Methods and compositions for stem cell differentiation

Abstract

The present disclosure provides transcription factors and methods for differentiating stem cells into liver cells or liver cells. In some examples, a pluripotent stem cell (PSC) comprises a nucleic acid. In some examples, the nucleic acid comprises an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator of the open reading frame encoding one or more transcription factors. In some examples, the nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator of the open reading frame encoding one or more transcription factors induces differentiation of the PSC into a hepatocyte or a liver cell.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 352,529, filed June 15, 2022, the entire contents of which are hereby incorporated by reference herein. Background Art

[0003] Hepatocytes play an important role in the liver: they can participate in the synthesis or storage of proteins; the conversion of carbohydrates; the synthesis of cholesterol, bile salts or phospholipids; and detoxification. Therapies to improve certain functions of the liver may involve the introduction or regulation of hepatocytes or hepatic cells. Stem cells can be programmed to various phenotypes by introducing one or more transcription factors or one or more molecules that regulate transcription or transcription factors. For example, stem cells can be programmed to hepatocytes or hepatocytes. Summary of the Invention

[0004] One aspect of the present disclosure is a pluripotent stem cell (PSC) 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 encoding 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 a 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 member, 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 a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the PSC further comprises at least two or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the PSC further comprises at least three or more transcription factors comprising a member 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, the PSC further comprises at least four or more transcription factors comprising a member 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, the PSC further comprises at least 5 or more transcription factors comprising a member 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, the PSC further comprises at least 6 or more transcription factors comprising a member 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, the PSC further comprises at least seven or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the PSC further comprises at least 8 or more 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 HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, the one or more FOX family members comprise FOXA1. In some embodiments, the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

[0005] Another aspect of the present disclosure is a method of generating a hepatocyte or a hepatocyte cell population comprising: providing one or more pluripotent stem cells (PSCs); 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 encoding 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; generating the hepatocyte or hepatocyte cell population from the one or more PSCs. 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 CEBPA, and the one or more transcription factors further comprise a 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 member, 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 a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least two or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least three or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least four or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 5 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 6 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 7 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least 8 or more 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 HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, the one or more FOX family members comprise FOXA1. In some embodiments, the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1. In some embodiments, the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ. In some embodiments, the at least one or more transcription factors induce the one or more PSCs to differentiate into hepatocytes or a population of hepatocytes in 30 days or less. In some embodiments, the one or more PSCs are provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or a population of hepatocytes. In some embodiments, the culture medium is not changed during delivery of at least one exogenous expression cassette to the one or more PSCs. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses CXCR-4. In some embodiments, at least 2% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 10% of the hepatocytes or population of hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or population of hepatocytes express CXCR-4. In some embodiments, at least 30% of the PSCs express CXCR-4.

[0006] Another aspect of the present disclosure is an exogenous expression cassette that induces pluripotent stem cells (PSCs) to differentiate into hepatocytes or liver cells, wherein the exogenous expression cassette comprises: 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 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 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 factor genes comprise CEBPA, and the one or more transcription factor genes further comprise a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the one or more additional transcription factor genes comprise RBPJ, and the one or more transcription factors further comprise a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette further comprises a 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 member, 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 comprising a 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 member, 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 comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least 5 or more transcription factor genes, the transcription factor genes comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least 6 or more transcription factor genes, the transcription factor genes comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the exogenous expression cassette comprises at least 7 or more transcription factor genes, comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the exogenous expression cassette comprises at least 8 or more transcription factor genes, the transcription factor genes 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 HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, the one or more FOX family members comprise FOXA1, or FOXA3, or any combination thereof. In some embodiments, the one or more transcription factor genes comprise HNF4A, HNF1A, ONECUT1 / HNF6 and FOXA1. In some embodiments, the one or more transcription factor genes include CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell. In some embodiments, the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 30 days or less. In some embodiments, the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 96 hours or less.

[0007] Another aspect of the present disclosure is a method for generating a hepatocyte or a hepatocyte colony, comprising: providing one or more pluripotent stem cells (PSCs); delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator encoding an open reading frame of one or more transcription factors to the one or more PSCs; and generating the hepatocyte or hepatocyte colony from the one or more PSCs within 30 days or less. In some embodiments, the one or more PSCs differentiate into the hepatocyte or hepatocyte colony within 96 hours or less. In some embodiments, the one or more PSCs are provided in a culture medium. In some embodiments, the culture medium is not changed during the differentiation of the one or more PSCs into the hepatocyte colony. 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) 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, 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) 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, the one or more transcription factors are transcription factors of a transcription factor family, such as a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, two or more transcription factors are transcription factors of a transcription factor family, such as a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the three or more transcription factors are transcription factors of a transcription factor family, e.g., a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the four or more transcription factors are transcription factors of a transcription factor family, e.g., a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the at least one or more transcription factors comprise SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a, and HNF6B.In some embodiments, the one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, the one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, and 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 culture medium. In some embodiments, the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or hepatocyte population. In some embodiments, the culture medium is not changed during delivery of a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcriptional activator encoding 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 in the hepatocyte or hepatocyte population expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population does not express CXCR-4. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses CXCR-4. In some embodiments, the hepatocyte or hepatocyte population comprises at least 2% of the hepatocytes or hepatocyte population expressing ASGR-1. In some embodiments, the hepatocyte or hepatocyte population comprises at least 3% of the hepatocytes or hepatocyte population expressing ASGR-1. In some embodiments, the hepatocyte or hepatocyte population comprises at least 5% of the hepatocytes or hepatocyte population expressing ASGR-1. In some embodiments, the hepatocytes or liver cell populations comprise at least 6% of the hepatocytes or liver cell populations expressing ASGR-1. In some embodiments, the hepatocytes or liver cell populations comprise at least 10% of the hepatocytes or liver cell populations expressing CXCR-4. In some embodiments, the hepatocytes or liver cell populations comprise at least 20% of the hepatocytes or liver cell populations expressing CXCR-4. In some embodiments, the hepatocytes or liver cell populations comprise at least 30% of the PSCs expressing CXCR-4.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 transcription activator encoding an open reading frame of 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 transcription activator encoding an open reading frame of one or more transcription factors induces the PSC to differentiate into hepatocytes or liver cells within 30 days or less. 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) 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) 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, 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) 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, the one or more transcription factors are transcription factors of a transcription factor family, such as 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 ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors comprise 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, 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 factors. 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, and 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 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. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocytes or liver cells. In some embodiments, the hepatocyte or liver cell population comprises two or more of the hepatocytes or liver cells. In some embodiments, the hepatocyte or liver cell population comprises at least 2% hepatocytes or liver cells expressing ASGR-1. In some embodiments, the hepatocyte or liver cell population comprises at least 3% hepatocytes or liver cells expressing ASGR-1. In some embodiments, the hepatocyte or liver cell population comprises at least 5% hepatocytes or liver cells expressing ASGR-1. In some embodiments, the hepatocyte or liver cell population comprises at least 6% hepatocytes or liver cells expressing ASGR-1. In some embodiments, the hepatocyte or liver cell population comprises at least 10% hepatocytes or liver cells expressing CXCR-4. In some embodiments, the hepatocyte or liver cell population comprises at least 20% hepatocytes or liver cells expressing CXCR-4. In some embodiments, the hepatocytes or liver cell population comprises at least 30% of the hepatocytes or liver cells expressing CXCR-4. In some embodiments, the hepatocytes or liver cell population comprises, without nutrient, or growth factor or microenvironment / matrix optimization.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: SPI1, HNF1A, FOXA2, CEBPA, ONECUT1, HNF4A and ONECUT2.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FOXA2 and FOXA1.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B and HNF1A.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A and RBPJ.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: HNF1A, CEBPA, ONECUT1, ONECUT2, FOS, HIF1A and TBX3.

[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 encoding an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

[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 encoding an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, HNF1A, CEBPA, ONECUT1 / HNF6, and HNF4A.

[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 encoding an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, 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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

[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 encoding an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, FOXA1, HNF1A, and HNF4A.

[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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, HNF1A, HNF4A, FOXA3, FOXA2, PXR and RXRA.

[0019] 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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: CEBPB, FOXA1, FOXA3, HLF, HNF1A, HNF4A, NR1I2 / PXR, NR1I3 / CAR, ONECUT1 / HNF6, PROX1, RBPJ, RORC and SALL4.

[0020] In another aspect of the present disclosure, a kit for inducing pluripotent stem cells (PSCs) to differentiate into hepatocytes or liver 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 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 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 a 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 member, 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 a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least two or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least three or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least four or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 5 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 6 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12. In some embodiments, the method further comprises at least 7 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.In some embodiments, the method further comprises at least 8 or more 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 HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof. In some embodiments, the one or more FOX family members comprise FOXA1, or FOXA3, or any combination thereof. In some embodiments, the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6 and FOXA1. In some embodiments, the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6 and RBPJ. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell. In some embodiments, the exogenous expression cassette induces differentiation of the PSC into the hepatocyte or liver cell in 30 days or less. In some embodiments, the exogenous expression cassette induces differentiation of the PSC into the hepatocyte or liver cell in 96 hours or less.

[0021] 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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: 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.

[0022] 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 transcription activator encoding an open reading frame of 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 transcription activator encoding an open reading frame of one or more transcription factors induces the PSC to differentiate into hepatocytes or liver cells within 30 days or less. 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) 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, 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) 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, the one or more transcription factors are transcription factors of a transcription factor family, such as 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 comprise ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors comprise 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, 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, wherein the one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, and GATA2 transcription factors. In some embodiments, wherein the one or more transcription factors are, for example, GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, wherein the one or more transcription factors are, for example, GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, and NFIX. In some embodiments, wherein the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, ​​and FoxA1. In some embodiments, wherein 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.

[0023] The present disclosure provides a hepatocyte or liver cell, wherein the hepatocyte or liver cell expresses ASGR-1. In some embodiments, the hepatocyte or liver cell does not express CXCR-4. In some embodiments, wherein the hepatocyte or liver cell expresses CXCR-4. In some embodiments, wherein the PSC is provided in culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell.

[0024] The present disclosure provides a hepatocyte or a population of hepatocytes comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0025] 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 encoding an open reading frame of 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 encoding an open reading frame of one or more transcription factors induces the PSC to differentiate into a hepatocyte or a liver cell, wherein the PSC is provided in a culture medium, and wherein the culture medium is not changed during the differentiation of the PSC into the hepatocyte or liver cell. In some embodiments, the one or more transcription factors induce the PSC to differentiate into the hepatocyte or liver cell in 30 days or less. 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) 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, 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, 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 factors. In some embodiments, the one or more transcription factors are, for example, GATA2 transcription factors, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors are, for example, GATA2 transcription factors, 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 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. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell.

[0026] The present disclosure provides a hepatocyte or a population of hepatocytes comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0027] 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 encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors comprise: HNF6, and 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) 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) 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, 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) domain, 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, the 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, the 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, four or more transcription factors are selected from the group consisting of the transcription factor family 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, nucleic acids encoding open reading frames of one or more transcription factors, one or more transcription factors, or transcriptional activators encoding open reading frames of one or more transcription factors induce the PSC to differentiate into hepatocytes or liver cells. In some embodiments, nucleic acids encoding open reading frames of one or more transcription factors, one or more transcription factors, or transcriptional activators encoding open reading frames of one or more transcription factors induce the PSC to differentiate into hepatocytes or liver cells in 30 days or less. 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 factors. 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, and 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 PSC is provided in culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocytes or liver cells.

[0028] The present disclosure provides a hepatocyte or liver cell, wherein the hepatocyte or liver cell expresses ASGR-1. In some embodiments, the hepatocyte or liver cell does not express CXCR-4. In some embodiments, the hepatocyte or liver cell expresses CXCR-4.

[0029] The present disclosure provides a cell population 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 cell population is provided in culture medium. In some embodiments, no nutrient, growth factor, microenvironment, or matrix optimization is performed.

[0030] Another aspect of the present disclosure provides a method for generating a hepatocyte or a hepatocyte colony, comprising: providing one or more pluripotent stem cells (PSCs); 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 transcription activator encoding an open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise an HNF6 gene; and generating the hepatocyte or hepatocyte colony from the one or more PSCs. In some embodiments, the method further comprises one or more transcription factors, such as 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, such as 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, the method further comprises three or more of one or more transcription factors, such as 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, the method further comprises four or more of one or more transcription factors, such as 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, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the at least one or more transcription factors include SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B. In some embodiments, the at least one or more transcription factors comprise HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1.In some embodiments, the at least one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the at least one or more transcription factors include FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ. In some embodiments, the at least one or more transcription factors include CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the at least one or more transcription factors include HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3. In some embodiments, the at least one or more transcription factors induce the one or more PSCs to express into hepatocytes or liver cell populations within 30 days or less. In some embodiments, the one or more PSCs are provided in culture medium. In some embodiments, the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or hepatocyte population. In some embodiments, the culture medium is not changed during delivery of at least one exogenous expression cassette to the one or more PSCs. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population does not express CXCR-4. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses CXCR-4. In some embodiments, at least 2% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocyte population express CXCR-4. In some embodiments, at least 20% of the hepatocytes or population of hepatocytes express CXCR-4. In some embodiments, at least 30% of the PSCs express CXCR-4.

