Methods and compositions for the differentiation of stem cells

Abstract

The present disclosure provides transcription factors and methods for the differentiation of stem cells into hepatocytes or liver cells. In some examples, pluripotent stem cells (PSCs) contain nucleic acids. In some examples, the nucleic acids include open reading frames encoding one or more transcription factors, one or more transcription factors, or transcriptional activators of open reading frames encoding one or more transcription factors. In some examples, nucleic acids containing open reading frames encoding one or more transcription factors, one or more transcription factors, or transcriptional activators of open reading frames encoding one transcription factor induce the differentiation of PSCs into hepatocytes or liver cells.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 352,529, filed on Jun. 15, 2022, the entire disclosure of which is incorporated herein by reference.

Background Art

[0002] Hepatocytes play an important role in the liver. Hepatocytes can be involved in protein synthesis or storage, carbohydrate transformation, synthesis of cholesterol, bile salts, or phospholipids, and detoxification. Treatments for improving specific functions of the liver can include the introduction or regulation of hepatocytes or liver cells. Stem cells can be programmed into various phenotypes through the introduction of one or more transcription factors, or one or more molecules that regulate transcription or transcription factors. For example, stem cells can be programmed into hepatocytes or liver cells.

Summary of the Invention

[0003] Aspects of the present disclosure are pluripotent stem cells (PSCs) engineered to express a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise one or more hepatocyte nuclear factor (“HNF”) family members, one or more forkhead box (“FOX”) family members, and one or more additional transcription factors comprising FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more transcription factors comprise two or more HNF family members. In some embodiments, the one or more transcription factors comprise three or more HNF family members. In some embodiments, the one or more additional transcription factors comprise CEBP, and the one or more transcription factors further comprise FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more additional transcription factors comprise RBPJ, and the one or more transcription factors further comprise FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the PSC further comprises at least two or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least three or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least four or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least five or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least six or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the PSC further comprises at least seven or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least eight or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, one or more HNF family members include HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, one or more FOX family members include FOXA1. In some embodiments, one or more transcription factors include HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, one or more transcription factors include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

[0004] Another aspect of the present disclosure is a method of generating a population of hepatocytes or hepatocytes, the method comprising providing one or more pluripotent stem cells (PSCs), and an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors. Delivering at least one nucleic acid to one or more PSCs, wherein the one or more transcription factors include one or more hepatocyte nuclear factor (HNF) family members, one or more forkhead box (FOX) family members, and FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12, and generating a population of hepatocytes or hepatocytes from the one or more PSCs. In some embodiments, the one or more transcription factors include two or more HNF family members. In some embodiments, the one or more transcription factors include three or more HNF family members. In some embodiments, the one or more additional transcription factors include CEBPA, and the one or more transcription factors further include FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more additional transcription factors include RBPJ, and the one or more transcription factors further include FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least two or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least three or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least four or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least five or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least six or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least seven or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least eight or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, one or more HNF family members include HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, one or more FOX family members include FOXA1. In some embodiments, one or more transcription factors include HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, one or more transcription factors include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ. In some embodiments, at least one or more transcription factors induce the expression of one or more PSCs into a population of hepatocytes or hepatocytes within 30 days. In some embodiments, one or more PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of one or more PSCs into a population of hepatocytes or hepatocytes. In some embodiments, the medium is not changed while delivering at least one exogenous expression cassette to one or more PSCs. In some embodiments, at least one of the one or more hepatocytes or hepatocytes of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes of the population of hepatocytes or hepatocytes expresses CXCR-4.In some embodiments, at least 2% of the population of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 3% of the population of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 5% of the population of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 6% of the population of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 10% of the population of hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, at least 20% of the population of hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, at least 30% of PSCs express CXCR-4.

[0005] Another aspect of the disclosure is an exogenous expression cassette that induces the differentiation of pluripotent stem cells (PSCs) into hepatocytes or hepatocytes. The exogenous expression cassette includes one or more hepatocyte nuclear factor (HNF) family member genes, one or more forkhead box (FOX) family member genes, and one or more additional transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more transcription factor genes include two or more HNF family members. In some embodiments, the one or more transcription factor genes include three or more HNF family members. In some embodiments, the one or more additional transcription factor genes include CEBPA, and the one or more transcription factor genes further include FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more additional transcription factor genes include RBPJ, and the one or more transcription factors further include FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette further includes FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the exogenous expression cassette comprises at least three or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least four or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least five or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least six or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least seven or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the exogenous expression cassette comprises at least eight or more transcription factor genes including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, one or more HNF family members include HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, one or more FOX family members include FOXA1 or FOXA3, or any combination thereof. In some embodiments, one or more transcription factor genes include HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, one or more transcription factor genes include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ. In some embodiments, the PSCs are provided in the medium. In some embodiments, the medium is not changed during differentiation of the PSCs into hepatocytes or liver parenchymal cells. In some embodiments, the exogenous expression cassette induces differentiation of the PSCs into hepatocytes or liver parenchymal cells within 30 days. In some embodiments, the exogenous expression cassette induces differentiation of the PSCs into hepatocytes or liver parenchymal cells within 96 hours.

[0006] Another aspect of the present disclosure is a method of generating a population of hepatocytes or liver parenchymal cells, the method comprising providing one or more pluripotent stem cells (PSCs), delivering to the one or more PSCs a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, and generating a population of hepatocytes or liver parenchymal cells from the one or more PSCs within 30 days. In some embodiments, the one or more PSCs differentiate into a population of hepatocytes or liver parenchymal cells within 96 hours. In some embodiments, the one or more PSCs are provided in a medium. In some embodiments, the medium is not changed during differentiation of the one or more PSCs into a population of hepatocytes. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, Plexin, Transcription factors) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, one or more transcription factors are members of a transcription factor family, such as those of the FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF transcription factor families, or one or more additional transcription factors from Table 12. In some embodiments, two or more transcription factors are members of a transcription factor family, such as those of the FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF transcription factor families, or one or more additional transcription factors from Table 12. In some embodiments, three or more transcription factors are members of a transcription factor family, such as those of the FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF transcription factor families, or one or more additional transcription factors from Table 12. In some embodiments, four or more transcription factors are members of a transcription factor family, such as those of the FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF transcription factor families, or one or more additional transcription factors from Table 12. In some embodiments, at least one or more transcription factors include SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors include the GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ. In some embodiments, one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors include HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, one or more PSCs are provided in a medium. In some embodiments, the medium is not changed during the differentiation of one or more PSCs into a population of hepatocytes or hepatocytes. In some embodiments, the medium does not change while delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors to one or more PSCs. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes does not express CXCR-4. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, the population of hepatocytes or hepatocytes comprises at least 2% of a population of hepatocytes or hepatocytes that express ASGR-1. In some embodiments, the population of hepatocytes or hepatocytes comprises at least 3% of a population of hepatocytes or hepatocytes that express ASGR-1. In some embodiments, the population of hepatocytes or hepatocytes comprises at least 5% of a population of hepatocytes or hepatocytes that express ASGR-1. In some embodiments, the population of hepatocytes or hepatocytes comprises at least 6% of a population of hepatocytes or hepatocytes that express ASGR-1. In some embodiments, the population of hepatocytes or hepatocytes comprises at least 10% of a population of hepatocytes or hepatocytes that express CXCR-4.In some embodiments, the population of hepatocytes or liver cells comprises at least 20% of the population of hepatocytes or liver cells that express CXCR-4. In some embodiments, the population of hepatocytes or liver cells comprises, and at least 30% of the PSCs express CXCR-4. A pluripotent stem cell (PSC) comprising a nucleic acid encoding one or more open reading frames of transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the nucleic acid encoding one or more open reading frames of transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces differentiation of the PSCs into hepatocytes or liver cells within 30 days. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domain, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domain, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription. factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more transcription factors are of a transcription factor family, such as, for example, members of the FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family, or one or more additional transcription factors from Table 12. In some embodiments, one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 transcription factor, GATA4, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, and GATA2 transcription factor. In some embodiments, one or more transcription factors are, for example, GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors are, for example, GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, NFIX. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1.In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 transcription factor, CEBPB, and NR1I3. In some embodiments, the one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express ASGR-1. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4. In some embodiments, PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver cells. In some embodiments, a population of hepatocytes or liver cells comprises two or more hepatocytes or liver cells. In some embodiments, a population of hepatocytes or liver cells comprises at least 2% of hepatocytes or liver cells that express ASGR-1. In some embodiments, a population of hepatocytes or liver cells comprises at least 3% of hepatocytes or liver cells that express ASGR-1. In some embodiments, a population of hepatocytes or liver cells comprises at least 5% of hepatocytes or liver cells that express ASGR-1. In some embodiments, a population of hepatocytes or liver cells comprises at least 6% of hepatocytes or liver cells that express ASGR-1. In some embodiments, a population of hepatocytes or liver cells comprises at least 10% of hepatocytes or liver cells that express CXCR-4.In some embodiments, the population of hepatocytes or hepatic parenchymal cells comprises at least 20% of hepatocytes or hepatic parenchymal cells that express CXCR-4. In some embodiments, the population of hepatocytes or hepatic parenchymal cells comprises at least 30% of hepatocytes or hepatic parenchymal cells that express CXCR-4. In some embodiments, a population of hepatocytes or hepatic parenchymal cells is implemented that does not include nutrient, or growth factor, or microenvironment / matrix optimization.

[0007] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include SPI1, HNF1A, FOXA2, CEBPA, ONECUT1, HNF4A, and ONECUT2.

[0008] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FOXA2, and FOXA1.

[0009] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A.

[0010] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, and RBPJ.

[0011] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include HNF1A, CEBPA, ONECUT1, ONECUT2, FOS, HIF1A, and TBX3.

[0012] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

[0013] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include FOXA1, HNF1A, CEBPA, ONECUT1 / HNF6, and HNF4A.

[0014] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include FOXA1, HNF4A, ONECUT1 / HNF6, and RBPJ.

[0015] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

[0016] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include CEBPA, FOXA1, HNF1A, and HNF4A.

[0017] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include CEBPA, HNF1A, HNF4A, FOXA3, FOXA2, PXR, and RXRA.

[0018] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include CEBPB, FOXA1, FOXA3, HLF, HNF1A, HNF4A, NR1I2 / PXR, NR1I3 / CAR, ONECUT1 / HNF6, PROX1, RBPJ, RORC, and SALL4.

