as described in Example 1 above, were transduced with lentiviral particles containing the transcription factor NFIC transcription variant 1 (NFIC-1) at an MOI of 50. After transduction, cells were cultured for 5 days in culture medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and 100 nM dexamethasone. Expression levels of mature hepatocyte markers CYP1A2 (Figure 5A), TAT (Figure 5B), and UGT1A1 (Figure 5C) were determined relative to the housekeeping gene RPL13A and normalized to uninfected negative control samples (absence of 8-Br-cAMP and dexamethasone). Primary human hepatocyte (PHH) expression values ​​represent the average of the expression values ​​of the PHH-AQL and PHH-TLY lots. PHH cells were obtained from BioIVT, and mRNA was extracted from frozen vials. The results of this study demonstrate that increasing the expression of NFIC in immature hepatocytes cultured in the presence of dexamethasone and 8-Br-cAMP increases the expression levels of mature hepatocyte markers, and thus enhances the production of mature hepatocytes, as shown in Figure 5.

[0429] Example 6: Increased expression of various transcription factors in immature hepatocytes Screening of transcription factors that promote hepatocyte maturation was performed in the HuH7-Tet-On3G cell line, which was generated as described in Example 1 above. Screening of transcription factors was performed by measuring the decreased expression of immature hepatocyte marker AFP (Figure 6A) and the increased expression of mature hepatocyte markers CYP1A2 (Figure 6B), TAT (Figure 6C), and CYP3A4 (Figure 6D) after transduction of cells with lentiviral particles containing various transcription factor candidates. Transduction with transcription factors was performed at a multiplicity of infection (MOI) of 10. After transduction, cells were cultured in culture medium containing 1 μg / ml doxycycline, 1 mM 8-Br-cAMP, and 100 nM dexamethasone. Expression of maturation markers was measured 5 days after transduction. Relative expression of maturation markers was normalized to transduction with NFIC transcript variant 1 (NFIC-1) in the presence of 1 μg / ml doxycycline as a control. Primary human hepatocyte (PHH) expression values ​​represent the average of the expression values ​​of PHH-AQL and PHH-TLY lots. PHH cells were obtained from BioIVT, and mRNA was extracted from frozen vials. Arrows in Figures 6A-6D represent different transcription factors that downregulated the expression levels of the immature hepatocyte marker AFP, and upregulated the expression levels of the mature hepatocyte markers CYP1A2, TAT, and CYP3A4.

[0430] Example 7: Increasing expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes increases expression of mature hepatocyte markers Pluripotent stem cell-derived immature hepatocytes were generated using a stage 4 stepwise differentiation process as detailed in Example 1 (FIG. 7A). At the end of stage 3, transduction was performed with lentiviral particles containing Tet-On3G (MOI of 5) ("TetOn") or with lentiviral particles containing Tet-On3G in combination with the transcription factors NFIC transcription variant 1 (NFIC-1); NFIX; or NFIC transcription variant 1 (NFIC-1) and NFIX (MOI of 3) on day 15 of differentiation towards hepatocyte-like cells (FIG. 7A). Cells were then cultured for 5 days in stage 4 medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-Br-cAMP and 100 nM dexamethasone as described in Example 1 above. Expression levels of mature hepatocyte markers CYP1A2 and TAT4 were determined relative to the housekeeping gene RPL13A and normalized to the non-infected negative control sample ("NI"). Primary human hepatocyte (PHH) expression values ​​represent the average of the expression values ​​of PHH-AQL and PHH-TLY lots. PHH cells were obtained from BioIVT, and mRNA was extracted from frozen vials. The results of this study demonstrate that increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes increases the expression levels of mature hepatocyte markers and, as a result, enhances mature hepatocyte production, as shown in Figure 7B.

