Methods and compositions for stem cell differentiation

Abstract

The present disclosure describes methods, kits, compositions, or pharmaceutical comprising engineered cells configured to express one or more transcription factors that promote cellular phenotypes comprising one or more improved liver functions, where such improved liver function is greater than that of a primary hepatocyte or a wild-type hepatocyte.

Description

METHODS AND COMPOSITIONS FOR STEM CELL DIFFERENTIATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 612,993, filed on December 20, 2023, which is incorporated by reference herein in its entirety for all purposes.SUMMARY

[0002] The present disclosure provides compositions, methods, kits, and formulations comprising an engineered cell configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the cell functionality in the engineered or genetically modified cell may be performed at levels that are much greater than the cell functionality of a native cell, a wild-type cell, or a cell that does not comprise the compositions, kits, or formulations of the present disclosure. The genetically engineered or genetically modified cell can enhance or improve the functionality of a cell in an organism.

[0003] This disclosure provides methods of generating hepatocytes or hepatocyte-like cells from induced pluripotent stem cells. An aspect of the present disclosure includes engineered cell configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte.

[0004] An aspect of the present disclosure is an engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise HNF1A, HNF4A, and FOXA1, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wildtype hepatocyte. In some embodiments, a cell is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell comprises a pluripotent stem cell (PSC). In some embodiment, a PSC comprises a mammalian stem cell. In some embodiments, a PSC comprises a human stem cell. In some embodiments, a PSC comprises a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC comprises a human progenitor cell. In some embodiments, a PSC comprises a human fetal stem cell. In some embodiments, a PSC comprises a hepatic stem cell. In some embodiments, a PSC comprises an embryonic stem cell. In some embodiments, a PSC comprises a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a human stem cell. In some embodiments, an engineered cell improves oxygen rate consumption compared to an uninduced cell type. In some embodiments, an engineered cell improves maximal oxygen rate consumption compared to an uninduced cell type. In some embodiments, an engineered cell improves spare respiratory capacity (SRC) compared to an uninduced cell type. In some embodiments, provided is an engineered cell for treating a liver dysfunction. In some embodiments, the liver dysfunction comprises a urea cycle disorders, a methylmalonic acidemia, a propionic acidemia, a glycogen storge disease type 1, a familiar hypercholesterolemia, or any combination thereof. In some embodiments, an engineered cell increases expression of urea cycle enzymes. In some embodiments, an engineered cell is configured to perform one or more hepatocyte functions at a rate of at least 30% greater than the primary hepatocyte or the wild-type hepatocyte. In some embodiments, the one or more hepatocyte functions comprise coagulation, copper transport, ion transport, retinol transport, protection against neutrophil elastase, inflammatory response, regulation of urea cycle, tyrosine metabolism, histidine metabolism, alanine metabolism, alcohol metabolism, transporting lipoproteins, fatty acid biosynthesis, albumin production, Al AT production, CYP3A4 activity, or any combination thereof. In some embodiments, an engineered cell comprises increased CYP3 A4 activity relative to the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell comprises one or more hepatocyte markers comprising SLC40A1, ATP7B, ARG1, HAL, ALDH6A1, APOM, Al AT, albumin or any combination thereof. In some embodiments, an engineered cell is configured to perform ion transport at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and an engineered cell expresses an equal to or greater amount of SLC40A1 relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform copper transport at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the cell expresses an equal to or greater amount of ATP7B relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform urea cycle at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the cell expresses an equal to or greater amount of ARG1 relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform histidine metabolism at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the cell expresses an equal to or greater amount of HAL relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform alcoholmetabolism at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the engineered cell expresses an equal to or greater amount of ALDH6A1 relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform transporting lipoproteins at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the engineered cell expresses an equal or greater amount of APOM relative to an amount of the hepatocyte marker expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell expresses ATP7B at 4x greater than a primary cell or a wild-type hepatocyte. In some embodiments, an engineered cell expresses SLC40A1 at least 2x greater than the primary cell or the wild-type hepatocyte. In some embodiments, an engineered cell expresses HAL at least 2.7x greater than the primary cell or the wild-type hepatocyte. In some embodiments, an engineered cell expresses ALDH6A1 at least 40% greater than the primary cell or the wild-type hepatocyte. In some embodiments, an engineered cell expresses APOM at least 2x greater than the primary cell or the wild-type hepatocyte. In some embodiments, an engineered cell expresses ARG1 at least 33% greater than the primary cell or the wild-type hepatocyte. In some embodiments, an engineered cell comprises at least one expression cassette comprising one or more transcription factors. In some embodiments, an engineered cell comprising at least one expression cassette comprising one or more transcription factors further comprises one or more additional transcription factors. In some embodiments, one or more transcription factors further comprise one or more additional transcription factors comprising: a FOS, JUN, PROX, GATA, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, an engineered cell comprises one or more transcription factors. In some embodiments, an engineered cell comprising the one or more transcription factors further comprise GATA6. In some embodiments, an engineered cell comprising the one or more transcription factors further comprise FOXA2. In some embodiments, an engineered cell comprising the one or more transcription factors further comprise CEBPA. In some embodiments, an engineered cell comprising one or more transcription factors further comprises one or more additional transcription factors. In some embodiments, the one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors comprise 2 or more HNF family members. In some embodiments, one or more additional transcription factors comprise 2 or more FOX family members. In some embodiments, the one or more transcription factors further comprises at least2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more transcription factors further comprises at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors further comprise one or more transcription factors from Table 3. In some embodiments, an engineered cell is configured to express CYP3 A4 activity that is at least 40% greater than in the primary hepatocyte or the wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, an engineered cell expresses at least 2x greater CYP3 A4 than the primary cell or a wild-type hepatocyte. In some embodiments, an engineered cell comprises a mammalian stem cell. In some embodiments, an engineered cell comprises a human stem cell. In some embodiments, an engineered cell comprises a human induced pluripotent stem cell (iPSC). In some embodiments, an engineered cell comprises a human progenitor cell. In some embodiments, an engineered cell comprises a human fetal stem cell. In some embodiments, an engineered cell comprises a hepatic stem cell. In some embodiments, an engineered cell comprises an embryonic stem cell. In some embodiments, an engineered cell is not derived from a patient. In some embodiments, a primary hepatocyte or a wild-type hepatocyte comprises a human hepatocyte cell. In some embodiments, an engineered cell secretes at least 4x more albumin than a primary cell or a wild-type hepatocyte. In some embodiments, an engineered cell secretes at least 5x more Al AT than in a primary cell or a wild-type hepatocyte. In some embodiments, an engineered cell further comprises an expression cassette comprising a nucleic acid molecule encoding one or more functional polypeptides. In some embodiments, an engineered cell is configured to comprise an activity of one or more functional polypeptides that is equal to or greater than that of a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell is configured to express one or more functional polypeptides comprising a green fluorescent protein (GFP). In some embodiments, an engineered cell is configured to express one or more functional polypeptides comprising an enzyme. In some embodiments, the one or more functional polypeptides improves functionality of an engineered cell. In some embodiments, an improved functionality of an engineered cell involves an enzymatic cellular activity. In some embodiments, an enzymatic cellular activity comprises metabolizing xenobiotics. In some embodiments, an engineered cell is configured to express one or more functional polypeptides comprising CYP3A4. In some embodiments, an engineered cell expresses one or more hepatocyte or hepatocyte-like markerscomprising SLC40A1, ARG1, HAL, ALDH6A1, APOM, ATP7B, CYP3A4 activity, AIAT production, or any combination thereof. In some embodiments, an engineered cell expresses one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, an engineered cell expresses one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, an engineered cell expresses one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, an engineered cell expresses one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2.

[0005] An aspect of the present disclosure is a pharmaceutical composition comprising an engineered cell disclosed herein. In some embodiments, a pharmaceutical composition further comprises an excipient. In some embodiments is a method of treating a liver condition in a subject in need thereof, the method comprising administering the engineered cell of the present disclosure to a subject in need thereof, thereby treating the liver condition.

[0006] An aspect of the present disclosure is a method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise HNF1 A, HNF4A, and FOXA1; inducing expression of the one or more transcription factors, thereby generating the engineered cell, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell comprises a pluripotent stem cell (PSC). In some embodiments, a PSC comprises a mammalian stem cell. In some embodiments, a PSC comprises a human stem cell. In some embodiments, a PSC comprises a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC comprises a human progenitor cell. In some embodiments, a PSC comprises a human fetal stem cell. In some embodiments, a PSC comprises a hepatic stem cell. In some embodiments, a PSC comprises an embryonic stem cell. In some embodiments, a PSC comprises a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocytelike cell is derived from a human stem cell. In some embodiments, a hepatocyte or hepatocytelike cell is not derived from a human stem cell. In some embodiments, an engineered is configured to perform ion transport at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses a greater amount of SLC40A1 relative to an amount expressedby the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered is configured to perform copper transport at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ATP7B relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered is configured to perform urea cycle at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ARG1 relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered is configured to perform histidine metabolism at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of HAL relative to an amount expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered is configured to perform alcohol metabolism at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ALDH6A1 relative to an amount of expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered is configured to perform transporting lipoproteins at a rate equal to or greater than a primary hepatocyte or a wildtype hepatocyte and the cell expresses an equal or greater amount of APOM relative to an amount expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, disclosed herein is a method of generating an engineered cell comprising one or more transcription factors further comprising GATA6. In some embodiments, disclosed herein is a method of generating an engineered cell comprising one or more transcription factors further comprising F0XA2. In some embodiments, disclosed herein is a method of generating an engineered cell comprising one or more transcription factors further comprising CEBPA. In some embodiments, the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GAT A, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors comprise 2 or more HNF family members. In some embodiments, the one or more additional transcription factors comprise 2 or more FOX family members. In some embodiments, a method comprises generating an engineered cell comprising at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GAT A, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, a method comprises generating an engineered cell comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT,SKI or HLF family member. In some embodiments, the one or more additional transcription factors further comprise one or more transcription factors from Table 3. In some embodiments, a method of generating an engineered cell comprises one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, a method of generating an engineered cell comprises one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, a method of generating an engineered cell comprises one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, a method of generating an engineered cell comprises one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2.

[0007] An aspect of the present disclosure is a method of treating a disease characterized by a liver dysfunction in a subject in need thereof, the method comprising: administering a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise HNF1A, HNF4A, and FOXA1. In some embodiments, a PSC comprises a mammalian stem cell. In some embodiments, a PSC comprises a human stem cell. In some embodiments, a PSC comprises a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC comprises a human progenitor cell. In some embodiments, a PSC comprises a human fetal stem cell. In some embodiments, a PSC comprises a hepatic stem cell. In some embodiments, a PSC comprises an embryonic stem cell. In some embodiments, a PSC comprises an engineered cell. In some embodiments, a PSC comprises or a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a mammalian stem cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a human stem cell. In some embodiments, a hepatocyte or hepatocyte-like cell is not derived from a human stem cell. In some embodiments, a PSC improves oxygen rate consumption compared to an uninduced cell type. In some embodiments, a PSC improves maximal oxygen rate consumption compared to an uninduced cell type. In some embodiments, a PSC improves spare respiratory capacity (SRC) compared to an uninduced cell type. In some embodiments, a method comprises treating a liver dysfunction comprising a urea cycle disorders, a methylmalonic acidemia, a propionic acidemia, a glycogen storge disease type 1, a familiar hypercholesterolemia, or any combination thereof. In some embodiments, a PSC increases expression of urea cycle enzymes. In some embodiments, a PSC comprises one or more transcription factors. In some embodiments, the one or more transcriptionfactors further comprise GATA6. In some embodiments, the one or more transcription factors further comprise F0XA2. In some embodiments, the one or more transcription factors further comprise CEBPA. In some embodiments, the one or more transcription factors further comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more transcription factors further comprise 2 or more HNF family members. In some embodiments, the one or more transcription factors further comprise 2 or more FOX family members. In some embodiments, the one or more transcription factors further comprise at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more transcription factors further comprise at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more transcription factors further comprise one or more additional transcription factors. In some embodiments, the one or more additional transcription factors further comprise one or more transcription factors from Table 3. In some embodiments, a method for generating an engineered cell comprises one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, a method for generating an engineered cell comprises one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, a method for generating an engineered cell comprises one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, a method for generating an engineered cell comprises one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, the method comprises a PSC. In some embodiments, a PSC is a mammalian stem cell. In some embodiments, a PSC is a human stem cell. In some embodiments, a PSC is a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC is a human progenitor cell. In some embodiments, a PSC is a human fetal stem cell. In some embodiments, a PSC is a hepatic stem cell. In some embodiments, a PSC is an embryonic stem cell. In some embodiments, a PSC is a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a human stem cell. In some embodiments, a PSC is not derived from the subject in need thereof. In some embodiments, a PSC is derived from the subject in need thereof.

[0008] An aspect of the present disclosure is method for preparing a population of cells for infusion into a subject in need thereof, wherein the population of cells comprises pluripotent stem cells (PSCs), wherein the PSCs are engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprising HNF1 A, HNF4A, and FOXA1, wherein the method comprises: (a) at least a first passage of the PSCs; (b) maintaining the PSCs on ice or at 0° C, and further passaging the PSCs at least one time; or (c) cryopreserving the PSCs at less than 0° C and thawing the PSCs; wherein after (a), (b), or (c), the PSCs secrete albumin at a rate of at least 4pg / day / million cells, wherein the PSCs produce hepatocytes or hepatocyte-like cells. In some embodiments, the PSCs comprise mammalian stem cells. In some embodiments, PSCs comprise human stem cells. In some embodiments, PSCs comprise human induced pluripotent stem cells (iPSCs). In some embodiments, PSCs comprise human progenitor cells. In some embodiments, PSCs comprise human fetal stem cells. In some embodiments, PSCs comprise hepatic stem cells. In some embodiments, PSCs comprise embryonic stem cells. In some embodiments, PSC comprise engineered cells. In some embodiments, PSC comprise hepatocytes or hepatocyte-like cells. In some embodiments, PSCs comprise hepatocytes or hepatocyte-like cells derived from human stem cells. In some embodiments, PSCs comprise hepatocytes or hepatocyte-like cells that are not derived from human stem cells. In some embodiments, the PSCs are maintained on ice or at 0°C for at least 6 hours prior to the first passage of the PSCs. In some embodiments, the PSCs are plated or cultured for 24 hours after (a) at least a first passage of the PSCs; (b) maintaining the PSCs on ice or at 0° C, and further passaging the PSCs at least one time; or (c) cry opreserving the PSCs at less than 0° C and thawing the PSCs. In some embodiments, the PSCs comprise distinct hepatocyte or hepatocyte-like morphology following (a), (b), (c) above. In some embodiments, the PSCs comprise at least 80% viable cells after (a), (b), (c) above, or any combination thereof. In some embodiments, PSCs can comprise one or more transcription factors. In some embodiments, the one or more transcription factors further comprise GATA6. In some embodiments, the one or more transcription factors further comprise FOXA2. In some embodiments, the one or more transcription factors further comprise CEBPA. In some embodiments, the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GAT A, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the PSCs comprising the one or more transcription factors further comprise one ormore additional transcription factors. In some embodiments, the one or more additional transcription factors comprise 2 or more HNF family members. In some embodiments, the one or more additional transcription factors comprise 2 or more FOX family members. In some embodiments, the one or more additional PSCs comprising one or more transcription factors comprise at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional PSCs comprising one or more transcription factors comprise at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors further comprise one or more transcription factors from Table 3. In some embodiments, a method comprising PSCs comprise one or more transcription factors comprising HNF4A, HNF1A, CEBP A, F0XA1, and GATA6. In some embodiments, a method comprising PSCs comprise one or more transcription factors consisting ofHNF4A, HNF1A, CEBP A, F0XA1, and GATA6. In some embodiments, a method comprising PSCs comprise one or more transcription factors comprising HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, a method comprising PSCs comprise one or more transcription factors consisting of HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2.

[0009] An aspect of the present disclosure is a method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acid molecules encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member; inducing expression of the expression cassette, thereby generating an engineered cell, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wildtype hepatocyte. In some embodiments, an engineered cell is configured to perform ion transport at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of SLC40A1 relative to an amount expressed by the primaryhepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform copper transport at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ATP7B relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform urea cycle at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ARG1 relative to an amount expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform histidine metabolism at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of HAL relative to an amount expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform alcohol metabolism at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ALDH6A1 relative to an amount of expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell is configured to perform transporting lipoproteins at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of APOM relative to an amount expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell can comprise one or more additional transcription factors further comprise 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. In some embodiments, an engineered cell can comprise one or more additional transcription factors comprising 2 or more HNF family members. In some embodiments, an engineered cell can comprise one or more additional transcription factors comprising 2 or more FOX family members. In some embodiments, an engineered cell can comprise one or more additional transcription factors comprising F0XA1. In some embodiments, an engineered cell can comprise one or more additional transcription factors comprising F0XA2. In some embodiments, an engineered cell can comprise one or more additional transcription factors comprising CEBPA. In some embodiments, an engineered cell improves oxygen rate consumption compared to an uninduced cell type. In some embodiments, an engineered cell improves maximal oxygen rate consumption compared to an uninduced cell type. In some embodiments, an engineered cell improves spare respiratory capacity (SRC) compared to an uninduced cell type. In some embodiments, a method comprises treating a liver dysfunction comprising a urea cycle disorders, a methylmalonic acidemia, a propionic acidemia,a glycogen storge disease type 1, a familiar hypercholesterolemia, or any combination thereof. In some embodiments, an engineered cell increases expression of urea cycle enzymes.

[0010] An aspect of the present disclosure is a pluripotent stem cell (PSC), wherein the PSC is engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise: GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member, wherein the PSC is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, a PSC can be configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, a PSC can comprise a mammalian stem cell. In some embodiments, a PSC can comprise human stem cell. In some embodiments, a PSC can comprise a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC can comprise a human progenitor cell. In some embodiments, a PSC can comprise a human fetal stem cell. In some embodiments, a PSC can comprise a hepatic stem cell. In some embodiments, a PSC can comprise an embryonic stem cell. In some embodiments, a PSC can comprise an engineered cell. In some embodiments, a PSC can comprise a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocyte-like cell can be derived from a human stem cell. In some embodiments, a hepatocyte or hepatocyte-like cell is not derived from a human stem cell. In some embodiments, a PSC improves oxygen rate consumption compared to an uninduced cell type. In some embodiments, a PSC improves maximal oxygen rate consumption compared to an uninduced cell type. In some embodiments, a PSC improves spare respiratory capacity (SRC) compared to an uninduced cell type. In some embodiments a method comprises a PSC. In some embodiments, a method comprises treating a liver dysfunction. In some embodiments, a liver dysfunction comprises a urea cycle disorders. In some embodiments, a liver dysfunction comprises a methylmalonic acidemia. In some embodiments, a liver dysfunction comprises a propionic acidemia. In some embodiments, a liver dysfunction comprises a glycogen storge disease type 1. In some embodiments, a liver dysfunction comprises a familiar hypercholesterolemia, or any combination of the above liver dysfunctions or known by an artisan. In some embodiments, an engineered cell increases expression of urea cycle enzymes. In some embodiments, a PSC increases expression of urea cycle enzymes.