[0019] In another aspect of the disclosure, a kit for inducing the differentiation of pluripotent stem cells (PSCs) into hepatocytes or hepatocyte-like cells is provided, the kit comprising one or more hepatocyte nuclear factor (HNF) family members, one or more forkhead box (FOX) family members, and one or more additional transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more transcription factor genes comprise two or more HNF family members. In some embodiments, the one or more transcription factor genes comprise three or more HNF family members. In some embodiments, the one or more additional transcription factors comprise CEBPA, and the one or more transcription factors further comprise FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the one or more additional transcription factors comprise RBPJ, and the one or more transcription factors further comprise FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least two or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least three or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least four or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least five or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least six or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least seven or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least eight or more transcription factors including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12. In some embodiments, one or more HNF family members include HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, one or more FOX family members include FOXA1 or FOXA3, or any combination thereof. In some embodiments, one or more transcription factors include HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, one or more transcription factors include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ. In some embodiments, the PSCs are provided in the medium. In some embodiments, the medium is not changed during differentiation of the PSCs into hepatocytes or liver parenchymal cells. In some embodiments, the exogenous expression cassette induces differentiation of the PSCs into hepatocytes or liver parenchymal cells within 30 days. In some embodiments, the exogenous expression cassette induces differentiation of the PSCs into hepatocytes or liver parenchymal cells within 96 hours.

[0020] Another aspect of the present disclosure is a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include ATF5, CEBPA, CEBPB, FOXA1, FOXA2, FOXA3, GATA6, HHEX, HIF1A, HLF, HNF1A, HNF4A, NR1H4 / FXR, NR1I2 / PXR, NR1I3 / CAR, NR5A2, ONECUT1 / HNF6, ONECUT2 / HNF6B, NR1C1 / PPARA, PROX1, RBPJ, RORC, RXRA, TBX3, and SALL4.

[0021] Another aspect of the present disclosure provides a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces differentiation of the PSC into hepatocytes or liver cells within 30 days. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, the one or more transcription factors are of transcription factor families, for example, those of the ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, AP-1 / JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP families. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B.In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 transcription factor, GATA4, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, and GATA2 transcription factor. In some embodiments, one or more transcription factors are GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors are, for example, GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, NFIX. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 transcription factor, CEBPB, and NR1I3. In some embodiments, one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.

[0022] The present disclosure provides hepatocytes or liver parenchymal cells, and the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, the hepatocytes or liver parenchymal cells do not express CXCR-4. In some embodiments, the hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0023] The present disclosure provides a population of hepatocytes or liver parenchymal cells comprising two or more of the hepatocytes or liver parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0024] Another aspect of the present disclosure provides a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces the differentiation of the PSC into hepatocytes or liver cells, the PSC is provided in a medium, and the medium is not changed during the differentiation of the PSC into hepatocytes or liver cells. In some embodiments, the one or more transcription factors induce the differentiation of the PSC into hepatocytes or liver cells within 30 days. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, the one or more transcription factors are selected from the group of transcription factor families consisting of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B.In some embodiments, one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 transcription factor, GATA4, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, and GATA2 transcription factor. In some embodiments, one or more transcription factors are, for example, GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors are, for example, GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, NFIX. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 transcription factor, CEBPB, and NR1I3. In some embodiments, one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express ASGR-1. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4. In some embodiments, PSC is provided in the medium.In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver cells.

[0025] The present disclosure provides a population of hepatocytes or liver cells comprising two or more of the hepatocytes or liver cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0026] Another aspect of the present disclosure provides a pluripotent stem cell (PSC) comprising a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include the HNF6 gene, as well as one or more additional hepatocyte programming factor genes, such as FoxA1, SPI1, FoxA2, CEBPA, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / FOS, JUN, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, and HIF1A. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, or TP53 family members. In some embodiments, two or more transcription factors are selected from the group of transcription factor families consisting of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more transcription factors are selected from the group of transcription factor families consisting of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, the four or more transcription factors are selected from the group of transcription factor families consisting of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, JUN, SKI, SALL, EGR, and TP. In some embodiments, a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one transcription factor induces the differentiation of PSCs into hepatocytes or liver cells. In some embodiments, a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces the differentiation of PSCs into hepatocytes or liver cells within 30 days. In some embodiments, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, the one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, the one or more transcription factors are, for example, SPI1, FoxA2, GATA2 transcription factor, GATA4, JUN / AP-1, and NFIX. In some embodiments, the one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, and GATA2 transcription factor. In some embodiments, the one or more transcription factors are, for example, GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors are, for example, GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A.In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 transcription factor, CEBPB, and NR1I3. In some embodiments, the one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0027] The present disclosure provides hepatocytes or liver parenchymal cells, and the hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, the hepatocytes or liver parenchymal cells do not express CXCR-4. In some embodiments, the hepatocytes or liver parenchymal cells express CXCR-4.

[0028] The present disclosure provides a population of cells comprising two or more of the hepatocytes or liver cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, the population of cells is provided in a culture medium. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment or matrix is performed.

[0029] Another aspect of the present disclosure provides a method for generating a population of hepatocytes or liver parenchymal cells, the method comprising providing one or more pluripotent stem cells (PSCs) and delivering to the one or more PSCs at least one nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors include the HNF6 gene, and generating a population of hepatocytes or liver parenchymal cells from the one or more PSCs. In some embodiments, the method further comprises one or more transcription factors that are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, the method further comprises two or more of one or more transcription factors that are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, the method further comprises three or more of one or more transcription factors such as, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, the method further comprises four or more of one or more transcription factors such as, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more transcription factors are of a transcription factor family such as, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more transcription factors are of a transcription factor family such as, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, four or more transcription factors are of transcription factor families such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of transcription factor families such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, at least one or more transcription factors include SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, at least one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, at least one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, at least one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ. In some embodiments, at least one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, at least one or more transcription factors include HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, at least one or more transcription factors induce the expression of one or more PSCs into a population of hepatocytes or hepatocytes within 30 days. In some embodiments, one or more PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of one or more PSCs into a population of hepatocytes or hepatocytes.In some embodiments, the medium is not altered while delivering at least one exogenous expression cassette to one or more PSCs. In some embodiments, at least one of one or more hepatocytes or a population of hepatocytes expresses ASGR-1. In some embodiments, at least one of one or more hepatocytes or a population of hepatocytes does not express CXCR-4. In some embodiments, at least one of one or more hepatocytes or a population of hepatocytes expresses CXCR-4. In some embodiments, at least 2% of a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 3% of a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 5% of a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 6% of a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 10% of a population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 20% of a population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 30% of PSCs expresses CXCR-4.

[0030] Another aspect of the present disclosure provides a method for generating a population of hepatocytes or hepatocytes, the method comprising providing one or more pluripotent stem cells (PSCs); delivering to the one or more PSCs a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors induce differentiation of the one or more PSCs into a population of hepatocytes or hepatocytes; further inducing expression of the one or more transcription factors in the one or more PSCs within 30 days; and generating a population of hepatocytes or hepatocytes from the one or more PSCs. In some embodiments, the one or more PSCs differentiate into a population of hepatocytes or hepatocytes within 96 hours. In some embodiments, the one or more PSCs are provided in a medium. In some embodiments, the medium is not changed during differentiation of the one or more PSCs into a population of hepatocytes. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, one or more transcription factors are of a transcription factor family such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, two or more transcription factors are of a transcription factor family such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more transcription factors are of a transcription factor family such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, JUN, SKI, SALL, EGR, and TP. In some embodiments, at least one or more transcription factors include SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors include the GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ.In some embodiments, the one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors include HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the one or more PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of the one or more PSCs into a population of hepatocytes or hepatocytes. In some embodiments, the medium does not change while delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors to the one or more PSCs. In some embodiments, at least one of the one or more hepatocytes or hepatocytes of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes of the population of hepatocytes or hepatocytes does not express CXCR-4. In some embodiments, at least one of the one or more hepatocytes or hepatocytes of the population of hepatocytes or hepatocytes expresses CXCR-4.

[0031] The present disclosure provides a population of hepatocytes or hepatocytes disclosed herein, wherein at least 2% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 3% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 5% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 6% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 10% of the population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 20% of the population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 30% of the PSCs expresses CXCR-4.

[0032] Another aspect of the present disclosure provides a method for generating a population of hepatocytes or liver parenchymal cells, the method comprising providing one or more pluripotent stem cells (PSCs) in a medium; delivering to the one or more PSCs a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors further induce the differentiation of the one or more PSCs into a population of hepatocytes or liver parenchymal cells; and generating a population of hepatocytes or liver parenchymal cells from the one or more PSCs, wherein the medium is not changed during the differentiation of the one or more PSCs into a population of hepatocytes or liver parenchymal cells. In some embodiments, the medium is not changed while delivering the one or more transcription factors to the one or more PSCs. In some embodiments, the one or more transcription factors include HNF6. In some embodiments, the one or more transcription factors induce the expression of the one or more PSCs into a population of hepatocytes or liver parenchymal cells within 30 days. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, two or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, at least one or more transcription factors include SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ. In some embodiments, one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors include HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.In some embodiments, at least one or more transcription factors induce the expression of one or more PSCs into a population of hepatocytes or hepatocytes within 30 days. In some embodiments, one or more PSCs are provided in the medium. In some embodiments, the medium is not changed during the differentiation of one or more PSCs into a population of hepatocytes or hepatocytes. In some embodiments, the medium is not changed while delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors to one or more PSCs. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes does not express CXCR-4. In some embodiments, at least one of one or more hepatocytes or hepatocytes in a population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 2% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 3% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 5% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 6% of the population of hepatocytes or hepatocytes expresses ASGR-1. In some embodiments, at least 10% of the population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 20% of the population of hepatocytes or hepatocytes expresses CXCR-4. In some embodiments, at least 30% of the PSCs express CXCR-4.

[0033] Another aspect of the present disclosure provides an exogenous expression cassette comprising one or more transcription factors that induce the differentiation of pluripotent stem cells (PSCs) into hepatocytes or liver parenchymal cells, and the exogenous expression cassette induces the expression of PSCs into hepatocytes or liver parenchymal cells within 30 days. In some embodiments, the PSCs are provided in a medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver parenchymal cells. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B. In some embodiments, two or more of the transcription factors are of a transcription factor family, for example, those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, the three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, the four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, the one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, the one or more transcription factors are, for example, FoxA1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, the one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, the one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors are, for example, additional FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A.In some embodiments, one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.

[0034] The present disclosure provides hepatocytes or liver cells disclosed herein, and the hepatocytes or liver cells express ASGR-1. In some embodiments, the hepatocytes or liver cells do not express CXCR-4. In some embodiments, the hepatocytes or liver cells express CXCR-4.

[0035] The present disclosure provides a population of hepatocytes or liver cells comprising two or more of the hepatocytes or liver cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0036] Another aspect of the present disclosure provides an exogenous expression cassette comprising one or more transcription factors that induce the differentiation of pluripotent stem cells (PSCs) into hepatocytes or hepatocytes, where the PSCs are provided in a medium that is not changed during the differentiation of the PSCs into hepatocytes or hepatocytes. In some embodiments, the exogenous expression cassette induces the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, the exogenous expression cassette induces the differentiation of PSCs into hepatocytes or hepatocytes within 96 hours. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the transcription factors are of a transcription factor family, for example, those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more of the transcription factors are of a transcription factor family, for example, those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, and HNF4A.In some embodiments, one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express ASGR-1. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4.