[0031] Another aspect of the present disclosure provides a method for generating a hepatocyte or a hepatocyte colony, comprising: providing one or more pluripotent stem cells (PSCs); delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator encoding an open reading frame of one or more transcription factors to the one or more PSCs, wherein the one or more transcription factors induce the one or more PSCs to differentiate into the hepatocytes or hepatocyte colonies; additionally inducing the one or more transcription factors to be expressed in the one or more PSCs within 30 days or less; and generating the hepatocytes or hepatocyte colonies from the one or more PSCs. In some embodiments, the one or more PSCs differentiate into the hepatocytes or hepatocyte colonies within 96 hours or less. In some embodiments, the one or more PSCs are provided in a culture medium. In some embodiments, the culture medium is not changed during the differentiation of the one or more PSCs into the hepatocyte colonies. 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) 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, 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 transcription factors 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, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, the four or more transcription factors are transcription factors of the 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, JUN, SKI, SALL, EGR and TP. In some embodiments, the at least one or more transcription factors include SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B. In some embodiments, the one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA and FoxM1. In some embodiments, the one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73 and HNF6. In some embodiments, the 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 a culture medium. In some embodiments, the culture medium is not changed during the differentiation of the one or more PSCs into the hepatocytes or hepatocyte population. In some embodiments, the culture medium is not changed during the delivery of nucleic acids encoding open reading frames of one or more transcription factors, one or more transcription factors, or transcriptional activators encoding open reading frames of one or more transcription factors to the one or more PSCs. In some embodiments, the hepatocyte or at least one of the one or more hepatocytes or hepatocytes of a population of hepatocytes expresses ASGR-1. In some embodiments, the hepatocyte or at least one of the one or more hepatocytes or hepatocytes of a population of hepatocytes does not express CXCR-4. In some embodiments, the hepatocyte or at least one of the one or more hepatocytes or hepatocytes of a population of hepatocytes expresses CXCR-4.

[0032] The present disclosure provides a hepatocyte or hepatocyte population as disclosed herein, wherein at least 2% of the hepatocyte or hepatocyte population express ASGR-1. In some embodiments, at least 3% of the hepatocyte or hepatocyte population express ASGR-1. In some embodiments, at least 5% of the hepatocyte or hepatocyte population express ASGR-1. In some embodiments, at least 6% of the hepatocyte or hepatocyte population express ASGR-1. In some embodiments, at least 10% of the hepatocyte or hepatocyte population express CXCR-4. In some embodiments, at least 20% of the hepatocyte or hepatocyte population express CXCR-4. In some embodiments, at least 30% of the PSCs express CXCR-4.

[0033] Another aspect of the present disclosure provides a method for generating a hepatocyte or a hepatocyte colony, comprising: providing one or more pluripotent stem cells (PSCs) in a culture medium; delivering a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator encoding an open reading frame of one or more transcription factors to the one or more PSCs, wherein the one or more transcription factors additionally induce the one or more PSCs to differentiate into the hepatocytes or hepatocyte colony; and generating the hepatocytes or hepatocyte colony from the one or more PSCs, wherein the culture medium is not changed during the differentiation of the one or more PSCs into the hepatocytes or hepatocyte colony. In some embodiments, the culture medium is not changed during the delivery of the one or more transcription factors to the one or more PSCs. In some embodiments, the one or more transcription factors comprise HNF6. In some embodiments, the one or more transcription factors induce the one or more PSCs to express into hepatocytes or hepatocyte colonies within 30 days or less. 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) 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, 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 transcription factors 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, the three or more transcription factors are transcription factors of the 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, the four or more transcription factors are transcription factors of the 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, the at least one or more transcription factors include SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B.In some embodiments, the one or more transcription factors include HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1. In some embodiments, the one or more transcription factors include GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the 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 at least one or more transcription factors induce the one or more PSCs to differentiate into hepatocytes or a population of hepatocytes in 30 days or less. In some embodiments, the one or more PSCs are provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or a population of hepatocytes. In some embodiments, the culture medium is not changed during delivery of nucleic acids comprising open reading frames encoding one or more transcription factors, one or more transcription factors, or transcriptional activators encoding open reading frames of 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 in the hepatocyte or hepatocyte population expresses ASGR-1. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population does not express CXCR-4. In some embodiments, at least one of the one or more hepatocytes or hepatocytes in the hepatocyte or hepatocyte population expresses CXCR-4. In some embodiments, at least 2% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocyte population express ASGR-1. In some embodiments, at least 5% of the hepatocytes or population of hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or population of hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or population of hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or population of hepatocytes express CXCR-4. In some embodiments, at least 30% of the PSCs express CXCR-4.

[0034] Another aspect of the present disclosure provides an exogenous expression cassette, which includes one or more transcription factors that induce pluripotent stem cells (PSC) to differentiate into hepatocytes or hepatocytes, wherein the exogenous expression cassette induces the PSC to express into the hepatocytes or hepatocytes within 30 days or less. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium does not change during the differentiation of the PSC into the hepatocytes or 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) 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) 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, 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 comprise ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors comprise ONECUT1 / HNF6 and ONECUT2 / HNF6B. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, the four or more transcription factors are transcription factors of the 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, 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, 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 transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.

[0035] The present disclosure provides a hepatocyte or liver cell as disclosed herein, wherein the hepatocyte or liver cell expresses ASGR-1. In some embodiments, the hepatocyte or liver cell does not express CXCR-4. In some embodiments, the hepatocyte or liver cell expresses CXCR-4.

[0036] The present disclosure provides a hepatocyte or a population of hepatocytes comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0037] Another aspect of the present disclosure provides an exogenous expression cassette comprising one or more transcription factors that induce pluripotent stem cells (PSC) to differentiate into hepatocytes or liver cells, wherein the PSC is provided in a culture medium, and wherein the culture medium is not changed during the differentiation of the PSC into the hepatocytes or liver cells. In some embodiments, the exogenous expression cassette induces the PSC to differentiate into the hepatocytes or liver cells in 30 days or less. In some embodiments, the exogenous expression cassette induces the PSC to differentiate into the hepatocytes or liver cells in 96 hours or less. 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) 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) 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, 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 transcription factors 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, the three or more transcription factors are transcription factors of the 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, the four or more transcription factors are transcription factors of the 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, the five or more transcription factors are transcription factors of the 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, 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 subtype P23769-2, GATA4, GATA2 subtype 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 subtype P23769-2, and GATA2 subtype P23769-1. In some embodiments, the one or more transcription factors are, for example, GATA2 subtype P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors are, for example, GATA2 subtype P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, and 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 transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, 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.

[0038] The present disclosure provides a hepatocyte or a population of hepatocytes comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0039] Another aspect of the present disclosure provides an exogenous expression cassette that induces pluripotent stem cells (PSCs) to differentiate into hepatocytes or liver cells, wherein the exogenous expression cassette comprises an HNF6 transcription factor and one or more additional transcription factors. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during differentiation of the PSC into the hepatocytes or liver cells. In some embodiments, the exogenous expression cassette induces the PSCs to differentiate into the hepatocytes or liver cells within 30 days or less. In some embodiments, the exogenous expression cassette induces the PSCs to differentiate into the hepatocytes or liver cells within 96 hours or less. 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) 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 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) 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, 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) 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, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more additional transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B. In some embodiments, the one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1.In some embodiments, the 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, the 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, the one or more additional transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more additional transcription factors are, for example, additional GATA2 isoform P23769-1, CEBPA, ATF3, GATA6, Fos, GATA3, and NFIX. In some embodiments, the one or more additional transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, ​​and FoxA1. In some embodiments, the one or more additional transcription factors are, for example, HHEX, SOX17, and HNF4A. In some embodiments, the one or more additional transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A. In some embodiments, the one or more additional transcription factors are, for example, FoxA1, HNF1A, FoxA2, ​​CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ. In some embodiments, the one or more additional 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 / HNF6, 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.

[0040] The present disclosure provides a hepatocyte or liver cell population comprising two or more hepatocytes or liver cells as 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, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0041] Another aspect of the present disclosure provides a hepatocyte or 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 transcription activators encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors induce pluripotent stem cells (PSC) to differentiate into the hepatocyte or liver cell in 30 days or less. 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) 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 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) 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, 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, the one or more transcription factors induce the PSC to differentiate into the hepatocytes or liver cells within 96 hours or less. In some embodiments, the PSC is provided in a culture medium. In some embodiments, the culture medium is not changed during the differentiation of the PSC into the hepatocytes or liver cells. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors 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, five or more transcription factors are transcription factors 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, the one or more transcription factors are, for example, HNF1A, SPI1, FoxA2, ​​CEBPA, HNF6, HNF4a, and HNF6B. In some embodiments, the one or more additional 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 isoforms P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6. In some embodiments, the one or more transcription factors are, for example, additional GATA2 isoforms 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.

[0042] The present disclosure provides a hepatocyte or liver cell population comprising two or more hepatocytes or liver cells as 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, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0043] Another aspect of the present disclosure provides a hepatocyte or 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 transcription activators encoding an open reading frame of one or more transcription factors, wherein the one or more transcription factors induce pluripotent stem cells (PSC) to differentiate into the hepatocyte or liver cell, and wherein the culture medium is not changed during the differentiation of the PSC into the hepatocyte or liver cell. 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) 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 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) 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, 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, the one or more exogenous expression cassettes induce the PSC to differentiate into the hepatocytes or liver cells in 30 days or less. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B.In some embodiments, the one or more additional 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 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 GATA2 isoforms 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.

[0044] The present disclosure provides a hepatocyte or liver cell population comprising two or more hepatocytes or liver cells as 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, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0045] Another aspect of the present disclosure provides a cell expressing ASGR-1, comprising: one or more nucleic acids comprising open reading frames encoding one or more transcription factors, one or more transcription factors, or one or more transcription activators encoding open reading frames of one or more transcription factors, wherein the one or more transcription factors induce pluripotent stem cells (PSCs) to differentiate into the cell expressing ASGR-1, and wherein the culture medium is not changed during the differentiation of the 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) 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 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) 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, 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, the one or more exogenous expression cassettes induce the PSC to differentiate into the hepatocytes or liver cells in 30 days or less. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B.In some embodiments, the one or more additional 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 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 GATA2 isoforms 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.

[0046] The present disclosure provides a hepatocyte or liver cell population comprising two or more hepatocytes or liver cells as 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, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0047] The present disclosure provides a cell expressing ASGR-1, comprising: one or more nucleic acids comprising open reading frames encoding one or more transcription factors, one or more transcription factors, or one or more transcription activators encoding open reading frames of one or more transcription factors, wherein the nucleic acids comprising open reading frames encoding one or more transcription factors, one or more transcription factors, or transcription activators encoding open reading frames of one or more transcription factors induce pluripotent stem cells (PSCs) to differentiate into the cell expressing ASGR-1 in 30 days or less.

[0048] The present disclosure provides a kind of hepatocyte or hepatocyte colony, it comprises two or more hepatocytes or hepatocytes as disclosed herein.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) 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, 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) 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, the one or more exogenous expression cassettes induce the PSC to differentiate into the hepatocytes or liver cells within 30 days or less.In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B. In some embodiments, the one or more additional 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 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 GATA2 isoforms 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.

[0049] The present disclosure provides a hepatocyte or a population of hepatocytes comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 3% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 5% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 6% of the hepatocytes or hepatocytes express ASGR-1. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.

[0050] Another aspect of the present disclosure provides a cell expressing CXCR-4, comprising: one or more nucleic acids comprising open reading frames encoding one or more transcription factors, one or more transcription factors, or one or more transcription activators encoding open reading frames of one or more transcription factors, wherein the one or more transcription factors induce pluripotent stem cells (PSCs) to differentiate into cells expressing CXCR-4, and wherein the culture medium is not changed during the differentiation of the 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) 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 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) 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, 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, the one or more exogenous expression cassettes induce the PSC to differentiate into the hepatocytes or liver cells in 30 days or less. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B.In some embodiments, the one or more additional transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, SPI1, FoxA2, ​​GATA2 subtype P23769-2, GATA4, GATA2 subtype P23769-1, JUN / AP-1, and NFIX, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos, GATA2 subtype P23769-2, and GATA2 subtype P23769-1, or any combination thereof. In some embodiments, the one or more transcription factors are, for example, GATA2 subtype 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.

[0051] The present disclosure provides a hepatocyte or a hepatocyte population comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 3% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 5% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 6% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor or microenvironment / matrix optimization is not performed.