[0011] An aspect of the present disclosure is a method of treating a disease characterized by a liver dysfunction in a subject in need thereof, the method comprising: administering a pluripotent stem cell (PSC) comprising one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame, to the subject in need thereof, wherein the one or more transcription factors comprise: GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GAT A, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member; and wherein the PSC is configured to perform one or more hepatocyte functions at a rate greater than or equal to a primary hepatocyte or a wild-type hepatocyte. In some embodiments, a PSC is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, a PSC comprises a mammalian stem cell. In some embodiments, a PSC comprises a human stem cell. In some embodiments, a PSC comprises a human induced pluripotent stem cell (iPSC). In some embodiments, a PSC comprises a human progenitor cell. In some embodiments, a PSC comprises a human fetal stem cell. In some embodiments, a PSC comprises a hepatic stem cell. In some embodiments, a PSC comprises an embryonic stem cell. In some embodiments, a PSC comprises an engineered cell. In some embodiments, a PSC comprises a hepatocyte or hepatocyte-like cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a mammalian stem cell. In some embodiments, a hepatocyte or hepatocyte-like cell is not derived from a human stem cell. In some embodiments, a hepatocyte or hepatocyte-like cell is derived from a human stem cell.INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0014] FIG. 1A-1B illustrates an example of H0033A-1A4 cells showing high efficiency of alpha- 1 -anti -trypsin and albumin expression after four days of differentiation. H0033A-1A4 cells were induced to differentiate for four days, fixed, and then stained for alpha- 1 -anti -trypsin (magenta), albumin (green), and DAPI (cyan). Scale bar: 50 pm. FIG. 1A shows confocal images following four days of differentiation. The percentage of cells positive for A1AT and albumin (ALB) was assessed (FIG. IB) among uninduced cells, induced cells, cells exposed to a secondary antibody (negative control) and primary hepatocytes (positive control).

[0015] FIG. 2A-FIG. 2E illustrates exemplary findings from gene expression experiments to compare gene expression between uninduced H0033A-1A4 cells and induced (differentiated) H0033 A-l A4 cells. Cells that were induced to express the transcription factor recipe demonstrated high expression of key hepatocyte markers and a loss of pluripotency markers POU5F1 / OCT4, NANOG and SOX2 (FIG. 2A). The induced hepatocytes or hepatocyte-like cells expressed hepatocyte markers APOB, ALB (albumin), RBP4 (FIG. 2B); SERPINA1 (alpha- 1 -anti -trypsin), SULT1A1, ASL (FIG. 2C); MTTP, APOA1, ALDH6A1 (FIG. 2D); SAA4, FGA (fibrinogen), and APOA2 (FIG. 2E).

[0016] FIG. 3A-FIG. 3D illustrates exemplary findings from gene expression experiments to compare gene expression differences between uninduced H0033A-1A4 cells, induced (differentiated) H0033A-1A4 cells, and primary human hepatocytes. The induced H0033A-1A4 cells expressed genes associated with several key hepatocyte functions such as, genes involved in coagulation and transport (FIG. 3A), genes involved amino acid and alcohol metabolism (FIG. 3B), genes involved in inflammation and detoxification (FIG. 3C), or lipoprotein carriers (FIG. 3D). Error bar: standard error of the mean (S.E.M.).

[0017] FIG. 4A-FIG. 4F illustrates exemplary findings from gene expression experiments to compare differences in gene expression between uninduced H0033A-1A4 cells, induced (differentiated) H0033A-1A4 cells, and primary human hepatocytes (control). The induced H0033A-1 A4 cells express a variety of genes associated with hereditary genetic diseases of the liver e.g., SERPINA1 (alpha- 1 -anti -trypsin deficiency; FIG. 4A), ATP7B (Wilson’s disease; FIG. 4B), FAH (tyrosinemia; FIG. 4C), SLC40A1 (hereditary haemochromatosis type 4; FIG. 4D), ARG1 (argininemia; FIG. 4E), and AGT (primary hyperoxaluria; FIG. 4F). Error bar: standard error of the mean (S.E.M.).

[0018] FIG. 5A-FIG. 5B illustrates exemplary findings from gene expression experiments to compare differences in gene expression between uninduced H0033A-1A4 cells, induced (differentiated) H0033A-1A4 cells, and primary human hepatocytes. The induced H0033A-1A4 cells express ASLI (FIG. 5A) and ARG1 (FIG. 5B) at higher or similar levels to primary human hepatocytes. Error bar: standard error of the mean (S.E.M.).

[0019] FIG. 6 illustrates exemplary findings when an unsupervised machine learning-based cell type classification system (a classifier) was used to automatically annotate gene expression data from H0033A-1 A4 cells. The output data from the classifier clustered and annotated sequencing data from uninduced versus induced, human induced pluripotent stem cells (hiPSCs) and primary hepatocyte controls, where the fractions of data from each cluster in hiPSC or hepatocytes and is shown in FIG. 6, almost all of the induced H0033A-1 A4 cells were classified as hepatocytes.

[0020] FIG. 7A-FIG. 7C illustrates exemplary assessment of CYP3A4 activity when H0033A- 1A4 and H0033A-2E11 cells were allowed to differentiate for seven days, and then 10 pM rifampicin was added to the cell culture media for 24 hours. CYP3A4 activity was assessed in induced cells before and after addition of rifampicin in induced H0033A-1A4 cells (FIG. 7A), H0033A-2E11 cells (FIG. 7B), and in two primary human hepatocyte lines not induced by rifampicin were assessed for comparison to a reference (FIG. 7C).

[0021] FIG. 8A-FIG. 8D: illustrates an exemplary study comparing functionality of induced H0033A-2E11 cells differentiated for seven days before being passaged, placed on ice, or cryopreserved (FIG. 8A). Induced H0033 A-2E11 cells were found to have superior functionality compared to primary hepatocytes when cells were tested for cell viability before plating (FIG. 8B), or when tested for albumin production (FIG. 8C) or Al AT production (FIG. 8D) by ELISA.

[0022] FIG. 9A-9B illustrates an exemplary study showing improvements to CYP3A4 activity using novel transcription factors or direct genetic modification. A) New cell lines were engineered with novel combinations of transcription factors. After 7 days of differentiation, CYP3A4 enzymatic activity was measured using a CYP3A4 luminescence assay (P450 Gio) (FIG. 9A). Combinations of transcription factors alongside CYP3A4 conferred increased CYP3A4 activity (FIG. 9B)

[0023] FIG. 10 illustrates an exemplary study showing albumin secretion can be increased using novel transcription factors. New cell lines were engineered that improved albumin secretion compared to primary human hepatocytes (PHH). After 7 days of differentiation, albumin secretion was measured by ELISA.

[0024] FIG. 11 illustrates an exemplary study showing alpha- 1 -anti -trypsin secretion can be increased using novel transcription factors. New cell lines were engineered that improved alpha- 1 -anti -trypsin secretion. After 7 days of differentiation, alpha- 1 -anti -trypsin secretion was measured by ELISA.

[0025] FIG. 12 illustrates an exemplary study showing cholesterol homeostasis using novel transcription factors. New cell lines were engineered and showed high cholesterol homeostasis. After 7 days of differentiation, the amount of cholesterol in the media was measured by chromogenic assay kit.

[0026] FIG. 13 illustrates an exemplary study showing triglycerides secretion using novel transcription factors. New cell lines were engineered and showed triglycerides secretion. After 7 days of differentiation, the quantity (amount) of triglycerides in the media was measured by chromogenic assay kit.

[0027] FIG. 14 illustrates an exemplary study showing bile acid secretion using novel transcription factors. New cell lines were engineered that increased bile acid secretion. After 7 days of differentiation, the amount of bile acids in the media was measured by chromogenic assay kit.

[0028] FIG. 15A-15C illustrates a comparison of an exemplary engineered hepatocyte or hepatocyte-like cell in various media conditions. A population of engineered hepatocyte or hepatocyte-like cells were cultured under different media conditions and assessed for albumin secretion (FIG. 15A), A1AT secretion (FIG. 15B) by ELISA, and for CYP3A4 activity (FIG. 15C) by a luminogenic substrate cleavage assay. Conditions: A) mTeSR for 7 days, B) mTeSR for 4 days, hepatocyte medium for 3 days, C) mTeSR for 4 days, DMEM / F12 for 3 days, D) mTeSR for 4 days, DMEM / F12 for 3 days, 4pM A83-01 throughout, E) mTeSR for 4 days, DMEM / F12 for 3 days, I M DZNeP throughout.

[0029] FIG. 16A-16B illustrates an engineered cell overexpressing an exemplary genetic cargo. HepG2 cells were transfected with pMaxGFP using either Lipofectamine 2000 or Lipofectamine 3000 at two concentrations. After 96 hours, the number of GFP+ cells were assessed using flow cytometry. 30% of the HepG2 cells were transfected with GFP using Lipofectamine 3000 + pMaxGFP (FIG. 16A). Transfected cells were also visualized under microscopy to assess GFP+ HepG2 cells (white) amongst non-transfected cells after 96 hours. Scale bar: 300 pm. Overexpression of genetic cargo in hepatocytes with the goal of delivering therapeutic cargo was next conducted. To do this, HepG2 hepatocytes were transfected with a construct to express GFP,a model genetic cargo (FIG. 16B). Using different transfection reagents (Lipofectamine 2000 and 3000) and at different concentrations (lx and 2x), expression of cargo based on flow cytometry was detected; few cells expressed the cargo in controls but up to 30% of cells expressed the cargo in the indicated conditions. Expression was further visualized by fluorescence imaging based on the presence of fluorescent cells.

[0030] FIG. 17A-FIG. 17D illustrates that mice transplanted with iPSC-hepatocytes (H33 A-l A4) gained weight normally and did not show signs of malignancy or tumor formation after 8 weeks. Healthy NOD scid gamma (NSG®) mice were injected intrasplenically with (1) 500,000 induced H33A-1A4 cells and (2) primary human hepatocytes (PHH) or received intravenously injections containing 75,000 induced H33A-1A4 cells. Mice were monitored for 4 and 8 weeks then monitored temporarily to collect data on (FIG. 17A) weight, (FIG. 17B) gross visual examination of the abdominal cavity, (FIG. 17C) expected lack of expression of human albumin in the lungs at 4 weeks post-transplantation, (FIG. 17D) gross examination of the lungs at 8 weeks posttransplantation.

[0031] FIG. 18A-FIG.18C illustrates that engrafted hepatocytes were not localized to any particular hepatic zone when the H33A-1A4 cells were injected. Healthy NSG® mice received intrasplenic or intravenous injections with engineered cells, primary hepatocytes, or vehicle shown in FIG. 17A-FIG. 17D. Tissue sections were obtained and stained for human albumin. The distances from human albumin-positive cells or cell clusters to the portal triad (FIG. 18A) and central vein (FIG. 18B) were measured, and human-specific albumin was detected without the need for continuous TF induction (FIG. 18C).

[0032] FIG. 19 illustrates that albumin positive cells could be found in mice transplanted with H33A-1A4 cells at 4 and 8 weeks. Tissue sections from transplanted mice were harvested at 4- and 8-weeks post-transplantation and stained for human albumin and propionyl-CoA carboxylase alpha (PCNA) markers (FIG. 19).

[0033] FIG. 20A-FIG. 20B illustrates an exemplary study showing that H33 A- 1A4 cells promote survival of mice in a carbon tetrachloride (CChj-induced model of acute liver failure. NSG mice were injected intraperitoneally with 2.5 mL / kg of 35% carbon tetrachloride (CCh) to induced acute liver injury. Twenty-four hours later, the mice were injected intravenously (through the tail vein) with (i) 2.5 x 105or 5 x 105H33A-1A4 cells, (ii) 2.5 x 105primary human hepatocytes (PHHs), or (iii) vehicle (saline). Mice were monitored to assess survival and any changes in body weights at the start of the study on day zero (0)) and day 1, 5, 10, and 15 after injection. A Kaplan-Meier curve showing survival of mice from day 0 (injection of CC14), day 1 when cells or vehicle was inj ected, and thereafter for the next two weeks is depicted in FIG. 20A. Monitoring of changes in body weight for the mice over the two-week period is depicted in FIG. 20B.

[0034] FIG. 21 illustrates a study showing transplantation of engineered cells promoted recovery of the liver two weeks after carbon tetrachloride (CC14)-induced acute liver failure similar to healthy liver whereas mice transplanted with vehicle only did not recover. NSG mice were injected intraperitoneally with 2.5 mL / kg of 35% carbon tetrachloride (CCh) to induced acute liver injury. Twenty-four hours later, the mice were injected intravenously with (i) 2.5 x 105or 5 x 105H33A- 1A4 cells, (ii) 2.5 x 105primary human hepatocytes (PHHs), or (iii) vehicle (saline). Mice were euthanized to assess condition of the whole livers (top; FIG. 21) and sections of the liver using hematoxylin and eosin-stained tissue sections (bottom; FIG. 21) for all conditions. Tissue regions with high immune cell recruitment (black star) and hepatocyte death (white triangle) are indicated.

[0035] FIG. 22A-FIG. 22D illustrates transplantation of engineered cells into mice resulting in promotion of recovery of the livers by two weeks. NSG mice were injected as disclosed above. Serum from all surviving mice in the CCL-induced acute liver injury was tested and results are shown for AST (FIG. 22A), ALT (FIG. 22B), total albumin (FIG. 22C), total bilirubin (FIG.22D)

[0036] FIG. 23A-FIG. 23D illustrates an exemplary metabolic profile induced by engineered H33A-1A4 cell line. The metabolic profiles of H33A-1A4 cells (both uninduced and induced) were assessed using an Agilent Seahorse XF96 instrument. Data was normalized by mass of protein using a bicinchoninic acid (BCA) assay. Shown is the metabolic profile detected for basal oxygen consumption rate (OCR) (FIG. 23A), and maximal OCR (FIG. 23B), spare respiratory capacity (SRC; FIG. 23C) and the oxygen consumption rate / extracellular acidification rate (ECAR) or OCR / ECAR ratio (FIG. 23D).

[0037] FIG. 24A-FIG. 24c illustrates transcription factor combinations or recipes that induced functional cell lines with improved urea production. The H85A top performing cell lines, H85A- 1B1 and H85-1B2 expressing HNF1A, HNF4A, CEBPA and GATA6 transcription factors were cultured and assessed for hepatocyte functionality. After 24 hours of culture, the cell lines were analyzed for urea production (FIG. 24 A) and for albumin (FIG. 24B) and A1AT (FIG. 24C) production in the cell culture media over the final 24 hours of culture using standard ELISA. Parental uninduced cells were included as comparator, reference, or control cell line(s). As seen in FIG. 24A-24C, engineered cells induced higher production of urea, albumin, or A1AT thanuninduced cells and within the range of primary human hepatocytes, suggesting the cells had differentiated into functional hepatocytes or hepatocyte-like cells.

[0038] FIG. 25A - FIG. 25E illustrates induction of the H85A-1B1 cells and expression of genes involved in the urea cycle. Gene expression profile was conducted using either uninduced or induced H85A-1B1 cells on day 7 of culture. Real time quantitative (reverse transcriptase) PCR (RT-qPCR) was conducted to assess expression transcripts involved in the urea cycle. All data was normalized to uninduced (undifferentiated) control or reference cells. Transcript expression profiling are shown for the induced and uninduced H85A-1B1 cells: (i) CPS1 mRNA fold change in expression (FIG. 25A), (ii) OTC mRNA fold change in expression (FIG. 25B), (III) ASS1 mRNA fold change in expression (FIG. 25C), (iv) ASL mRNA fold change in expression (FIG. 25D) or (v) ARG1 mRNA fold change in expression (FIG. 25E).

[0039] FIG. 26 illustrates induction of H85A-1B1 cells promoted mice survival following carbon tetrachloride (CC14)-induced acute liver failure (described above). Twenty -four hours later, mice were injected intravenously (IV) with three different lots of (i) primary hepatocytes (PHH) or engineered H85A-1B1 cells that had been differentiated for seven days or with vehicle (media). The survival of mice was assessed using a Kaplan-Meier curve. The probability of survival of the mice starting from day 0 (injection of CC14), day 1 (Tx) and for the next two weeks thereafter is shown in FIG. 26.

[0040] FIG. 27A - FIG. 27C illustrates engineered H85A-1B1 cells induced a metabolic profile. The metabolic profiles of uninduced or induced H85A-1B1 cells was investigated following four days or seven days of culture. Cells were assessed using an Agilent Seahorse XF96 instrument. Data was normalized by mass of protein using a bicinchoninic acid (BCA) assay. Shown is the metabolic profile detected for (i) the oxygen consumption rate (OCR) generated by uninduced and induced H85A-1B1 ( IB 1 ) cells at either four days (D4) or seven days (D7) of culture (FIG. 27A), or (ii) the OCR generated by uninduced and induced H85 A-1B 1 (IB 1) cells at either four (D4) or seven days (D7) of culture when quantified for basal (FIG. 27B) and maximal states. The ratio of the oxygen consumption rate / extracellular acidification rate (ECAR) or OCR to ECAR ratio is shown in FIG. 27C.

[0041] FIG. 28A - FIG. 28C illustrates that the H98B-derived cell line was capable of inducing improved urea production. The H98B-derived cell lines was used to assess urea production (FIG. 28A), albumin secretion (FIG. 28B) or Al AT secretion (FIG. 28C) into the media over the final 24 hours of culture.

[0042] FIG. 29A - FIG. 29E illustrates study that quantified expression of transcripts involved in the urea cycle. A real time quantitative (reverse transcriptase) PCR (RT-qPCR) was conducted using the induced or uninduced H98B-6A5 cells on day 7 of cell culture. Data was normalized to uninduced (undifferentiated) control cells. Relative transcript expression on day 7 relative to the control was determined and is depicted as: ornithine transcarbamylase, OTC mRNA fold change in expression (FIG. 29A), argininosuccinate synthase 1, AS SI mRNA fold change in expression (FIG. 29B), carbamoyl-phosphate synthase 1, CPS1 mRNA fold change in expression (FIG. 29C), argininosuccinate lyase, ASL mRNA fold change in expression (FIG. 29D) and arginase 1, ARG1 mRNA fold change in expression (FIG. 29E).