[0037] The present disclosure provides a population of hepatocytes or liver cells comprising two or more of the hepatocytes or liver cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0038] Another aspect of the present disclosure provides an exogenous expression cassette that induces the differentiation of pluripotent stem cells (PSCs) into hepatocytes or hepatocytes, and the exogenous expression cassette includes the HNF6 transcription factor and one or more additional transcription factors. In some embodiments, the PSCs are provided in a medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or hepatocytes. In some embodiments, the exogenous expression cassette induces the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, the exogenous expression cassette induces the differentiation of PSCs into hepatocytes or hepatocytes within 96 hours. In some embodiments, the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the transcription factors are of transcription factor families, for example, those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, three or more of the transcription factors are of transcription factor families, for example, those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, four or more transcription factors are of transcription factor families such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of transcription factor families such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more additional transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more additional transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more additional transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more additional transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, one or more additional transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, one or more additional transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1.In some embodiments, one or more additional transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, one or more additional transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, one or more additional transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, one or more additional transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express ASGR-1. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4.

[0039] The present disclosure provides a population of hepatocytes or liver cells comprising two or more hepatocytes or liver cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0040] Another aspect of the present disclosure provides a hepatocyte or a liver cell comprising one or more nucleic acids comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or one or more transcriptional activators of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors induce the differentiation of pluripotent stem cells (PSCs) into hepatocytes or liver cells within 30 days. In some embodiments, the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more transcription factors induce the differentiation of PSCs into hepatocytes or liver cells within 96 hours. In some embodiments, the PSCs are provided in a medium. In some embodiments, the medium is not changed during the differentiation of PSCs into hepatocytes or liver cells. In some embodiments, two or more transcription factors are of a transcription factor family, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, HNF1A, SPI1, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, FoxA1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the hepatocytes or liver cells express ASGR-1. In some embodiments, the hepatocytes or liver cells do not express CXCR-4. In some embodiments, the hepatocytes or liver cells express CXCR-4.

[0041] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0042] Another aspect of the present disclosure provides a hepatocyte or a liver cell comprising one or more nucleic acids comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or one or more transcriptional activators of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors induce differentiation of pluripotent stem cells (PSCs) into hepatocytes or liver cells, and the medium is not changed during differentiation of PSCs into hepatocytes or liver cells. In some embodiments, the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more exogenous expression cassettes induce the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, two or more transcription factors are of a transcription factor family, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express ASGR-1. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4.

[0043] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0044] Another aspect of the present disclosure provides ASGR-expressing cells comprising one or more nucleic acids comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or one or more transcriptional activators of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors induce differentiation of pluripotent stem cells (PSCs) into ASGR-1-expressing cells, and the medium is not changed during differentiation of PSCs into hepatocytes or liver parenchymal cells. In some embodiments, the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more exogenous expression cassettes induce the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, two or more transcription factors are of a transcription factor family, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the hepatocytes or liver cells express ASGR-1. In some embodiments, the hepatocytes or liver cells do not express CXCR-4. In some embodiments, the hepatocytes or liver cells express CXCR-4.

[0045] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0046] The present disclosure provides ASGR-1-expressing cells comprising one or more nucleic acids comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or one or more transcriptional activators of an open reading frame encoding one or more transcription factors, wherein the nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces differentiation of pluripotent stem cells (PSCs) into ASGR-1-expressing cells within 30 days.

[0047] The present disclosure provides a population of hepatocytes or hepatocyte parenchymal cells comprising two or more hepatocytes or hepatocyte parenchymal cells disclosed herein. In some embodiments, one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more exogenous expression cassettes induce the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, two or more transcription factors are of a transcription factor family, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the hepatocytes or liver cells express ASGR-1. In some embodiments, the hepatocytes or liver cells do not express CXCR-4. In some embodiments, the hepatocytes or liver cells express CXCR-4.

[0048] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more of the hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0049] Another aspect of the present disclosure provides CXCR-4-expressing cells comprising one or more nucleic acids comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or one or more transcriptional activators of an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors induce differentiation of pluripotent stem cells (PSCs) into CXCR-4-expressing cells, and the medium is not changed during differentiation of PSCs into hepatocytes or hepatocytes. In some embodiments, the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, two or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more additional transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more exogenous expression cassettes induce the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, two or more transcription factors are of a transcription factor family, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family, such as those of ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1, or any combination thereof. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX, or any combination thereof. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1, or any combination thereof.In some embodiments, the one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3, or any combination thereof. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3, or any combination thereof. In some embodiments, the hepatocytes or liver cells express CXCR-4. In some embodiments, the hepatocytes or liver cells do not express CXCR-4. In some embodiments, the hepatocytes or liver cells express CXCR-4.

[0050] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more of the hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0051] Another aspect of the present disclosure provides CXCR-4-expressing cells, the CXCR-4-expressing cells comprising: (a) one or more nucleic acids, each comprising an open reading frame encoding one or more transcription factors; (b) one or more transcription factors; and / or (c) one or more transcriptional activators of the open reading frame encoding one or more transcription factors, wherein the nucleic acid comprising the open reading frame encoding one or more transcription factors, the one or more transcription factors, or the transcriptional activator of the open reading frame encoding one or more transcription factors induces differentiation of pluripotent stem cells (PSCs) into CXCR-4-expressing cells within 30 days.

[0052] In some embodiments, a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator of an open reading frame encoding one or more transcription factors induces the differentiation of pluripotent stem cells (PSCs) into CXCR-4 expressing cells within 96 hours. In some embodiments, the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, TP53 family members, or any combination thereof. In some embodiments, two or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members.In some embodiments, three or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, four or more of the one or more transcription factors are, for example, basic leucine zipper transcription factors, forkhead box (FOX) transcription factors, HNF hepatocyte nuclear factor transcription factors, GATA zinc finger transcription factors, NKL subclass homeobox and pseudogene transcription factors, basic helix-loop-helix transcription factors, CUT homeobox transcription factors, NR nuclear receptor family members, IPT (Ig-like, plexin, transcription factor) domains, nuclear factor I (NFI) family transcription factors, SMAD proteins, SRY-box family members, helix-turn-helix ETS family members, T-box family members, and TP53 family members. In some embodiments, one or more exogenous expression cassettes induce the differentiation of PSCs into hepatocytes or hepatocytes within 30 days. In some embodiments, two or more transcription factors are of transcription factor families, for example, ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP.In some embodiments, three or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, four or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, five or more transcription factors are of a transcription factor family such as ATF, CEBP, FOS, FOX, GATA, HHEX, HIF, HNF, JUN, MYC, NFE, NFI, NR, ONECUT, RBPJ, RXR, SMAD, SOX, SPI, TBX, PPAR, FOS, JUN, SKI, SALL, EGR, and TP. In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, and NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 isoform P23769-2, and GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, and FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, and NR1I3. In some embodiments, the transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / , ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, hepatocytes or liver cells express CXCR-4. In some embodiments, hepatocytes or liver cells do not express CXCR-4. In some embodiments, hepatocytes or liver cells express CXCR-4.

[0053] The present disclosure provides a population of hepatocytes or hepatic parenchymal cells comprising two or more of the hepatocytes or hepatic parenchymal cells disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 3% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 5% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 6% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 10% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatic parenchymal cells express CXCR-4. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed.

[0054] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, where only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other embodiments and different embodiments, and various details thereof can be modified in various obvious respects without departing from the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

[0055] Incorporation by reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure herein, the present specification is intended to take precedence over and / or supersede any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures and (FIG.)").

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Mode for Carrying Out the Invention

[0057] Although embodiments of the present invention are shown and described in this specification, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be utilized.

[0058] Pluripotent stem cells (PSCs) are characterized by their ability to self-renew while maintaining efficacy for therapeutic use. PSCs further have the ability to differentiate into a vast number of different phenotypes (e.g., hepatocytes). Disclosed herein are various compositions, formulations, and methods that promote efficient differentiation of PSCs into hepatocytes (and / or liver cells).

[0059] Hepatocytes or liver cells generated from the use of the compositions, formulations, and methods disclosed herein can be used for therapeutic applications in which classical lineage hepatocytes have conventionally been used. Thus, the various compositions, formulations, and methods disclosed herein represent a significant improvement over hepatocyte-based therapeutics because the compositions, formulations, and methods disclosed herein enable efficient and reproducible differentiation of PSCs into robust hepatocytes and / or liver cells.

[0060] PSCs can divide indefinitely and produce identical daughter cells. When signals are provided, PSCs can differentiate into various phenotypes. In one example, PSCs are embryonic stem cells (ESCs). In another example, PSCs are induced pluripotent stem cells (iPSCs).

[0061] In some aspects, the disclosure provides methods and compositions for efficient and reproducible differentiation of PSCs (defined, e.g., by cell surface marker expression signature, function, etc.) into hepatocytes or liver cells. These methods and compositions can represent a significant improvement over current cell therapies that utilize hepatocyte or liver cell administration (e.g., availability, dosage, efficacy, etc.).

[0062] Pluripotent stem cells Pluripotent stem cells (PSCs) can be characterized by self-renewal and potency. PSCs can divide indefinitely and produce identical daughter cells. When signals are provided, PSCs can differentiate into various phenotypes. In one example, PSCs are embryonic stem cells (ESCs). In another example, PSCs are induced pluripotent stem cells (iPSCs). PSCs (e.g., ESCs and iPSCs) express TRA-1-60. TRA-1-60 expression indicates the ability of the cells to differentiate.

[0063] iPSCs are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed into an ES cell-like state by the expression of genes and factors important for the maintenance of definitive characteristics of embryonic stem (ES) cells. iPSCs are similar to ES cells in many aspects, including the expression of ES cell markers, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, pluripotency, and the ability to contribute to many different tissues in vitro. Studies have reported the directed differentiation of iPSCs into various functional cell types in vitro, and the cell therapy effects of transplanted iPSCs have been demonstrated in several animal models of disease. Directed differentiation is a biotechnological method that utilizes the potential of stem cells by restricting the differentiation of stem cells in vitro towards a specific cell type or tissue of interest. Directed differentiation can be applied primarily to PSCs of mammalian origin, such as mouse and human cells for biomedical research purposes. The differentiation of cells can involve the transformation from a proliferative mode to a differentiated mode. Directed differentiation can involve mimicking the developmental culture under controlled conditions that include specific substrates or extracellular matrices that promote cell adhesion and differentiation, and defining the culture medium composition. Signaling factors such as growth factors or small molecules can be applied in various doses and exposure times, either continuously or in a combinatorial manner, to regulate differentiation. Direct reprogramming, also known as differentiation conversion or direct conversion, can involve overexpressing one or more factors introduced into the cells. In one example, the one or more factors can be transcription factors. Appropriate differentiation of the cell type of interest can be verified by analyzing cell type-specific markers, gene expression profiles, and functional assays.