[0431] Example 8: Time course analysis of expression of mature hepatocyte markers by increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes Pluripotent stem cell-derived immature hepatocytes were generated using a stage 4 stepwise differentiation process as detailed in Example 1 (FIG. 8A). At the end of stage 3, transduction was performed with lentiviral particles containing Tet-On3G (MOI of 5) ("TetOn") or with lentiviral particles containing Tet-On3G in combination with the transcription factors NFIC transcription variant 1 (NFIC-1); NFIX; or NFIC transcription variant 1 (NFIC-1) and NFIX (MOI of 3) on day 15 of differentiation towards hepatocyte-like cells (FIG. 8A). Cells were then cultured for 5 or 9 days in stage 4 medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-Br-cAMP and 100 nM dexamethasone as described in Example 1 above. Cells were harvested on the 20th and 24th days of cell culture, and the expression levels of the immature hepatocyte marker AFP and the mature hepatocyte marker CYP1A2 were determined relative to the housekeeping gene RPL13A and normalized to the non-infected ("NI") negative control sample. Primary human hepatocyte (PHH) expression values ​​represent the average of the expression values ​​of the PHH-AQL and PHH-TLY lots. PHH cells were obtained from BioIVT, and mRNA was extracted from frozen vials. The results of this study demonstrate that increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes reduces the expression levels of immature hepatocyte markers and increases the expression levels of mature hepatocyte markers, and thus enhances mature hepatocyte production, as shown in FIG. 8B.

[0432] Example 9: Increasing expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes shifts the transcriptome towards that of mature hepatocytes Principal component analysis (PCA) was performed on pluripotent stem cell-derived immature hepatocytes. Pluripotent stem cell-derived immature hepatocytes were generated using a stage 4 stepwise differentiation process as detailed in Example 1. At the end of stage 3, transduction was performed with lentiviral particles containing Tet-On3G (MOI of 5) or with lentiviral particles containing Tet-On3G in combination with the transcription factors NFIC transcription variant 1 (NFIC-1); NFIX; or NFIC transcription variant 1 (NFIC-1) and NFIX (MOI of 3) on day 15 of differentiation towards hepatocyte-like cells. Cells were then cultured for 5 or 9 days in stage 4 medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-Br-cAMP and 100 nM dexamethasone as described in Example 1 above. Cells were harvested on days 20 and 24 of cell culture. Ten different primary human hepatocyte (PHH) data sets corresponding to ten different individuals were used for PCA analysis. PHH cells were obtained from BioIVT and mRNA was extracted from frozen vials. The results of this study demonstrate that increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes leads to the shift of 30-34% of the transcriptome to that of primary human hepatocytes, as shown in Figure 9.

[0433] Example 10: Functional assay of pluripotent stem cell-derived immature hepatocytes containing increased expression of transcription factors NFIC and / or NFIX Pluripotent stem cell-derived immature hepatocytes (GMP1-Hep) were generated using a stage 4 stepwise differentiation process as detailed in Example 1. At the end of stage 3, transduction was performed with lentiviral particles containing Tet-On3G (MOI of 5) or with lentiviral particles containing Tet-On3G in combination with the transcription factors NFIC transcription variant 1 (NFIC); NFIX; or NFIC transcription variant 1 (NFIC) and NFIX (MOI of 3) on day 15 of differentiation towards hepatocyte-like cells (Figure 8A). Cells were then cultured for 5 or 9 days in stage 4 medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-Br-cAMP and 100 nM dexamethasone as described in Example 1 above. Cells were harvested on days 20 and 24 of cell culture. Functional activity assays were performed as detailed in Example 1 to determine CYP1A2 activity (FIG. 10A), ALB secretion (FIG. 10B), AFP secretion (FIG. 10C), and urea secretion (FIG. 10D). The results of this study demonstrate that increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes increases the activity of CYP1A2, increases the secretion of ALB, and decreases the secretion of AFP, and thus enhances the production of mature hepatocytes, as shown in FIG.

[0434] Example 11: Increased expression of various combinations of transcription factors in immature hepatocytes HuH7-Tet-On3G cells, generated as described in Example 1 above, were transduced with lentiviral particles containing various transcription factors as described in FIG. 11A at an MOI of 10. After transduction, cells were cultured for 5 days in culture medium containing 1 μg / ml doxycycline. The expression levels of mature hepatocyte markers CYP1A2 and TCYP3A4 (FIG. 11B) were determined relative to the housekeeping gene RPL13A and normalized to the non-infected ("NI") negative control sample. PHH cells from lots AQL and TLY were obtained from BioIVT, and mRNA was extracted from frozen vials. The results of this study, as shown in FIG. 11B, demonstrate that increasing the expression of various transcription factor combinations does not further increase the expression levels of mature hepatocyte markers in immature hepatocytes compared to the increase observed when increasing the expression of NFIC alone.