[0052] Another aspect of the present disclosure provides a cell expressing CXCR-4, comprising: (a) one or more nucleic acids, wherein each of the one or more nucleic acids comprises an open reading frame encoding one or more transcription factors, (b) one or more transcription factors, and / or (c) one or more transcription activators encoding open reading frames of 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 transcription activators encoding open reading frames of one or more transcription factors induces pluripotent stem cells (PSCs) to differentiate into the cell expressing CXCR-4 in 30 days or less.

[0053] In some embodiments, a nucleic acid comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or a transcription activator encoding an open reading frame encoding one or more transcription factors induces pluripotent stem cells (PSCs) to differentiate into the cells expressing CXCR-4 within 96 hours or less. 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) 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) 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, 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) 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, the one or more exogenous expression cassettes induce the PSC to differentiate into the hepatocytes or liver cells within 30 days or less. In some embodiments, the two or more transcription factors are transcription factors of the 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, the three or more transcription factors are transcription factors of the 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, four or more transcription factors are transcription factors of the 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, five or more transcription factors are transcription factors of the 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, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a and HNF6B.In some embodiments, the one or more additional 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 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 GATA2 isoforms 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, 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.

[0054] The present disclosure provides a hepatocyte or a hepatocyte population comprising two or more of the hepatocytes or hepatocytes disclosed herein. In some embodiments, at least 2% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 3% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 5% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 6% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 10% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 20% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, at least 30% of the hepatocytes or hepatocytes express CXCR-4. In some embodiments, nutrient, growth factor or microenvironment / matrix optimization is not performed.

[0055] Other aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive.

[0056] Incorporation by reference

[0057] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that a publication, patent, or patent application incorporated by reference conflicts with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "Figures" and "FIG.") which set forth illustrative embodiments utilizing the principles of the invention, in which:

[0059] Figure 1 Depicted is an exemplary screen showing the ability of a collection of transcription factors to induce hepatocyte formation. iPSCs are transfected and integrants are selected using specific antibiotics.

[0060] Figure 2Depicted are exemplary experiments showing morphological changes following differentiation in thirteen unique cell lines.

[0061] Figure 3 The expression of CK18, albumin, and ASGR-1 by cells without and with transcription factor induction is depicted.

[0062] Figures 4A-4C The expression or lack thereof of hepatocyte-specific markers and / or definitive endoderm markers of various mixtures is depicted. Positive expression of the hepatocyte-specific marker ASGR-1 ( Figure 4A ), definitive endoderm marker CXCR4 ( Figure 4B ) and stem cell marker TRA-1-60 ( Figure 4C ) of cells.

[0063] Figure 5A-5B The expression of different cell markers, ASGR-1 and albumin are depicted. The expression of transcription factor (TF) formula or combination H0008 ( Figure 5A )、H0011( Figure 5B ) and the percentage of cells expressing both CK18 and albumin in the cell lines expressing the two TF formulations ( Figure 5C ).

[0064] Figure 6 Staining for CK18 and albumin of two independent experiments is depicted.

[0065] Figure 7 Depicted are the results of immunofluorescence imaging of H0002.

[0066] Figure 8 Depicted are the results of immunofluorescence imaging of H0003.

[0067] Figure 9 Depicted are the results of immunofluorescence imaging of H0004.

[0068] Figure 10 Depicted are the results of immunofluorescence imaging of H0005.

[0069] Figure 11 Depicted are the results of immunofluorescence imaging of H0006.

[0070] Figure 12 Depicted are the results of immunofluorescence imaging of H0008.

[0071] Figure 13 Depicted are the results of immunofluorescence imaging of H0009.

[0072] Figure 14 Depicted are the results of immunofluorescence imaging of H0010.

[0073] Figure 15 Depicted are the results of immunofluorescence imaging of H0011.

[0074] Figure 16 Depicted are the results of immunofluorescence imaging of H0012.

[0075] Figure 17 Depicted are the results of immunofluorescence imaging of H0013.

[0076] Figures 18A-18B Depicted are primary hepatocytes and induced cell lines expressing the H0001 cocktail that differentiated into hepatocytes. The ability to store lipids is depicted with lipid droplets, which are stained red, as expected in primary cells ( Figure 18A ) and recapitulated by induced cells ( Figure 18B ).

[0077] Figures 19A-19C The lipid storage capacity of iPSCs was demonstrated when they were modified to express a specific cocktail, H0011. Oil Red O staining was used to stain primary cells (positive control; Figure 19A ) and induced iPSCs differentiated into hepatocytes and stained for lipid droplets ( Figure 19B ), but lipid storage was detected in hiPSCs without any TF induction (negative control; Figure 19C ), no signs of lipid accumulation were detected in the 1% WT ( ) , as expected.

[0078] Figure 20A-Figure 20B The percentage of cells that differentiated into hepatocytes and therefore positively expressed the markers ASGR-1, CK18, albumin and had the ability to store lipids is depicted. Figure 20A ) and H0011( Figure 20B ) to confirm the percentage of cells that positively express markers and store lipids.

[0079] Figure 21 Describes an experimental protocol for screening transcription factors (TFs) that are critical for hepatocyte differentiation.

[0080] Figures 22A-22B The use of flow cytometry to assess the percentage of differentiated cells expressing the hepatocyte marker ASGR-1 on their surface is illustrated. An example graph of a mixture of transcription factors induced in modified cells is shown on the x-axis ( Figure 22A); the runs of the transcription factor cocktail were identical (e.g., run 29 was cell line H0064). ASGR-1 expression was assessed in the run 29 / H0064 cell line by flow cytometry; undifferentiated cells remained positive for the TRA-1-60 marker ( Figure 22B ).

[0081] Figure 23 An assay to measure the amount of albumin secreted by differentiated cells by ELISA is described.

[0082] Figures 24A-24C The Unified Manifold Approximation and Projection ("UMAP") of induced H0011 cells was demonstrated, which revealed two cell clusters expressing hepatocyte genes and down-regulation of pluripotency genes. Clustering of induced H0011 cells revealed six clusters of gene expression profiles ( Figure 24A ). The gene expression heat map showed that the expression of hepatocyte genes in clusters 4 and 5 was upregulated, including APOA2, SERPINA1, APOA1, RBP4, CEBPA, HP and APOB ( Figure 24B Mapping the gene expression of the selected genes on UMAP showed that clusters 4 and 5 had lower expression of the stem cell marker POU5F1 and higher expression of hepatocyte markers such as APOA2, fibrinogen (FGA), vitronectin (VTN), and albumin (ALB) ( Figure 24C ).

[0083] Figures 25A-25C Illustrated is the machine learning classification of hepatocyte-like cells in induced H0011 cells. UMAP shows the four samples studied: primary human hepatocytes, iPSCs, induced H0011, and uninduced H0011. Uninduced H0011 cells cluster more closely with iPSCs than with induced H0011 ( Figure 25A The hepatocyte-like cells classified by the classification algorithm include primary hepatocytes and iPSC-derived hepatocytes ( Figure 25B ). Graph showing the number of hepatocyte-like cells and iPSC-like cells in each sample ( Figure 25C ).

[0084] Figure 26A-Figure 2 6.B shows that predicted hepatocyte-like cells in induced H0011 cells express specific immature and mature hepatocyte markers. Predicted hepatocyte-like cells cluster together in the UMAP of induced H0011 cells ( Figure 26A Relative gene expression of a selected panel of hepatocyte markers was assessed in all samples; hepatocyte-like cells predicted by the induced H0011 population had enriched expression of many key mature hepatocyte genes, including albumin, ASGR-1, apolipoprotein, and SERPINA1 (alpha-1-antitrypsin) ( Figure 26B The predicted hepatocyte-like cells also expressed some immature hepatocyte markers, such as DLK1 and AFP.

[0085] Figure 27 The description shows that the predicted hepatocyte-like cells in induced H0011 have abundant expression of many hepatocyte-related genes, including CYP3A5, CYP2B6, CYP2A7, SULT1A1, ABCC3, ABCC6, FAH, ALDH6A1, HAL, TAT, ABCC2, LEPR, ASL, HPR, CP, DEFB1, ACSL1, PLIN1, MTTP, APOB, and APOM.

[0086] Figure 28 Description Describes an assay for identifying a combination of transcription factors that produces a high proportion of hepatocyte-like cells in induced H0011 cells.

[0087] Figure 29 Illustrated are induced cells from the engineered line H0014-16 stained with antibodies to alpha-fetoprotein (AFP), cytokeratin 18 (CK18), albumin, and DAPI to mark the nuclei. Scale bar: 50 μm.

[0088] Figure 30 Illustrated are induced cells from engineered lines H0020, H0021, and H0025 stained with antibodies to AFP, CK18, albumin, and DAPI to mark the nucleus. Scale bar: 100 μm.

[0089] Figure 31 Illustrated are induced cells from engineered lines H0015C and H0026A-30A stained with antibodies to A1AT, AFP, CK18, and albumin. Scale bar: 100 μm.

[0090] Figure 32 Illustrated are induced cells from engineered lines H0011C and H0031A-35A stained with antibodies to A1AT, AFP, CK18, and albumin. Scale bar: 100 μm.

[0091] Figures 33A-33C Quantification of immunofluorescence staining on lines H0011C and H0031A-35A stained with antibodies to albumin, CK18, and ASGR-1 is illustrated. Figure 33A ), CK18( Figure 33B ) or ASGR-1( Figure 33C ) as indicated by the mean object intensity exceeding a set threshold.

[0092] Figure 34 Illustrated in H0014-35 cells induced for 96 hours, and then measured via flow cytometry the transcription factor Tra-1-60 expression (a marker of pluripotency). Isotype shows background fluorescence. Non-engineered iPSCs are positive controls, and HepG2 cells are negative controls.

[0093] Figure 35 The figure shows that the transcription factor ASGR1 (a marker of mature hepatocytes) was induced in H0014-35 cells for 96 hours and then measured by flow cytometry. The isotype shows background fluorescence. Non-engineered iPSCs are negative controls and HepG2 cells are positive controls.

[0094] Figure 36 The figure shows the percentage of ASGR1+ cells in the TRA-1-60- population after 96 hours of induction in H0014-35 cells and subsequent measurement of ASGR1 and TRA-1-60 expression by flow cytometry.

[0095] Figure 37 Illustrated in H0014-35 cells induced for 96 hours, and then measured via flow cytometry the transcription factor CXCR4 expression (a marker of definitive endoderm). Isotype shows background fluorescence. Non-engineered iPSCs are negative controls, and HepG2 cells are positive controls.

[0096] Figures 38A-38C RT-qPCR data are presented showing increased induction of liver-specific marker transcription factors. RT-qPCR was performed on uninduced and induced engineered hepatocyte-like cells and primary human hepatocytes, normalized by housekeeping genes. ALB ( Figure 38A )、CYP3A7( Figure 38B ) and CYP3A4( Figure 38C ) Fold changes in gene expression. Significance was determined by unpaired t-test; *p<0.5, **p<0.01.

[0097] Figure 39 Depicted are the amounts of albumin produced by uninduced and induced engineered cell lines H0027B, H0028B, H0031A, and H0033A, as measured by ELISA and normalized to time and total cell number.

[0098] Figure 40Depicted are the amounts of A1AT produced by uninduced and induced engineered cell lines H0027B, H0028B, H0031A, and H0033A, as measured by ELISA and normalized to time and total cell number.

[0099] Figure 41 Schematic diagram depicting H0028B and H0033A cells that were isolated and differentiated for four days (96 hours) and seven days and analyzed by several indicators, including immunofluorescence staining (for A1AT, CK18, and albumin), ELISA for detection of secreted A1AT and albumin, chromogenic assay for detection of secreted urea, and measurement of luminescent substrate cleavage rate for cytochrome p450 enzyme activity. The best performers, as assessed by these indicators, exhibited the most hepatocyte-like phenotype and were selected for expansion and further validation.

[0100] Figures 42A-42B The top performers from H0028 (H28B) and H0033 (H33A) cell lines cultured in 0.5 μM A83-01 for 4 days (96 hours) in mTeSR Plus stem cell medium with transcription factor induction are shown. The cells were then stained with antibodies to detect A1AT, CK18, and albumin. Several performers functioned well in multiple assays, indicating that they are hepatocyte-like ( Figures 42A-42B When comparing A1AT, CK18, and albumin in the best performers to the original cells, a higher percentage of cells expressed A1AT, CK18, and albumin compared to the uninduced and parental cells ( Figures 42A-42B ). Numbers below the images indicate the percentage of cells expressing each marker, averaged across nine fields of view. Scale bar: 100 μm.

[0101] Figure 43A-43B Cell lines expressing H0028B and H0033A transcription factors were described, which were also cultured in mTeSR Plus with transcription factor induction for 4 days (96 hours). After 96 hours of staining, cells were next stained with antibodies to A1AT, CK18, and albumin. Several performers functioned well in multiple assays, indicating that they were hepatocyte-like ( Figures 43A-43B When comparing A1AT, CK18, and albumin in the best performers to the original cells, a higher percentage of cells expressed A1AT, CK18, and albumin compared to the uninduced and parental cells ( Figures 43A-43B ). Numbers below the images indicate the percentage of cells expressing each marker, averaged across nine fields of view. Scale bar: 100 μm.