[0043] FIG. 30A - FIG. 30B illustrates metabolic profiles induced by engineered H98B-6A5 cells. The metabolic profiles of uninduced or uninduced H98B-6A5 (6A5) cells were investigated after 7 days of cell culture using an Agilent Seahorse XF96 instrument. Data was normalized by mass of protein using a bicinchoninic acid (BCA) assay. Data showed the basal and maximal oxygen consumption rate (OCR; FIG. 30A) and the ratio of the oxygen consumption rate / extracellular acidification rate (ECAR) or OCR to ECAR ratio observed in each cell line at 7 days of culture (FIG. 30B) in engineered induced H98B-6A5 (6A5) cells relative to uninduced controls.DETAILED DESCRIPTION

[0044] While various embodiments of the invention have been shown and described herein, it will be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. As such, it should be obvious that various alternatives to the embodiments of the invention described herein may be employed. Additionally, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

[0045] The present disclosure provides compositions, methods, kits, and formulations comprising an engineered cell configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the cell functionality in the engineered or genetically modified cell may be performed at levels that are much greater than the cell functionality of a native cell, a wild-type cell, or a cell that does not comprise the compositions, kits, or formulations of the present disclosure. The geneticallyengineered or genetically modified cell can enhance or improve the functionality of a cell in an organism. In some embodiments, the improved cell functionality may be a cell function performed at an organ level, at a systemic level, at a tissue level, at a cellular level, or at any one or more combination of levels. From hereinafter, an engineered cell as used herein can be referred to interchangeably, as a genetically modified cell, a modified cell. As provided herein when referencing an engineered or genetically modified cell, it should be understood to mean, a cell comprising an exogenous nucleic acid sequence, amino acid sequence, an expression cassette or a cell comprising new genetic information inserted into the cell that may modify a cell or an organism for the purpose of changing any desired characteristics. The genetically engineered cell can be in naked within a cell, a vector, or in a capsule or a biocompatible polymer (e.g., to protect them against an immune response or protect release until it is delivered to site of action or protect it and retrieve as an encapsulated engineered cell can be removed if it is toxic or has any side effects). Non-limiting example of a reason to genetically modify a cell may include, to improve cell functionality, modify a cell so it is able to deliver a nucleic acid molecule, a protein, an agent (therapeutic or not therapeutic) to another cell, tissue, organ, system, or to another organism for any number of functional activities as desired. In some embodiments, the engineered cell disclosed herein can be further engineered to express at least one expression cassette comprising one or more transcription factors, one or more transcription factor family members, or one or enhancers of transcription. In some embodiments, the engineered cell comprising the one or more transcription factor family members, one or more transcription factors, or one or more enhancers of transcription can be induced to drive desired expression in the engineered cell. In some embodiments, the desired expression can be for the engineered cell to differentiate to any desired cellular phenotype. In some embodiments, the desired expression can comprise improved expression of one or more cellular markers, genes, or gene products e.g., one or more hepatocyte markers, hepatocyte or hepatocyte-like genes or gene products. In some embodiments, a marker may comprise a gene or gene product. In some embodiments, the marker may comprise a secreted gene product or a gene product with cellular activity. In some embodiments, the desired expression may be one or more improved cellular functional activities. For example, the engineered cell disclosed can be induced to differentiate to a hepatocyte or hepatocyte-like cell or to any desired cellular enzymatic activity e.g., enzymatic activity et cetera.

[0046] In some embodiments, the engineered cell of the present disclosure can be a mammalian cell. In some cases, the mammalian cell can be an animal cell. In some embodiments, the animal cell can be a non-primate cell e.g., a human cell. In some embodiments, the engineered cell ofthe present disclosure can a stem cell or an adult cell. For example, the engineered cell can be a human stem cell or a human adult cell, or a human fetal stem cell. In some embodiments, the cell can be a stem cell. In some embodiments, the cell can be a pluripotent stem cell. In some embodiments, the cell can be a progenitor cell, a fetal stem cell, an umbilical cord stem cell, a stem cell from an infant or child, or an adult stem cell. In some cases, the engineered cell can be a hepatic stem cell or a hepatic progenitor cell. In some embodiments, the cell can be a progenitor cell. In some embodiments, the pluripotent stem cell can be an embryonic cell or an induced pluripotent stem cell (iPSC). In some embodiments, the cell can be a mesenchymal stem cells, endodermal cells, a hematopoietic stem cell, a neural stem cell or any cell disclosed herein or known in the art. The source cell of the engineered cell can be a pluripotent stem cell, a fetal stem cell or an adult stem cell. Non-limiting examples of adult stem cell include: an oval cell, a mesenchymal stem cell, blood stem cells, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuron and glia cell, a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, a muscle cell, a dermal fibroblast, an adipocyte among others. In some cases, the source cell of the engineered cell can be a parenchymal or a non-parenchymal cell.

[0047] In some embodiments, the engineered cell of the present disclosure can be a hepatocyte or hepatocyte-like cell. In some embodiments, the engineered cell of the present disclosure can comprise one or more functions. In some embodiments, the one or more functions of the engineered cell can perform at improved or enhanced levels relative to a cell that has not been genetically modified as disclosed herein. In some embodiments, the engineered cell disclosed can be configured to perform one or more hepatocyte functions at improved or enhanced cell functionality relative to cell functionality of an uninduced cell, a non-modified native or a wildtype hepatocyte. In some embodiments, the engineered cell disclosed is configured to perform one or more improved or enhanced hepatocyte functions. Non-limiting examples of the one or more improved or enhanced can comprise coagulation, copper transport, ion transport, retinol transport, protection against neutrophil elastase, inflammatory response, regulation of urea cycle, tyrosine metabolism, histidine metabolism, alanine metabolism, alcohol metabolism, transporting lipoproteins, fatty acid biosynthesis, albumin production, Al AT production, CYP3A4 activity, or any combination thereof. For example, the engineered cell disclosed can perform improved or enhanced enzymatic activities at higher levels than in a primary hepatocyte, a native cell, or a wild-type hepatocyte. The enzymatic activity can include one or more enzymatic activities comprising hepatocyte-specific enzymatic cellular activities or nonhepatocyte enzymatic cellular activities. In some embodiments, the engineered cell of thepresent disclosure can express a greater expression of one or more hepatocyte genes or gene products relative to the expression of the hepatocyte genes or gene products expressed by a primary hepatocyte, a native hepatocyte, or a wild-type hepatocyte. In some embodiments, the engineered cell disclosed can express a greater amount of one or more hepatocyte markers relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. In non-limiting examples, the hepatocyte markers that can be expressed at greater amount by the engineered cell disclosed can include Alpha- 1 antitrypsin (ai-antitrypsin), albumin, or both.

[0048] Pluripotent stem cells or stem cells in general can express high amounts of genes or gene products involved in retaining pluripotency. Such genes or gene products may comprise pluripotency-reinforcing cues. In some embodiments, the engineered cell provided can be further engineered such that the cell produces a low or reduced expression of one or more pluripotency-reinforcing cues. In other cases, the engineered cell provided can be further engineered such that the provided cell does not produce the one or more pluripotencyreinforcing cues. In some embodiments, the cell can be engineered to genetically knockdown or genetically knockout genes or gene product comprising one or more pluripotency-reinforcing cues. In some embodiments, the engineered cell can be genetically manipulated such that the cell produces reduced or no expression of the one or more pluripotency -reinforcing cues comprising NANOG, POU5F1 / OCT4, TGF- S, ALKs, or any combination thereof. In some embodiments, the engineered cell can be genetically manipulated such that the cell produces reduced or no expression of NANOG. In some embodiments, the engineered cell can be genetically manipulated such that the cell produces reduced or no expression of POU5F1 / OCT4. In some embodiments, the engineered cell can be genetically manipulated such that the cell produces reduced or no expression of one or more TGF- S family members. In some embodiments, the engineered cell can be genetically manipulated such that the cell produces reduced or no expression of one or more of ALKs family members.

[0049] In some embodiments, overexpression of histone acetyltransferases can be directly linked to accelerated cell proliferation and survival. The engineered cell provided herein can further be engineered to over express one or more histone acetyltransferases. In some embodiments, the cell can be engineered to genetically knockdown or genetically knockout genes or gene product comprising one or more histone acetyltransferases. In some embodiments, the engineered cell can be genetically manipulated such that the cell overexpresses the one or more histone acetyltransferases. Non-limiting examples of the one or more histoneacetyltransferases can include comprise HDAC1, HDAC2, HDAC3, HDAC4, HD AC 5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7.

[0050] In another embodiment, the present disclosure provides at least one method of generating the engineered cell. In some embodiments, the method provided can allow generation of an engineered cells that can be configured to perform one or more functions. In some embodiments, the engineered cell is configured to perform the one or more (cellular) functions at a rate greater than a primary cell or a wild-type cell. In a non-limiting example, the engineered cell can be configured to perform one or more hepatocyte or hepatocyte-like functions at a rate greater than a primary hepatocyte, a native hepatocyte, or a wild-type hepatocyte. In some embodiments, the one or more cellular functions comprises any cellular functions disclosed herein. In some embodiments, the one or more functions performed by a hepatocyte e.g., a primary hepatocyte, a native or a wild-type hepatocyte. In some embodiments, the one or more functions can be performed by a hepatocyte-like cell. In some embodiments, the method further comprises engineering an expression cassette comprising one or more transcription factors. In some embodiments, the at least one expression cassette can comprise one or more transcription factors, one or more transcription family members or one or more enhancers of transcription. In some embodiments, the at least one expression cassette can comprise one or more polynucleotides that encodes one or more functional polypeptides. In some embodiments, the methods provided can be used to generate any engineered cell disclosed herein. The engineered cell generated by the disclosed methods can be a hepatocyte, a hepatocyte-like cell. The methods of disclosed that can be used to generate the engineered cell disclosed herein. In another embodiment, the present disclosure provides methods or kits for generating an engineered cell comprising one or more transcription factor family members, one or more transcription factors or enhancers of transcription and further comprising an expression cassette comprising a polynucleotide encoding one or more functional polypeptides. The polynucleotide can be a recombinant polynucleotide, a synthetic polynucleotide, or a wild-type native polynucleotide. In some embodiments, the polynucleotide can comprise one or more genes, gene fragments, portions of genes or a combination thereof. In some embodiments, the one or more genes can be synthetic gene, an exogenous gene, a wild-type gene, a native gene, a heterologous gene, a homologous gene, or a combination thereof.

[0051] In yet another embodiment, the present disclosure provides a method of treating one or more disease. In some embodiments, the method of treating a disease can be characterized bydysfunction of an organ. In some embodiments, the organ can be a liver, for example, a dysfunction of the liver which can cause disease in a patient. In some embodiments, the one or more diseases can comprise a liver disease. The present disclosure provides methods of treating one or more liver diseases in a subject suffering from a dysfunction of a liver. In one non-limiting example, the method of treating a disease, such as a liver dysfunction or liver disease can comprise administering a therapeutic composition comprising the engineered cells described herein. In other non-limiting examples, the therapeutic composition comprising the engineered cells described herein can comprise engineered cells further comprising one or more functional polypeptides, therapeutic cargo and / or a payload as disclosed herein.

[0052] In another embodiment, the present disclosure provides compositions, kits, formulations, methods comprising a genetically engineered cell that is modified to express one or more functional recombinant polypeptides. In some embodiments, the one or more functional recombinant polypeptides can be delivered via an expression cassette comprising all the structural components of an expression vector. In some embodiments, the functional polypeptide is expressed on an expression cassette comprising a nucleic acid molecule that encodes the one or more functional recombinant polypeptide. In some embodiments, the engineered cell modified to express the one or more functional recombinant polypeptide can be configured to perform one or more non-hepatocyte functions. In some embodiments, the engineered cell modified to express the one or more functional recombinant polypeptide can be configured to perform one or more hepatocyte functions. In some embodiments, the engineered cell modified to express a functional recombinant polypeptide can be configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte, a native cell, or a wild-type hepatocyte. In some embodiments, the one or more functional recombinant polypeptides improves functionality of the engineered cell. The improved functionality can comprise any known function of a cell, e.g., a primary hepatocyte or a wild-type hepatocyte. For example, the improved functionality can comprise enhanced functional activity of a hepatocyte enzyme.

[0053] In other embodiments, the present disclosure provides methods of generating an engineered cell comprising a cell modified to express one or more functional recombinant polypeptide, wherein the cell is contacted with an expression cassette comprising a nucleic acid molecule that encodes the one or more functional recombinant polypeptide. In some embodiments, the engineered cell is configured to perform one or more cellular functions, for instance, one or more hepatocyte functions where the function of the engineered hepatocyte or hepatocyte-like cell is at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, theengineered cell can further be engineered to express at least one expression cassette comprising one or more transcription factors. In some embodiments, the genetically engineered cell can be modified to overexpress a genetic cargo in a hepatocyte with the goal of delivering a therapeutic cargo. In some embodiments, the genetic cargo can be delivered by an expression cassette. In some embodiments, the genetic cargo can be delivered by a hepatocyte.

[0054] Also provided herein in some embodiments, is a method of treating a disease in a cell, tissue, organ, or system of a subject or patient in need thereof. In some embodiments, the method comprising treating the disease can be characterized by dysfunction of a liver in a patient suffering therefrom. In some embodiments, the method comprises administering to the subject or patient an engineered cell comprising a cell modified to express one or more functional recombinant polypeptide. In some embodiments, the engineered cell can be contacted with an expression cassette comprising a nucleic acid molecule that encodes the one or more functional recombinant polypeptide in which the engineered cell can be configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some cases, the one or more functional recombinant polypeptide improves functionality of the engineered cell. In some embodiments, the functional activity of the one or more functional recombinant polypeptide in the engineered cell can be greater than an activity of the one or more functional recombinant polypeptide in a primary hepatocyte or a wild-type hepatocyte. The functional activity of the one or more functional recombinant polypeptide of the engineered cell can comprise an enzymatic cellular activity, a non-enzymatic cellular activity, or any other cellular activity disclosed herein or known in the art.

[0055] In some embodiments, the engineered cell may be derived from a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, or a human embryonic stem cell. In some embodiments, the engineered cell can be a hepatocyte or hepatocyte-like cell. In some embodiments, the engineered cell can be a hepatocyte or hepatocyte-like cell or a non-hepatocyte or non-hepatocyte-like cell. In some embodiments, the engineered cell disclosed may be derived from a subject or a patient. In some embodiments, the subject can comprise a healthy subject. The patient may comprise a subject with an illness, a disease, a condition, a symptom, or syndrome of any ailments disclosed herein or known in the medical field. In some embodiments, the subject can comprise a patient. In some embodiments, the subject can comprise a healthy subject. The subject can be a mammal, or a non-mammal as disclosed herein. In some embodiments, the engineered cell can be derived from a subject. In some embodiments, the engineered cell may not be derived from a subject. In some instances, the humanstem cell, the human induced pluripotent stem cell, the human progenitor cell, the human fetal stem cell, the hepatic stem cell, or the embryonic stem cell may not be derived from a subject or a patient. In some embodiments, the engineered cell can be a hepatocyte or hepatocyte-like cell. In some embodiments, the hepatocyte or hepatocyte-like cell may not be derived from a subject or a patient. In some embodiments, the primary hepatocyte or the wild-type hepatocyte is a human hepatocyte cell, wherein the human hepatocyte cell may not be derived from a subject or a patient. In some embodiments, the hepatocyte or hepatocyte-like cell may be derived from a human induced pluripotent stem cell.

[0056] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.Compositions

[0057] Engineered cells or genetically modified cells: Although liver disease is a leading cause of morbidity and mortality, there are hardly any proven treatments besides liver transplantation, in which a diseased liver is replaced with a healthy liver. However, liver transplantation is a treatment for end-stage liver failure that is limited by the availability of donor organs. Nonetheless, there are no approved alternative treatments for end-stage liver diseases, however, cell-based therapies have gained traction in the last few years. Hepatocytes play an essential role in liver function and make up 70-80% of the cytoplasmic mass of the liver. The present disclosure provides compositions, kits, formulations, pharmaceutical compositions, cell therapeutics, or vectors comprising the present compositions may comprise a genetically engineered or genetically modified cell configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, an engineered or genetically modified cell can be a pluripotent stem cell (PSC). In non-limiting examples, a PSC can be a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell. In some embodiment, an engineered cell can be configured to perform one or more hepatocyte functions at a rate equal to or greater than that found in an uninduced cell, a naive cell, a primary hepatocyte, or a wild-type hepatocyte. In some embodiment, an engineered cell can be configured to perform one or more hepatocyte functions at greater than that found in an uninduced cell, a naive cell, a primary hepatocyte, or a wild-type hepatocyte. Insome embodiments, the one or more hepatocyte functions can be any function disclosed herein. In some embodiments, the one or more hepatocyte functions can be any function known by a skilled artisan. Non-exemplary hepatocyte functions are presented herein in Table 4 and cover any known markers or endpoint measure to determine any functionality disclosed herein. In some embodiments, the markers can be any hepatocyte markers that designate functionality of a cell thereby confirming cellular differentiation or stage. In some embodiments, a measure of cellular functionality can confirm non-differentiation or differentiation of a cell. In some embodiments, the cellular functionality can be from an introduced exogenous gene product, e.g., green fluorescent protein (GFP) or an endogenous marker of a hepatocyte e.g., CYP3A4. In some embodiments, an engineered cell can comprise an exogenous gene or synthetic gene. In some embodiments, the synthetic gene is a marker gene. In some embodiments, the synthetic gene can be a representative of an endogenous gene. In some embodiments, any gene, marker, or gene product can be selected for use with the present disclosure.