[0435] Example 12: Time course analysis of expression of mature hepatocyte markers by increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes Pluripotent stem cell-derived immature hepatocytes were generated using a stage 4 stepwise differentiation process as detailed in Example 1 (FIG. 8A). At the end of stage 3, transduction was performed with lentiviral particles containing Tet-On3G (MOI of 5) ("TetOn") or with lentiviral particles containing Tet-On3G in combination with the transcription factors NFIC transcription variant 1 (NFIC); NFIX; or NFIC transcription variant 1 (NFIC) and NFIX (MOI of 3) on day 15 of differentiation towards hepatocyte-like cells (FIG. 8A). Cells were then cultured for 5 or 9 days in stage 4 medium containing 1 μg / ml doxycycline in the presence or absence of 1 mM 8-Br-cAMP and 100 nM dexamethasone as described in Example 1 above. Cells were harvested on days 20 and 24 of cell culture, and the expression levels of mature hepatocyte markers ALB (FIG. 12A), CYP3A4 (FIG. 12B), and UGT1A1 (FIG. 12C) were determined relative to the housekeeping gene RPL13A and normalized to the non-infected ("NI") negative control sample. Primary human hepatocyte (PHH) expression values ​​represent the average of the expression values ​​of PHH-AQL and PHH-TLY lots. PHH cells were obtained from BioIVT, and mRNA was extracted from frozen vials. The results of this study demonstrate that increasing the expression of transcription factors NFIC and / or NFIX in pluripotent stem cell-derived immature hepatocytes increases the expression levels of mature hepatocyte markers and, as a result, enhances mature hepatocyte production, as shown in FIGS. 12A-12C.

[0436] Informal sequence listing TIFF2024518409000005.tif228159TIFF2024518409000006.tif239159TIFF2024518409000007.tif239149TIFF2024518409000008.tif238149TIFF2024518409000009.tif239149TIFF2024518409000010.tif236149TIFF2024518409000011.tif239149TIFF2024518409000012.tif238149TIFF2024518409000013.tif239149TIFF2024518409000014.tif236149TIFF2024518409000015.tif239149TIFF2024518409000016.tif238149TIFF2024518409000017.tif238155TIFF2024518409000018.tif239149TIFF2024518409000019.tif238149TIFF2024518409000020.tif236149TIFF2024518409000021.tif239149TIFF2024518409000022.tif236149TIFF2024518409000023.tif236149TIFF2024518409000024.tif236156TIFF2024518409000025.tif239149TIFF2024518409000026.tif236156TIFF2024518409000027.tif236149TIFF2024518409000028.tif239149TIFF2024518409000029.tif239149TIFF2024518409000030.tif236149TIFF2024518409000031.tif236149TIFF2024518409000032.tif239149TIFF2024518409000033.tif236149TIFF2024518409000034.tif239149TIFF2024518409000035.tif236149TIFF2024518409000036.tif236149TIFF2024518409000037.tif236149TIFF2024518409000038.tif239149TIFF2024518409000039.tif239149TIFF2024518409000040.tif239149TIFF2024518409000041.tif236149TIFF2024518409000042.tif239149TIFF2024518409000043.tif236149TIFF2024518409000044.tif220149TIFF2024518409000045.tif236149TIFF2024518409000046.tif236149TIFF2024518409000047.tif239149TIFF2024518409000048.tif239149TIFF2024518409000049.tif238149TIFF2024518409000050.tif236149TIFF2024518409000051.tif238149TIFF2024518409000052.tif223149TIFF2024518409000053.tif238161TIFF2024518409000054.tif239149TIFF2024518409000055.tif238159TIFF2024518409000056.tif236159TIFF2024518409000057.tif21158.