[0064] The present disclosure provides pluripotent stem cells that can be induced to differentiate into one or more hepatocytes or liver cells. The pluripotent stem cells can be induced to express one or more transcription factors and can be induced to differentiate into one or more hepatocytes or liver cells by various methods including contacting with one or more transcription factor proteins or nucleic acids encoding the transcription factor proteins, or by other means disclosed herein. The one or more nucleic acids can be expressed in one or more exogenous expression cassettes. The one or more exogenous expression cassettes can contain one or more transcription factors. The one or more transcription factors can induce the differentiation of PSCs. The one or more transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells. The one or more transcription factors can induce the differentiation of PSCs into hepatocytes, or liver cells, or cells expressing ASGR-1, or cells expressing CXCR-4, or cells expressing cytokeratin-18 (CK18), or cells expressing α-fetoprotein (AFP), or cells expressing α-1-antitrypsin (A1AT), or cells expressing albumin, or cells expressing the cytochrome p450 subfamily CYP3A7, or cells expressing the cytochrome p450 subfamily CYP3A4, or any combination thereof. The pluripotent stem cells can be induced to differentiate into one or more hepatocytes or liver cells within 30 days. The pluripotent stem cells can be induced to differentiate into one or more hepatocytes or liver cells within 96 hours. The exogenous expression cassette can induce the differentiation of PSCs into hepatocytes or liver cells within 30 days. The exogenous expression cassette can induce the differentiation of PSCs into hepatocytes or liver cells within 96 hours. The PSCs can be provided in a medium. The medium may not need to be changed during the differentiation of PSCs into hepatocytes or liver cells. The medium can be changed during the differentiation of PSCs into hepatocytes or liver cells. A population of cells containing one or more hepatocytes or liver cells can be generated.The cell population includes one or more hepatocytes, or liver cells, cells expressing ASGR-1, cells expressing CXCR-4, or cells expressing cytokeratin-18 (CK18), or cells expressing α-fetoprotein (AFP), or cells expressing α-1-antitrypsin (A1AT), or cells expressing albumin, or cells expressing cytochrome p450. The cell population may include adherent cells. The cell population may include suspension cells. The cell population may include adherent cells and suspension cells. The cell population may be provided in a medium. The medium may not need to be changed during the differentiation of PSCs into hepatocytes or liver cells. The medium may not require any optimization of nutrients, growth factors, or microenvironment or matrix. The medium may require optimization of any nutrients, growth factors, or microenvironment or matrix. In some embodiments, no optimization of any nutrients, growth factors, or microenvironment / matrix is performed. In some embodiments, optimization of nutrients, growth factors, or microenvironment / matrix is performed. In some embodiments, optimization of nutrients, growth factors, and microenvironment / matrix is performed. In some embodiments, no optimization of any nutrients is performed. In some embodiments, no optimization of any growth factors is performed. In some embodiments, no optimization of any microenvironment / matrix is performed. In some embodiments, no optimization of any microenvironment is performed. In some embodiments, no optimization of any matrix is performed. In some embodiments, no optimization of any nutrients or growth factors is performed. In some embodiments, no optimization of any nutrients or microenvironment / matrix is performed. In some embodiments, no optimization of any growth factors or microenvironment / matrix is performed. In some embodiments, no optimization of any nutrients or microenvironment is performed. In some embodiments, no optimization of any growth factors or microenvironment is performed. In some embodiments, no optimization of any nutrients or matrix is performed.In some embodiments, no optimization of any growth factor or matrix is performed.

[0065] Hepatocytes Hepatocytes can be present in the liver. Hepatocytes can be involved in protein synthesis, protein storage, carbohydrate transformation, cholesterol synthesis, bile salt synthesis, phospholipid synthesis, detoxification, and bile secretion. Hepatocytes can be cubical. In one example, hepatocytes can have a side length of 20 - 30 microns. In another example, hepatocytes can have a volume of 3.4×10 -9 cm 3 . Hepatocytes can be used to explore the mechanisms of drug metabolism. Hepatocytes can be used to predict in vivo drug metabolism. Hepatocytes can be isolated by collagenase digestion.

[0066] In some embodiments, the hepatocytes can express asialoglycoprotein receptor 1 (ASGR-1), also known as ASGR1, ASGPR, ASGPR1, CLEC4H1, HL-1, or asialoglycoprotein receptor 1. ASGR-1 (e.g., its subunits) can be encoded by the ASGR-1 gene. The ASGR-1 protein can act as a receptor protein. ASGR-1 can be a transmembrane protein. ASGR-1 can play a role in serum glycoprotein homeostasis. ASGR-1 can mediate the endocytosis of glycoproteins. ASGR-1 can mediate the endocytosis of glycoproteins having exposed terminal galactose. ASGR-1 can mediate the endocytosis of glycoproteins having N-acetylgalactosamine residues. ASGR-1 can promote liver infection. ASGR-1 can promote viral liver infection. ASGR-1 can promote hepatitis B-induced liver infection. ASGR-1 can be a target for liver-specific drug delivery. ASGR-1 can be a hetero-oligomeric protein. ASGR-1 can include a main subunit and a minor subunit. The main subunit and the minor subunit can be encoded by different genes. Multiple isoforms of ASGR-1 can be encoded via alternative splicing.

[0067] In some embodiments, the hepatocytes can express, for example, fusin, CXCR4, CD184, D2S201E, FB22, HM89, HSY3RR, LAP-3, LAP3, LCR1, LESTR, NPY3R, NPYR, NPYRL, NPYY3R, WHIM, WHIMS, chemokine receptor type 4 protein CXCR-4, also known as WHIMS1. CXCR-4 can be classified as a CXC chemokine receptor. CXCR-4 can be regarded as an α-chemokine receptor. CXCR-4 can be specific for stromal cell-derived factor-1 (SDF-1). SDF-1 can exhibit chemotaxis for lymphocytes. SDF-1 can be important in hematopoietic stem cell homing to the bone marrow. SDF-1 can be important in hematopoietic stem cell quiescence. CXCR-4 can help with liver regeneration. CXCR-4 can be fibrotic-promoting. CXCR-4 can be fibrotic-promoting through the activation of hepatic stellate cells. CXCR-4 signaling can be detrimental to liver recovery and regeneration. Clinical therapies using CXCR-4 antagonists can improve liver recovery after acute liver injury. In liver diseases, abnormal CXCR-4 expression can be related to the migration and movement of liver-specific cells. In liver diseases, abnormal CXCR-4 expression can be related to the migration and movement of liver-specific cells through crosstalk with other pathways. CXCR-4 and its ligand can play a role in hepatitis. The mechanism of the inflammatory response mediated by CXCR-4 signaling can affect (e.g., detrimentally) the chemotaxis of inflammatory cells (e.g., lymphocytes, neutrophils, and monocytes). CXCR-4 can be used by HIV to infect T cells. The CXCR-4 receptor can be involved in the adhesion phase of human transplantation. CXCR-4 signaling can regulate the expression of CD20 on B cells. Ubiquitin can be a natural ligand of CXCR-4. The dimerization of CXCR-4 can be dynamic. The dimerization of CXCR-4 can increase with concentration. CXCR-4 can be expressed in cancers including breast cancer, ovarian cancer, melanoma, or prostate cancer.

[0068] In one aspect, the present disclosure provides one or more hepatocytes, or one or more liver cells, or one or more cells expressing ASGR-1, or one or more cells expressing CXCR-4, or one or more cells expressing cytokeratin-18 (CK18), or one or more cells expressing alpha-fetoprotein (AFP), or one or more cells expressing alpha-1-antitrypsin (A1AT), or one or more cells expressing albumin, or one or more cells expressing cytokine p450 subfamily CYP3A7 or cytokine p450 subfamily CYP3A4, or any other hepatocyte-like marker, hepatocyte lineage marker or hepatocyte-related marker disclosed herein, or cells expressing any other marker disclosed herein related to the (TF recipe) non-natural exogenous induction of the differentiation of iPSCs along a differentiation trajectory. In some embodiments, the hepatocytes, or liver cells, or cells expressing ASGR-1, cells expressing CXCR-4, cells expressing cytokeratin-18 (CK18), or cells expressing alpha-fetoprotein (AFP), or cells expressing alpha-1-antitrypsin (A1AT), or cells expressing albumin, or cells expressing cytokine p450 subfamily CYP3A7, or cells expressing cytokine p450 subfamily CYP3A4 may comprise one or more exogenous expression cassettes. In some embodiments, the one or more exogenous expression cassettes comprise one or more transcription factors that induced the differentiation of one or more PSCs.Exogenous expression cassettes can induce the differentiation of PSCs into cells that express hepatocytes, hepatocytes, cells expressing ASGR-1, cells expressing CXCR-4, cells expressing cytokeratin-18 (CK18), or cells expressing α-fetoprotein (AFP), or cells expressing α-1-antitrypsin (A1AT), or cells expressing albumin, or cells expressing cytochrome p450 subfamily CYP3A7, or cells expressing cytochrome p450 subfamily CYP3A4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the non-natural exogenous induction of the differentiation of iPSCs along a differentiation trajectory (TF recipe). In some embodiments, the PSCs can be provided in a medium. The medium need not be changed during the differentiation of PSCs into cells that express hepatocytes, hepatocytes, or cells expressing ASGR-1, cells expressing CXCR-4, cells expressing cytokeratin-18 (CK18), or cells expressing α-fetoprotein (AFP), or cells expressing α-1-antitrypsin (A1AT), or cells expressing albumin, or cells expressing cytochrome p450 subfamily CYP3A7, or cells expressing cytochrome p450 subfamily CYP3A4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the non-natural exogenous induction of the differentiation of iPSCs along a differentiation trajectory (TF recipe). In some embodiments, the PSCs can be provided in a medium.

[0069] The present disclosure provides PSCs that can be induced to differentiate into one or more hepatocytes or hepatocytes. Pluripotent stem cells can be induced to express one or more transcription factors, by various methods including contacting with one or more transcription factor proteins or nucleic acids encoding transcription factor proteins, or by other means disclosed herein, to differentiate into one or more hepatocytes or hepatocytes. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes within 30 days. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes within up to about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes within up to about 1 day. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes in at least up to 4 days. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes within up to about 96 hours. PSCs can be induced to differentiate into one or more hepatocytes or hepatocytes within up to about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10 hours. One or more nucleic acids can be expressed in one or more exogenous expression cassettes. One or more exogenous expression cassettes can include one or more transcription factors. One or more transcription factors can induce the differentiation of PSCs. One or more transcription factors can induce the differentiation of PSCs into hepatocytes or hepatocytes. PSCs can include one or more exogenous expression cassettes. One or more exogenous cassettes can include one or more transcription factors that induce the differentiation of PSCs into cells expressing hepatocytes, hepatocytes, or ASGR-1, CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of iPSC differentiation along the differentiation trajectory. The exogenous expression cassette can induce the differentiation of PSCs into hepatocytes, hepatocytes, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4 within 30 days.Exogenous expression cassettes can induce the differentiation of PSCs into hepatocytes, hepatocytes, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4 within 96 hours. The PSCs can be provided in the medium. The medium may not need to be changed during the differentiation of PSCs into hepatocytes, hepatocytes, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4.