[0102] Figures 44A-44BCell lines expressing H0028B and H0033A were described, which were also cultured in mTeSR Plus without transcription factor induction for 96 hours. They were then stained using antibodies to A1AT, CK18, and albumin. Several expressors functioned well in multiple assays, indicating that they are hepatocyte-like ( Figures 44A-42B When comparing A1AT, CK18, and albumin in the best performers to the original cells, a higher percentage of cells expressed A1AT, CK18, and albumin compared to the uninduced and parental cells ( Figures 44A-44B ). Numbers below the images indicate the percentage of cells expressing each marker, averaged across nine fields of view. Scale bar: 100 μm. DETAILED DESCRIPTION

[0103] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, variations, and substitutions will occur to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0104] Pluripotent stem cells (PSCs) are characterized by their ability to self-renew while maintaining potency for therapeutic applications. PSCs also have the ability to differentiate into a large number of different phenotypes (e.g., hepatocytes). Disclosed herein are various compositions, formulations, and methods for promoting the efficient differentiation of PSCs into hepatocytes (and / or liver cells).

[0105] Hepatocytes or liver cells generated using the compositions, formulations, and methods disclosed herein can be used in therapeutic applications where classical lineage hepatocytes have traditionally been used. Thus, the various compositions, formulations, and methods disclosed herein provide robust improvements to hepatocyte-based therapeutics because the compositions, formulations, and methods disclosed herein are capable of efficiently and reproducibly differentiating PSCs into potent hepatocytes and / or liver cells.

[0106] PSCs are able to divide indefinitely and produce identical daughter cells. When provided with a signal, 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).

[0107] In some aspects, the present disclosure provides methods and compositions for efficiently and reproducibly differentiating PSCs into hepatocytes or liver cells (e.g., as defined by cell surface marker expression characteristics, function, etc.). These methods and compositions can provide robust improvements (e.g., availability, dosage, efficacy, etc.) to current cell therapies administered using hepatocytes or liver cells.

[0108] pluripotent stem cells

[0109] Pluripotent stem cells (PSCs) can be characterized by self-renewal and efficacy. PSCs are able to divide indefinitely and produce identical daughter cells. When provided with a signal, 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 differentiation ability of cells.

[0110] iPSC is a pluripotent stem cell derived from adult cells that has been genetically reprogrammed to an embryonic stem (ES) cell-like state by expressing genes and factors that are essential for maintaining the defining properties of embryonic stem cells. iPSCs are similar to ES cells in many ways, including 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. Research reports indicate that iPSCs are directed to differentiate into different functional cell types in vitro, and the cell therapy effects of implanted iPSCs have been demonstrated in several animal models of disease. Directed differentiation is a bioengineering method that harnesses the potential of stem cells by restricting their differentiation into specific cell types or target tissues in vitro. Directed differentiation may be primarily applied to PSCs of mammalian origin, such as mouse and human cells for biomedical research applications. Cell differentiation may involve a transition from a proliferation mode to a differentiation mode. Directed differentiation can include mimicking developmental culture under controlled conditions, involving specific substrates or extracellular matrices that promote cell adhesion and differentiation, and determining the composition of the culture medium. Signal transduction factors, such as growth factors or small molecules, can be applied in different dosages and exposure time sequences or in combination to regulate differentiation. Direct reprogramming, also referred to as transdifferentiation or direct conversion, can include overexpression of one or more factors introduced into the cell. In one example, one or more factors can be transcription factors. By analyzing cell type specific markers, gene expression profiles, and functional assays, the appropriate differentiation of the target cell type can be verified.

[0111] 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 differentiate into one or more hepatocytes or liver cells by various methods, including being induced to express one or more transcription factors, contacting with one or more transcription factor proteins or nucleic acids encoding transcription factor proteins, or by other means as 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 PSC. The one or more transcription factors can induce PSC to differentiate into hepatocytes or liver cells. One or more transcription factors can induce the PSC to differentiate into hepatocytes or liver cells, or cells expressing ASGR-1, or cells expressing CXCR-4, or 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, or any combination thereof. The pluripotent stem cells can be induced to differentiate into one or more hepatocytes or liver cells in 30 days or less. The pluripotent stem cells can be induced to differentiate into one or more hepatocytes or liver cells in 96 hours or less. The exogenous expression cassette can induce the PSC to differentiate into hepatocytes or liver cells in 30 days or less. The exogenous expression cassette can induce the PSC to differentiate into hepatocytes or liver cells in 96 hours or less. The PSC can be provided in culture medium. The culture medium does not need to be changed during the differentiation of the PSC into hepatocytes or liver cells. During the differentiation of PSCs into hepatocytes or liver cells, the culture medium may be altered. A cell population comprising one or more hepatocytes or liver cells may be generated. The cell population may comprise one or more hepatocytes or liver cells, cells expressing ASGR-1, cells expressing CXCR-4, cells expressing cytokeratin-18 (CK18), cells expressing alpha-fetoprotein (AFP), cells expressing alpha-1-antitrypsin (A1AT), cells expressing albumin, or cells expressing cytokine p450. The cell population may comprise adherent cells. The cell population may comprise suspension cells. The cell population may comprise adherent cells and suspension cells. The cell population may be provided in a culture medium. During the differentiation of PSCs into hepatocytes or liver cells, the culture medium may not need to be altered. The culture medium may not require any nutrients, growth factors, or microenvironment or matrix optimization. The culture medium may require any nutrients, growth factors, or microenvironment or matrix optimization. In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is not performed.In some embodiments, nutrient, growth factor, or microenvironment / matrix optimization is performed. In some embodiments, nutrient, growth factor, and microenvironment / matrix optimization is performed. In some embodiments, nutrient optimization is not performed. In some embodiments, growth factor optimization is not performed. In some embodiments, microenvironment / matrix optimization is not performed. In some embodiments, microenvironment optimization is not performed. In some embodiments, matrix optimization is not performed. In some embodiments, nutrient or growth factor optimization is not performed. In some embodiments, nutrient or microenvironment / matrix optimization is not performed. In some embodiments, growth factor or microenvironment / matrix optimization is not performed. In some embodiments, nutrient or microenvironment optimization is not performed. In some embodiments, growth factor or microenvironment optimization is not performed. In some embodiments, nutrient or matrix optimization is not performed. In some embodiments, growth factor or matrix optimization is not performed.

[0112] Hepatocytes

[0113] Hepatocytes can be found in the liver. Hepatocytes can be involved in protein synthesis, protein storage, carbohydrate conversion, cholesterol synthesis, bile salt synthesis, phospholipid synthesis, detoxification, and bile secretion. Hepatocytes can be cuboidal. In one example, a hepatocyte can have a side length of 20-30 microns. In another example, a hepatocyte can have a side length of 3.4 x 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.

[0114] In some embodiments, hepatocytes may express the protein asialoglycoprotein receptor 1 (ASGR-1), also known as ASGR1, ASGPR, ASGPR1, CLEC4H1, HL-1, or asialoglycoprotein receptor 1. ASGR-1 (e.g., a subunit thereof) may be encoded by the ASGR-1 gene. The ASGR-1 protein may function as a receptor protein. ASGR-1 may be a transmembrane protein. ASGR-1 may play a role in serum glycoprotein homeostasis. ASGR-1 may mediate endocytosis of glycoproteins. ASGR-1 may mediate endocytosis of glycoproteins with exposed terminal galactose. ASGR-1 may mediate endocytosis of glycoproteins with N-acetylgalactosamine residues. ASGR-1 may promote liver infection. ASGR-1 may promote viral infection of the liver. ASGR-1 may promote hepatitis B infection of the liver. ASGR-1 may serve as a target for liver-specific drug delivery. ASGR-1 may be a hetero-oligomeric protein. ASGR-1 may comprise a major subunit and a minor subunit. The major and minor subunits can be encoded by different genes. Multiple isoforms of ASGR-1 can be encoded through alternative splicing.

[0115] In some embodiments, hepatocytes may express the chemokine receptor type 4 protein CXCR-4, also known as, for example, fusin, CXCR4, CD184, D2S201E, FB22, HM89, HSY3RR, LAP-3, LAP3, LCR1, LESTR, NPY3R, NPYR, NPYRL, NPYY3R, WHIM, WHIMS, CXC motif chemokine receptor 4, or WHIMS1. CXCR-4 can be classified as a CXC chemokine receptor. CXCR-4 can be considered an α-chemokine receptor. CXCR-4 can be specific for stromal-derived factor-1 (SDF-1). SDF-1 can exhibit chemotaxis for lymphocytes. SDF-1 may be important in the homing of hematopoietic stem cells to the bone marrow. SDF-1 may be important in the quiescence of hematopoietic stem cells. CXCR-4 can help liver regeneration. CXCR-4 can be pro-fibrotic. CXCR-4 can promote fibrosis by activating hepatic stellate cells. CXCR-4 signaling may be detrimental to liver recovery and regeneration. Clinical therapy using CXCR-4 antagonists may improve liver recovery after acute liver injury. In liver disease, abnormal CXCR-4 expression may be associated with the migration and motility of liver-specific cells. In liver disease, abnormal CXCR-4 expression may be associated with the migration and motility of liver-specific cells through crosstalk with other pathways. CXCR-4 and its ligands may play a role in hepatitis. Inflammatory response mechanisms mediated by CXCR-4 signaling may affect (e.g., adversely) the chemotaxis of inflammatory cells (e.g., lymphocytes, neutrophils, and monocytes). CXCR-4 may be used by HIV to infect T cells. The CXCR-4 receptor may be involved in the adhesion phase of human implantation. CXCR-4 signaling can regulate the expression of CD20 on B cells. Ubiquitin may be a natural ligand for CXCR-4. CXCR-4 dimerization may be dynamic. CXCR-4 dimerization may increase with concentration. CXCR-4 can be expressed in cancers including breast, ovarian, melanoma or prostate cancer.

[0116] 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 cells expressing any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein that is associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. In some embodiments, the hepatocytes, or liver cells, or cells expressing ASGR-1, or cells expressing CXCR-4, or 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 induce differentiation of one or more PSCs. The exogenous expression cassette can induce PSCs to differentiate into hepatocytes, liver cells, 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, or cells expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. In some embodiments, the PSCs can be provided in culture.The culture medium may not need to be changed during differentiation of PSCs into hepatocytes, liver cells, or cells expressing ASGR-1, cells expressing CXCR-4, or 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, or cells expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. In some embodiments, the PSCs may be provided in culture medium.

[0117] The present disclosure provides PSCs that can be induced to differentiate into one or more hepatocytes or liver cells. The pluripotent stem cells can be induced to differentiate into one or more hepatocytes or liver cells by various methods, including being induced to express one or more transcription factors, contacting with one or more transcription factor proteins or nucleic acids encoding transcription factor proteins, or by other means as disclosed herein. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in 30 days or less. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 1 day. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to 4 days. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 96 hours. The PSC can be induced to differentiate into one or more hepatocytes or liver cells in up to about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10 hours or less. 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 the PSC. The one or more transcription factors can induce the PSC to differentiate into hepatocytes or liver cells. The PSC can contain one or more exogenous expression cassettes. The one or more exogenous cassettes can contain one or more transcription factors that induce the PSC to differentiate into hepatocytes, liver cells, or cells expressing ASGR-1, cells expressing CXCR-4, or cells expressing any other hepatocyte-like, hepatocyte lineage or hepatocyte-related markers disclosed herein, or any other markers disclosed herein that are associated with the non-natural, exogenous induction of iPSCs along a differentiation trajectory. The exogenous expression cassette can induce the PSC to differentiate into hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4 in 30 days or less. The exogenous expression cassette can induce the PSC to differentiate into hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4 in 96 hours or less. The PSC can be provided in a culture medium. The culture medium does not need to be changed during the differentiation of the PSC into hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, cells not expressing ASGR-1, or cells not expressing CXCR-4.

[0118] The present disclosure provides a cell population comprising one or more hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, or cells expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. The cell population may comprise adherent cells. The cell population may comprise suspension cells. The cell population may comprise adherent cells and suspension cells. The cell population may be provided in a culture medium. The culture medium may not need to be changed during differentiation of PSCs into hepatocytes, liver cells, cells expressing ASGR-1, cells expressing CXCR-4, or cells expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. The cell population may be provided in a culture medium. The culture medium may not require any nutrients, growth factors, or microenvironmental or matrix optimization. At least 5% of the cells may 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, or cells that can express any other hepatocyte-like, hepatocyte-lineage or hepatocyte-associated marker disclosed herein, or any other marker associated with differentiation of (TF formulation) non-natural, exogenous induction of iPSCs along a differentiation trajectory disclosed herein, can express ASGR-1. At least 5% of the cells, or cells that can express any other hepatocyte-like, hepatocyte-lineage or hepatocyte-associated marker disclosed herein, or any other marker associated with differentiation of (TF formulation) non-natural, exogenous induction of iPSCs along a differentiation trajectory disclosed herein, can express CXCR-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 may express ASGR-1.