Claims

1. 1. A method for producing mature hepatocytes, comprising: Increasing the expression of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) in immature hepatocytes, thereby generating mature hepatocytes. A method comprising:

2. Increasing expression of at least one transcription factor in the immature hepatocytes (a) contacting an immature hepatocyte with at least one transcription factor; (b) transducing immature hepatocytes with a viral vector encoding at least one transcription factor; (c) transfecting immature hepatocytes with an expression vector encoding at least one transcription factor; and / or (d) using a gene switch construct encoding at least one transcription factor, optionally wherein the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct; 2. The method of claim 1, comprising:

3. The method of claim 1, wherein the immature hepatocytes comprise an expression vector comprising a nucleic acid encoding at least one transcription factor.

4. The method of claim 1, wherein the immature hepatocytes are derived from pluripotent stem cells, which are embryonic stem cells or induced pluripotent stem cells.

5. A method for producing mature hepatocytes derived from pluripotent stem cells, comprising: (a) differentiating pluripotent stem cells into immature hepatocytes, wherein the pluripotent stem cells comprise an expression vector comprising a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC); and (b) increasing expression of at least one transcription factor from the expression vector in the immature hepatocytes, thereby generating mature hepatocytes. A method comprising:

6. (a) The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. (b) the immature liver cells comprise hepatoblasts or hepatic stem cells; and / or (c) the immature hepatocytes contain an expression vector comprising a nucleic acid encoding at least one transcription factor; The method of claim 5.

7. 6. The method of claim 1 or 5, wherein the transcription factor is NFIX, NFIC, or NFIX and NFIC.

8. 6. The method of claim 1 or 5, wherein NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; NFIC transcript variant 5; and NFIC transcript variant 1 and NFIC transcript variant 3. (a) increasing expression of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 in immature hepatocytes; and (b) culturing the immature hepatocytes in a culture medium containing dexamethasone, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or a combination thereof; 6. The method of claim 1 or 5, further comprising: (a) the culturing step is carried out for at least 2, 3, 4, 5, 6, 7, 8, or 9 days. (b) the concentration of 8-Br-cAMP is at least 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 nM, or 1 mM; and / or (c) the concentration of dexamethasone is at least 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, or 100 nM; The method of claim 9. (a) The expression vector is a viral vector or a non-viral vector. (b) the expression vector is an inducible expression vector; (c) the expression vector comprises a promoter operably linked to a nucleic acid encoding at least one transcription factor, the promoter being an endogenous promoter, an artificial promoter, or an inducible promoter; and / or (d) The expression vector contains a self-cleaving sequence. The method according to claim 3 or 5.

12. The method of claim 5, wherein the step of increasing the expression of at least one transcription factor in the immature hepatocyte comprises inducing expression of at least one transcription factor in the immature hepatocyte.

13. 13. The method of claim 12, wherein inducing expression of at least one transcription factor in the immature hepatocyte comprises using a gene switch construct encoding at least one transcription factor. (a) the pluripotent stem cells are transduced with a viral vector encoding at least one transcription factor; (b) the pluripotent stem cells are transfected with an expression vector encoding at least one transcription factor, and / or (c) step (a) comprises culturing the pluripotent stem cells in a first differentiation medium comprising activin A, a second differentiation medium comprising at least one of BMP4 and FGF2, and a third differentiation medium comprising HGF, thereby generating immature hepatocytes; The method of claim 5.

15. The method of claim 15, wherein: (a) the cells are cultured in each of a first differentiation medium, a second differentiation medium, and a third differentiation medium for at least 5 days. (b) the immature hepatocytes are cultured in a culture medium containing hepatocyte growth factor (HGF), and / or (c) immature hepatocytes are cultured in a culture medium containing oncostatin M (OSM); The method of claim 14. (a) the immature hepatocytes are cultured for at least 2, 3, 4, or 5 days before increasing the expression of at least one transcription factor; and / or (b) the immature hepatocytes are cultured for at least 2, 3, 4, 5, 6, 7, 8, or 9 days after increasing expression of at least one transcription factor; The method according to claim 1 or 5.