[0070] The present disclosure provides a population of cells comprising one or more hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of differentiation of iPSCs along a differentiation trajectory. The population of cells can comprise adherent cells. The population of cells can comprise suspension cells. The population of cells can comprise adherent cells and suspension cells. The population of cells can be provided in a medium. The medium may not need to be changed during the differentiation of PSCs into cells expressing a hepatocyte, liver cell, cell expressing ASGR-1, cell expressing CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of differentiation of iPSCs along a differentiation trajectory. The population of cells can be provided in a medium. The medium may not require any nutrient, growth factor, or optimization of the microenvironment or matrix. At least 5% of the cells can express ASGR-1. At least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the cells can express cells expressing ASGR-1, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of differentiation of iPSCs along a differentiation trajectory.At least 5% of the cells can express cells that can express CXCR-1, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein that is related to the (TF recipe) non-natural exogenous induction of the differentiation of iPSCs along the differentiation trajectory. At least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the cells can express ASGR-1.

[0071] In some embodiments, at least 0.01% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 0.1% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 0.5% of hepatocytes or liver parenchymal cells express ASGR-1, or any other liver parenchymal cell-like marker, liver parenchymal cell lineage marker, or liver parenchymal cell-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-natural exogenous induction of differentiation of iPSCs along a differentiation trajectory. In some embodiments, at least 1% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 1.5% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 2% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 3% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 4% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 5% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 6% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 7% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 8% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 9% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 10% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 12% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 15% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 17% of hepatocytes or liver parenchymal cells express ASGR-1. In some embodiments, at least 20% of hepatocytes or liver parenchymal cells express ASGR-1.In some embodiments, at least 25% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 30% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 35% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 40% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 45% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 50% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 60% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 70% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 80% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 90% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 95% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 99% of the hepatocytes or liver cells express ASGR-1.

[0072] In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of the differentiation of iPSCs along a differentiation trajectory, is at least about 0.01%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 12%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 is up to about 0.01%, up to about 0.1%, up to about 0.5%, up to about 1%, up to about 1.5%, up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 12%, up to about 15%, up to about 17%, up to about 20%, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 91%, up to about 92%, up to about 93%, up to about 94%, up to about 95%, up to about 96%, up to about 97%, up to about 98%, up to about 99%, up to about 99.55, or up to about 99.9%.

[0073] In some embodiments, the percentage of hepatocytes or liver parenchymal cells expressing ASGR-1 is from about 1% to about 90%. In some embodiments, the percentage of hepatocytes or liver parenchymal cells expressing ASGR-1, or any other liver parenchymal cell-like marker, liver parenchymal cell lineage marker, or liver parenchymal cell-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-natural exogenous induction of differentiation of iPSCs along a differentiation trajectory, of the cells expressing the marker is at least about 1%. In some embodiments, the percentage of hepatocytes or liver parenchymal cells expressing ASGR-1 is up to about 90%. In some embodiments, the percentage of hepatocytes or liver parenchymal cells expressing ASGR-1 is about 1% to about 2%, about 1% to about 3%, about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 50%, about 1% to about 90%, about 2% to about 3%, about 2% to about 5%, about 2% to about 6%, about 2% to about 7%, about 2% to about 8%, about 2% to about 10%, about 2% to about 20%, about 2% to about 30%, about 2% to about 50%, about 2% to about 90%, about 3% to about 5%, about 3% to about 6%, about 3% to about 7%, about 3% to about 8%, about 3% to about 10%, about 3% to about 20%, about 3% to about 30%, about 3% to about 50%, about 3% to about 90%, about 5% to about 6%, about 5% to about 7%, about 5% to about 8%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 50%, about 5% to about 90%, about 6% to about 7%, about 6% to about 8%, about 6% to about 10%, about 6% to about 20%, about 6% to about 30%, about 6% to about 50%, about 6% to about 90%, about 7% to about 8%, about 7% to about 10%, about 7% to about 20%, about 7% to about 30%, about 7% to about 50%, about 7% to about 90%, about 8% to about 10%, about 8% to about 20%, about 8% to about 30%, about 8% to about 50%, about 8% to about 90%, about 10% to about 20%, about 10% to about 30%, about 10% to about 50%, about 10% to about 90%, about 20% to about 30%, about 20% to about 50%, about 20% to about 90%, about 30% to about 50%, about 30% to about 90%, or about 50% to about 90%.In some embodiments, the percent of hepatocytes or liver parenchymal cells expressing ASGR-1 is about 1%, about 2%, about 3%, about 5%, about 6%, about 7%, about 8%, about 10%, about 20%, about 30%, about 50%, or about 90%.

[0074] In some embodiments, at least 0.01% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 0.1% of the hepatocytes or liver cells express cells that express CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-natural exogenous induction of the differentiation of iPSCs along a differentiation trajectory. In some embodiments, at least 0.5% of the hepatocytes or liver cells express cells that express CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-natural exogenous induction of the differentiation of iPSCs along a differentiation trajectory. In some embodiments, at least 1% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 1.5% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 2% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 3% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 4% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 5% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 6% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 7% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 8% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 9% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 10% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 12% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 15% of the hepatocytes or liver cells express CXCR-4.In some embodiments, at least 17% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 20% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 25% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 30% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 35% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 40% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 45% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 50% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 60% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 70% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 80% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 90% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 95% of the hepatocytes or liver cells express CXCR-4. In some embodiments, at least 99% of the hepatocytes or liver cells express CXCR-4.

[0075] In some embodiments, hepatocytes or liver parenchymal cells that express CXCR-4 are at least about 0.01%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 12%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, hepatocytes or liver parenchymal cells that express CXCR-4, or any other liver parenchymal cell-like marker, liver parenchymal cell lineage marker, or liver parenchymal cell-related marker disclosed herein, or any other marker disclosed herein related to the (TF recipe) non-native exogenous induction of differentiation of iPSCs along a differentiation trajectory, are at most about 0.01%, at most about 0.1%, at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 3%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 12%, at most about 15%, at most about 17%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at most about 99.55, or at most about 99.9%.

[0076] In some embodiments, the percent of hepatocytes or liver parenchymal cells that express CXCR-4 is from about 1% to about 90%. In some embodiments, the percent of hepatocytes or liver parenchymal cells that express CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein that is associated with non-native exogenous induction of (TF recipe) differentiation of iPSCs along a differentiation trajectory, is at least about 1%. In some embodiments, the percent of hepatocytes or liver parenchymal cells that express CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein that is associated with non-native exogenous induction of (TF recipe) differentiation of iPSCs along a differentiation trajectory, is at most about 90%.In some embodiments, the percent of hepatocytes or liver parenchymal cells that express CXCR-4 is from about 1% to about 2%, from about 1% to about 3%, from about 1% to about 5%, from about 1% to about 6%, from about 1% to about 7%, from about 1% to about 8%, from about 1% to about 10%, from about 1% to about 20%, from about 1% to about 30%, from about 1% to about 50%, from about 1% to about 90%, from about 2% to about 3%, from about 2% to about 5%, from about 2% to about 6%, from about 2% to about 7%, from about 2% to about 8%, from about 2% to about 10%, from about 2% to about 20%, from about 2% to about 30%, from about 2% to about 50%, from about 2% to about 90%, from about 3% to about 5%, from about 3% to about 6%, from about 3% to about 7%, from about 3% to about 8%, from about 3% to about 10%, from about 3% to about 20%, from about 3% to about 30%, from about 3% to about 50%, from about 3% to about 90%, from about 5% to about 6%, from about 5% to about 7%, from about 5% to about 8%, from about 5% to about 10%, from about 5% to about 20%, from about 5% to about 30%, from about 5% to about 50%, from about 5% to about 90%, from about 6% to about 7%, from about 6% to about 8%, from about 6% to about 10%, from about 6% to about 20%, from about 6% to about 30%, from about 6% to about 50%, from about 6% to about 90%, from about 7% to about 8%, from about 7% to about 10%, from about 7% to about 20%, from about 7% to about 30%, from about 7% to about 50%, from about 7% to about 90%, from about 8% to about 10%, from about 8% to about 20%, from about 8% to about 30%, from about 8% to about 50%, from about 8% to about 90%, from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 50%, from about 10% to about 90%, from about 20% to about 30%, from about 20% to about 50%, from about 20% to about 90%, from about 30% to about 50%, from about 30% to about 90%, or from about 50% to about 90%. In some embodiments, the percent of hepatocytes or liver parenchymal cells that express CXCR-4 is about 1%, about 2%, about 3%, about 5%, about 6%, about 7%, about 8%, about 10%, about 20%, about 30%, about 50%, or about 90%.

[0077] The present disclosure provides one or more hepatocytes. The hepatocytes may be similar to hepatocytes. The hepatocytes may be polygonal. For example, the hepatocytes may be hexagonal. The hepatocytes may be cubic. The stem cells may be pluripotent stem cells. The pluripotent stem cells may be induced pluripotent stem cells. The hepatocytes may express ASGR-1. The hepatocytes may express CXCR-4. The hepatocytes may express both ASGR-1 and CXCR-4. In a population of two or more hepatocytes, at least 2% of the hepatocytes express ASGR-1, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the non-natural exogenous induction of (TF recipe) differentiation of iPSCs along a differentiation trajectory. In a population of two or more hepatocytes, at least 10% of the hepatocytes or hepatocytes express CXCR-4, or any other hepatocyte-like marker, hepatocyte lineage marker, or hepatocyte-related marker disclosed herein, or any other marker disclosed herein related to the non-natural exogenous induction of (TF recipe) differentiation of iPSCs along a differentiation trajectory.

[0078] Transcription factor The present disclosure provides transcription factors (TFs), the expression of which can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The transcription factors can induce differentiation in various ways, such as through the expression of one or more nucleic acids encoding one or more transcription factors in PSCs, contacting one or more transcription factor proteins or nucleic acids encoding transcription factor proteins with PSCs, or through other means as disclosed herein. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within 30 days. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within at most about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within at most about 1 day. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within at least at most 4 days. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within at most about 96 hours. The PSCs can be induced to differentiate into one or more hepatocytes or liver parenchymal cells within at most about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10 hours. One or more nucleic acids encoding one or more transcription factors can be expressed in one or more exogenous expression cassettes.

[0079] TFs can be used to initiate a differentiation program. Certain transcription factors may be able to induce stem cells to differentiate into a specific lineage, such as hepatocytes or liver parenchymal cells. In some embodiments, combinations of transcription factors can be used to achieve differentiation into a specific cell lineage. The combination can achieve a cell type or cell subtype that cannot be achieved by any single transcription factor alone. The combination can achieve the same cell type as one of the individual transcription factors but can achieve it more efficiently.