[0119] In some embodiments, at least 0.01% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 0.1% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 0.5% of the hepatocytes or liver cells or cells expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory, express ASGR-1. In some embodiments, at least 1% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 1.5% of the hepatocytes or liver cells express ASGR-1. 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 4% 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 7% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 8% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 9% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 10% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 12% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 15% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 17% of the hepatocytes or liver cells express ASGR-1. In some embodiments, at least 20% of the hepatocytes or liver 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.

[0120] In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1, or cells expressing any other hepatocyte-like, hepatocyte-lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation 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, in some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 is 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 18%, at most about 20%, at most about 21%, at most about 22%, at most about 23%, at most about 24%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34%, at most about 35%, at most about 36%, at most about 37%, at most about 38%, at most about 39%, at most about 40%, at most about 41%, at most about 42%, at most about 43%, at most about 44%, at most about 45%, at most about 46%, at most about 47%, at most about 48%, at most about 49%, at most about 50%, at most about 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%.

[0121] In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 is from about 1% to about 90%. In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 or expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory is at least about 1%. In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 is up to about 90%. In some embodiments, the percentage of hepatocytes or liver cells expressing ASGR-1 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%, 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 In some embodiments, the percentage of hepatocytes or liver 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%, 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%.

[0122] 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 or cells expressing any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with (TF formula) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory express CXCR-4. In some embodiments, at least 0.5% of the hepatocytes or liver cells or cells expressing any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with (TF formula) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory express CXCR-4. 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.

[0123] In some embodiments, the percentage of hepatocytes or hepatocytes that express CXCR-4 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, in some embodiments, the percentage of hepatocytes or liver cells expressing CXCR-4 or cells expressing any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formulation) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory is 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 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 17%, at most about 18%, at most about 19%, at most about 20%, at most about 21%, at most about 22%, at most about 23%, at most about 24%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34%, at most about 35%, at most about 36%, at most about 37%, at most about 38%, at most about 39%, at most about 40%, at most about 41%, at most about 42%, at most about 43%, at most about 44%, at most about 45%, at most about 46%, at most about 47%, at most about 48%, at most about 49%, at most about 50%, at most about 51%, at most about 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%.

[0124] In some embodiments, the percentage of hepatocytes or liver cells expressing CXCR-4 is from about 1% to about 90%. In some embodiments, the percentage of hepatocytes or liver cells expressing CXCR-4 or expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with differentiation of (TF formulation) non-natural, exogenous induction of iPSCs along a differentiation trajectory is at least about 1%. In some embodiments, the percentage of hepatocytes or liver cells expressing CXCR-4 or expressing any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein as associated with differentiation of (TF formulation) non-natural, exogenous induction of iPSCs along a differentiation trajectory is at most about 90%. In some embodiments, the percentage of hepatocytes or liver cells that express CXCR-4 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 ... 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 In some embodiments, the percentage of hepatocytes or liver 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%, 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 percentage of hepatocytes or liver 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%.

[0125] The present disclosure provides one or more liver cells. The liver cells may be similar to hepatocytes. The liver cells may be polygonal. For example, the liver cells may be hexagonal. The liver cells may be cubic. The stem cells may be pluripotent stem cells. The pluripotent stem cells may be induced pluripotent stem cells. The liver cells may express ASGR-1. The liver cells may express CXCR-4. The liver cells may express both ASGR-1 and CXCR-4. In a population of two or more liver cells, at least 2% of the liver cells may express ASGR-1 or any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formula) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory. In a population of two or more liver cells, at least 10% of the liver cells or liver cells express CXCR-4 or any other hepatocyte-like, hepatocyte lineage, or hepatocyte-associated marker disclosed herein, or any other marker disclosed herein associated with (TF formula) non-natural, exogenous induction of iPSC differentiation along a differentiation trajectory.

[0126] transcription factors

[0127] The present disclosure provides transcription factors (TFs) whose expression can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. Transcription factors can induce differentiation by various methods, such as expressing one or more nucleic acids encoding one or more transcription factors in PSCs, contacting PSCs with one or more transcription factor proteins or nucleic acids encoding transcription factor proteins, or by other means as disclosed herein. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in 30 days or less. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 1 day. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to 4 days. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 96 hours. The PSCs can be induced to differentiate into one or more hepatocytes or liver cells in up to about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10 hours or less. One or more nucleic acids encoding one or more transcription factors can be expressed in one or more exogenous expression cassettes.

[0128] TFs can be used to trigger differentiation programs. Certain transcription factors can be able to induce stem cells to differentiate into specific lineages, such as hepatocytes or liver cells. In some aspects, combinations of transcription factors can be used to achieve differentiation into specific cell lineages. The combination can achieve cell types or cell subtypes that cannot be achieved by any single transcription factor. The combination can achieve the same cell type as one of the transcription factors alone, but can achieve it more efficiently.

[0129] The present disclosure provides cells, exogenous expression cassettes and methods, including the use of one or more transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can induce the differentiation of one or more PSCs. One or more transcription factors can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. A combination comprising one or more transcription factors can be produced. The combination of transcription factors can be introduced into one or more expression cassettes. One or more expression cassettes can be induced in one or more PSCs. Inducing one or more expression cassettes in one or more PSCs can cause one or more PSCs to differentiate into hepatocytes or liver cells. Some transcription factors may require a critical amount of expression to effectively induce differentiation, such as the equivalent of at least 5, 10, 15, 20, 25 or 50 open reading frame (ORF) copies / cell. Other factors may need to be below a certain expression threshold because there may be a high level of toxicity, such as less than 20, 10 or 5 copies / cell. An increase in expression level can also be achieved by increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon.

[0130] In some embodiments, the 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, the one or more transcription factors comprise FoxA1. In some embodiments, the one or more transcription factors comprise one or more members, such as 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, the one or more transcription factors comprise FoxA1. In some embodiments, the one or more transcription factors comprise HNF1A. In some embodiments, the one or more transcription factors comprise SPI1. In some embodiments, the one or more transcription factors comprise FoxA2. In some embodiments, the one or more transcription factors comprise CEBPA. In some embodiments, the one or more transcription factors comprise ONECUT1 / HNF6. In some embodiments, the one or more transcription factors comprise ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors comprise HNF4a. In some embodiments, the one or more transcription factors comprise Atf3. In some embodiments, the one or more transcription factors comprise Fos. In some embodiments, the one or more transcription factors comprise CEBPB. In some embodiments, the one or more transcription factors comprise FoxM1. In some embodiments, the one or more transcription factors comprise GATA2 isoform P23769-2. In some embodiments, the one or more transcription factors comprise GATA4. In some embodiments, the one or more transcription factors comprise GATA2 isoform P23769-1.In some embodiments, the one or more transcription factors comprise JUN / AP-1. In some embodiments, the one or more transcription factors comprise NFIX. In some embodiments, the one or more transcription factors comprise GATA6. In some embodiments, the one or more transcription factors comprise TBX3. In some embodiments, the one or more transcription factors comprise RXRB. In some embodiments, the one or more transcription factors comprise NFE2L2. In some embodiments, the one or more transcription factors comprise Myc. In some embodiments, the one or more transcription factors comprise TP73. In some embodiments, the one or more transcription factors comprise HHEX. In some embodiments, the one or more transcription factors comprise SOX17. In some embodiments, the one or more transcription factors comprise ATF5. In some embodiments, the one or more transcription factors comprise RBPJ. In some embodiments, the one or more transcription factors comprise Smad3. In some embodiments, the one or more transcription factors comprise NR5A2. In some embodiments, the one or more transcription factors comprise HIF1A. In some embodiments, the one or more transcription factors comprise PROX. In some embodiments, the one or more transcription factors comprise FOXA3. In some embodiments, the one or more transcription factors comprise EGR1. In some embodiments, the one or more transcription factors comprise NR1I2 / PXR. In some embodiments, the one or more transcription factors comprises SKI.

[0131] In some embodiments, the one or more transcription factors comprise ONECUT1 / HNF6 or ONECUT2 / HNF6B. In some embodiments, the one or more transcription factors comprise ONECUT1 / HNF6 and ONECUT2 / HNF6B.

[0132] In some embodiments, one or more of the one or more transcription factors is, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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.

[0133] In some embodiments, the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a, or 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 isoform P23769-2, GATA4, GATA2 isoform P23769-1, JUN / AP-1, or NFIX. In some embodiments, the 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, the one or more transcription factors are, for example, GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, or HNF6. In some embodiments, the one or more transcription factors are, for example, 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, ​​or FoxA1. In some embodiments, the one or more transcription factors are, for example, HHEX, SOX17, or HNF4A. In some embodiments, the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, or HNF1A. In some embodiments, the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, ​​CEBPA, ONECUT1 / HNF6, HNF4a, or RBPJ. In some embodiments, the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 isoform P23769-1, CEBPB, or NR113. In some embodiments, the one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, or TBX3.

[0134] Forkhead box (FOX) transcription factors

[0135] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. The transcription factor can be a member of the FOX transcription factor family. The FOX transcription factor can be, for example, FoxA1, FoxA2, ​​FoxA3, or FoxM1.

[0136] FoxA1

[0137] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. In some embodiments, one or more transcription factors can be used. In some embodiments, the transcription factor can be FoxA1, which can also be referred to 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 3alpha.

[0138] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include FoxA1. FoxA1 may be introduced into the expression cassette. The expression cassette comprising FoxA1 may be introduced into a PSC. The expression cassette comprising FoxA1 may be expressed in the PSC. The expression cassette comprising FoxA1 may induce the PSC to differentiate into hepatocytes or liver cells.

[0139] FoxA1 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising FoxA1 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0140] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise FoxA1. An expression cassette comprising FoxA1 can be expressed in PSCs. An expression cassette comprising FoxA1 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of FoxA1 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of FoxA1 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of FoxA1 can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0141] In some embodiments, the number of copies of the introduced FoxA1 ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced FoxA1 ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced FoxA1 ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced FoxA1 ORF 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 or 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 number of introduced FoxA1 ORF copies 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 one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include FoxA1. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0143] FoxM1

[0144] The present disclosure provides cells, expression cassettes, and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be FoxM1, which may also be referred to 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 responsive 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.

[0145] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may include FoxM1. FoxM1 may be introduced into the expression cassette. FoxM1 may be expressed in the expression cassette. The expression cassette comprising FoxM1 may be introduced into a PSC. The expression cassette comprising FoxM1 may be expressed in the PSC. The expression cassette comprising FoxM1 may induce the PSC to differentiate into hepatocytes or liver cells.

[0146] FoxM1 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising FoxM1 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0147] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise FoxM1. An expression cassette comprising FoxM1 can be expressed in PSC. An expression cassette comprising FoxM1 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of FoxM1 can be introduced into PSC. Different amounts of FoxM1 can be introduced into PSC. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of FoxM1 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of FoxM1 can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0148] In some embodiments, the number of copies of the introduced FoxM1 ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced FoxM1 ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced FoxM1 ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced FoxM1 ORF 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 or 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 number of introduced FoxM1 ORF copies 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.

[0149] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include FoxM1. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0150] FoxA2

[0151] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be FoxA2, ​​which may also be referred to as forkhead box A2; HNF3B; hepatocyte nuclear factor 3-beta; forkhead box protein A2; transcription factor 3B; HNF-3-beta; TCF3B; hepatocyte nuclear factor 3 beta; hepatic nuclear factor-3-beta; HNF-3B; or TCF-3B.

[0152] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise FoxA2. FoxA2 may be introduced into the expression cassette. The expression cassette comprising FoxA2 may be introduced into a PSC. FoxA2 may be introduced into the expression cassette. FoxA2 may be expressed in the expression cassette. The expression cassette comprising FoxA2 may be introduced into a PSC. The expression cassette comprising FoxA2 may be expressed in a PSC. The expression cassette comprising FoxA2 may induce PSCs to differentiate into hepatocytes or liver cells.

[0153] FoxA2 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising FoxA2 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0154] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise FoxA2. An expression cassette comprising FoxA2 can be expressed in PSCs. An expression cassette comprising FoxA2 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of FoxA2 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of FoxA2 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of FoxA2 can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0155] In some embodiments, the number of copies of the introduced FoxA2 ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced FoxA2 ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced FoxA2 ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced FoxA2 ORF 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 or 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 number of introduced FoxA2 ORF copies 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.