17. (a) increasing expression of NFIX comprises an increase of at least 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold compared to endogenous expression levels of NFIX in immature hepatocytes. (b) increasing expression of NFIC comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold increase compared to the endogenous expression level of NFIC in immature hepatocytes; (c) mature hepatocytes exhibit increased expression of albumin (ALB), cytochrome P450 enzyme 1A2 (CYP1A2), cytochrome P450 enzyme 3A4 (CYP3A4), tyrosine aminotransferase (TAT), and / or UDP-glucuronosyltransferase 1A-1 (UGT1A1) compared to immature hepatocytes; (d) Mature hepatocytes exhibit decreased expression of alpha-fetoprotein (AFP) compared to immature hepatocytes; (e) mature hepatocytes exhibit increased albumin (ALB) secretion, decreased AFP secretion, and / or increased CYP1A2 activity compared to immature hepatocytes; and / or (f) increasing the expression of at least one transcription factor shifts at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the transcriptome of the immature hepatocyte to that of a mature hepatocyte. The method according to claim 1 or 5.

18. (a) increased expression of CYP1A2 includes at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes. (b) increased expression of CYP3A4 includes at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes; (c) increased expression of TAT, including at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase in expression compared to immature hepatocytes; (d) increased expression of UGT1A1, including at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 2,000-fold, 5,000-fold, or 10,000-fold increase compared to immature hepatocytes; (e) the increase in ALB secretion comprises at least a 5%, 10%, 15%, 20%, or 25% increase compared to immature hepatocytes; (f) an increase in CYP1A2 activity, including at least a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold increase compared to immature hepatocytes; (g) the reduction in expression of AFP comprises at least a 0.1-fold, 0.2-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, or 4-fold reduction compared to immature hepatocytes; and / or (h) the reduction in AFP secretion comprises at least a 5%, 10%, 20%, 40%, or 60% reduction compared to immature hepatocytes; The method of claim 17.

19. A composition comprising a mature hepatocyte population produced by the method of claim 1 or 5.

20. 13. A pharmaceutical composition comprising a mature hepatocyte population produced by the method of claim 1 or 5 and a pharma- ceutically acceptable carrier.

21. A composition comprising a liver cell population, the liver cell population comprising an increased expression level of at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC) compared to the endogenous expression level of the transcription factor in the liver cell population.

22. The composition of claim 21, wherein the hepatocytes further comprise an increased expression level of one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1 compared to the endogenous expression level of the one or more transcription factors in the hepatocyte population.

23. The method of claim 22, wherein the increased expression comprises exogenous expression of at least one transcription factor; and / or (b) the hepatocytes contain an expression vector comprising a nucleic acid encoding at least one transcription factor; 22. The composition of claim 21. (a) The expression vector is a viral vector or a non-viral vector. (b) the expression vector is an inducible expression vector; (c) the expression vector comprises a promoter operably linked to a nucleic acid encoding at least one transcription factor; (d) the expression vector comprises a gene switch construct encoding at least one transcription factor; and / or (e) the expression vector further comprises a self-cleaving sequence; 24. The composition of claim 23.

25. (a) the viral vector is selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a herpes simplex viral vector, a Sendai viral vector, and a retroviral vector; (b) the non-viral vector is selected from the group consisting of plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA); (c) the non-viral vector comprises naked nucleic acid, liposome, dendrimer, nanoparticle, lipid-polymer system, solid lipid nanoparticle, and / or liposome-protamine / DNA lipoplex (LPD); (d) the promoter is an endogenous promoter, an artificial promoter, or an inducible promoter; (e) the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct, and / or (f) the self-cleaving sequence is selected from the group consisting of T2A, P2A, E2A, and F2A; 25. The composition of claim 24.

26. The method of claim 25, wherein the hepatocyte population is an immature hepatocyte population. (b) the hepatocyte population is a mature hepatocyte population; (c) the composition further comprises cells other than hepatocytes (d) the hepatocyte population is in the form of an organoid; (e) the hepatocytes are derived from pluripotent stem cells, the pluripotent stem cells being embryonic stem cells or induced pluripotent stem cells; and / or (r) the hepatocyte population comprises at least 10 6 hepatocytes; 22. The composition of claim 21.

27. 22. A pharmaceutical composition comprising the population of hepatocytes described in claim 21 and a pharma- ceutically acceptable carrier.

28. A composition comprising a pluripotent stem cell population comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding at least one transcription factor selected from the group consisting of nuclear factor IX (NFIX) and nuclear factor IC (NFIC).

29. 30. The composition of claim 21 or 28, wherein the transcription factor is NFIX, NFIC, or NFIX and NFIC.