[0080] The present disclosure provides methods that include the use of cells, exogenous expression cassettes, and one or more transcription factors, or molecules that increase transcription or increase transcriptional activators. The one or more transcription factors can induce the differentiation of one or more PSCs. The one or more transcription factors can induce the differentiation of one or more PSCs into one or more hepatocytes or liver cells. Combinations can be made that include one or more transcription factors. The combinations of transcription factors can be introduced into one or more expression cassettes. The one or more expression cassettes can be induced in one or more PSCs. Induction of the one or more expression cassettes in one or more PSCs can differentiate the one or more PSCs into hepatocytes or liver cells. Some transcription factors may require an amount of expression essential for effectively inducing differentiation, such as the equivalent of at least 5, 10, 15, 20, 25, or 50 copies of an open reading frame (ORF) per cell. Other factors may require expression below a particular threshold due to toxicity that can occur at high levels, such as less than 20, 10, or 5 copies per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0081] In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, one or more transcription factors include FoxA1. In some embodiments, one or more transcription factors include one or more members, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, one or more transcription factors include FoxA1. In some embodiments, one or more transcription factors include HNF1A. In some embodiments, one or more transcription factors include SPI1. In some embodiments, one or more transcription factors include FoxA2. In some embodiments, one or more transcription factors include CEBPA. In some embodiments, one or more transcription factors include ONECUT1 / HNF6. In some embodiments, one or more transcription factors include ONECUT2 / HNF6B. In some embodiments, one or more transcription factors include HNF4a. In some embodiments, one or more transcription factors include Atf3. In some embodiments, one or more transcription factors include Fos. In some embodiments, one or more transcription factors include CEBPB.In some embodiments, one or more transcription factors include FoxM1. In some embodiments, one or more transcription factors include GATA2 isoform P23769-2. In some embodiments, one or more transcription factors include GATA4. In some embodiments, one or more transcription factors include GATA2 isoform P23769-1. In some embodiments, one or more transcription factors include JUN / AP-1. In some embodiments, one or more transcription factors include NFIX. In some embodiments, one or more transcription factors include GATA6. In some embodiments, one or more transcription factors include TBX3. In some embodiments, one or more transcription factors include RXRB. In some embodiments, one or more transcription factors include NFE2L2. In some embodiments, one or more transcription factors include Myc. In some embodiments, one or more transcription factors include TP73. In some embodiments, one or more transcription factors include HHEX. In some embodiments, one or more transcription factors include SOX17. In some embodiments, one or more transcription factors include ATF5. In some embodiments, one or more transcription factors include RBPJ. In some embodiments, one or more transcription factors include Smad3. In some embodiments, one or more transcription factors include NR5A2. In some embodiments, one or more transcription factors include HIF1A. In some embodiments, one or more transcription factors include PROX. In some embodiments, one or more transcription factors include FOXA3. In some embodiments, one or more transcription factors include EGR1. In some embodiments, one or more transcription factors include NR1I2 / PXR. In some embodiments, one or more transcription factors include SKI.

[0082] In some embodiments, one or more transcription factors include ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, one or more transcription factors include ONECUT1 / HNF6 and ONECUT2 / HNF6B.

[0083] In some embodiments, one or more of the transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, two or more of the one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, three or more of the one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A.In some embodiments, four or more of the one or more transcription factors are, for example, FoxA1, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, or PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, HIF1A. In some embodiments, five or more of the one or more transcription factors are selected from the group consisting of, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, six or more of the one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, or HIF1A.In some embodiments, seven or more of the one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A. In some embodiments, eight or more of the one or more transcription factors are, for example, FoxA1, HNF1A, SPI1, FoxA2, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, HNF4a, Atf3, Fos, CEBPB, FoxM1, GATA2 transcription factor, GATA4, JUN / AP-1, NFIX, GATA6, TBX3, RXRB, NFE2L2, Myc, TP73, HHEX, SOX17, ATF5, RBPJ, Smad3, NR5A2, NR1I3, PROX, FOXA3, EGR1, NR1I2 / PXR, SKI, or HIF1A.

[0084] In some embodiments, one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, CEBPA, HNF6, HNF4a, or HNF6B. In some embodiments, one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, one or more transcription factors are, for example, SPI1, FoxA2, GATA2 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, or NFIX. In some embodiments, one or more transcription factors are, for example, HNF4a, CEBPA, HNF1A, TBX3, Fos, GATA2 isoform P23769-2, or GATA2 isoform P23769-1. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, or HNF6. In some embodiments, one or more transcription factors are, for example, GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, NFIX. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, or FoxA1. In some embodiments, one or more transcription factors are, for example, HHEX, SOX17, or HNF4A. In some embodiments, one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, or HNF1A. In some embodiments, one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, CEBPA, ONECUT1 / HNF6, HNF4a, or RBPJ. In some embodiments, one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, or NR1I3.In some embodiments, the one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, or TBX3.

[0085] Forkhead box (FOX) transcription factor The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the FOX family of transcription factors. The FOX transcription factor can be, for example, FoxA1, FoxA2, FoxA3, FoxM1.

[0086] FoxA1

[0087] The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. In some embodiments, one or more transcription factors can be used. In some embodiments, the transcription factor can be FoxA1, also known as HNF3A; hepatocyte nuclear factor 3-alpha; forkhead box protein A1; transcription factor 3A; HNF-3-alpha; HNF-3A; TCF-3A; TCF3A, or hepatocyte nuclear factor 3, alpha.

[0088] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors can include FoxA1. FoxA1 can be introduced into the expression cassette. The expression cassette comprising FoxA1 can be introduced into PSCs. The expression cassette comprising FoxA1 can be expressed in PSCs. The expression cassette comprising FoxA1 can induce the differentiation of PSCs into hepatocytes or hepatocyte-like cells.

[0089] FoxA1 can be introduced into an expression cassette together with one or more other transcription factors. Combinations containing one or more transcription factors can be made. The combination of transcription factors can be introduced into an expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette containing FoxA1 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0090] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include FoxA1. An expression cassette containing FoxA1 can be expressed in PSCs. An expression cassette containing FoxA1 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of FoxA1 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of FoxA1 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of FoxA1 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0091] In some embodiments, the copy number of the ORF for FoxA1 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for FoxA1 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for FoxA1 introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for FoxA1 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for FoxA1 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0092] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include FoxA1. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0093] FoxM1 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be FoxM1, also known as forkhead box M1; HFH-11; MPP2; M-phase phosphoprotein 2; MPHOSPH2; FKHL16; HNF-3; INS-1; hepatocyte nuclear factor 3 forkhead homolog 11; winged helix factor from INS-1 cells; MPM-2 reactive phosphoprotein 2; forkhead-related protein FKHL16; transcription factor Trident; HNF-3 / forkhead homolog 11; forkhead box protein M1; Trident; HFH11; TGT3; forkhead, Drosophila, homolog-like 16; forkhead box M1-D; Trident; FOXM1A; FOXM1B; FOXM1C; MPP-2; PIG29; or WIN.

[0094] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include FoxM1. FoxM1 can be introduced into the expression cassette. FoxM1 can be expressed in the expression cassette. The expression cassette comprising FoxM1 can be introduced into PSCs. The expression cassette comprising FoxM1 can be expressed in PSCs. The expression cassette comprising FoxM1 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0095] FoxM1 can be introduced into an expression cassette together with one or more other transcription factors. Combinations containing one or more transcription factors can be made. The combination of transcription factors can be introduced into an expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette containing FoxM1 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0096] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include FoxM1. An expression cassette containing FoxM1 can be expressed in PSCs. An expression cassette containing FoxM1 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of FoxM1 can be introduced into PSCs. Various amounts of FoxM1 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of FoxM1 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of FoxM1 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0097] In some embodiments, the copy number of the ORF for FoxM1 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for FoxM1 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for FoxM1 introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for FoxM1 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for FoxM1 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0098] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or hepatic cells. The one or more transcription factors may include FoxM1. The one or more hepatocytes or hepatic cells can be differentiated from the one or more PSCs. The one or more hepatocytes or hepatic cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or hepatic cells.

[0099] FoxA2 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be FoxA2, also known as forkhead box A2; HNF3B; hepatocyte nuclear factor 3-β; forkhead box protein A2; transcription factor 3B; HNF-3-β; TCF3B; hepatocyte nuclear factor 3, β; hepatic nuclear factor-3-β; HNF-3B; or TCF-3B.

[0100] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include FoxA2. FoxA2 can be introduced into the expression cassette. The expression cassette comprising FoxA2 can be introduced into PSCs. FoxA2 can be introduced into the expression cassette. FoxA2 can be expressed in the expression cassette. The expression cassette comprising FoxA2 can be introduced into PSCs. The expression cassette comprising FoxA2 can be expressed in PSCs. The expression cassette comprising FoxA2 can induce the differentiation of PSCs into hepatocytes or hepatic cells.

[0101] FoxA2 can be introduced into the expression cassette together with one or more other transcription factors. A combination comprising one or more transcription factors can be made. The combination of transcription factors can be introduced into the expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. The expression cassette comprising FoxA2 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or hepatic cells.

[0102] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors can include FoxA2. An expression cassette containing FoxA2 can be expressed in PSCs. An expression cassette containing FoxA2 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells. Various amounts of FoxA2 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of FoxA2 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of FoxA2 can be introduced per cell. An increase in the expression level can be further achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0103] In some embodiments, the copy number of the ORF for FoxA2 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for FoxA2 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for FoxA2 introduced per cell can be up to about 90. In some embodiments, the copy number of the ORF for FoxA2 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for FoxA2 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0104] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include FoxA2. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0105] FOXA3 The FOXA3 gene encodes a member of the forkhead class of DNA-binding proteins. These hepatocyte nuclear factors are transcriptional activators of liver-specific transcripts such as albumin and transthyretin and also interact with chromatin. The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. In some embodiments, one or more transcription factors can be used. In some embodiments, the transcription factor can be FoxA3, also known as forkhead box A3, HNF3G, hepatocyte nuclear factor 3-gamma, forkhead-related protein FKH H3, forkhead box protein A3, or transcription factor 3G.

[0106] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include FoxA3. FoxA3 can be introduced into the expression cassette. The expression cassette comprising FoxA3 can be introduced into PSCs. FoxA3 can be introduced into the expression cassette. FoxA3 can be expressed in the expression cassette. The expression cassette comprising FoxA3 can be introduced into PSCs. The expression cassette comprising FoxA3 can be expressed in PSCs. The expression cassette comprising FoxA3 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0107] FoxA3 can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. The combination of transcription factors can be introduced into an expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising FoxA3 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0108] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include FoxA3. An expression cassette comprising FoxA3 can be expressed in PSCs. An expression cassette comprising FoxA3 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of FoxA3 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of FoxA3 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of FoxA3 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0109] In some embodiments, the copy number of the ORF for FoxA3 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for FoxA3 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for FoxA3 introduced per cell can be up to about 90. In some embodiments, the copy number of the ORF for FoxA3 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for FoxA3 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0110] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or hepatic parenchymal cells. The one or more transcription factors can include FoxA3. The one or more hepatocytes or hepatic parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or hepatic parenchymal cells can include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or hepatic parenchymal cells.