[0156] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include FoxA2. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0157] FOXA3

[0158] 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 they also interact with chromatin. The present disclosure provides cells, expression cassettes, and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. In some embodiments, one or more transcription factors may be used. In some embodiments, the transcription factor may be FoxA3, which may also be referred to as forkhead box A3; HNF3G, hepatocyte nuclear factor 3-gamma; forkhead-related protein FKH H3; forkhead box protein A3; or transcription factor 3G.

[0159] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise FoxA3. FoxA3 may be introduced into the expression cassette. The expression cassette comprising FoxA3 may be introduced into a PSC. FoxA3 may be introduced into the expression cassette. FoxA3 may be expressed in the expression cassette. The expression cassette comprising FoxA3 may be introduced into a PSC. The expression cassette comprising FoxA3 may be expressed in a PSC. The expression cassette comprising FoxA3 may induce PSCs to differentiate into hepatocytes or liver cells.

[0160] FoxA3 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising FoxA3 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0161] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise FoxA3. An expression cassette comprising FoxA3 can be expressed in PSCs. An expression cassette comprising FoxA3 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of FoxA3 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of FoxA3 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of FoxA3 can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon.

[0162] In some embodiments, the number of copies of the introduced FoxA3 ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced FoxA3 ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced FoxA3 ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced FoxA3 ORF 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 or 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 number of introduced FoxA3 ORF copies 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.

[0163] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include FoxA3. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0164] HNF hepatocyte nuclear factor transcription factor

[0165] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. The transcription factor can be a member of the HNF hepatocyte nuclear factor transcription factor family. The HNF hepatocyte nuclear factor transcription factor can be, for example, HNF1A or HNF4A.

[0166] HNF1A

[0167] The present disclosure provides cells, expression cassettes, and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. 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, which can also be referred to as HNF1 homeobox A; LFB1; HNF1; TCF1; liver-specific transcription factor LF-B1; hepatocyte nuclear factor 1-alpha; HNF-1-alpha; HNF-1A; MODY3; TCF-1; hepatic transcription factor 1; LF-B1, hepatic nuclear factor (HNF1), albumin-proximal factor; truncated hepatocyte nuclear factor 1alpha; interferon production regulatory factor; hepatic transcription factor 1; hepatic nuclear factor 1; albumin-proximal factor; transcription factor 1; HNF1alpha; IDDM20; HNF1α; or HNF1A.

[0168] 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 a PSC. The expression cassette comprising HNF1A may be expressed in the PSC. The expression cassette comprising HNF1A may induce the PSC to differentiate into hepatocytes or liver cells.

[0169] HNF1A can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising HNF1A and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0170] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include HNF1A. An expression cassette comprising HNF1A can be expressed in PSCs. An expression cassette comprising HNF1A can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of HNF1A can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of HNF1A can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of HNF1A can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon.

[0171] In some embodiments, the number of copies of the introduced HNF1A ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced HNF1A ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced HNF1A ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced HNF1A ORF 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 or 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 number of copies of the introduced HNF1A ORF 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 one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include HNF1A. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0173] HNF4a

[0174] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. 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, which may also be referred to as hepatocyte nuclear factor 4 alpha; NR2A1; HNF4; TCF14; nuclear receptor subfamily 2A group 2 member 1; hepatocyte nuclear factor 4-alpha; transcription factor HNF-4; transcription factor 14; TCF-14; MODY1; MODY; hepatic nuclear factor 4 alpha; HNF4 alpha 10 / 11 / 12; HNF-4-alpha; HNF4 alpha; HNF4a7; HNF4a8; HNF4a9; NR2A21; FRTS4; or TCF.

[0175] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise HNF4a. HNF4a may be introduced into the expression cassette. HNF4a may be expressed in the expression cassette. The expression cassette comprising HNF4a may be introduced into a PSC. The expression cassette comprising HNF4a may be expressed in the PSC. The expression cassette comprising HNF4a may induce the PSC to differentiate into hepatocytes or liver cells.

[0176] HNF4a can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising HNF4a and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0177] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver 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 PSCs to differentiate into hepatocytes or liver cells. Different amounts of HNF4a can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of HNF4a can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of HNF4a can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0178] In some embodiments, the number of copies of the introduced HNF4a ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced HNF4a ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced HNF4a ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced HNF4a ORF 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 or 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 number of copies of the introduced HNF4a ORF 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.

[0179] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include HNF4a. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0180] CUT homeobox transcription factor

[0181] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. The transcription factor can be a member of the CUT homeobox transcription factor family. The transcription factor can be, for example, HNF6A / ONECUT1 or HNF6B / ONECUT2.

[0182] ONECUT1 / HNF6

[0183] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be a ONECUT transcription factor, which may also be referred to as ONECUT1; One Cut homeobox 1; HNF-6; HNF6A; HNF6; One Cut domain family member 1; hepatocyte nuclear factor 6; hepatocyte nuclear factor 6α; or One Cut domain family member 1. The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise ONECUT1 / HNF6. ONECUT1 / HNF6 can be introduced into an expression cassette. ONECUT1 / HNF6 can be expressed in an expression cassette. An expression cassette comprising ONECUT1 / HNF6 can be introduced into a PSC. An expression cassette comprising ONECUT1 / HNF6 can be expressed in a PSC. An expression cassette comprising ONECUT1 / HNF6 can induce PSC differentiation into hepatocytes or liver cells.

[0184] ONECUT1 / HNF6 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an 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 PSCs to differentiate into hepatocytes or liver cells.

[0185] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more 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 PSCs to differentiate into hepatocytes or liver cells. Different amounts of ONECUT1 / HNF6 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of ONECUT1 / HNF6 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of ONECUT1 / HNF6 can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0186] In some embodiments, the number of copies of the ORF of ONECUT1 / HNF6 introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of ONECUT1 / HNF6 introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of ONECUT1 / HNF6 introduced per cell may be at most about 90. In some embodiments, the number of copies of the introduced ONECUT1 / HNF6 ORF 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 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the number of copies of the introduced ONECUT1 / HNF6 ORF 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.

[0187] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include ONECUT1 / HNF6. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0188] ONECUT2 / HNF6B

[0189] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be a ONECUT transcription factor, which may also be referred to as ONECUT2; One Cut homeobox 2; OC-2; hepatocyte nuclear factor 6-beta; One Cut domain family member 2; transcription factor ONECUT-2; HNF-6-beta; ONECUT-2 homeodomain transcription factor; One Cut domain family member 2; Onecut 2; HNF6B; or OC2.

[0190] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise ONECUT2 / HNF6B. ONECUT2 / HNF6B may be introduced into the expression cassette. ONECUT2 / HNF6B may be expressed in the expression cassette. The expression cassette comprising ONECUT2 / HNF6B may be introduced into a PSC. The expression cassette comprising ONECUT2 / HNF6B may be expressed in a PSC. The expression cassette comprising ONECUT2 / HNF6B may induce PSCs to differentiate into hepatocytes or liver cells.

[0191] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include ONECUT2 / HNF6B. ONECUT2 / HNF6B can be introduced into an expression cassette along 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 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 PSCs to differentiate into hepatocytes or liver cells.

[0192] Different amounts of ONECUT2 / HNF6B can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of ONECUT2 / HNF6B can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of ONECUT2 / HNF6B can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0193] In some embodiments, the number of copies of the ORF of ONECUT2 / HNF6B introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of ONECUT2 / HNF6B introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of ONECUT2 / HNF6B introduced per cell may be at most about 90. In some embodiments, the number of copies of the introduced ONECUT2 / HNF6B ORF 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 2 or 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 number of copies of the introduced ONECUT2 / HNF6B ORF 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.

[0194] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include ONECUT2 / HNF6B. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0195] basic leucine zipper transcription factor

[0196] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. 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.

[0197] CEBPA

[0198] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be CCAAT enhancer binding protein (CEBP), which may also be referred to as CCAAT enhancer binding protein alpha; C / EBP-alpha; CEBP; CCAAT / enhancer binding protein (C / EBP) alpha; CCAAT / enhancer binding protein alpha; CCAAT / enhancer binding protein alpha; or C / EBP alpha.

[0199] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise CEBPA. CEBPA may be introduced into the expression cassette. CEBPA may be expressed in the expression cassette. The expression cassette comprising CEBPA may be introduced into a PSC. The expression cassette comprising CEBPA may be expressed in a PSC. The expression cassette comprising CEBPA may induce PSCs to differentiate into hepatocytes or liver cells.

[0200] CEBPA can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising CEBPA and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0201] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise CEBPA. An expression cassette comprising CEBPA can be expressed in a PSC. An expression cassette comprising CEBPA can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of CEBPA can be introduced into a PSC. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of CEBPA can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of CEBPA can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon.

[0202] In some embodiments, the number of copies of the ORF of CEBPA introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of CEBPA introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of CEBPA introduced per cell may be at most about 90. In some embodiments, the number of copies of the introduced ORF of CEBPA 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 or 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 number of copies of the introduced CEBPA ORF 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.

[0203] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include CEBPA. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0204] Atf3

[0205] The present disclosure provides cells, expression cassettes and methods that use transcription factors, or increase transcription or increase molecules activated by transcription factors. One or more transcription factors can be used. The transcription factor can be a transcription activator (ATF). In some embodiments, ATF can be Atf3, which can also be referred to as transcription activator 3, cyclic AMP-dependent transcription factor ATF-3 or cAMP-dependent transcription factor ATF-3.

[0206] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise Atf3. Atf3 may be expressed in the expression cassette. The expression cassette comprising Atf3 may be introduced into a PSC. The expression cassette comprising Atf3 may be expressed in the PSC. The expression cassette comprising Atf3 may induce the PSC to differentiate into hepatocytes or liver cells.

[0207] Atf3 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising Atf3 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0208] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise Atf3. An expression cassette comprising Atf3 can be expressed in PSC. An expression cassette comprising Atf3 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of Atf3 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of Atf3 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of Atf3 can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0209] In some embodiments, the number of ORF copies of Atf3 introduced per cell may be from about 1 to about 90. In some embodiments, the number of ORF copies of Atf3 introduced per cell may be at least about 1. In some embodiments, the number of ORF copies of Atf3 introduced per cell may be at most about 90. In some embodiments, the number of copies of the introduced Atf3 ORF 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 or 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 number of introduced Atf3 ORF copies 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.

[0210] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include Atf3. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0211] Fos

[0212] The present disclosure provides cells, expression cassettes and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be a member of the Fos family. The Fos family member can be Fos, which can also be referred to as Fos proto-oncogene AP-1 transcription factor subunit; AP-1; FBJ murine osteosarcoma viral oncogene homolog; G0 / G1 switch regulatory protein 7; proto-oncogene C-Fos; C-Fos; FBJ murine; osteosarcoma viral (V-Fos) oncogene homolog (oncogene FOS); V-Fos FBJ murine osteosarcoma viral 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.

[0213] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise Fos. Fos may be introduced into the expression cassette. Fos may be expressed in the expression cassette. The expression cassette comprising Fos may be introduced into a PSC. The expression cassette comprising Fos may be expressed in the PSC. The expression cassette comprising Fos may induce the PSC to differentiate into hepatocytes or liver cells.

[0214] Fos can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. 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 PSCs to differentiate into hepatocytes or liver cells.

[0215] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. One or more transcription factors can include Fos. Different amounts of Fos can be introduced into PSC. An expression cassette comprising Fos can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of Fos can be introduced into PSC. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frames (ORF) copies / cell of Fos can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frames (ORF) copies / cell of Fos can be introduced. By increasing the copy number of ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0216] In some embodiments, the number of copies of the ORF of Fos introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of Fos introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of Fos introduced per cell may be at most about 90. In some embodiments, the number of copies of the ORF of 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 or 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 number of copies of the introduced Fos ORF 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.

[0217] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include Fos. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0218] CEBPB

[0219] The present disclosure provides cells, expression cassettes and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be CCAAT enhancer binding protein (CEBP), which can also be referred to as CCAAT enhancer binding protein β; 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 activating protein; C / EBPβ; NF-IL6; TCF-5; or LIP.

[0220] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise CEBPB. CEBPB may be expressed in the expression cassette. The expression cassette comprising CEBPB may be introduced into a PSC. The expression cassette comprising CEBPB may be expressed in the PSC. The expression cassette comprising CEBPB may induce the PSC to differentiate into hepatocytes or liver cells.

[0221] CEBPB can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising CEBPB and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0222] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise CEBPB. An expression cassette comprising CEBPB can be expressed in PSC. An expression cassette comprising CEBPB can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of CEBPB can be introduced into PSC. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of CEBPB can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of CEBPB can be introduced. By increasing the copy number of the ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0223] In some embodiments, the number of copies of the ORF of CEBPB introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of CEBPB introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of CEBPB introduced per cell may be at most about 90. In some embodiments, the number of copies of the introduced ORF of CEBPB 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 or 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 number of copies of the introduced CEBPB ORF 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.

[0224] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include CEBPB. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0225] JUN / AP-1

[0226] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. 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, which may also be referred to as Jun proto-oncogene 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.