30. 30. The composition of claim 21 or 28, wherein the NFIC is at least one NFIC alternative splice variant selected from the group consisting of NFIC transcript variant 1; NFIC transcript variant 2; NFIC transcript variant 3; NFIC transcript variant 4; NFIC transcript variant 5; and NFIC transcript variant 1 and NFIC transcript variant 3.

31. 29. The composition of claim 28, wherein the pluripotent stem cells further comprise an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1. (a) The expression vector is a viral vector or a non-viral vector. (b) the expression vector is an inducible expression vector; (c) the expression vector comprises a promoter operably linked to a nucleic acid encoding at least one transcription factor; (d) the expression vector comprises a gene switch construct encoding at least one transcription factor; and / or (e) the expression vector further comprises a self-cleaving sequence; 29. The composition of claim 28.

33. (a) the viral vector is selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a herpes simplex viral vector, a Sendai viral vector, and a retroviral vector; (b) the non-viral vector is selected from the group consisting of plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA); (c) the non-viral vector comprises naked nucleic acid, liposome, dendrimer, nanoparticle, lipid-polymer system, solid lipid nanoparticle, and / or liposome-protamine / DNA lipoplex (LPD); (d) the promoter is an endogenous promoter, an artificial promoter, or an inducible promoter; (e) the gene switch construct is a transcriptional gene switch construct or a post-transcriptional gene switch construct, and / or (f) the self-cleaving sequence is selected from the group consisting of T2A, P2A, E2A, and F2A; 33. The composition of claim 32.

34. (a) the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells, and / or (b) the pluripotent stem cell population comprises at least 10 6 pluripotent stem cells; 29. The composition of claim 28.

35. 29. Use of a composition according to claim 21 or 28, or a pharmaceutical composition according to claim 27, in the manufacture of a medicament for treating a disease in a subject.

36. The disease may be fulminant hepatic failure of any cause, viral hepatitis, drug-induced liver damage, cirrhosis, hereditary liver dysfunction (e.g., Wilson's disease, Gilbert's syndrome, or α1-antitrypsin deficiency), hepatobiliary cancer, autoimmune liver disease (e.g., autoimmune chronic hepatitis or primary biliary cirrhosis), urea cycle disorders, factor VII deficiency, glycogen storage disease type 1, childhood Refsum's disease, phenylketonuria, severe childhood oxalosis, hepatic cirrhosis 36. The use of claim 35, wherein the patient is selected from the group consisting of: hepatic malformation, liver damage, acute liver failure, hepatobiliary cancer, hepatocellular carcinoma, hereditary cholestasis (PFIC and Alagille syndrome), hereditary hemochromatosis, tyrosinemia type 1, argininosuccinic aciduria (ASL), Crigler-Najjar syndrome, familial amyloidotic polyneuropathy, atypical hemolytic uremic syndrome-1, primary hyperoxaluria type 1, maple syrup urine disease (MSUD), acute intermittent porphyria, coagulation disorders, GSD type Ia (in metabolic control), homozygous familial hypercholesterolemia, organic acidurias, and any other pathology that causes liver dysfunction.

37. 28. A kit comprising the composition of claim 21 or 28, or the pharmaceutical composition of claim 27.

38. A kit comprising nuclear factor IX (NFIX), nuclear factor IC (NFIC), and an expression vector comprising a nucleic acid encoding at least one transcription factor selected from the group consisting of NFIX and NFIC.

39. 39. The kit of claim 38, further comprising an expression vector comprising a nucleic acid encoding one or more transcription factors selected from the group consisting of RORC, NR0B2, ESR1, THRSP, TBX15, HLF, ATOH8, NR1I2, CUX2, ZNF662, TSHZ2, ATF5, NFIA, NFIB, NPAS2, FOS, ONECUT2, PROX1, NR1H4, MLXIPL, ETV1, AR, CEBPB, NR1D1, HEY2, ARID3C, KLF9, and DMRTA1.

40. (a) NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:

1. (b) the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; (c) NFIX comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:40; or (d) the NFIC comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45; 39. The method of claim 1 or 5, the composition of claim 21 or 28, or the kit of claim 38.

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