[0058] The compositions of the present disclosure can comprise the present disclosure presented in various ways, methods etcetera. Non-limiting examples of the composition of the present disclosure can comprise, but are not limited to, for example formulations (in any form), pharmaceuticals, cell therapeutics or vectors bearing the compositions in liquid, gel, paste, tablets, caplets, solid, nanoparticles, mRNA or any formulations disclosed herein or that may be suitable to prepare the current disclosure. The engineered cell can comprise a homogenous population of cells. The engineered cell can comprise a mixed population of cells. In some embodiments, the homogenous population of cells comprises engineered cells comprising the same recombinant constructs. In some embodiments, the population of engineered cells may not comprise a homogenous population of cells. For example, the population of engineered cells can comprise a fraction that comprises engineered cells, that may be differentiated cell, or on the differentiation trajectory. For example, the population of engineered cells can comprise a fraction that comprises a population of cells closely associated with pluripotency (undifferentiated or relatively undifferentiated or largely undifferentiated). In some embodiments, the population of engineered cells disclosed may comprise partly differentiated cells and partly undifferentiated, stem-cell like cells. In some embodiments, the population of engineered cell comprising partly differentiated cells and partly undifferentiated, stem-cell like cell can comprise various proportions of each. In non-limiting examples, this mixed population of partly differentiated and partly undifferentiated cell can comprise, at least about 99% differentiated cells or 98%, 97%, 96%, 95%, 94%, all the way to 40% differentiated cells while the remainder portion could comprise the partly undifferentiated cell. In some embodiments, the proportion of differentiated cells could be lessthan 40% and the other percentage could comprise partly undifferentiated cells, and the like. In some embodiments, the engineered cells can differentiate to hepatocyte or hepatocyte-like cell. In some embodiments, the differentiated engineered cell can be characterized to comprise a hepatocyte or hepatocyte-like cell using any known metrics e.g., in an in vitro assay. In some embodiments, the in vitro assay may comprise a morphological characteristic, the levels of expression of RNA, protein, DNA, or the activity level of the gene product / protein. The engineered or genetically modified cell may comprise any number of exogenous, foreign, recombinant, heterologous, native or wild-type nucleic acid or amino acid sequences. In some embodiments, the engineered cell can comprise one or more polynucleotide sequences that can or cannot encode at least one full length functional protein. In some embodiments, the engineered cell can comprise full sequences, portions of sequences or fragments of sequences, e.g., full nucleic acid or amino acid sequences in any form without any limitations. In some embodiments, the sequence can be a polynucleotide. In some embodiments, full sequences, portions of a sequence or fragments of a sequence can comprise modified bases, unmodified bases, or both. In some cases, the sequences of the engineered cell can comprise any type of nucleic acid sequence disclosed herein. In other embodiments, the engineered cell can comprise any type of amino acid sequence disclosed or known in the art. In other embodiments, as desired, the engineered cell can comprise one or more polynucleotides that can encode a non-functional, truncated, or mutated protein. In some embodiments, the one or more polynucleotide sequence can comprise a scrambled or shuffled DNA sequence. In other cases, the engineered cell can comprise at least one polypeptide sequence. In some embodiments, the at least one polypeptide sequence can comprise one or more scrambled peptide sequences. The engineered cell can be a stem cell. In some instances, the engineered cell can be a progenitor cell or a pluripotent stem cell. The engineered cell can comprise an induced pluripotent stem cell, an embryonic stem cell, a fetal stem cell, a hepatic stem cell, or any other stem cell. In some embodiments, the engineered cell can be further engineered to express at least one expression cassette comprising one or more transcription factors. In some embodiments, a cell of the present disclosure can be engineered and configured to express one or more hepatocyte biomarkers disclosed herein in the figures, examples, or in the detailed description. Non-limiting examples of SLC40A1, ATP7B, ARG1, HAL, ALDH6A1, APOM, Al AT, albumin, CYP3A4, or any combination thereof. In non-limiting examples, an engineered cell can express any cytochrome p450 protein. In some embodiments, a cell of the present disclosure can be engineered and configured to express one or more hepatocyte functions In some embodiments, a cell expressing one or more transcription factors disclosed herein can be configured to perform one or more hepatocyte functions at a rate equal to or greater than a primaryhepatocyte or a wild-type hepatocyte. In some embodiments, an engineered cell of the present disclosure is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, a cell is engineered to comprise one or more transcription factors of the present disclosure. In some embodiments, the one or more transcription factors can comprise HNF4A, HNF1 A, CEBPA, F0XA1, and GATA6. In some embodiments, the one or more transcription factors can consist of HNF4A, HNF1A, CEBPA, F0XA1, and GATA6. In some embodiments, the one or more transcription factors can comprise HNF4A, HNF1 A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, the one or more transcription factors can consist of HNF4A, HNF1 A, CEBPA, F0XA1, GATA6, and F0XA2. In some embodiments, the one or more transcription factors can comprise GATA6. In some embodiments, the one or more transcription factors can comprise F0XA2. In some embodiments, the one or more transcription factors can comprise CEBPA. In some embodiments, the one or more transcription factors can comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GAT A, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors can comprise F0XA2. In some embodiments, the one or more additional transcription factors can comprise CEBPA. In some embodiments, the one or more additional transcription factors can comprise In some embodiments, the one or more additional transcription factors can comprise F0XA1. In some embodiments, the one or more additional transcription factors can comprise 2 or more HNF family members. In some embodiments, the one or more additional transcription factors can comprise 2 or more FOX family members. In some embodiments, the one or more transcription factors can further comprise at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GAT A, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more transcription factors can further comprise further comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member. In some embodiments, the one or more additional transcription factors can further comprise one or more transcription factors from Table 3. In some embodiments, the one or more transcription factors can comprise GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member In some embodiments, the one ormore additional transcription factors further comprise 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. In some embodiments, the one or more transcription factors can comprise CEBPA, HNF1 A, F0XA1, HNF4A, ONECUT 1, RBPJ, or any combination thereof. In some embodiments, the one or more transcription factors consist of: CEBPA, HNF1A, F0XA1, HNF4A, ONECUT 1 and RBPJ. In some embodiments, the one or more transcription factors consist of: CEBPA, HNF1 A, F0XA1, ONECUT 1 and HNF4A. In some embodiments, the one or more transcription factors consist of: CEBPA, HNF1 A, F0XA1, and HNF4A. In some embodiments, the engineered cell comprising the one or more transcription factors (TFs) can be induced to differentiate into any of the different cellular lineages. In some embodiments, the engineered cell comprising the one or more transcription factors in any combination may differentiate into a hepatocyte or hepatocytelike cell. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate of about 1 %, greater than in a primary hepatocyte or a wild-type hepatocyte to about 45 % greater than in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate about 1 % to about 2 %, about 1 % to about 3 %, 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 30 %, about 1 % to about 40 %, about 1 % to about 45 %, about 2 % to about 3 %, 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 30 %, about 2 % to about 40 %, about 2 % to about 45 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 10 %, about 3 % to about 15 %, about 3 % to about 20 %, about 3 % to about 25 %, about 3 % to about 30 %, about 3 % to about 40 %, about 3 % to about 45 %, 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 30 %, about 4 % to about 40 %, about 4 % to about 45 %, about 5 % to about 10 %, about 5 % to about 15 %, about 5 % to about 20 %, about 5 % to about 25 %, about 5 % to about 30 %, about 5 % to about 40 %, about 5 % to about 45 %, about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 45 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 40 %, about 15 % to about 45 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 45 %,about 25 % to about 30 %, about 25 % to about 40 %, about 25 % to about 45 %, about 30 % to about 40 %, about 30 % to about 45 %, or about 40 % to about 45 % greater than in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 40 %, or about 45 % % greater than in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate at least about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, or about 40 % % greater than in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell is configured to perform the one or more hepatocyte functions at a rate at most about 2 %, about 3 %, about 4 %, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 40 %, or about 45 % % greater than in a primary hepatocyte or a wild-type hepatocyte.

[0059] In some embodiments, the one or more hepatocyte functions can comprise coagulation, copper transport, ion transport, retinol transport, protection against neutrophil elastase, inflammatory response, regulation of urea cycle, tyrosine metabolism, histidine metabolism, alanine metabolism, alcohol metabolism, transporting lipoproteins, fatty acid biosynthesis, albumin production, A1AT production, CYP3A4 activity, or any combination thereof. In some embodiments, the one or more hepatocyte functions comprise coagulation. In some embodiments, the one or more hepatocyte functions comprise copper transport. In some embodiments, the one or more hepatocyte functions comprise ion transport. In some embodiments, the one or more hepatocyte functions comprise retinol transport. In some embodiments, the one or more hepatocyte functions comprise protection against neutrophil elastase. In some embodiments, the one or more hepatocyte functions comprise inflammatory response. In some embodiments, the one or more hepatocyte functions comprise regulation of urea cycle. In some embodiments, the one or more hepatocyte functions comprise tyrosine metabolism. In some embodiments, the one or more hepatocyte functions comprise histidine metabolism. In some embodiments, the one or more hepatocyte functions comprise alanine metabolism. In some embodiments, the one or more hepatocyte functions comprise alcohol metabolism. In some embodiments, the one or more hepatocyte functions comprise transporting lipoproteins. In some embodiments, the one or more hepatocyte functions comprise fatty acid biosynthesis. In some embodiments, the one or more hepatocyte functions comprise albumin production. In some embodiments, the one or more hepatocyte functions comprise A1AT production. In some embodiments, the one or more hepatocyte functions comprise CYP3 A4 activity.

[0060] In some embodiments, the engineered cell can express a greater amount of one or more hepatocyte gene product relative to the expression detected in a primary hepatocyte or a wild-type hepatocyte. The engineered cell may also express a greater amount of one or more hepatocyte gene relative to the expression detected in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the one or more hepatocyte gene can be SLC40A1, ATP7B, ARG1, HAL, ALDH6A1, or APOM. In some embodiments, the engineered cell can be configured to perform ion transport at the rate greater than a primary hepatocyte and the engineered cell expresses a greater amount of SLC40A1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least 5% more, 10% more, 20% more, 40% more, 50% more, 100% more, 150% more, 200% more, 250% more or at least 300% more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 5 % to about 120 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 5 % to about 10 %, about 5 % to about 20 %, about 5 % to about 30 %, about 5 % to about 40 %, about 5 % to about 50 %, about 5 % to about 60 %, about 5 % to about 70 %, about 5 % to about 80 %, about 5 % to about 90 %, about 5 % to about 100 %, about 5 % to about 120 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 10 % to about 100 %, about 10 % to about 120 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 100 %, about 20 % to about 120 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 100 %, about 30 % to about 120 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 100 %, about 40 % to about 120 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 100 %, about 50 % to about 120 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 100 %, about 60 % to about 120 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 100 %, about 70 % to about 120 %, about 80 % to about 90 %, about 80 % to about 100 %, about 80 % to about 120 %, about 90 % to about 100 %, about 90 % to about 120 %, or about 100 % to about 120 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, theengineered cell can express about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, or about 120 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 100 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, or about 120 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 130 % to about 250 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wildtype hepatocyte. In some embodiments, the engineered cell can express about 130 % to about 140 %, about 130 % to about 150 %, about 130 % to about 160 %, about 130 % to about 170 %, about 130 % to about 180 %, about 130 % to about 190 %, about 130 % to about 200 %, about 130 % to about 220 %, about 130 % to about 230 %, about 130 % to about 240 %, about 130 % to about 250 %, about 140 % to about 150 %, about 140 % to about 160 %, about 140 % to about 170 %, about 140 % to about 180 %, about 140 % to about 190 %, about 140 % to about 200 %, about 140 % to about 220 %, about 140 % to about 230 %, about 140 % to about 240 %, about 140 % to about 250 %, about 150 % to about 160 %, about 150 % to about 170 %, about 150 % to about 180 %, about 150 % to about 190 %, about 150 % to about 200 %, about 150 % to about 220 %, about 150 % to about 230 %, about 150 % to about 240 %, about 150 % to about 250 %, about 160 % to about 170 %, about 160 % to about 180 %, about 160 % to about 190 %, about 160 % to about 200 %, about 160 % to about 220 %, about 160 % to about 230 %, about 160 % to about 240 %, about 160 % to about 250 %, about 170 % to about 180 %, about 170 % to about 190 %, about 170 % to about 200 %, about 170 % to about 220 %, about 170 % to about 230 %, about 170 % to about 240 %, about 170 % to about 250 %, about 180 % to about 190 %, about 180 % to about 200 %, about 180 % to about 220 %, about 180 % to about 230 %, about 180 % to about 240 %, about 180 % to about 250 %, about 190 % to about 200 %, about 190 % to about 220 %, about 190 % to about 230 %, about 190 % to about 240 %, about 190 % to about 250 %, about 200 % to about 220 %, about 200 % to about 230 %, about 200 % to about 240 %, about 200 % to about 250 %, about 220 % to about 230 %, about 220 % to about 240 %, about 220 % to about 250 %, about 230 % to about 240 %, about 230 % to about 250 %, or about 240 % to about 250 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wildtype hepatocyte. In some embodiments, the engineered cell can express about 130 %, about 140 %,about 150 %, about 160 %, about 170 %, about 180 %, about 190 %, about 200 %, about 220 %, about 230 %, about 240 %, or about 250 % . In some embodiments, the engineered cell can express at least about 130 %, about 140 %, about 150 %, about 160 %, about 170 %, about 180 %, about 190 %, about 200 %, about 220 %, about 230 %, or about 240 %. In some embodiments, the engineered cell can express at most about 140 %, about 150 %, about 160 %, about 170 %, about 180 %, about 190 %, about 200 %, about 220 %, about 230 %, about 240 %, or about 250 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 260 % to about 350 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 260 % to about 280 %, about 260 % to about 300 %, about 260 % to about 320 %, about 260 % to about 350 %, about 280 % to about 300 %, about 280 % to about 320 %, about 280 % to about 350 %, about 300 % to about 320 %, about 300 % to about 350 %, or about 320 % to about 350 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 260 %, about 280 %, about 300 %, about 320 %, or about 350 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 260 %, about 280 %, about 300 %, or about 320 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 280 %, about 300 %, about 320 %, or about 350 % more SLC40A1 relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte.

[0061] In some embodiments, the engineered cell can be configured to perform copper transport at a rate greater than a primary hepatocyte or a wild-type hepatocyte and the engineered cell expresses a greater amount of ATP7B relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. The engineered cell can express at least 100% more, 5% more, 10% more, 20% more, 40% more, 50% more, 100% more, 120% more, 130% more, 140% more, 150% more, 200% more, 250% more, 300% more, 400% more, 450% more, 500% more, 550% more, or at least 600% more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 30 % to about 140 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 100 %, about 30 % to about 110 %, about 30 % to about 120 %, about 30 % to about 130 %, about 30 % toabout 140 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 100 %, about 40 % to about 110 %, about 40 % to about 120 %, about 40 % to about 130 %, about 40 % to about 140 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 100 %, about 50 % to about 110 %, about 50 % to about 120 %, about 50 % to about 130 %, about 50 % to about 140 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 100 %, about 60 % to about 110 %, about 60 % to about 120 %, about 60 % to about 130 %, about 60 % to about 140 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 100 %, about 70 % to about 110 %, about 70 % to about 120 %, about 70 % to about 130 %, about 70 % to about 140 %, about 80 % to about 90 %, about 80 % to about 100 %, about 80 % to about 110 %, about 80 % to about 120 %, about 80 % to about 130 %, about 80 % to about 140 %, about 90 % to about 100 %, about 90 % to about 110 %, about 90 % to about 120 %, about 90 % to about 130 %, about 90 % to about 140 %, about 100 % to about 110 %, about 100 % to about 120 %, about 100 % to about 130 %, about 100 % to about 140 %, about 110 % to about 120 %, about 110 % to about 130 %, about 110 % to about 140 %, about 120 % to about 130 %, about 120 % to about 140 %, or about 130 % to about 140 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wildtype hepatocyte. In some embodiments, the engineered cell can express about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, about 110 %, about 120 %, about 130 %, or about 140 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, about 110 %, about 120 %, or about 130 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, about 110 %, about 120 %, about 130 %, or about 140 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 150 % to about 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 150 % to about 180 %, about 150 % to about 200 %, about 150 % to about 250 %, about 150 % to about 300 %, about 150 % to about 350 %, about 150 % to about 400 %, about 150 % to about 450 %, about 150 % to about 500 %, about 150 % to about 550 %, about 150 % to about 600 %, about 180 % to about 200 %, about 180 % to about 250 %, about 180 % to about 300 %, about 180 % to about 350 %, about 180 % to about 400 %, about180 % to about 450 %, about 180 % to about 500 %, about 180 % to about 550 %, about 180 % to about 600 %, about 200 % to about 250 %, about 200 % to about 300 %, about 200 % to about 350 %, about 200 % to about 400 %, about 200 % to about 450 %, about 200 % to about 500 %, about 200 % to about 550 %, about 200 % to about 600 %, about 250 % to about 300 %, about 250 % to about 350 %, about 250 % to about 400 %, about 250 % to about 450 %, about 250 % to about 500 %, about 250 % to about 550 %, about 250 % to about 600 %, about 300 % to about 350 %, about 300 % to about 400 %, about 300 % to about 450 %, about 300 % to about 500 %, about 300 % to about 550 %, about 300 % to about 600 %, about 350 % to about 400 %, about 350 % to about 450 %, about 350 % to about 500 %, about 350 % to about 550 %, about 350 % to about 600 %, about 400 % to about 450 %, about 400 % to about 500 %, about 400 % to about 550 %, about 400 % to about 600 %, about 450 % to about 500 %, about 450 % to about 550 %, about 450 % to about 600 %, about 500 % to about 550 %, about 500 % to about 600 %, or about 550 % to about 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 150 %, about 180 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 150 %, about 180 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, or about 550 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 180 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least 0.5%, 1%, 2%, 3%, 4%, 5% all the way up to at least 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte (expression levels include all integers and decimals included from about 0.5% for example, 1%, 2%, 3% all the way to at least 600%). In some embodiments, the engineered cell can express over 600 % more ATP7B relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte.