[0227] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise JUN / AP-1. JUN / AP-1 may be expressed in the expression cassette. The expression cassette comprising JUN / AP-1 may be introduced into a PSC. The expression cassette comprising JUN / AP-1 may be expressed in a PSC. The expression cassette comprising JUN / AP-1 may induce PSCs to differentiate into hepatocytes or liver cells.

[0228] JUN / AP-1 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an 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 PSCs to differentiate into hepatocytes or liver cells.

[0229] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise JUN / AP-1. An expression cassette comprising JUN / AP-1 can be expressed in a PSC. An expression cassette comprising JUN / AP-1 can induce a PSC to differentiate into a hepatocyte or liver cell. Various amounts of JUN / AP-1 can be introduced into a PSC. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of JUN / AP-1 can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of JUN / AP-1 can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0230] In some embodiments, the number of copies of the introduced JUN / AP-1 ORF per cell may be from about 1 to about 90. In some embodiments, the number of copies of the introduced JUN / AP-1 ORF per cell may be at least about 1. In some embodiments, the number of copies of the introduced JUN / AP-1 ORF per cell may be at most about 90. In some embodiments, the number of copies of the introduced JUN / AP-1 ORF 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 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 or 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 number of copies of the introduced JUN / AP-1 ORF 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.

[0231] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include JUN / AP-1. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0232] NFE2L2

[0233] The present disclosure provides cells, expression cassettes and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. 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, which can also be referred to as NFE2-like BZIP transcription factor 2; NRF2; nuclear factor erythroid 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 2-like 2; NFE2-related factor 2; IMDDHH; or NRF-2.

[0234] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise NFE2L2. NFE2L2 may be introduced into the expression cassette. NFE2L2 may be expressed in the expression cassette. The expression cassette comprising NFE2L2 may be introduced into a PSC. The expression cassette comprising NFE2L2 may be expressed in the PSC. The expression cassette comprising NFE2L2 may induce the PSC to differentiate into hepatocytes or liver cells.

[0235] NFE2L2 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising NFE2L2 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0236] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise NFE2L2. An expression cassette comprising NFE2L2 can be expressed in a PSC. An expression cassette comprising NFE2L2 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of NFE2L2 can be introduced into a PSC. At least about 5, about 10, about 15, about 20, about 25, or about 50 open reading frame (ORF) copies / cell of NFE2L2 can be introduced. Alternatively or in addition, up to about 50, about 25, about 20, about 15, about 10, or about 5 open reading frame (ORF) copies / cell of NFE2L2 can be introduced. Increased expression levels can also be achieved by increasing the copy number of the ORF, for example, by using a higher copy number vector or by using a transposon.

[0237] In some embodiments, the number of copies of the ORF of NFE2L2 introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of NFE2L2 introduced per cell may be at least about 1. In some embodiments, the number of copies of the ORF of NFE2L2 introduced per cell may be at most about 90. In some embodiments, the number of ORF copies of the introduced NFE2L2 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 or 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 number of copies of the introduced NFE2L2 ORF 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.

[0238] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include NFE2L2. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0239] GATA zinc finger transcription factors

[0240] The present disclosure provides cells, expression cassettes and methods that use transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be a member of the GATA zinc finger transcription factor family, which can be, 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.

[0241] GATA2 isoform P23769-2

[0242] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be GATA2 isoform P23769-2, which may also be referred to as GATA binding protein 2; NFE1B; endothelial transcription factor GATA-2; GATA binding protein 2; MONOMAC; IMD21; or DCML.

[0243] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise GATA2 isoform P23769-2. GATA2 isoform P23769-2 may be introduced into the expression cassette. GATA2 isoform P23769-2 may be expressed in the expression cassette. The expression cassette comprising GATA2 isoform P23769-2 may be introduced into a PSC. The expression cassette comprising GATA2 isoform P23769-2 may be expressed in a PSC. The expression cassette comprising GATA2 isoform P23769-2 may induce PSCs to differentiate into hepatocytes or liver cells.

[0244] GATA2 isoform P23769-2 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising GATA2 isoform P23769-2 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0245] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. One or more transcription factors can include GATA2 subtype P23769-2. An expression cassette comprising GATA2 subtype P23769-2 can be expressed in PSCs. An expression cassette comprising GATA2 subtype P23769-2 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of GATA2 subtype P23769-2 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of GATA2 subtype P23769-2 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of GATA2 subtype P23769-2 can be introduced. Increased expression levels may also be achieved by increasing the copy number of the ORF, for example by using a higher copy number vector or by using a transposon.

[0246] In some embodiments, the number of ORF copies / cell of the introduced GATA2 isoform P23769-2 can be from about 1 to about 90. In some embodiments, the number of ORF copies / cell of the introduced GATA2 isoform P23769-2 can be at least about 1. In some embodiments, the number of ORF copies / cell of the introduced GATA2 isoform P23769-2 can be at most about 90. In some embodiments, the number of ORF copies / cell of the introduced GATA2 isoform P23769-2 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 or 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 number of ORF copies of the introduced GATA2 isoform P23769-2 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.

[0247] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can include GATA2 isoform P23769-2. The one or more hepatocytes or liver cells can be differentiated from the one or more PSCs. The one or more hepatocytes or liver cells can include one or more transcription factors that can induce one or more PSCs to differentiate into hepatocytes or liver cells.

[0248] GATA4

[0249] The present disclosure provides cells, expression cassettes, and methods that utilize transcription factors, or molecules that increase transcription or increase transcription factor activation. One or more transcription factors can be used. In some embodiments, the transcription factor can be GATA4, which may also be referred to as GATA binding protein 4; transcription factor GATA-4; GATA binding factor 4; GATA binding protein 4; TACHD; ASD2; VSD1; or TOF.

[0250] The present disclosure provides an expression cassette comprising one or more transcription factors. The one or more transcription factors may comprise GATA4. GATA4 may be introduced into the expression cassette. GATA4 may be expressed in the expression cassette. The expression cassette comprising GATA4 may be introduced into a PSC. The expression cassette comprising GATA4 may be expressed in the PSC. The expression cassette comprising GATA4 may induce the PSC to differentiate into hepatocytes or liver cells.

[0251] GATA4 can be introduced into an expression cassette along with one or more other transcription factors. Combinations comprising one or more transcription factors can be generated. Combinations of transcription factors can be introduced into an expression cassette. Combinations of transcription factors can be expressed in an expression cassette. The expression cassette can be introduced into PSCs. An expression cassette comprising GATA4 and one or more other transcription factors can induce PSCs to differentiate into hepatocytes or liver cells.

[0252] The present disclosure provides one or more transcription factors that can induce one or more PSCs to differentiate into one or more hepatocytes or liver cells. The one or more transcription factors can comprise GATA4. An expression cassette comprising GATA4 can be expressed in PSC. An expression cassette comprising GATA4 can induce PSCs to differentiate into hepatocytes or liver cells. Different amounts of GATA4 can be introduced into PSCs. At least about 5, about 10, about 15, about 20, about 25 or about 50 open reading frame (ORF) copies / cell of GATA4 can be introduced. Alternatively or in addition, at most about 50, about 25, about 20, about 15, about 10 or about 5 open reading frame (ORF) copies / cell of GATA4 can be introduced. By increasing the copy number of ORF, for example, by using a vector with a higher copy number or by using a transposon, an increase in expression level can also be achieved.

[0253] In some embodiments, the number of copies of the ORF of GATA4 introduced per cell may be from about 1 to about 90. In some embodiments, the number of copies of the ORF of GATA4 introduced per cell may be at least about 1. In some embodiments,...

Claims

1. A pluripotent stem cell (PSC) 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 the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: a. one or more hepatocyte nuclear factor ("HNF") family members; b. one or more forkhead box ("FOX") family members; and one or more additional transcription factors comprising a 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 member, or One or more additional transcription factors from Table 12.

2. The PSC of claim 1, wherein the one or more transcription factors comprise 2 or more HNF family members.

3. The PSC of claim 1 or claim 2, wherein the one or more transcription factors comprise 3 or more HNF family members.

4. A PSC as described in any of the preceding claims, wherein 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 an HLF family member, or one or more additional transcription factors from Table 12.

5. A PSC as described in any of the preceding claims, wherein 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.

6. The PSC of any of the preceding claims, further comprising at least two or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

7. A PSC as described in any of the preceding claims, further comprising at least 3 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

8. The PSC of any of the preceding claims, further comprising at least four or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

9. A PSC as described in any of the preceding claims, further comprising at least 5 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

10. A PSC as described in any of the preceding claims, further comprising at least 6 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

11. A PSC as described in any of the preceding claims, further comprising at least 7 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

12. A PSC as described in any of the preceding claims, further comprising at least 8 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

13. The PSC of any of the preceding claims, wherein the one or more HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof.

14. The PSC of any of the preceding claims, wherein the one or more FOX family members comprises FOXA1.

15. The PSC of any of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

16. The PSC of any of the preceding claims, wherein the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

17. A method for generating hepatocytes or a hepatocyte population, comprising: a. Providing one or more pluripotent stem cells (PSC); b. 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 the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: i. one or more hepatocyte nuclear factor ("HNF") family members; ii. one or more forkhead box ("FOX") family members; and iii. one or more additional transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12; c. generating the hepatocyte or hepatocyte population from the one or more PSCs.

18. The method of claim 17, wherein the one or more transcription factors comprise two or more HNF family members.

19. The method of claim 17 or claim 18, wherein the one or more transcription factors comprise three or more HNF family members.

20. The method of any of the preceding claims, wherein the one or more additional transcription factors comprises CEBPA and the one or more transcription factors further comprises a 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 member, or one or more additional transcription factors from Table 12.

21. The method of any of the preceding claims, wherein the one or more additional transcription factors comprises RBPJ and the one or more transcription factors further comprises a 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 member, or one or more additional transcription factors from Table 12.

22. The method of any of the preceding claims, further comprising at least two or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

23. The method of any of the preceding claims, further comprising at least three or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

24. The method of any of the preceding claims, further comprising at least 4 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

25. The method of any of the preceding claims, further comprising at least 5 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

26. The method of any of the preceding claims, further comprising at least 6 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

27. The method of any of the preceding claims, further comprising at least 7 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

28. The method of any of the preceding claims, further comprising at least 8 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

29. The method of any of the preceding claims, wherein the one or more HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof.

30. The method of any of the preceding claims, wherein the one or more FOX family members comprises FOXA1.

31. The method of any of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

32. The method of any of the preceding claims, wherein the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

33. The method of any of the preceding claims, wherein the at least one or more transcription factors induce the one or more PSCs to express into a hepatocyte or liver cell population in 30 days or less.

34. The method of any one of the preceding claims, wherein the one or more PSCs are provided in culture medium.

35. The method of claim 34, wherein the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or hepatocyte population.

36. The method of claim 34, wherein the culture medium is not changed during delivery of the at least one exogenous expression cassette to the one or more PSCs.

37. The method of any one of the preceding claims, wherein the one or more hepatocytes or at least one of the hepatocytes of the hepatocyte or hepatocyte population expresses ASGR-1.

38. The method of any one of the preceding claims, wherein the one or more hepatocytes or at least one of the hepatocytes of the hepatocyte or hepatocyte population expresses CXCR-4.

39. The method of any one of the preceding claims, wherein at least 2% of the hepatocytes or population of hepatocytes express ASGR-1.

40. The method of any one of the preceding claims, wherein at least 3% of the hepatocytes or population of hepatocytes express ASGR-1.

41. The method of any one of the preceding claims, wherein at least 5% of the hepatocytes or population of hepatocytes express ASGR-1.

42. The method of any one of the preceding claims, wherein at least 6% of the hepatocytes or population of hepatocytes express ASGR-1.

43. The method of any one of the preceding claims, wherein at least 10% of the hepatocytes or population of hepatocytes express CXCR-4.

44. The method of any one of the preceding claims, wherein at least 20% of the hepatocytes or population of hepatocytes express CXCR-4.

45. The method of any of the preceding claims, wherein at least 30% of the PSCs express CXCR-4.

46. ​​An exogenous expression cassette that induces pluripotent stem cells (PSCs) to differentiate into hepatocytes or liver cells, wherein the exogenous expression cassette comprises: a. one or more hepatocyte nuclear factor ("HNF") family member genes; b. one or more forkhead box ("FOX") family member genes; and c. one or more additional transcription factor genes, the additional transcription factor genes 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.

47. The exogenous expression cassette of claim 46, wherein the one or more transcription factor genes comprise two or more HNF family members.

48. The exogenous expression cassette of claim 46 or claim 47, wherein the one or more transcription factor genes comprise three or more HNF family members.

49. An exogenous expression cassette as described in any of the preceding claims, wherein the one or more additional transcription factor genes comprise CEBPA, and the one or more transcription factor genes 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.

50. An exogenous expression cassette as described in any of the preceding claims, wherein the one or more additional transcription factor genes 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.