[0111] HNF hepatocyte nuclear factor transcription factor The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the hepatocyte nuclear factor (HNF) family of transcription factors. The HNF hepatocyte nuclear factor transcription factor can be, for example, HNF1A or HNF4A.

[0112] HNF1A The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the HNF family. In some embodiments, the transcription factor can be HNF1A, also known as hepatocyte nuclear factor 1 homeobox A; LFB1; HNF1; TCF1; liver-specific transcription factor LF-B1; hepatocyte nuclear factor 1-alpha; HNF-1-alpha; HNF-1A; MODY3; TCP-1; transcription factor 1, liver; LF-B1, hepatocyte nuclear factor (HNF1), albumin proximal factor; truncated hepatocyte nuclear factor 1-alpha; interferon production regulatory factor; transcription factor 1, liver; hepatocyte nuclear factor 1; albumin proximal factor; transcription factor 1; HNF1α; IDDM20; HNF1I±; HNF1α; or, HNF1A.

[0113] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include HNF1A. HNF1A may be introduced into the expression cassette. HNF1A may be expressed in the expression cassette. The expression cassette comprising HNF1A may be introduced into PSCs. The expression cassette comprising HNF1A may be expressed in PSCs. The expression cassette comprising HNF1A may induce the differentiation of PSCs into hepatocytes or liver cells.

[0114] HNF1A may be introduced into the expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be made. The combination of transcription factors may be introduced into the expression cassette. The combination of transcription factors may be expressed in the expression cassette. The expression cassette may be introduced into PSCs. The expression cassette comprising HNF1A and one or more other transcription factors may induce the differentiation of PSCs into hepatocytes or liver cells.

[0115] The present disclosure provides one or more transcription factors that may induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors may include HNF1A. The expression cassette comprising HNF1A may be expressed in PSCs. The expression cassette comprising HNF1A may induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of HNF1A may be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of HNF1A may be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of HNF1A may be introduced per cell. An increase in the expression level can be further achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0116] In some embodiments, the copy number of the ORF for HNF1A introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for HNF1A introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for HNF1A introduced per cell can be up to about 90. In some embodiments, the copy number of the ORF for HNF1A introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for HNF1A introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0117] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include HNF1A. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0118] HNF4a The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the HNF family. In some embodiments, the transcription factor can be HNF4a, also known as hepatocyte nuclear factor 4α; NR2A1; HNF4; TCF14; nuclear receptor subfamily 2 group A member 1; hepatocyte nuclear factor 4α; transcription factor HNF-4; transcription factor 14; TCP-14; MODY1; MODY; hepatocyte nuclear factor 4α; HNF4α10 / 11 / 12; HNF-4-α; HNF4α; HNF4a7; HNF4a8; HNF4a9; NR2A21; FRTS4; or, TCF.

[0119] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include HNF4a. HNF4a can be introduced into the expression cassette. HNF4a can be expressed in the expression cassette. The expression cassette comprising HNF4a can be introduced into PSCs. The expression cassette comprising HNF4a can be expressed in PSCs. The expression cassette comprising HNF4a can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0120] HNF4a can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. The combinations of transcription factors can be introduced into an expression cassette. The combinations of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising HNF4a and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or hepatic cells.

[0121] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or hepatic cells. The one or more transcription factors can include HNF4a. An expression cassette comprising HNF4a can be expressed in PSCs. An expression cassette comprising HNF4a can induce the differentiation of PSCs into hepatocytes or hepatic cells. Various amounts of HNF4a can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of HNF4a can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of HNF4a can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0122] In some embodiments, the copy number of the ORF relative to HNF4a introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF relative to HNF4a introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF relative to HNF4a introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF relative to HNF4a introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF relative to HNF4a introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0123] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include HNF4a. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may contain one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0124] CUT homeobox transcription factor The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the CUT homeobox family of transcription factors. The transcription factor can be, for example, HNF6A / ONECUT1 or HNF6B / ONECUT2.

[0125] ONECUT1 / HNF6 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be the ONECUT transcription factor, also known as ONECUT1; ONECUT1 homeobox 1; HNF-6; HNF6A; HNF6; ONECUT domain family member 1; hepatocyte nuclear factor 6; hepatocyte nuclear factor 6, alpha; or ONECUT domain, family member 1. The present disclosure provides an expression cassette containing one or more transcription factors. The one or more transcription factors may include ONECUT1 / HNF6. ONECUT1 / HNF6 can be introduced into the expression cassette. ONECUT1 / HNF6 can be expressed in the expression cassette. The expression cassette containing ONECUT1 / HNF6 can be introduced into PSCs. The expression cassette containing ONECUT1 / HNF6 can be expressed in PSCs. The expression cassette containing ONECUT1 / HNF6 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0126] ONECUT1 / HNF6 can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. The combinations of transcription factors can be introduced into an expression cassette. The combinations of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising ONECUT1 / HNF6 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0127] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include ONECUT1 / HNF6. An expression cassette comprising ONECUT1 / HNF6 can be expressed in PSCs. An expression cassette comprising ONECUT1 / HNF6 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of ONECUT1 / HNF6 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of ONECUT1 / HNF6 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of ONECUT1 / HNF6 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0128] In some embodiments, the copy number of the ORF for ONECUT1 / HNF6 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for ONECUT1 / HNF6 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for ONECUT1 / HNF6 introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for ONECUT1 / HNF6 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for ONECUT1 / HNF6 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0129] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include ONECUT1 / HNF6. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0130] ONECUT2 / HNF6B The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be the ONECUT transcription factor, also known as ONECUT2; ONECUT2 homeobox 2; OC-2; hepatocyte nuclear factor 6-beta; ONECUT domain family member 2; transcription factor ONECUT-2; HNF-6-beta; ONECUT-2 homeodomain transcription factor; ONECUT domain family member 2; Onecut 2; HNF6B; or OC2.

[0131] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include ONECUT2 / HNF6B. ONECUT2 / HNF6B can be introduced into the expression cassette. ONECUT2 / HNF6B can be expressed in the expression cassette. The expression cassette comprising ONECUT2 / HNF6B can be introduced into PSCs. The expression cassette comprising ONECUT2 / HNF6B can be expressed in PSCs. The expression cassette comprising ONECUT2 / HNF6B can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0132] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into hepatocytes or liver cells. The one or more transcription factors can include ONECUT2 / HNF6B. ONECUT2 / HNF6B can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be made. The combination of transcription factors can be introduced into an expression cassette. The combination of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising ONECUT2 / HNF6B and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0133] Various amounts of ONECUT2 / HNF6B can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of ONECUT2 / HNF6B can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of ONECUT2 / HNF6B can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0134] In some embodiments, the copy number of the ORF for ONECUT2 / HNF6B introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for ONECUT2 / HNF6B introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for ONECUT2 / HNF6B introduced per cell can be up to about 90. In some embodiments, the copy number of the ORF for ONECUT2 / HNF6B introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for ONECUT2 / HNF6B introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0135] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include ONECUT2 / HNF6B. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells may contain one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0136] basic leucine zipper transcription factor The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a basic leucine zipper transcription factor. The basic leucine zipper transcription factor can be, for example, ATF3, ATF5, CEBPA, CEBPB, FOS, JUN, or NFE2L2.

[0137] CEBPA The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be CCAAT enhancer-binding protein (CEBP) α, also known as CCAAT enhancer-binding protein (CEBP); C / EBP-α; CEBP; CCAAT / enhancer-binding protein (C / EBP), α; CCAAT / enhancer-binding protein α; CCAAT / enhancer-binding protein α; or C / EBPα.

[0138] The present disclosure provides an expression cassette containing one or more transcription factors. The one or more transcription factors may include CEBPA. CEBPA can be introduced into the expression cassette. CEBPA can be expressed in the expression cassette. The expression cassette containing CEBPA can be introduced into PSCs. The expression cassette containing CEBPA can be expressed in PSCs. The expression cassette containing CEBPA can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0139] CEBPA can be introduced into an expression cassette together with one or more other transcription factors. Combinations containing one or more transcription factors can be made. The combination of transcription factors can be introduced into the expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. The expression cassette containing CEBPA and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0140] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include CEBPA. The expression cassette containing CEBPA can be expressed in PSCs. The expression cassette containing CEBPA can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of CEBPA can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of CEBPA can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of CEBPA can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0141] In some embodiments, the copy number of the ORF for CEBPA introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for CEBPA introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for CEBPA introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for CEBPA introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for CEBPA introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0142] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include CEBPA. The one or more hepatocytes or liver parenchymal cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0143] Atf3 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be an activating transcription factor (ATF). In some embodiments, the ATF can be Atf3, also known as activating transcription factor 3, cyclic AMP-dependent transcription factor ATF-3, or cAMP-dependent transcription factor ATF-3.

[0144] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include Atf3. Atf3 can be expressed in the expression cassette. The expression cassette comprising Atf3 can be introduced into PSCs. The expression cassette comprising Atf3 can be expressed in PSCs. The expression cassette comprising Atf3 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0145] Atf3 can be introduced into the expression cassette together with one or more other transcription factors. A combination comprising one or more transcription factors can be made. The combination of transcription factors can be introduced into the expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. The expression cassette comprising Atf3 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0146] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver cells. The one or more transcription factors can include Atf3. An expression cassette containing Atf3 can be expressed in PSCs. An expression cassette containing Atf3 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of Atf3 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of Atf3 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of Atf3 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0147] In some embodiments, the copy number of the ORF for Atf3 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for Atf3 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for Atf3 introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for Atf3 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for Atf3 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0148] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors can include Atf3. The one or more hepatocytes or liver parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or liver parenchymal cells can include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0149] Fos The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be a member of the Fos family. Fos family members can be Fos, also known as the Fos proto-oncogene, an AP-1 transcription factor subunit; AP-1; FBJ murine osteosarcoma virus oncogene homolog; G0 / G1 switch regulatory protein 7; cellular proto-oncogene C-Fos; C-Fos; FBJ mouse; osteosarcoma virus (V-Fos) oncogene homolog (oncogene FOS); V-Fos FBJ murine osteosarcoma virus oncogene homolog; Fos proto-oncogene, AP-1 transcription factor subunit; cellular oncogene C-Fos; cellular oncogene Fos; activator protein 1; C-FOS; G0S7; or, P55.