[0062] The engineered cell can be configured to perform urea cycle at the rate greater than a primary hepatocyte or a wild-type hepatocyte and the engineered cell expresses a greater amount of ARG1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, or at least 43% higher levelsof ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 % to about 35 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 % to about 30 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 % to about 1 %, about 0.5 % to about 2 %, about 0.5 % to about 3 %, about 0.5 % to about 4 %, about 0.5 % to about 5 %, about 0.5 % to about 6 %, about 0.5 % to about 10 %, about 0.5 % to about 15 %, about 0.5 % to about 20 %, about 0.5 % to about 25 %, about 0.5 % to about 30 %, about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 4 %, about 1 % to about 5 %, about 1 % to about 6 %, about 1 % to about 10 %, about 1 % to about 15 %, about 1 % to about 20 %, about 1 % to about 25 %, about 1 % to about 30 %, about 2 % to about 3 %, about 2 % to about 4 %, about 2 % to about 5 %, about 2 % to about 6 %, about2 % to about 10 %, about 2 % to about 15 %, about 2 % to about 20 %, about 2 % to about 25 %, about 2 % to about 30 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 6 %, about 3 % to about 10 %, about 3 % to about 15 %, about 3 % to about 20 %, about 3 % to about 25 %, about 3 % to about 30 %, about 4 % to about 5 %, about 4 % to about 6 %, about 4 % to about 10 %, about 4 % to about 15 %, about 4 % to about 20 %, about 4 % to about 25 %, about 4 % to about 30 %, about 5 % to about 6 %, about 5 % to about 10 %, about 5 % to about 15 %, about 5 % to about 20 %, about 5 % to about 25 %, about 5 % to about 30 %, about 6 % to about 10 %, about 6 % to about 15 %, about 6 % to about 20 %, about 6 % to about 25 %, about 6 % to about 30 %, about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 20 % to about 25 %, about 20 % to about 30 %, or about 25 % to about 30 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 %, about 1 %, about 2 %, about3 %, about 4 %, about 5 %, about 6 %, about 10 %, about 15 %, about 20 %, about 25 %, or about 30 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 0.5 %, about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 6 %, about 10 %, about 15 %, about 20 %, or about 25 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wildtype hepatocyte. In some embodiments, the engineered cell can express at most about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 6 %, about 10 %, about 15 %, about 20 %, about 25 %, or about 30 %. In some embodiments, the engineered cell can express about 31 % to about 50 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-typehepatocyte. In some embodiments, the engineered cell can express about 31 % to about 32 %, about 31 % to about 33 %, about 31 % to about 34 %, about 31 % to about 35 %, about 31 % to about 36 %, about 31 % to about 37 %, about 31 % to about 40 %, about 31 % to about 42 %, about 31 % to about 43 %, about 31 % to about 45 %, about 31 % to about 50 %, about 32 % to about 33 %, about 32 % to about 34 %, about 32 % to about 35 %, about 32 % to about 36 %, about 32 % to about 37 %, about 32 % to about 40 %, about 32 % to about 42 %, about 32 % to about 43 %, about 32 % to about 45 %, about 32 % to about 50 %, about 33 % to about 34 %, about 33 % to about 35 %, about 33 % to about 36 %, about 33 % to about 37 %, about 33 % to about 40 %, about 33 % to about 42 %, about 33 % to about 43 %, about 33 % to about 45 %, about 33 % to about 50 %, about 34 % to about 35 %, about 34 % to about 36 %, about 34 % to about 37 %, about 34 % to about 40 %, about 34 % to about 42 %, about 34 % to about 43 %, about 34 % to about 45 %, about 34 % to about 50 %, about 35 % to about 36 %, about 35 % to about 37 %, about 35 % to about 40 %, about 35 % to about 42 %, about 35 % to about 43 %, about 35 % to about 45 %, about 35 % to about 50 %, about 36 % to about 37 %, about 36 % to about 40 %, about 36 % to about 42 %, about 36 % to about 43 %, about 36 % to about 45 %, about 36 % to about 50 %, about 37 % to about 40 %, about 37 % to about 42 %, about 37 % to about 43 %, about 37 % to about 45 %, about 37 % to about 50 %, about 40 % to about 42 %, about 40 % to about 43 %, about 40 % to about 45 %, about 40 % to about 50 %, about 42 % to about 43 %, about 42 % to about 45 %, about 42 % to about 50 %, about 43 % to about 45 %, about 43 % to about 50 %, or about 45 % to about 50 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 31 %, about 32 %, about 33 %, about 34 %, about 35 %, about 36 %, about 37 %, about 40 %, about 42 %, about 43 %, about 45 %, or about 50 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 31 %, about 32 %, about 33 %, about 34 %, about 35 %, about 36 %, about 37 %, about 40 %, about 42 %, about 43 %, or about 45 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 32 %, about 33 %, about 34 %, about 35 %, about 36 %, about 37 %, about 40 %, about 42 %, about 43 %, about 45 %, or about 50 % higher levels of ARG1 than the levels expressed in a primary hepatocyte or a wildtype hepatocyte.

[0063] In some embodiments, the engineered cell can be configured to perform histidine metabolism at the rate greater than a primary hepatocyte and the engineered cell expresses a greater amount of HAL relative to an amount of the hepatocyte marker expressed by a primaryhepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell expresses at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 100%, 150%, 170% 200%, 250%, 270%, 300%, or at least 350% more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 % to about 110 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 1 % to about 80 %, about 1 % to about 90 %, about 1 % to about 100 %, about 1 % to about 110 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 10 % to about 100 %, about 10 % to about 110 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 100 %, about 20 % to about 110 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 100 %, about 30 % to about 110 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 100 %, about 40 % to about 110 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 100 %, about 50 % to about 110 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 100 %, about 60 % to about 110 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 100 %, about 70 % to about 110 %, about 80 % to about 90 %, about 80 % to about 100 %, about 80 % to about 110 %, about 90 % to about 100 %, about 90 % to about 110 %, or about 100 % to about 110 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, or about 110 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 100 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %, or about 110 % more HAL relativeto the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 120 % to about 400 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 120 % to about 150 %, about 120 % to about 180 %, about 120 % to about 200 %, about 120 % to about 230 %, about 120 % to about 250 %, about 120 % to about 270 %, about 120 % to about 290 %, about 120 % to about 300 %, about 120 % to about 330 %, about 120 % to about 350 %, about 120 % to about 400 %, about 150 % to about 180 %, about 150 % to about 200 %, about 150 % to about 230 %, about 150 % to about 250 %, about 150 % to about 270 %, about 150 % to about 290 %, about 150 % to about 300 %, about 150 % to about 330 %, about 150 % to about 350 %, about 150 % to about 400 %, about 180 % to about 200 %, about 180 % to about 230 %, about 180 % to about 250 %, about 180 % to about 270 %, about 180 % to about 290 %, about 180 % to about 300 %, about 180 % to about 330 %, about 180 % to about 350 %, about 180 % to about 400 %, about 200 % to about 230 %, about 200 % to about 250 %, about 200 % to about 270 %, about 200 % to about 290 %, about 200 % to about 300 %, about 200 % to about 330 %, about 200 % to about 350 %, about 200 % to about 400 %, about 230 % to about 250 %, about 230 % to about 270 %, about 230 % to about 290 %, about 230 % to about 300 %, about 230 % to about 330 %, about 230 % to about 350 %, about 230 % to about 400 %, about 250 % to about 270 %, about 250 % to about 290 %, about 250 % to about 300 %, about 250 % to about 330 %, about 250 % to about 350 %, about 250 % to about 400 %, about 270 % to about 290 %, about 270 % to about 300 %, about 270 % to about 330 %, about 270 % to about 350 %, about 270 % to about 400 %, about 290 % to about 300 %, about 290 % to about 330 %, about 290 % to about 350 %, about 290 % to about 400 %, about 300 % to about 330 %, about 300 % to about 350 %, about 300 % to about 400 %, about 330 % to about 350 %, about 330 % to about 400 %, or about 350 % to about 400 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 120 %, about 150 %, about 180 %, about 200 %, about 230 %, about 250 %, about 270 %, about 290 %, about 300 %, about 330 %, about 350 %, or about 400 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 120 %, about 150 %, about 180 %, about 200 %, about 230 %, about 250 %, about 270 %, about 290 %, about 300 %, about 330 %, or about 350 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 150 %, about 180 %, about 200 %, about 230 %, about 250 %, about 270 %, about 290 %, about 300 %, about 330 %, about350 %, or about 400 % more HAL relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte.

[0064] The engineered cell can be configured to perform alcohol metabolism at the rate greater than a primary hepatocyte and the engineered cell expresses a greater amount of ALDH6A1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least 0.5 %, 1 %, 2 %, 3 %,4 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 40%, 45%, 50%, 55%, or at least 60% higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 % to about 35 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 % to about 1 %, about 0.5 % to about 2 %, about 0.5 % to about 3 %, about 0.5 % to about 4 %, about 0.5 % to about 5 %, about 0.5 % to about 10 %, about 0.5 % to about 15 %, about 0.5 % to about 20 %, about 0.5 % to about 25 %, about 0.5 % to about 30 %, about 0.5 % to about 35 %, about 1 % to about 2 %, about 1 % to about 3 %, 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 30 %, about 1 % to about 35 %, about 2 % to about 3 %, 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 30 %, about 2 % to about 35 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 10 %, about 3 % to about 15 %, about 3 % to about 20 %, about 3 % to about 25 %, about 3 % to about 30 %, about 3 % to about 35 %, 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 30 %, about 4 % to about 35 %, about 5 % to about 10 %, about 5 % to about 15 %, about 5 % to about 20 %, about 5 % to about 25 %, about 5 % to about 30 %, about5 % to about 35 %, about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 35 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 25 % to about 30 %, about 25 % to about 35 %, or about 30 % to about 35 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 0.5 %, about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, or about 35 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 0.5 %, about 1 %, about 2 %, about 3 %, about 4 %,about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, or about 30 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, or about 35 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 36 % to about 60 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 36 % to about 37 %, about 36 % to about 38 %, about 36 % to about 39 %, about 36 % to about 40 %, about 36 % to about 43 %, about 36 % to about 45 %, about 36 % to about 47 %, about 36 % to about 50 %, about 36 % to about 55 %, about 36 % to about 60 %, about 37 % to about 38 %, about 37 % to about 39 %, about 37 % to about 40 %, about 37 % to about 43 %, about 37 % to about 45 %, about 37 % to about 47 %, about 37 % to about 50 %, about 37 % to about 55 %, about 37 % to about 60 %, about 38 % to about 39 %, about 38 % to about 40 %, about 38 % to about 43 %, about 38 % to about 45 %, about 38 % to about 47 %, about 38 % to about 50 %, about 38 % to about 55 %, about 38 % to about 60 %, about 39 % to about 40 %, about 39 % to about 43 %, about 39 % to about 45 %, about 39 % to about 47 %, about 39 % to about 50 %, about 39 % to about 55 %, about 39 % to about 60 %, about 40 % to about 43 %, about 40 % to about 45 %, about 40 % to about 47 %, about 40 % to about 50 %, about 40 % to about 55 %, about 40 % to about 60 %, about 43 % to about 45 %, about 43 % to about 47 %, about 43 % to about 50 %, about 43 % to about 55 %, about 43 % to about 60 %, about 45 % to about 47 %, about 45 % to about 50 %, about 45 % to about 55 %, about 45 % to about 60 %, about 47 % to about 50 %, about 47 % to about 55 %, about 47 % to about 60 %, about 50 % to about 55 %, about 50 % to about 60 %, or about 55 % to about 60 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 36 %, about 37 %, about 38 %, about 39 %, about 40 %, about 43 %, about 45 %, about 47 %, about 50 %, about 55 %, or about 60 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 36 %, about 37 %, about 38 %, about 39 %, about 40 %, about 43 %, about 45 %, about 47 %, about 50 %, or about 55 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 37 %, about 38 %, about 39 %, about 40 %, about 43 %, about 45 %, about 47 %, about 50 %, about 55 %, or about 60 % higher levels of ALDH6A1 than the levels expressed in a primary hepatocyte or a wild-type hepatocyte.

[0065] The engineered cell can be configured to perform transporting lipoproteins at the rate greater than a primary hepatocyte and the engineered cell expresses a greater amount of APOM relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 0.5 %, 1 %, 2 %, 3 %, 4 %, 5 %, 10 %, 15 %, 20 %, 25%, 30%, 33%, 35%, about 40%, about 50% more, 100% more, 150% more, 200% more, 250% more, 300% more, 350% or at least 400% more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 % to about 80 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 5 %, about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 1 % to about 80 %, about 2 % to about 3 %, about 2 % to about 5 %, about 2 % to about 10 %, about 2 % to about 20 %, about 2 % to about 30 %, about 2 % to about 40 %, about 2 % to about 50 %, about 2 % to about 60 %, about 2 % to about 70 %, about 2 % to about 80 %, about 3 % to about 5 %, about 3 % to about 10 %, about 3 % to about 20 %, about 3 % to about 30 %, about 3 % to about 40 %, about 3 % to about 50 %, about 3 % to about 60 %, about 3 % to about 70 %, about 3 % to about 80 %, about 5 % to about 10 %, about 5 % to about 20 %, about 5 % to about 30 %, about 5 % to about 40 %, about 5 % to about 50 %, about 5 % to about 60 %, about 5 % to about 70 %, about 5 % to about 80 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 60 % to about 70 %, about 60 % to about 80 %, or about 70 % to about 80 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 1 %, about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 1 %, about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, or about 70 % more APOM relativeto the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 81 % to about 180 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 81 % to about 82 %, about 81 % to about 85 %, about 81 % to about 90 %, about 81 % to about 100 %, about 81 % to about 120 %, about 81 % to about 130 %, about 81 % to about 140 %, about 81 % to about 150 %, about 81 % to about 160 %, about 81 % to about 170 %, about 81 % to about 180 %, about 82 % to about 85 %, about 82 % to about 90 %, about 82 % to about 100 %, about 82 % to about 120 %, about 82 % to about 130 %, about 82 % to about 140 %, about 82 % to about 150 %, about 82 % to about 160 %, about 82 % to about 170 %, about 82 % to about 180 %, about 85 % to about 90 %, about 85 % to about 100 %, about 85 % to about 120 %, about 85 % to about 130 %, about 85 % to about 140 %, about 85 % to about 150 %, about 85 % to about 160 %, about 85 % to about 170 %, about 85 % to about 180 %, about 90 % to about 100 %, about 90 % to about 120 %, about 90 % to about 130 %, about 90 % to about 140 %, about 90 % to about 150 %, about 90 % to about 160 %, about 90 % to about 170 %, about 90 % to about 180 %, about 100 % to about 120 %, about 100 % to about 130 %, about 100 % to about 140 %, about 100 % to about 150 %, about 100 % to about 160 %, about 100 % to about 170 %, about 100 % to about 180 %, about 120 % to about 130 %, about 120 % to about 140 %, about 120 % to about 150 %, about 120 % to about 160 %, about 120 % to about 170 %, about 120 % to about 180 %, about 130 % to about 140 %, about 130 % to about 150 %, about 130 % to about 160 %, about 130 % to about 170 %, about 130 % to about 180 %, about 140 % to about 150 %, about 140 % to about 160 %, about 140 % to about 170 %, about 140 % to about 180 %, about 150 % to about 160 %, about 150 % to about 170 %, about 150 % to about 180 %, about 160 % to about 170 %, about 160 % to about 180 %, or about 170 % to about 180 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 81 %, about 82 %, about 85 %, about 90 %, about 100 %, about 120 %, about 130 %, about 140 %, about 150 %, about 160 %, about 170 %, or about 180 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 81 %, about 82 %, about 85 %, about 90 %, about 100 %, about 120 %, about 130 %, about 140 %, about 150 %, about 160 %, or about 170 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express atmost about 82 %, about 85 %, about 90 %, about 100 %, about 120 %, about 130 %, about 140 %, about 150 %, about 160 %, about 170 %, or about 180 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 181 % to about 400 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 181 % to about 185 %, about 181 % to about 195 %, about 181 % to about 200 %, about 181 % to about 230 %, about 181 % to about 250 %, about 181 % to about 300 %, about 181 % to about 350 %, about 181 % to about 400 %, about 185 % to about 195 %, about 185 % to about 200 %, about 185 % to about 230 %, about 185 % to about 250 %, about 185 % to about 300 %, about 185 % to about 350 %, about 185 % to about 400 %, about 195 % to about 200 %, about 195 % to about 230 %, about 195 % to about 250 %, about 195 % to about 300 %, about 195 % to about 350 %, about 195 % to about 400 %, about 200 % to about 230 %, about 200 % to about 250 %, about 200 % to about 300 %, about 200 % to about 350 %, about 200 % to about 400 %, about 230 % to about 250 %, about 230 % to about 300 %, about 230 % to about 350 %, about 230 % to about 400 %, about 250 % to about 300 %, about 250 % to about 350 %, about 250 % to about 400 %, about 300 % to about 350 %, about 300 % to about 400 %, or about 350 % to about 400 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express about 181 %, about 185 %, about 195 %, about 200 %, about 230 %, about 250 %, about 300 %, about 350 %, or about 400 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at least about 181 %, about 185 %, about 195 %, about 200 %, about 230 %, about 250 %, about 300 %, or about 350 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the engineered cell can express at most about 185 %, about 195 %, about 200 %, about 230 %, about 250 %, about 300 %, about 350 %, or about 400 % more APOM relative to the amount expressed in a primary hepatocyte or a wild-type hepatocyte.