51. The exogenous expression cassette of any of the preceding claims, further comprising at least two or more transcription factor genes comprising a 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 member, or one or more additional transcription factors from Table 12.

52. The exogenous expression cassette of any of the preceding claims, further comprising at least three or more transcription factor genes 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.

53. The exogenous expression cassette of any of the preceding claims, further comprising at least 4 or more transcription factor genes 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.

54. The exogenous expression cassette of any of the preceding claims, further comprising at least 5 or more transcription factor genes 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.

55. The exogenous expression cassette of any of the preceding claims, further comprising at least 6 or more transcription factor genes 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.

56. An exogenous expression cassette as described in any of the preceding claims, further comprising at least 7 or more transcription factor genes, wherein the transcription factor genes 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.

57. An exogenous expression cassette as described in any of the preceding claims, further comprising at least 8 or more transcription factor genes, wherein the transcription factor genes 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.

58. The exogenous expression cassette of any of the preceding claims, wherein the one or more HNF family members comprise HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof.

59. The exogenous expression cassette of any of the preceding claims, wherein the one or more FOX family members comprise FOXA1, or FOXA3, or any combination thereof.

60. The exogenous expression cassette of any of the preceding claims, wherein the one or more transcription factor genes comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

61. The exogenous expression cassette of any of the preceding claims, wherein the one or more transcription factor genes comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

62. The exogenous expression cassette of any one of the preceding claims, wherein the PSC is provided in culture medium.

63. The exogenous expression cassette of any of the preceding claims, wherein the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell.

64. The exogenous expression cassette of any one of the preceding claims, wherein the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 30 days or less.

65. The exogenous expression cassette of any one of the preceding claims, wherein the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 96 hours or less.

66. A method of generating a hepatocyte or a hepatocyte population, comprising: a. Providing one or more pluripotent stem cells (PSC); b. 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 the open reading frame encoding one or more transcription factors; and c. generating the hepatocytes or hepatocyte populations from the one or more PSCs in 30 days or less.

67. The method of claim 66, wherein the one or more PSCs differentiate into the hepatocytes or hepatocyte population in 96 hours or less.

68. The method of claim 66 or 67, wherein the one or more PSCs are provided in culture medium.

69. The method of any of the preceding claims, wherein the culture medium is not changed during differentiation of the one or more PSCs into the hepatocyte population.

70. The method of any of the preceding claims, wherein 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.

71. A method as described in any of the preceding claims, wherein 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.

72. A method as described in any of the preceding claims, wherein 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.

73. A method as described in any of the preceding claims, wherein 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.

74. The method of any of the preceding claims, wherein the one or more transcription factors are transcription factors of a transcription factor family, such as: 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.

75. The method of any of the preceding claims, wherein the two or more transcription factors are transcription factors of a transcription factor family, such as: 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.

76. The method of any of the preceding claims, wherein the three or more transcription factors are transcription factors of a transcription factor family, such as: 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.

77. The method of any of the preceding claims, wherein the four or more transcription factors are transcription factors of a transcription factor family, such as: 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.

78. The method of any of the preceding claims, wherein the at least one or more transcription factors comprise SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a, and HNF6B.

79. The method of any of the preceding claims, wherein the one or more transcription factors comprise HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1.

80. The method of any of the preceding claims, wherein the one or more transcription factors comprise GATA2 isoform P23769-2, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.

81. The method of any of the preceding claims, wherein the one or more transcription factors comprise FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A, RBPJ.

82. The method of any of the preceding claims, wherein the one or more transcription factors comprise CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A.

83. The method of any of the preceding claims, wherein the one or more transcription factors comprise HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.

84. The method of any of the preceding claims, wherein the one or more PSCs are provided in culture medium.

85. The method of any of the preceding claims, wherein the culture medium is not changed during differentiation of the one or more PSCs into the hepatocytes or liver cell population.

86. The method of any of the preceding claims, wherein the culture medium is not changed during delivery of the nucleic acid comprising an open reading frame encoding one or more transcription factors, the one or more transcription factors, or the transcriptional activator encoding an open reading frame of one or more transcription factors to the one or more PSCs.

87. The method of any of the preceding claims, wherein the one or more hepatocytes or at least one of the hepatocytes of the hepatocyte or hepatocyte population expresses ASGR-1.

88. The method of any of the preceding claims, wherein the one or more hepatocytes or at least one of the hepatocytes of the hepatocyte or hepatocyte population does not express CXCR-4.

89. The method of any of the preceding claims, wherein the one or more hepatocytes or at least one of the hepatocytes of the hepatocyte or hepatocyte population expresses CXCR-4.

90. The method of any one of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 2% of the hepatocytes or a population of hepatocytes expressing ASGR-1.

91. The method of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 3% of the hepatocytes or a population of hepatocytes expressing ASGR-1.

92. The method of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 5% of the hepatocytes or a population of hepatocytes expressing ASGR-1.

93. The method of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 6% of the hepatocytes or a population of hepatocytes expressing ASGR-1.

94. The method of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 10% of the hepatocytes or a population of hepatocytes expressing CXCR-4.

95. The method of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 20% of the hepatocytes or a population of hepatocytes expressing CXCR-4.

96. The method of any of the preceding claims, wherein the hepatocytes or population of liver cells comprises at least 30% of the PSCs expressing CXCR-4.

97. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the nucleic acid comprising an open reading frame encoding one or more transcription factors, the one or more transcription factors, or the transcription activator of the open reading frame encoding one or more transcription factors induces the PSC to differentiate into hepatocytes or liver cells within 30 days or less.

98. The PSC of claim 97, wherein 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.

99. A PSC as described in any of the preceding claims, wherein 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.

100. A PSC as described in any of the preceding claims, wherein 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) 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.

101. A PSC as described in any of the preceding claims, wherein 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) 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.

102. A PSC as described in any of the preceding claims, wherein the one or more transcription factors are transcription factors of a transcription factor family, such as: 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.

103. The PSC of any of the preceding claims, wherein the one or more transcription factors comprise ONECUT1 / HNF6 or ONECUT2 / HNF6B.

104. The PSC of any of the preceding claims, wherein the one or more transcription factors comprise ONECUT1 / HNF6 and ONECUT2 / HNF6B.

105. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, SPI1, HNF1A, FoxA2, ​​CEBPA, HNF6, HNF4a, and HNF6B.

106. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, HNF1A, Atf3, HNF6, HNF6B, Fos, CEBPA, and FoxM1.

107. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, SPI1, FoxA2, ​​GATA2 transcription factor, GATA4, JUN / FOS, JUN, and NFIX.

108. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, HNF4a, CEBPA, GATA6, TBX3, Fos and GATA2 transcription factors.

109. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, GATA2 transcription factor, HNF4a, RXRB, NFE2L2, Myc, TP73, and HNF6.

110. The PSC of any of the preceding claims, wherein the one or more transcription factors are, e.g., GATA2 transcription factor, CEBPA, ATF3, Fos, GATA3, NFIX.

111. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FoxA2, ​​and FoxA1.

112. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, HHEX, SOX17, and HNF4A.

113. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B, and HNF1A.

114. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, FoxA1, HNF1A, FoxA2, ​​CEBPA, ONECUT1 / HNF6, HNF4a, and RBPJ.

115. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, Smad3, HHEX, ATF5, NR5A2, GATA2 transcription factor, CEBPB, and NR113.

116. The PSC of any of the preceding claims, wherein the one or more transcription factors are, for example, HNF1A, CEBPA, ONECUT1 / HNF6, ONECUT2 / HNF6B, Fos, HIF1A, and TBX3.

117. The PSC of any of the preceding claims, wherein the hepatocyte or the liver cell expresses ASGR-1.

118. The PSC of any of the preceding claims, wherein the hepatocyte or the liver cell does not express CXCR-4.

119. The PSC of any of the preceding claims, wherein the hepatocyte or the liver cell expresses CXCR-4.

120. The PSC of any of the preceding claims, wherein the PSC is provided in culture medium.

121. The PSC of claim 120, wherein the culture medium is not changed during differentiation of the PSC into the hepatocyte or liver cell.

122. The PSC of any of the preceding claims, wherein the hepatocyte or population of hepatocytes comprises two or more of the hepatocytes or the hepatocytes.

123. A PSC as described in any of the preceding claims, wherein the hepatocytes or liver cell population comprises at least 2% of the hepatocytes or liver cells expressing ASGR-1.

124. The PSC of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 3% of the hepatocytes or the hepatocytes expressing ASGR-1.

125. The PSC of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 5% of the hepatocytes or the hepatocytes expressing ASGR-1.

126. The PSC of any of the preceding claims, wherein the hepatocytes or a population of liver cells comprises at least 6% of the hepatocytes or the liver cells expressing ASGR-1.

127. The PSC of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 10% of the hepatocytes or the hepatocytes expressing CXCR-4.

128. The PSC of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 20% of the hepatocytes or the hepatocytes expressing CXCR-4.

129. The PSC of any of the preceding claims, wherein the hepatocytes or a population of hepatocytes comprises at least 30% of the hepatocytes or the hepatocytes expressing CXCR-4.

130. The PSC of any of the preceding claims, wherein the hepatocyte or liver cell population comprises, without nutrient, or growth factor, or microenvironment / matrix optimization.

131. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: SPI1, HNF1A, FOXA2, CEBPA, ONECUT1, HNF4A and ONECUT2.

132. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: HHEX, SOX17, HNF4A, HNF1A, ONECUT1 / HNF6, FOXA2 and FOXA1.

133. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, ATF5, HHEX, SOX17, HNF4A, ONECUT2 / HNF6B and HNF1A.

134. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, HNF1A, FOXA2, CEBPA, ONECUT1 / HNF6, HNF4A and RBPJ.

135. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: HNF1A, CEBPA, ONECUT1, ONECUT2, FOS, HIF1A and TBX3.

136. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: HNF4A, HNF1A, ONECUT1 / HNF6 and FOXA1.

137. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, HNF1A, CEBPA, ONECUT1 / HNF6 and HNF4A.

138. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: FOXA1, HNF4A, ONECUT1 / HNF6 and RBPJ.

139. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6 and RBPJ.

140. 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 the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, FOXA1, HNF1A and HNF4A.

141. 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 transcription activator of the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPA, HNF1A, HNF4A, FOXA3, FOXA2, PXR and RXRA.

142. 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 the open reading frame encoding one or more transcription factors, wherein the one or more transcription factors comprise: CEBPB, FOXA1, FOXA3, HLF, HNF1A, HNF4A, NR1I2 / PXR, NR1I3 / CAR, ONECUT1 / HNF6, PROX1, RBPJ, RORC and SALL4.

143. A kit for inducing pluripotent stem cells (PSCs) to differentiate into hepatocytes or liver cells, the kit comprising: a. one or more hepatocyte nuclear factor ("HNF") family members; b. one or more forkhead box ("FOX") family members; and c. one or more additional transcription factors, comprising a 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 member, or one or more additional transcription factors from Table 12.

144. The kit of claim 143, wherein the one or more transcription factor genes comprise two or more HNF family members.

145. The kit of any of the preceding claims, wherein the one or more transcription factor genes comprise three or more HNF family members.

146. A kit as described in any of the preceding claims, wherein 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.

147. A kit as described in any of the preceding claims, wherein 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.

148. A kit as described in any of the preceding claims, further comprising at least 2 or more transcription factors comprising a 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 member, or one or more additional transcription factors from Table 12.

149. A kit as described in any of the preceding claims, further comprising at least 3 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

150. A kit as described in any of the preceding claims, further comprising at least 4 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

151. A kit as described in any of the preceding claims, further comprising at least 5 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

152. A kit as described in any of the preceding claims, further comprising at least 6 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

153. A kit as described in any of the preceding claims, further comprising at least 7 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

154. A kit as described in any of the preceding claims, further comprising at least 8 or more transcription factors, wherein the transcription factors comprise a 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 member, or one or more additional transcription factors from Table 12.

155. The kit of any of the preceding claims, wherein the one or more HNF family members comprises HNF4A, HNF1A, ONECUT1 / HNF6, or any combination thereof.

156. The kit of any of the preceding claims, wherein the one or more FOX family members comprises FOXA1, or FOXA3, or any combination thereof.

157. The kit of any of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, ONECUT1 / HNF6, and FOXA1.

158. The kit of any of the preceding claims, wherein the one or more transcription factors comprise CEBPA, FOXA1, HNF1A, HNF4A, ONECUT1 / HNF6, and RBPJ.

159. The kit of any of the preceding claims, wherein the PSCs are provided in culture medium.

160. The kit of any of the preceding claims, wherein the culture medium is not changed during differentiation of the PSCs into the hepatocytes or liver cells.

161. The kit of any of the preceding claims, wherein the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 30 days or less.

162. The kit of any of the preceding claims, wherein the exogenous expression cassette induces the PSC to differentiate into the hepatocyte or liver cell in 96 hours or less.

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