[0150] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors can include Fos. Fos can be introduced into the expression cassette. Fos can be expressed in the expression cassette. The expression cassette comprising Fos can be introduced into PSCs. The expression cassette comprising Fos can be expressed in PSCs. The expression cassette comprising Fos can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0151] Fos can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be made. The combinations of transcription factors can be introduced into an expression cassette. The combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising Fos and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0152] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into one or more hepatocytes or liver cells. The one or more transcription factors can include Fos. Various amounts of Fos can be introduced into PSCs. An expression cassette comprising Fos can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of Fos can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of Fos can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of Fos can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0153] In some embodiments, the copy number of the ORF relative to Fos introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF relative to Fos introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF relative to Fos introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF relative to Fos introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF relative to Fos introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0154] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors can include Fos. The one or more hepatocytes or liver parenchymal cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver parenchymal cells can include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0155] CEBPB The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be CCAAT enhancer-binding protein (CEBP); C / EBP-β; IL6DBP; TCF5; LAP; CCAAT / enhancer-binding protein (C / EBP), β; interleukin 6-dependent DNA-binding protein; CCAAT / enhancer-binding protein β; nuclear factor of interleukin 6; transcription factor 5; nuclear factor NF-IL6; NFIL6; CRP2; liver-enriched transcriptional activator protein; CCAAT / enhancer-binding protein β; liver-enriched inhibitory protein; transcription factor C / EBPβ; liver activator protein; C / EBPβ; NF-IL6; TCP-5; or LIP.

[0156] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors can include CEBPB. CEBPB can be expressed in the expression cassette. The expression cassette comprising CEBPB can be introduced into PSCs. The expression cassette comprising CEBPB can be expressed in PSCs. The expression cassette comprising CEBPB can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0157] CEBPB can be introduced into an expression cassette together with one or more other transcription factors. Combinations containing one or more transcription factors can be made. The combinations of transcription factors can be introduced into an expression cassette. The combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette containing CEBPB and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0158] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into one or more hepatocytes or liver cells. The one or more transcription factors can include CEBPB. An expression cassette containing CEBPB can be expressed in PSCs. An expression cassette containing CEBPB can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of CEBPB can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of CEBPB can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of CEBPB can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0159] In some embodiments, the copy number of the ORF for CEBPB introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for CEBPB introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for CEBPB introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for CEBPB introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for CEBPB introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0160] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver parenchymal cells. The one or more transcription factors may include CEBPB. The one or more hepatocytes or liver parenchymal cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver parenchymal cells.

[0161] JUN / AP-1 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the JUN family. In some embodiments, the transcription factor can be JUN / AP-1. JUN / AP-1 may also be known as JUN proto-oncogene, JUN / AP-1 as an AP-1 transcription factor subunit; V-JUN avian sarcoma virus 17 oncogene homolog; C-Jun; AP-1; transcription factor AP-1; proto-oncogene C-Jun; activator protein 1; Jun oncogene; AP1; P39; V-Jun sarcoma virus 17 oncogene homolog; Jun activation domain-binding protein; enhancer-binding protein AP1; proto-oncogene CJun; or CJUN.

[0162] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include JUN / AP-1. JUN / AP-1 can be expressed in the expression cassette. The expression cassette containing JUN / AP-1 can be introduced into PSCs. The expression cassette containing JUN / AP-1 can be expressed in PSCs. The expression cassette containing JUN / AP-1 can induce the differentiation of PSCs into hepatocytes or liver parenchymal cells.

[0163] JUN / AP-1 can be introduced into an expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. The combinations of transcription factors can be introduced into an expression cassette. The combinations of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising JUN / AP-1 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or liver cells.

[0164] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or liver cells. The one or more transcription factors can include JUN / AP-1. An expression cassette comprising JUN / AP-1 can be expressed in PSCs. An expression cassette comprising JUN / AP-1 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of JUN / AP-1 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of JUN / AP-1 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of JUN / AP-1 can be introduced per cell. An increase in the expression level can further be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0165] In some embodiments, the copy number of the ORF for JUN / AP-1 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for JUN / AP-1 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for JUN / AP-1 introduced per cell can be up to about 90. In some embodiments, the copy number of the ORF for JUN / AP-1 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for JUN / AP-1 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0166] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or hepatic parenchymal cells. The one or more transcription factors can include JUN / AP-1. The one or more hepatocytes or hepatic parenchymal cells can be differentiated from one or more PSCs. The one or more hepatocytes or hepatic parenchymal cells can include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or hepatic parenchymal cells.

[0167] NFE2L2 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. The transcription factor can be a member of the NFE family. In some embodiments, the transcription factor can be NFE2L2, also known as nuclear factor, erythroid 2-like 2; NRF2; nuclear factor, erythroid-derived 2-like 2; nuclear factor erythroid 2-related factor 2; NF-E2-related factor 2; HEBP1; Nrf-2; nuclear factor (erythroid-derived 2)-like 2; nuclear factor, erythroid-derived 2, like 2; nuclear factor erythroid-derived 2-like 2; nuclear factor, erythroid-derived 2-like 2; NFE2-related factor 2; IMDDHH; or NRF-2.

[0168] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors can include NFE2L2. NFE2L2 can be introduced into the expression cassette. NFE2L2 can be expressed in the expression cassette. The expression cassette comprising NFE2L2 can be introduced into PSCs. The expression cassette comprising NFE2L2 can be expressed in PSCs. The expression cassette comprising NFE2L2 can induce the differentiation of PSCs into hepatocytes or hepatic parenchymal cells.

[0169] NFE2L2 can be introduced into the expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be made. The combination of transcription factors can be introduced into the expression cassette. The combination of transcription factors can be expressed in the expression cassette. The expression cassette can be introduced into PSCs. The expression cassette comprising NFE2L2 and one or more other transcription factors can induce the differentiation of PSCs into hepatocytes or hepatic parenchymal cells.

[0170] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or liver cells. The one or more transcription factors can include NFE2L2. An expression cassette containing NFE2L2 can be expressed in PSCs. The expression cassette containing NFE2L2 can induce the differentiation of PSCs into hepatocytes or liver cells. Various amounts of NFE2L2 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) of NFE2L2 can be introduced per cell. Alternatively or additionally, up to about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) of NFE2L2 can be introduced per cell. An increase in the expression level can be further achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0171] In some embodiments, the copy number of the ORF for NFE2L2 introduced per cell can be from about 1 to about 90. In some embodiments, the copy number of the ORF for NFE2L2 introduced per cell can be at least about 1. In some embodiments, the copy number of the ORF for NFE2L2 introduced per cell can be at most about 90. In some embodiments, the copy number of the ORF for NFE2L2 introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the copy number of the ORF for NFE2L2 introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about 20, about 25, about 40, about 50, about 60, or about 90.

[0172] The present disclosure provides one or more transcription factors that can induce the differentiation of one or more pluripotent stem cells (PSCs) into one or more hepatocytes or hepatic parenchymal cells. The one or more transcription factors may include NFE2L2. The one or more hepatocytes or hepatic parenchymal cells can be differentiated from the one or more PSCs. The one or more hepatocytes or hepatic parenchymal cells may include one or more transcription factors that can induce the differentiation of one or more PSCs into hepatocytes or hepatic parenchymal cells.

[0173] GATA zinc finger transcription factor The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor may be a member of the GATA zinc finger family of transcription factors, such as, for example, GATA2, GATA4, or GATA6, ODAG, RGO83M05.2, GATA zinc finger domain-containing protein 1, eye development-related gene protein, FJ22489, eye development-related gene, CMD2B. The GATA2 transcription factor can be, for example, isoform P23769-1 or P23769-2.

[0174] GATA2 isoform P23769-2 The present disclosure provides methods of using cells, expression cassettes, and transcription factors, or molecules that increase transcription or increase transcriptional activators. One or more transcription factors can be used. In some embodiments, the transcription factor can be GATA2 isoform P23769-2, also known as GATA binding protein 2; NFE1B; endothelial transcription factor GATA-2; GATA binding protein 2; MONOMAC; IMD21; or DCML.

[0175] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include the GATA2 isoform P23769-2. The GATA2 isoform P23769-2 may be introduced into the expression cassette. The GATA2 isoform P23769-2 may be expressed in the expression cassette. The expression cassette comprising the GATA2 isoform P23769-2 may be introduced into PSCs. The expression cassette comprising the GATA2 isoform P23769-2 may be expressed in PSCs. The expression cassette comprising the GATA2 isoform P23769-2 may induce the differentiation of PSCs into hepatocytes or liver cells.

[0176] The GATA2 isoform P23769-2 may be introduced into the expression cassette together with one or more other transcription factors. Combinations comprising one or more transcription factors can be made. The combination of transcription factors may be introduced into the e...

Claims

1. A method for generating hepatocytes or a population of hepatocytes, (a) A step of delivering at least one nucleic acid containing one or more transcription factors to one or more cells, (b) A step of generating hepatocytes or the population of hepatocytes from one or more cells, wherein the one or more cells express at least three hepatocyte markers within 96 hours after induction of the one or more transcription factors. Methods that include...

2. The method according to claim 1, wherein the one or more transcription factors include (a) one or more hepatocyte nuclear factor ("HNF") family members, (b) one or more forkhead box ("FOX") family members, or both.

3. The method according to claim 1, further comprising at least two transcription factors, including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.

4. The method according to claim 1, further comprising at least three transcription factors, including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.

5. The method according to claim 1, further comprising at least four transcription factors, including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.

6. The method according to claim 1, further comprising at least five transcription factors, including FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI, or HLF family members, or one or more additional transcription factors from Table 12.

7. The method according to claim 2, wherein the one or more HNF family members include HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof.

8. The method according to claim 2, wherein the one or more HNF family members include three or fewer HNF family members.

9. The method according to claim 2, wherein the one or more FOX family members include FOXA1, FOXA2, FOXA3, or any combination thereof.

10. The method according to claim 2, wherein the one or more FOX family members include two or fewer FOX family members.

11. The method according to claim 1, wherein the one or more transcription factors include HNF4A, HNF1A, and FOXA1.

12. The method according to claim 1, wherein the one or more transcription factors further comprise one or more of FOXA2, CEBPA, ONECUT1 / HNF6, RBPJ, or GATA6.

13. The method according to claim 1, wherein at least one transcription factor induces the expression of the one or more cells in hepatocytes or a population of hepatocytes within 30 days.

14. The method according to claim 1, wherein the at least three hepatocyte markers include one or more of CK18, albumin, ASGR-1, and CXCR4.

15. The method according to claim 14, wherein 4% or more of the one or more cells express CK18.

16. The method according to claim 14, wherein 2% or more of the one or more cells express albumin.

17. The method according to claim 16, wherein a portion of the one or more cells expressing albumin also express CK18.

18. The method according to claim 14, wherein 2% or more of the one or more cells express ASGR-1.

19. The method according to claim 14, wherein 2% or more of the one or more cells express CXCR-4.

20. The method according to claim 1, wherein at least one of the one or more cells expresses CYP3A7.

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