[0066] Enzymes: The engineered cell of the present disclosure may be configured to comprise higher activity of one or more enzymes. In some embodiments, the one or more enzymes may be an organ or tissue-specific enzyme. In some embodiments, the one or more enzymes may comprise enzymes that function within a liver (hepatic), an intestine, pancreas, heart, lungs, stomach, or any other bodily organs. In some embodiments, the one or more enzymes can function within the systemic regions of a body. In some embodiments, the one or more enzymes may perform various functions in the body. Non-limiting examples of enzymes and / or functions of enzymes include those involved in signal transduction (kinases, phosphatases), those involved in generatingmovement with myosin (muscle protein) hydrolyzing ATP to generate a muscle contraction, those involved in transporting cargo around the cell (part of cytoskeleton), enzymes involved in digestion, metabolism, respiration, hormone production, nutrient absorption and transport, detoxification, cellular repair and division, digestive enzymes e.g., amylases and proteases that break down large molecules of starches or proteins into smaller ones for proper absorption in the intestine. In some embodiments, the enzymatic functions may comprise metabolic, oxidoreductases, transferases, hydrolases, lyases, ligases, isomerases. In some embodiments, the one or more enzymes may be engineered for controlled activity. In some embodiments, the activity of the one or more enzymes can be controlled using for example, regulatory molecules (turn activity up or down by e.g., a transcription regulator, small molecule or an activator, inhibitor molecules that may bind the enzyme), cofactors, compartmentalization of enzymes or using feedback inhibition. In some embodiments, the one or more enzymes can be a lipase, an amylase, a maltase, a trypsin (any type of trypsin), a lactase, an acetylcholinesterase, or a helicase, a DNA polymerase. In other cases, the one or more enzymes may be a function-related enzyme e.g., a detoxification enzyme, or a metabolic enzyme. In some embodiments, the one or more enzymes can function in the liver, liver cell or any other site in the body. In some embodiments, the enzyme can comprise cytochrome p450 (CYP3 A4). Cytochrome P450 (CYP450), a large superfamily of heme-thiolate proteins, are involved in the metabolism of both exogenous and endogenous compounds. The highest concentration of these enzymes is found in the liver and small intestine. Many forms of CYP450 enzymes exist in nature and are responsible for phase I metabolism of xenobiotics. In particular, CYP3A4 can be involved in liver or hepatic metabolism. In some embodiments, the present disclosure provides an engineered cell comprising a higher CYP3A4 activity than a CYP3A4 activity in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. The engineered cell of the present disclosure may be configured to comprise a higher CYP3 A4 activity than a CYP3 A4 activity in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the absence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of 0.5 pmol / hr / million cells, 1 pmol / hr / million cells, 2 pmol / hr / million cells, 3 pmol / hr / million cells, 5 pmol / hr / million cells, 6 pmol / hr / million cells, 7 pmol / hr / million cells, 8 pmol / hr / million cells, 9 pmol / hr / million cells, 10 pmol / hr / million cells, 11 pmol / hr / million cells, 12 pmol / hr / million cells, 13 pmol / hr / million cells, or at least 15pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of about 0.5 pmol / hr / million cells to about 11 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprisesCYP3A4 activity of about 0.5 pmol / hr / million cells to about 1 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 2 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 3 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 4 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 5 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 6 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 7 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 8 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 9 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 10 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 11 pmol / hr / million cells, about 1 pmol / hr / million cells to about 2 pmol / hr / million cells, about 1 pmol / hr / million cells to about 3 pmol / hr / million cells, about 1 pmol / hr / million cells to about 4 pmol / hr / million cells, about 1 pmol / hr / million cells to about 5 pmol / hr / million cells, about 1 pmol / hr / million cells to about 6 pmol / hr / million cells, about 1 pmol / hr / million cells to about 7 pmol / hr / million cells, about 1 pmol / hr / million cells to about 8 pmol / hr / million cells, about 1 pmol / hr / million cells to about 9 pmol / hr / million cells, about 1 pmol / hr / million cells to about 10 pmol / hr / million cells, about 1 pmol / hr / million cells to about 11 pmol / hr / million cells, about 2 pmol / hr / million cells to about 3 pmol / hr / million cells, about 2 pmol / hr / million cells to about 4 pmol / hr / million cells, about 2 pmol / hr / million cells to about 5 pmol / hr / million cells, about 2 pmol / hr / million cells to about 6 pmol / hr / million cells, about 2 pmol / hr / million cells to about 7 pmol / hr / million cells, about 2 pmol / hr / million cells to about 8 pmol / hr / million cells, about 2 pmol / hr / million cells to about 9 pmol / hr / million cells, about 2 pmol / hr / million cells to about 10 pmol / hr / million cells, about 2 pmol / hr / million cells to about 11 pmol / hr / million cells, about 3 pmol / hr / million cells to about 4 pmol / hr / million cells, about 3 pmol / hr / million cells to about 5 pmol / hr / million cells, about 3 pmol / hr / million cells to about 6 pmol / hr / million cells, about 3 pmol / hr / million cells to about 7 pmol / hr / million cells, about 3 pmol / hr / million cells to about 8 pmol / hr / million cells, about 3 pmol / hr / million cells to about 9 pmol / hr / million cells, about 3 pmol / hr / million cells to about 10 pmol / hr / million cells, about 3 pmol / hr / million cells to about 11 pmol / hr / million cells, about 4 pmol / hr / million cells to about 5 pmol / hr / million cells, about 4 pmol / hr / million cells to about 6 pmol / hr / million cells, about 4 pmol / hr / million cells to about 7 pmol / hr / million cells, about 4 pmol / hr / million cells to about 8 pmol / hr / million cells, about 4 pmol / hr / million cells to about 9 pmol / hr / million cells, about 4 pmol / hr / million cells to about 10 pmol / hr / million cells, about 4 pmol / hr / million cells to about 11 pmol / hr / million cells, about 5 pmol / hr / million cells to about 6 pmol / hr / million cells, about 5 pmol / hr / million cells to about 7 pmol / hr / million cells, about 5 pmol / hr / million cells to about 8 pmol / hr / million cells, about 5 pmol / hr / million cells to about 9 pmol / hr / million cells, about 5 pmol / hr / million cells to about 10 pmol / hr / million cells, about 5 pmol / hr / million cells to about 11pmol / hr / million cells, about 6 pmol / hr / million cells to about 7 pmol / hr / million cells, about 6 pmol / hr / million cells to about 8 pmol / hr / million cells, about 6 pmol / hr / million cells to about 9 pmol / hr / million cells, about 6 pmol / hr / million cells to about 10 pmol / hr / million cells, about 6 pmol / hr / million cells to about 11 pmol / hr / million cells, about 7 pmol / hr / million cells to about 8 pmol / hr / million cells, about 7 pmol / hr / million cells to about 9 pmol / hr / million cells, about 7 pmol / hr / million cells to about 10 pmol / hr / million cells, about 7 pmol / hr / million cells to about 11 pmol / hr / million cells, about 8 pmol / hr / million cells to about 9 pmol / hr / million cells, about 8 pmol / hr / million cells to about 10 pmol / hr / million cells, about 8 pmol / hr / million cells to about 11 pmol / hr / million cells, about 9 pmol / hr / million cells to about 10 pmol / hr / million cells, about 9 pmol / hr / million cells to about 11 pmol / hr / million cells, or about 10 pmol / hr / million cells to about 11 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of about 0.5 pmol / hr / million cells, about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, or about 11 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3 A4 activity of at least about 0.5 pmol / hr / million cells, about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about9 pmol / hr / million cells, or about 10 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3 A4 activity of at most about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about10 pmol / hr / million cells, or about 11 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3 A4 activity of about 12 pmol / hr / million cells to about 20 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of about 12 pmol / hr / million cells to about 13 pmol / hr / million cells, about 12 pmol / hr / million cells to about 14 pmol / hr / million cells, about 12 pmol / hr / million cells to about 15 pmol / hr / million cells, about 12 pmol / hr / million cells to about 16 pmol / hr / million cells, about 12 pmol / hr / million cells to about 17 pmol / hr / million cells, about 12 pmol / hr / million cells to about 18 pmol / hr / million cells, about 12 pmol / hr / million cells to about 19 pmol / hr / million cells, about 12pmol / hr / million cells to about 20 pmol / hr / million cells, about 13 pmol / hr / million cells to about 14 pmol / hr / million cells, about 13 pmol / hr / million cells to about 15 pmol / hr / million cells, about 13 pmol / hr / million cells to about 16 pmol / hr / million cells, about 13 pmol / hr / million cells to about 17 pmol / hr / million cells, about 13 pmol / hr / million cells to about 18 pmol / hr / million cells, about 13 pmol / hr / million cells to about 19 pmol / hr / million cells, about 13 pmol / hr / million cells to about 20 pmol / hr / million cells, about 14 pmol / hr / million cells to about 15 pmol / hr / million cells, about 14 pmol / hr / million cells to about 16 pmol / hr / million cells, about 14 pmol / hr / million cells to about 17 pmol / hr / million cells, about 14 pmol / hr / million cells to about 18 pmol / hr / million cells, about 14 pmol / hr / million cells to about 19 pmol / hr / million cells, about 14 pmol / hr / million cells to about 20 pmol / hr / million cells, about 15 pmol / hr / million cells to about 16 pmol / hr / million cells, about 15 pmol / hr / million cells to about 17 pmol / hr / million cells, about 15 pmol / hr / million cells to about 18 pmol / hr / million cells, about 15 pmol / hr / million cells to about 19 pmol / hr / million cells, about 15 pmol / hr / million cells to about 20 pmol / hr / million cells, about 16 pmol / hr / million cells to about 17 pmol / hr / million cells, about 16 pmol / hr / million cells to about 18 pmol / hr / million cells, about 16 pmol / hr / million cells to about 19 pmol / hr / million cells, about 16 pmol / hr / million cells to about 20 pmol / hr / million cells, about 17 pmol / hr / million cells to about 18 pmol / hr / million cells, about 17 pmol / hr / million cells to about 19 pmol / hr / million cells, about 17 pmol / hr / million cells to about 20 pmol / hr / million cells, about 18 pmol / hr / million cells to about 19 pmol / hr / million cells, about 18 pmol / hr / million cells to about 20 pmol / hr / million cells, or about 19 pmol / hr / million cells to about 20 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of about 12 pmol / hr / million cells, about 13 pmol / hr / million cells, about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16 pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, about 19 pmol / hr / million cells, or about 20 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of at least about 12 pmol / hr / million cells, about 13 pmol / hr / million cells, about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16 pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, or about 19 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the engineered cell comprises CYP3A4 activity of at most about 13 pmol / hr / million cells, about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16 pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, about 19 pmol / hr / million cells, or about 20 pmol / hr / million cells when the engineered cell is cultured in the presence of rifampicin.

[0067] In some embodiments, the engineered cell can comprise an activity of CYP3A4 that is greater than an activity in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, induction of the activity of CYP3A4 in engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) can comprise about 20 times induction upon rifampicin addition. Induction by rifampicin of the engineered cells so that they produce such an activity of CYP3 A4 can indicate a physiological response to drugs. In some embodiments, the induced activity of CYP3A4 can be at least 1.5 times, 2.0 times or 2.4 time greater than activity in primary hepatocytes at uninduced levels. In some embodiments, the CYP3A4 activity in the engineered cell is at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 100%, 150%, 160%, 200%, 240%, 280%, or at least 300% greater than in a primary hepatocyte or a wildtype hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is about 0.5 % to about 70 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3 A4 activity in the engineered cell is about 0.5 % to about 1 %, about 0.5 % to about 2 %, about 0.5 % to about 3 %, about 0.5 % to about 5 %, about 0.5 % to about 10 %, about 0.5 % to about 20 %, about 0.5 % to about 30 %, about 0.5 % to about 40 %, about 0.5 % to about 50 %, about 0.5 % to about 60 %, about 0.5 % to about 70 %, about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 5 %, about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 2 % to about 3 %, about 2 % to about 5 %, about 2 % to about 10 %, about 2 % to about 20 %, about 2 % to about 30 %, about 2 % to about 40 %, about 2 % to about 50 %, about 2 % to about 60 %, about 2 % to about 70 %, about 3 % to about 5 %, about 3 % to about 10 %, about 3 % to about 20 %, about 3 % to about 30 %, about 3 % to about 40 %, about 3 % to about 50 %, about 3 % to about 60 %, about 3 % to about 70 %, about 5 % to about 10 %, about 5 % to about 20 %, about 5 % to about 30 %, about 5 % to about 40 %, about 5 % to about 50 %, about 5 % to about 60 %, about 5 % to about 70 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 50 % to about 60 %, about 50 % to about 70 %, or about 60 % to about 70 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in theengineered cell is about 0.5 %, about 1 %, about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, or about 70 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is at least about 0.5 %, about 1 %, about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, or about 60 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is at most about 1 %, about 2 %, about 3 %, about 5 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, or about 70 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3 A4 activity in the engineered cell is about 80 % to about 300 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is about 80 % to about 90 %, about 80 % to about 100 %, about 80 % to about 120 %, about 80 % to about 140 %, about 80 % to about 160 %, about 80 % to about 180 %, about 80 % to about 200 %, about 80 % to about 220 %, about 80 % to about 240 %, about 80 % to about 280 %, about 80 % to about 300 %, about 90 % to about 100 %, about 90 % to about 120 %, about 90 % to about 140 %, about 90 % to about 160 %, about 90 % to about 180 %, about 90 % to about 200 %, about 90 % to about 220 %, about 90 % to about 240 %, about 90 % to about 280 %, about 90 % to about 300 %, about 100 % to about 120 %, about 100 % to about 140 %, about 100 % to about 160 %, about 100 % to about 180 %, about 100 % to about 200 %, about 100 % to about 220 %, about 100 % to about 240 %, about 100 % to about 280 %, about 100 % to about 300 %, about 120 % to about 140 %, about 120 % to about 160 %, about 120 % to about 180 %, about 120 % to about 200 %, about 120 % to about 220 %, about 120 % to about 240 %, about 120 % to about 280 %, about 120 % to about 300 %, about 140 % to about 160 %, about 140 % to about 180 %, about 140 % to about 200 %, about 140 % to about 220 %, about 140 % to about 240 %, about 140 % to about 280 %, about 140 % to about 300 %, about 160 % to about 180 %, about 160 % to about 200 %, about 160 % to about 220 %, about 160 % to about 240 %, about 160 % to about 280 %, about 160 % to about 300 %, about 180 % to about 200 %, about 180 % to about 220 %, about 180 % to about 240 %, about 180 % to about 280 %, about 180 % to about 300 %, about 200 % to about 220 %, about 200 % to about 240 %, about 200 % to about 280 %, about 200 % to about 300 %, about 220 % to about 240 %, about 220 % to about 280 %, about 220 % to about 300 %, about 240 % to about 280 %, about 240 % to about 300 %, or about 280 % to about 300 % greater than in a primary hepatocyte or a wild-type hepatocytewhen the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is about 80 %, about 90 %, about 100 %, about 120 %, about 140 %, about 160 %, about 180 %, about 200 %, about 220 %, about 240 %, about 280 %, or about 300 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3A4 activity in the engineered cell is at least about 80 %, about 90 %, about 100 %, about 120 %, about 140 %, about 160 %, about 180 %, about 200 %, about 220 %, about 240 %, or about 280 % greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiments, the CYP3 A4 activity in the engineered cell is at most about 90 %, about 100 %, about 120 %, about 140 %, about 160 %, about 180 %, about 200 %, about 220 %, about 240 %, about 280 %, or about 300 % greater than in a primary hepatocyte or a wildtype hepatocyte when the engineered cell is cultured in the presence of rifampicin. In some embodiment, the engineered cell expressing CYP3 A4 activity at higher levels than the expression in a primary hepatocyte or a wild-type hepatocyte (when the engineered cell is cultured in the presence of rifampicin) may comprise an induced stem cell, an induced pluripotent stem cell or induced progenitor cell that differentiates into a cell lineage able to express CYP3 A4 activity. In some embodiments, the engineered cell can comprise a hepatocyte or a hepatocyte-like cell. In some embodiments, the hepatocyte or hepatocyte-like cell may not be derived from a patient. In some embodiments, the hepatocyte or hepatocyte-like cell may not be derived from a human induced pluripotent stem cell. In some embodiments, the cell may be a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, or an embryonic stem cell. In some embodiments, the human stem cell, the human induced pluripotent stem cell, the human progenitor cell, the human fetal stem cell, the hepatic stem cell, or the embryonic stem cell may be derived from a patient or may not be derived from a patient. In some embodiments, a primary hepatocyte or the wild-type hepatocyte can be a human hepatocyte cell, wherein the human stem cell or iPSC may or may not be derived from a patient. In some embodiments, the patient is a subject. The subject may be a healthy subject, e.g., a study control subject or study participant. In some embodiments, the subject may be a patient. In some embodiments, the subject or patient can be a donor or donate cells, a cell, organ, tissue, blood, etcetera. In other embodiments, the patient may refer to a subject who may be suffering from a hepatic or liver disease.

[0068] In some embodiments, the engineered cell disclosed herein may have improved efficiency of differentiation. The improved efficiency of differentiation may be detectable by higher amounts of secreted hepatocyte markers such as albumin, Al AT among others. In non-limiting examples, the engineered cell can secrete alpha- 1 -trypsin (A1AT) levels such that between at least 50-99%of the differentiated engineered cells express A1AT. In some embodiments, the level of A1AT expressed may be similar to that detected in primary hepatocytes. In some embodiments, the percent of cells expressing Al AT can be about 50 % to about 99 % of the differentiated engineered cells express A1AT. In some embodiments, the percent of cells expressing A1AT can be about 50 % to about 55 %, about 50 % to about 60 %, about 50 % to about 65 %, about 50 % to about 70 %, about 50 % to about 75 %, about 50 % to about 76 %, about 50 % to about 77 %, about 50 % to about 80 %, about 50 % to about 85 %, about 50 % to about 90 %, about 50 % to about 99 %, about 55 % to about 60 %, about 55 % to about 65 %, about 55 % to about 70 %, about 55 % to about 75 %, about 55 % to about 76 %, about 55 % to about 77 %, about 55 % to about 80 %, about 55 % to about 85 %, about 55 % to about 90 %, about 55 % to about 99 %, about 60 % to about 65 %, about 60 % to about 70 %, about 60 % to about 75 %, about 60 % to about 76 %, about 60 % to about 77 %, about 60 % to about 80 %, about 60 % to about 85 %, about 60 % to about 90 %, about 60 % to about 99 %, about 65 % to about 70 %, about 65 % to about 75 %, about 65 % to about 76 %, about 65 % to about 77 %, about 65 % to about 80 %, about 65 % to about 85 %, about 65 % to about 90 %, about 65 % to about 99 %, about 70 % to about 75 %, about 70 % to about 76 %, about 70 % to about 77 %, about 70 % to about 80 %, about 70 % to about 85 %, about 70 % to about 90 %, about 70 % to about 99 %, about 75 % to about 76 %, about 75 % to about 77 %, about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 99 %, about 76 % to about 77 %, about 76 % to about 80 %, about 76 % to about 85 %, about 76 % to about 90 %, about 76 % to about 99 %, about 77 % to about 80 %, about 77 % to about 85 %, about 77 % to about 90 %, about 77 % to about 99 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 99 %, about 85 % to about 90 %, about 85 % to about 99 %, or about 90 % to about 99 % of the differentiated engineered cells express Al AT. In some embodiments, the percent of cells expressing Al AT can be about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 76 %, about 77 %, about 80 %, about 85 %, about 90 %, or about 99 % of the differentiated engineered cells express Al AT. In some embodiments, the percent of cells expressing Al AT can be at least about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 76 %, about 77 %, about 80 %, about 85 %, or about 90 % of the differentiated engineered cells express A1AT. In some embodiments, the percent of cells expressing Al AT can be at most about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 76 %, about 77 %, about 80 %, about 85 %, about 90 %, or about 99 % of the differentiated engineered cells express Al AT.

[0069] The engineered cell may also express a greater amount of one or more hepatocyte gene product relative to the expression detected in an uninduced control cell. In some embodiments,the engineered cell disclosed herein may have improved efficiency of differentiation. The improved efficiency of differentiation may be detectable by higher amounts of secreted hepatocyte markers such as albumin among others. In non-limiting examples, the engineered cell can secrete albumin such that between at least about 50-99% of the differentiated engineered cells express albumin. In some embodiments, the percent of cells expressing albumin can be about 50 % to about 99 % of the differentiated engineered cells express albumin. In some embodiments, the percent of cells expressing albumin can be about 50 % to about 55 %, about 50 % to about 60 %, about 50 % to about 65 %, about 50 % to about 70 %, about 50 % to about 75 %, about 50 % to about 76 %, about 50 % to about 77 %, about 50 % to about 80 %, about 50 % to about 85 %, about 50 % to about 90 %, about 50 % to about 99 %, about 55 % to about 60 %, about 55 % to about 65 %, about 55 % to about 70 %, about 55 % to about 75 %, about 55 % to about 76 %, about 55 % to about 77 %, about 55 % to about 80 %, about 55 % to about 85 %, about 55 % to about 90 %, about 55 % to about 99 %, about 60 % to about 65 %, about 60 % to about 70 %, about 60 % to about 75 %, about 60 % to about 76 %, about 60 % to about 77 %, about 60 % to about 80 %, about 60 % to about 85 %, about 60 % to about 90 %, about 60 % to about 99 %, about 65 % to about 70 %, about 65 % to about 75 %, about 65 % to about 76 %, about 65 % to about 77 %, about 65 % to about 80 %, about 65 % to about 85 %, about 65 % to about 90 %, about 65 % to about 99 %, about 70 % to about 75 %, about 70 % to about 76 %, about 70 % to about 77 %, about 70 % to about 80 %, about 70 % to about 85 %, about 70 % to about 90 %, about 70 % to about 99 %, about 75 % to about 76 %, about 75 % to about 77 %, about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 99 %, about 76 % to about 77 %, about 76 % to about 80 %, about 76 % to about 85 %, about 76 % to about 90 %, about 76 % to about 99 %, about 77 % to about 80 %, about 77 % to about 85 %, about 77 % to about 90 %, about 77 % to about 99 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 99 %, about 85 % to about 90 %, about 85 % to about 99 %, or about 90 % to about 99 % of the differentiated engineered cells express albumin. In some embodiments, the percent of cells expressing albumin can be about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 76 %, about 77 %, about 80 %, about 85 %, about 90 %, or about 99 % of the differentiated engineered cells express albumin. In some embodiments, the percent of cells expressing albumin can be at least about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 76 %, about 77 %, about 80 %, about 85 %, or about 90 % of the differentiated engineered cells express albumin. In some embodiments, the percent of cells expressing albumin can be at most about 55 %, about 60 %, about 65 %, about 70 %, about 75 %,about 76 %, about 77 %, about 80 %, about 85 %, about 90 %, or about 99 % of the differentiated engineered cells express albumin.

[0070] The engineered cell may also express a higher activity of one or more hepatocyte marker or gene product relative to the expression detected in an uninduced control cell. In some embodiments, the engineered cell disclosed herein may have improved efficiency of differentiation. The improved efficiency of differentiation may be detectable by higher activity of hepatocyte marker or enzyme such as CYP3A4, among others. In non-limiting examples, the engineered cell can express high levels of activity of CYP3A4 of between at least about 50 pmol / hr / million cells to 99 pmol / hr / million cells.

[0071] In some embodiments, the engineered cell can express significant levels of activity of CYP3A4 ranging from about 50 pmol / hr / million cells to about 99 pmol / hr / million cells. In some embodiments, the engineered cell can express significant levels of activity of CYP3A4 ranging from about 50 pmol / hr / million cells to about 55 pmol / hr / million cells, about 50 pmol / hr / million cells to about 60 pmol / hr / million cells, about 50 pmol / hr / million cells to about 65 pmol / hr / million cells, about 50 pmol / hr / million cells to about 70 pmol / hr / million cells, about 50 pmol / hr / million cells to about 75 pmol / hr / million cells, about 50 pmol / hr / million cells to about 76 pmol / hr / million cells, about 50 pmol / hr / million cells to about 77 pmol / hr / million cells, about 50 pmol / hr / million cells to about 80 pmol / hr / million cells, about 50 pmol / hr / million cells to about 85 pmol / hr / million cells, about 50 pmol / hr / million cells to about 90 pmol / hr / million cells, about 50 pmol / hr / million cells to about 99 pmol / hr / million cells, about 55 pmol / hr / million cells to about 60 pmol / hr / million cells, about 55 pmol / hr / million cells to about 65 pmol / hr / million cells, about 55 pmol / hr / million cells to about 70 pmol / hr / million cells, about 55 pmol / hr / million cells to about 75 pmol / hr / million cells, about 55 pmol / hr / million cells to about 76 pmol / hr / million cells, about 55 pmol / hr / million cells to about 77 pmol / hr / million cells, about 55 pmol / hr / million cells to about 80 pmol / hr / million cells, about 55 pmol / hr / million cells to about 85 pmol / hr / million cells, about 55 pmol / hr / million cells to about 90 pmol / hr / million cells, about 55 pmol / hr / million cells to about 99 pmol / hr / million cells, about 60 pmol / hr / million cells to about 65 pmol / hr / million cells, about 60 pmol / hr / million cells to about 70 pmol / hr / million cells, about 60 pmol / hr / million cells to about 75 pmol / hr / million cells, about 60 pmol / hr / million cells to about 76 pmol / hr / million cells, about 60 pmol / hr / million cells to about 77 pmol / hr / million cells, about 60 pmol / hr / million cells to about 80 pmol / hr / million cells, about 60 pmol / hr / million cells to about 85 pmol / hr / million cells, about 60 pmol / hr / million cells to about 90 pmol / hr / million cells, about 60 pmol / hr / million cells to about 99 pmol / hr / million cells, about 65 pmol / hr / million cells to about 70 pmol / hr / million cells, about 65 pmol / hr / millioncells to about 75 pmol / hr / million cells, about 65 pmol / hr / million cells to about 76 pmol / hr / million cells, about 65 pmol / hr / million cells to about 77 pmol / hr / million cells, about 65 pmol / hr / million cells to about 80 pmol / hr / million cells, about 65 pmol / hr / million cells to about 85 pmol / hr / million cells, about 65 pmol / hr / million cells to about 90 pmol / hr / million cells, about 65 pmol / hr / million cells to about 99 pmol / hr / million cells, about 70 pmol / hr / million cells to about 75 pmol / hr / million cells, about 70 pmol / hr / million cells to about 76 pmol / hr / million cells, about 70 pmol / hr / million cells to about 77 pmol / hr / million cells, about 70 pmol / hr / million cells to about 80 pmol / hr / million cells, about 70 pmol / hr / million cells to about 85 pmol / hr / million cells, about 70 pmol / hr / million cells to about 90 pmol / hr / million cells, about 70 pmol / hr / million cells to about 99 pmol / hr / million cells, about 75 pmol / hr / million cells to about 76 pmol / hr / million cells, about 75 pmol / hr / million cells to about 77 pmol / hr / million cells, about 75 pmol / hr / million cells to about 80 pmol / hr / million cells, about 75 pmol / hr / million cells to about 85 pmol / hr / million cells, about 75 pmol / hr / million cells to about 90 pmol / hr / million cells, about 75 pmol / hr / million cells to about 99 pmol / hr / million cells, about 76 pmol / hr / million cells to about 77 pmol / hr / million cells, about 76 pmol / hr / million cells to about 80 pmol / hr / million cells, about 76 pmol / hr / million cells to about 85 pmol / hr / million cells, about 76 pmol / hr / million cells to about 90 pmol / hr / million cells, about 76 pmol / hr / million cells to about 99 pmol / hr / million cells, about 77 pmol / hr / million cells to about 80 pmol / hr / million cells, about 77 pmol / hr / million cells to about 85 pmol / hr / million cells, about 77 pmol / hr / million cells to about 90 pmol / hr / million cells, about 77 pmol / hr / million cells to about 99 pmol / hr / million cells, about 80 pmol / hr / million cells to about 85 pmol / hr / million cells, about 80 pmol / hr / million cells to about 90 pmol / hr / million cells, about 80 pmol / hr / million cells to about 99 pmol / hr / million cells, about 85 pmol / hr / million cells to about 90 pmol / hr / million cells, about 85 pmol / hr / million cells to about 99 pmol / hr / million cells, or about 90 pmol / hr / million cells to about 99 pmol / hr / million cells. In some embodiments, the engineered cell can express significant levels of activity of CYP3A4 ranging from about 50 pmol / hr / million cells, about 55 pmol / hr / million cells, about 60 pmol / hr / million cells, about 65 pmol / hr / million cells, about 70 pmol / hr / million cells, about 75 pmol / hr / million cells, about 76 pmol / hr / million cells, about 77 pmol / hr / million cells, about 80 pmol / hr / million cells, about 85 pmol / hr / million cells, about 90 pmol / hr / million cells, or about 99 pmol / hr / million cells. In some embodiments, the engineered cell can express significant levels of activity of CYP3A4 ranging from at least about 50 pmol / hr / million cells, about 55 pmol / hr / million cells, about 60 pmol / hr / million cells, about 65 pmol / hr / million cells, about 70 pmol / hr / million cells, about 75 pmol / hr / million cells, about 76 pmol / hr / million cells, about 77 pmol / hr / million cells, about 80 pmol / hr / million cells, about 85 pmol / hr / million cells, or about 90 pmol / hr / million cells. In some embodiments, the engineered cell can express significant levels ofactivity of CYP3 A4 ranging from at most about 55 pmol / hr / million cells, about 60 pmol / hr / million cells, about 65 pmol / hr / million cells, about 70 pmol / hr / million cells, about 75 pmol / hr / million cells, about 76 pmol / hr / million cells, about 77 pmol / hr / million cells, about 80 pmol / hr / million cells, about 85 pmol / hr / million cells, about 90 pmol / hr / million cells, or about 99 pmol / hr / million cells.

[0072] In some embodiments, the engineered cell provided can further be engineered to have reduced or no expression of one or more pluripotency-reinforcing cues. Pluripotency is controlled by master transcription factors (TFs). Non-limiting examples of the master TF includes OCT4, SOX2 and NANOG. In some cases, the master TFs may partition into condensates in the nucleus of embryonic stem cells. In some cases, the partitioned condensates may be relevant in the regulation of gene expression and the maintenance of pluripotency. In some embodiments, one or more differentiation cues may initiate a redistribution of TFs during replication. In other cases, the one or more differentiation cues may initiate gradual changes in the TF chromatin interactions. Disclosed herein, in some embodiments, the engineered cell or population of engineered cell may further be modified to reduce or hinder expression of one or more pluripotency -reinforcing cues. The one or more pluripotency -reinforcing cues can comprise signals, genes, gene products, agent, or small molecule. In some embodiments, the one or more pluripotency-reinforcing cues can comprise NANOG, POU5F1 / OCT4, TGF-0S, ALKs, or any combination thereof. In some instances, the one or more pluripotency-reinforcing cues comprise one or more TGF-fJs. Nonlimiting examples of the one or more TGF- S can comprise TGFB1, TGFB2, TGFB3. In some instances, the one or more pluripotency-reinforcing cues can comprise one or more ALKs. Nonlimiting examples of the one or more ALKs can include one or more ALKs comprising ALK1, ALK3, ALK4, ALK5, ALI .

[0073] In some embodiments, the engineered cell can further be engineered to comprise one or more histone acetyltransferases. Histone acetyltransferases (HATs) are epigenetic enzymes which can acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form 8-N-acetyllysine. In some cases HATs can install acetyl groups onto lysine resideues of cellular proteins e.g., histones, transcription factors, nuclear receptors, or enzymes. For example, DNA may be wrapped around histones such that by transferring an acetyl group to the histones, genes can be turned on or turned off. The present engineered cell can further be genetically modified to comprise any histone acetyltransferases. Non-limiting examples of histone acetyltransferases includes, for example, histone deacetylase 1 (HDAC1), histone deacetylase 2 (HDAC2), histone deacetylase 3 (HDAC3), histone deacetylase 4 (HDAC4), histonedeacetylase 5 (HDAC5), histone deacetylase 6 (HDAC6), histone deacetylase 7 (HDAC7), histone deacetylase 8 (HDAC8), histone deacetylase 9 (HDAC9), histone deacetylase 10 (HD AC 10), histone deacetylase 11 (HDAC11), sirtuin 1 (SIRT1), sirtuin 2 (SIRT2), sirtuin 3 (SIRT3), sirtuin 4 (SIRT4), sirtuin 5 (SIRT5), sirtuin 6 (SIRT6), or sirtuin 7 (SIRT7). In some instance, sirtuin (silent mating type information regulation 2 homolog) e.g., SIRT1, may be a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase. In some cases, sirtuin e.g., sirtl, an enzyme in cell nucleus, may deacetylate certain TFs. In some embodiments, the engineered cell provided herein may comprise at least one histone deacetylase enzyme. In some embodiments, the engineered cell provided herein may comprise at least one SIRT enzyme. In some embodiments, the engineered cell provided herein may comprise at least one any enzyme selected from the groups disclosed herein or known to a person of skill in the art. In some embodiments, the engineered cell can further be engineered to comprise over express one or more histone acetyltransferases. The expression of the one or more histone acetyltransferases, histone deacetylases or other enzymes may comprise expression of at least one polynucleotide comprising cDNA sequence. In some embodiments, the one or more histone acetyltransferases, histone deacetylases or other enzymes can comprise an overexpression of these enzymes or an active inhibition of expression of any of the enzymes. The overexpression of the one or more histone acetyltransferases may comprise overexpression of at least one polynucleotide comprising cDNA sequence. The overexpression of the one or more histone acetyltransferases may comprise overexpression of at least one polynucleotide comprising one or more open reading frames (ORFs). The overexpression of the one or more histone acetyltransferases may comprise overexpression of at least one polynucleotide comprising at least one CRISPR activator sequence. In some cases, the overexpression construct can directly be introduced into a stem cell e.g., an embryonic stem cell or an induced pluripotent stem cell using any gene or sequence delivery system available to one of skill in the art. Introduction of genetic traits such as the one or more genes, ORFs, fragments or portions thereof, can be comprise gene manipulation techniques, genome editing tools in order to introduce or enforce an expression of exogenous DNA, gene, ORFs or fragments or portions thereof. In some cases, the overexpression may be conducted using at least one expression cassette. The expression cassette can be delivered by any vector or any method that can deliver an expression cassette to a cell e.g., delivery methods are known to a skilled person in the art. Gene expression can be modulated by reversibly targeting the endogenous promoter. In some embodiments, genetic information in a stem cell or iPSC can be controlled, e.g., deleted or inverted. In other embodiments, the genetic information in a stem cell or iPSC can be modified minimally as for example when applying single-nucleotide changes which can be introduced to an engineered cellto mimic disease-associated mutations. In some embodiments, the genetic modification can be implemented to reset a mutation for which the generated engineered cell with the corrected mutation may be created.

[0074] Disclosed herein in some embodiments is an engineered cell comprising a cell line. In some embodiments, a cell line can be a high performing cell line or a top performer cell line, e.g., the TF recipes are expressed at high levels in one cell line over another. An engineered cell line can be composed of a population of cells. In some embodiments, a population of cells can be used to generate or isolate a single cell. In some embodiments, a single cell can be an isogenic cell. In some embodiments, a single cell can be isolated from any one of the cell lines disclosed herein e.g., any cell line disclosed in Table 1 in the example section or in the figure descriptions. In some embodiments, the cell line can be a top performer or a non-top performer. In some embodiments, a top performer can be a cell line that expresses one or more transcription factors at a certain level e.g., at a high level in comparison to another cell line, or to a naive cell line, an uninduced cell line or a wild-type cell line. In some embodiments, a cell line can be evaluated to assess the specific transcription factors that contribute to cellular functionality. Various cellular functionality that can be of interest are disclosed herein and in the example section. Non-limiting exemplary transcription factors, engineered cell functionality, or transcription factor recipes are shown in Table 1-4. In some embodiments, a cell line can be isolated from a single clone. In some embodiments, the single clone can have a particular site where the transcription of one or more transcription factors (TFs) are integrated. In some embodiments, the expression level of one or more transcription factors can depend on an integration site. In some embodiments, the site can be a locus e.g., a gene locus. In some embodiments a gene locus can be an active regulatory region e.g., around, or nearby to a transcription site, a translation site, or other regulatory region. In some embodiments, an expression level of one or more transcription factors can depend on an integration site in the genome. In some embodiments, an expression level of one or more transcription factors in a clone can depend on a particular vector that is expressing the one or more TFs.

[0075] The present disclosure can involve administering an engineered cell, a composition, a pharmaceutical or any formulation disclosed herein. In some embodiments, delivery of the therapeutic can be accomplished using any method known to the artisan or any method disclosed herein. In some embodiments, a subject receiving a therapeutic disclosed can be evaluated to assess efficacy of the therapeutic, safety, survival (e.g., percent probability of survival) weight, or any other indicators used in the art. In some embodiments, a metabolic profile of the cell or cellline can be conducted to assess cellular functionality of an engineered cell in relation to a wildtype cell, a naive cell, or an uninduced cell. In some embodiments, the cell can comprise an engineered cell of the present disclosure following induction. In some embodiments, the engineered cell may not be induced (e.g., an uninduced cell). The present disclosure is nonlimiting to the kind and type of metabolic profile that can be conducted on a subject, a cell, or a cell line. The present disclosure is non-limiting to the kind and type of cellular functionality that can be conducted on a subject, a cell, or a cell line. In some embodiments, the cellular functionality can be targeted to a hepatocyte, hepatocyte-like cell, uninduced cell (e.g., differentiated cell), an uninduced cell (undifferentiated cell), or to a wild-type (e.g., naive cell). Non-limiting examples of cellular functionality of hepatocyte or hepatocyte-like cells are disclosed herein and exemplified throughout the disclosure e.g., in the examples and elsewhere without limitations.Therapeutic cargo compositions:

[0076] Provided herein in some embodiments is an engineered cell genetically modified to further express one or more functional polypeptide in which the engineered cell is contacted with an expression cassette comprising a nucleic acid molecule may be able to encode the one or more functional polypeptide. In some embodiments, the one or more functional polypeptide can elicit an immune response. The immune response that can be elicited can comprise any physiological response in the subject or patient arising from or due to administration of the functional polypeptide. In some embodiments, the therapeutic composition can comprise a plurality of compounds. In some embodiments, the functional polypeptide can be one or more therapeutic compositions or fragments or portions thereof. The composition described herein can be for use to prevent or treat at least one disease in a subject. In some embodiments, the one or more therapeutic composition can comprise one or more therapeutic molecules, payloads, cargos or one or more particles. Non-limiting examples of the one or more therapeutic molecules can include one or more drugs, small molecules, therapeutic compounds, therapeutic molecules, therapeutic proteins, peptides, peptide epitopes, therapeutic mRNA molecules, therapeutic nucleic acid molecules or any one or more macromolecules known in the art. some embodiments, the therapeutic compounds can comprise a composition comprising delivery vehicle e.g., a particle, a particle, such as a lipid-based particle The one or more therapeutic compositions disclosed can be in various forms e.g., without any limitations the forms can include: solid, liquid, form, tablet, caplets, gel, paste, capsule, vapors etcetera. In some embodiments, the particle can comprise any suitable form known to an artisan of skill in the field. For example, the particle can be as a liposome. In some embodiments, the particle can comprise nanoparticle or nanoparticles of anysuitable diameter. The therapeutic composition e.g., drug, compound, molecule, particle, cargo, nanoparticle, lipid-based particle etcetera can comprise any molar ratio or a particle of any suitable diameter. In some embodiments, the particle, payload, and the like, can comprise any suitable modulator or suitable particle encapsulation. In some embodiments, the engineered cell can be configured to perform one or more hepatocyte functions at a rate greater than a control cell. In some embodiments, the function of the engineered hepatocyte or hepatocyte-like cell of the present disclosure can be higher or improved relative to a control cell. Non-limiting examples of a control cell can include an uninduced cell, an iPSC or stem cell or a primary hepatocyte or a wild-type hepatocyte of any function disclosed herein. Non-limiting examples of mature hepatocyte functions can include, for example, be able to perform one or more of detoxification, glycogen storage and LDL, secretion of AAT or albumin, bile production, thrombopoietin production, metabolize drugs via the CYP3A4 pathway (or other drug metabolizing enzymes), hepatocyte angiotensinogen production, conversion of ammonia to urea (urea cycle), cholesterol synthesis, glycogenolysis, glycogenesis and lipogenesis, expression of GFP protein under hepatocyte or non-hepatocyte promoter (e.g., as shown in FIG. 16, or any other figure or tables disclosed herein). In some embodiments, new cell lines (Table 5 in the example sections, in the example section, or elsewhere as described) can be engineered with novel combinations of transcription factors (TFs) which can be observed by 7 days of differentiation. In some embodiments, the novel combinations of TFs can include any TF recipe disclosed herein for instance in the TF recipes or cocktails shown in the figures or in Table 1, 2 or elsewhere in any part of the present disclosure. In some embodiments, the engineered cells e.g., engineered hepatocytes or hepatocyte-like cells can be found to comprise improved cell functionality in a physiological or functional pathway and engineered cells can comprise different TF recipes (nonlimiting examples of cell functionality assessed can be seen in the present disclosure for example, in Table 1, Table 2, Table 4, ). In some cases, cell functionality of the engineered cell, e.g., engineered hepatocyte or hepatocyte-like cell can be improved relative to a control comparator. Different control cells or comparators can be utilized as disclosed or known in the art. In some embodiments, the engineered cell that differentiates to the hepatocyte or hepatocyte-like cells may display one or more of the following characteristics: occasional binucleity; glycogen deposits; apical microprotrusions; rough and smooth endoplasmic reticulum (ER) and a prominent Golgi body. In some embodiments, the engineered differentiated cell comprising the hepatocyte or hepatocyte-like may comprise expression of genes that may be involved in protein storage, transformation of carbohydrates, synthesis of cholesterol, bile salts, phospholipids, and are involved in detoxification, modification, and excretion of exogenous and endogenous substancesin the liver. In some embodiments, the engineered differentiated cell comprising the hepatocyte or hepatocyte-like may comprise expression of genes that may initiate the formation and secretion of bile or manufacture serum albumin. In some embodiments, the engineered differentiated cell comprising the hepatocyte or hepatocyte-like may comprise expression of genes that may be involved in fibrinogen and the prothrombin group of clotting factors. The engineered differentiated hepatocyte or hepatocyte-like cell may show tendencies as the main site for the synthesis of lipoproteins, ceruloplasmin, transferrin, complement and glycoproteins. In some embodiments, the engineered differentiated hepatocyte or hepatocyte-like cell may have the ability to metabolize, detoxify, and inactivate exogenous compounds e.g., drugs, insecticides, and endogenous compounds e.g., steroids.

[0077] In other embodiments, the engineered cell that may further express at least one expression cassette comprising one or more polynucleotides comprising one or more genes, gene fragments, or portions of genes. In some embodiments, the one or more genes may be one or more exogenous genes, or transgenes, or recombinant genes, or non-recombinant genes, or copies of wild-type copies of genes. The one or more genes may be homologous or heterologous. In some embodiments, the engineered cell can further comprise at least one expression cassette comprising one or more genes. In some embodiments, the one or more genes can reduce or inhibit expression of immunopathology-related disorders, disease, or conditions. In some embodiments, the immunopathology-related disorders, conditions, or diseases comprise instant blood-mediated inflammatory reaction, graft versus host disease, inflammatory bowel disease, asthma, rheumatoid arthritis, psoriasis, NK cell response-related disorders, an immune rejection disorder, or celiac condition, liver failure or liver steatosis. The genes may be exogenous, transgenes, or not. In some embodiments, expression of the one or more genes may promote regeneration of a liver tissue. In some embodiments, the expression of the one or more genes that promotes regeneration of the liver tissue can comprise hepatocyte growth factor (HGF) or epidermal growth factor (EGF). In some embodiments, the expression of the one or more genes that promote survival after engraftment. In some embodiments, the survival after engraftment comprises survival of B-cell lymphoma 2 (Bcl-2). In some embodiments, the one or more genes can encode at least one polypeptide. In some embodiments, the at least one polypeptide may comprise at least one therapeutic polypeptide. In some embodiments, the one or more the one or more polypeptide may comprise one or more peptides, oligomeric proteins, antibodies, antibody -based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins and thrombolytics, or any combination thereof. In some embodiments, the one or more polynucleotides of the engineeredcell may modulate expression of lipopolysaccharide binding protein (LBP). In some embodiments, the one or more polynucleotides of the engineered cell may induce overexpress of one or more metabolic pathway genes or gene products. In some embodiments, the one or more polynucleotides of the engineered cell may induce overexpression of one or more gene or gene products that facilitate cell engulfment. In some embodiments, the one or more gene or gene products that facilitate cell engulfment comprise soluble factors, growth factors, or cell adhesion molecules. In some embodiments, the one or more soluble factors comprise A1AT, CD14, lipopolysaccharide binding protein, factor II, V, VII, VIII, IX, X, or vWF. In some embodiments, the one or more soluble factors is Al AT. In some embodiments, the one or more polynucleotides or the one or more exogenous genes may alter, modify, or induce increased hepatocyte functionality or maturation. In some embodiments, the hepatocyte functionality or maturation comprises Oncostatin M Receptor (OSMR) function. In some embodiments, the hepatocyte functionality or maturation may comprise CYP3A4 activity. In some embodiments, the one or more polynucleotides of the engineered cell may reduce or inhibit expression of immunopathology-related disorders, diseases, or conditions. Non-limiting examples of immunopathology -related disorders, diseases, or conditions can comprise instant blood-mediated inflammatory reaction, graft versus host disease, inflammatory bowel disease, asthma, rheumatoid arthritis, psoriasis, NK cell response-related disorders, an immune rejection disorder, or celiac condition, liver failure or liver steatosis. In some embodiments, the one or more polynucleotides of the engineered cell may promote regeneration of a liver tissue. In some embodiments, the one or more polynucleotides of the engineered cell may promote regeneration of a liver tissue comprising hepatocyte growth factor (HGF) or epidermal growth factor (EGF). In some embodiments, the one or more polynucleotides of the engineered cell may promote survival after engraftment e.g., survival of B cell lymphoma-2 (Bcl-2). In some embodiments, the one or more genes may comprise one or more gene products. In some embodiments, the one or more genes or gene products can be involved in a number of cellular functions which may be hepatocyte associated or non-hepatocyte associated functions. Non-limiting examples of cellular functions may include metabolic pathway, inflammatory response, growth response, cancer, infection, digestion, vitamin storage, immunity, detoxification, homeostasis, bile production, drug metabolism, dysregulation of plasma proteins (albumin, binding globulins, protein C, protein S, clotting factors), bilirubin metabolism, production of cholesterol, protein metabolism (ammonia), breakdown / storage of glycogen, production of blood plasma, production of bile, regulation of blood clotting, processing of hemoglobin, metabolic-associated fatty liver disease. In some embodiments, the engineered cell further expressing at least one expression cassette encoding afunctional polypeptide may function with improved cell functionality that is higher, greater, superior to that of a primary hepatocyte or a wild-type hepatocyte.

[0078] In some embodiments, the engineered cell further express at least one expression cassette comprising a nucleic acid molecule that encodes one or more functional polypeptide. In some embodiments, the engineered cell comprising the one or more functional polypeptide may be configured to perform one or more mature hepatocyte functions. In some embodiments, the engineered cell comprising the one or more functional polypeptide may be configured to perform one or more mature hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte of any function disclosed herein.

[0079] In some embodiments, the engineered cell further comprising at least one exogenous expression cassette may comprise transcription factors, or molecules that increase transcription or increase activation of transcription factors. In some embodiments, one or more transcription factors may be used. In some embodiments, the engineered cell comprising the at least one or more exogenous expression cassette comprising one or more transcription factors (TFs), one or more transcription factor family members, or one or more transcription factor recipes or cocktails disclosed. In some embodiments, the engineered cell comprising the at least one or more exogenous expression cassette comprising one or more transcription factors (TFs), one or more transcription factor family members, or any combination thereof, can be induced so that the particular one or more TFs, and / or TF family members can be expressed. Induction of the one or more TFs and / or TF family members expression can trigger the engineered cell to differentiate into a different cellular lineage. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can comprise CEBPA, HNF1A, HNF4A, FOXA1, PROXI, ATF5, CYP3A4, RXRA NR1I3, or any combination thereof. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can consist of: CEBPA, HNF1 A, HNF4A, FOXA1, PROXI, ATF5, CYP3A4, RXRA and NR1I3. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can comprise: CEBPA, HNF1 A, HNF4A, FOXA1, NR1I3, HNF4A, FOXA2, ONECUT1 or any combination thereof. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can consist of: CEBPA, HNF1 A, HNF4A, FOXA1, NR1I3, HNF4A, FOXA2, and ONECUT1. In some embodiments the NR1I3 disclosed herein can comprise NR1I3 comprises constitutive androstane receptor (CAR). In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors (TFs) cancomprise FOXA1. In some embodiments, the one or more transcription factors (TFs) can comprise a codon optimized TF. In non-limiting examples, a transcription factor of the present disclosure can be a codon optimized transcription factor. In some embodiments a transcription factor of present disclosure can lack codon-optimization e.g., a transcription factor that may not be codon optimized. In some embodiments, the codon optimization can occur at the DNA level. In non-limiting examples, a F0XA1 can be codon optimized at the DNA level. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors (TFs) can comprise F0XA1 comprising F0XA1 (Uniprot P55317). In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can comprise HNF4A. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can comprise: CEBPA, HNF1A, HNF4A, F0XA1, RXR, RORA, NR1I3, NR1H3 or a combination thereof. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors can consist of: CEBPA, HNF1A, HNF4A, F0XA1, RXR, RORA, NR1I3, and NR1H3. In some embodiments the NR1I3 disclosed herein can comprise NR1I3 comprises constitutive androstane receptor (CAR). In some embodiments F0XA1 can comprise F0XA1 Uniprot ID P55317. In some embodiments, the at least one exogenous expression cassette comprising the one or more transcription factors (TFs) can comprise (Uniprot P55317). In some embodiments, a combination of one or more transcription factors, or transcription factor family members may be expressed in a PSC. Any combination of TF or TF families may be combined. Any combination of TF or TF families may be introduced in a PSC and may be induced and expressed in the PSC. In some embodiments, introduction of the at least one exogenous expression cassette comprising the one or more TFs disclosed in the present application in a PSC may induce differentiation of the iPSC into a hepatocyte or hepatocyte-like cell. In some embodiments, the one or more transcription factors that may induce differentiation of one or more PSCs into one or more hepatocytes or hepatocyte-like cell may comprise, one or more TFs or one or more members of a TF family from among the TFs that are disclosed herein. The TF family members are known to a person of skill in the art (a skilled artisan). Where the TF family members are not listed in this application, but the one or more transcription factor is disclosed, is hereby claimed as though added in the application because such TF family members are known in the art or to a person of skill in the art (a skilled artisan).

[0080] Disclosed herein in some embodiments, a transcription factor expressed in a cell of the present disclosure can take any form. In some embodiments, a transcription factor (TF) of the present disclosure can comprise a variant form of a TF. In some embodiments a variant form ofa TF can comprise a TF having a mutation. Non-limiting examples of a mutation can comprise a deletion, a SNP, an indel, or any other genetic mutation. In some embodiments, a mutation can comprise more than one mutation in the TF. In some embodiments, a mutation can be engineered e.g., engineered by an artisan. In some embodiments, a mutation may not be engineered. In some embodiments, a mutation can comprise as a change in amino acid or nucleic acid sequence. In some embodiments, a mutation can be created by addition of a genetic fragment or sequence as is used by a skilled artisan. In some embodiments, a mutation can comprise point mutations. In some embodiments, a point mutation or mutations, (by additions or deletions) can comprise a mutation in a synthetic domain. In some embodiments, a synthetic domain or domains can comprise an activation domain or domains. In some embodiments, an activation domain(s) can boost or enhance transcription. In some embodiments, synthetic domain can be a repression domain(s). In some embodiments, a repression domain can be used to repress transcription. In some embodiments, a synthetic domain can comprise a conditional binding domain(s). In some embodiments, a conditional binding domain can be used so that its use can be regulated e.g., by activation upon addition of a binder. In some embodiments, a binder can comprise a small molecule or any other binder. In some embodiments, synthetic mutations, synthetic domains, genetic fragments, or sequences can be added to a TF, nearby in a regulatory region of a TF, a protein, a gene sequence, or in a target site in an engineered cell. In some embodiments, any additions or deletions of synthetic nucleic acid or amino acid sequences can generate one or more truncations. In some embodiments, one or more truncations can be added in an internal region or at the end of a protein. In some embodiments, an isoform of a gene, gene product, TF or TF recipe can be generated for various uses of the present disclosure. In some embodiments, an isoform can comprise a variant e.g. splice variant.

[0081] Provided herein are compositions, formulations, kits, pharmaceuticals, or therapeutics including cell therapeutics comprising the present disclosure. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may express an activity of the one or more functional polypeptide in the engineered cell may be greater than an activity of the one or more functional recombinant polypeptide in a primary hepatocyte or a wild-type hepatocyte. In some embodiments, the one or more functional polypeptides comprises a recombinant, exogenous, transgenic polypeptide, or a copy of a wild-type polypeptide. In some embodiments, the functional polypeptide may be an enzyme. In some embodiments, the one or more functional polypeptides improves functionality of the engineered cell. In some embodiments, the improved functionality of the engineered cell can be much higher or superior to that of a primary hepatocyte or a wild-type hepatocyte. Insome embodiments, the improved functionality of the engineered cell involves an enzymatic cellular activity. In some embodiments, the cellular activity may involve activity of one or more other enzymes. The other enzyme may not comprise a metabolizing enzyme activity. In some embodiments, the functional polypeptide can comprise any other polypeptide of interest that is engineered and expressed by the engineered cell. In some embodiments, the enzymatic cellular activity may involve activity of one or more metabolizing xenobiotics. In some embodiments, the one or more metabolizing xenobiotics may comprise CYP3 A4 enzyme. In some embodiments, the functional polypeptide can comprise CYP3A4. In some embodiments, the engineered cell may further be engineered to express one or more functional polypeptide, which is CYP3 A4. In some embodiments, the CYP3 A4 may not be a recombinant, exogenous or transgenic polypeptide or it may comprise a copy of a wild-type CYP3 A4 polypeptide. Such an engineered cell may be an iPSC or a stem cell that may be induced to express the one or more functional polypeptide. For example, the iPSC may be induced to express CYP3A4. In some embodiments, the induced expression of CYP3 A4 activity can comprise at least 1 pmol / hr / million cells , 2 pmol / hr / million cells , 3 pmol / hr / million cells , 5 pmol / hr / million cells, 7 pmol / hr / million cells, 10 pmol / hr / million cells, 12 pmol / hr / million cells, 13 pmol / hr / million cells, 14 pmol / hr / million cells, 16 pmol / hr / million cells, 18 pmol / hr / million cells, or at least 20 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1 pmol / hr / million cells to about 12 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1 pmol / hr / million cells to about 2 pmol / hr / million cells, about 1 pmol / hr / million cells to about 3 pmol / hr / million cells, about 1 pmol / hr / million cells to about 4 pmol / hr / million cells, about 1 pmol / hr / million cells to about 5 pmol / hr / million cells, about 1 pmol / hr / million cells to about 6 pmol / hr / million cells, about 1 pmol / hr / million cells to about 7 pmol / hr / million cells, about 1 pmol / hr / million cells to about 8 pmol / hr / million cells, about 1 pmol / hr / million cells to about 9 pmol / hr / million cells, about 1 pmol / hr / million cells to about 10 pmol / hr / million cells, about 1 pmol / hr / million cells to about 11 pmol / hr / million cells, about 1 pmol / hr / million cells to about 12 pmol / hr / million cells, about 2 pmol / hr / million cells to about 3 pmol / hr / million cells, about 2 pmol / hr / million cells to about 4 pmol / hr / million cells, about 2 pmol / hr / million cells to about 5 pmol / hr / million cells, about 2 pmol / hr / million cells to about 6 pmol / hr / million cells, about 2 pmol / hr / million cells to about 7 pmol / hr / million cells, about 2 pmol / hr / million cells to about 8 pmol / hr / million cells, about 2 pmol / hr / million cells to about 9 pmol / hr / million cells, about 2 pmol / hr / million cells to about 10 pmol / hr / million cells, about 2 pmol / hr / million cells to about 11 pmol / hr / million cells, about 2 pmol / hr / million cells to about 12 pmol / hr / million cells, about 3 pmol / hr / million cells to about 4pmol / hr / million cells, about 3 pmol / hr / million cells to about 5 pmol / hr / million cells, about 3 pmol / hr / million cells to about 6 pmol / hr / million cells, about 3 pmol / hr / million cells to about 7 pmol / hr / million cells, about 3 pmol / hr / million cells to about 8 pmol / hr / million cells, about 3 pmol / hr / million cells to about 9 pmol / hr / million cells, about 3 pmol / hr / million cells to about 10 pmol / hr / million cells, about 3 pmol / hr / million cells to about 11 pmol / hr / million cells, about 3 pmol / hr / million cells to about 12 pmol / hr / million cells, about 4 pmol / hr / million cells to about 5 pmol / hr / million cells, about 4 pmol / hr / million cells to about 6 pmol / hr / million cells, about 4 pmol / hr / million cells to about 7 pmol / hr / million cells, about 4 pmol / hr / million cells to about 8 pmol / hr / million cells, about 4 pmol / hr / million cells to about 9 pmol / hr / million cells, about 4 pmol / hr / million cells to about 10 pmol / hr / million cells, about 4 pmol / hr / million cells to about 11 pmol / hr / million cells, about 4 pmol / hr / million cells to about 12 pmol / hr / million cells, about 5 pmol / hr / million cells to about 6 pmol / hr / million cells, about 5 pmol / hr / million cells to about 7 pmol / hr / million cells, about 5 pmol / hr / million cells to about 8 pmol / hr / million cells, about 5 pmol / hr / million cells to about 9 pmol / hr / million cells, about 5 pmol / hr / million cells to about 10 pmol / hr / million cells, about 5 pmol / hr / million cells to about 11 pmol / hr / million cells, about 5 pmol / hr / million cells to about 12 pmol / hr / million cells, about 6 pmol / hr / million cells to about 7 pmol / hr / million cells, about 6 pmol / hr / million cells to about 8 pmol / hr / million cells, about 6 pmol / hr / million cells to about 9 pmol / hr / million cells, about 6 pmol / hr / million cells to about 10 pmol / hr / million cells, about 6 pmol / hr / million cells to about 11 pmol / hr / million cells, about 6 pmol / hr / million cells to about 12 pmol / hr / million cells, about 7 pmol / hr / million cells to about 8 pmol / hr / million cells, about 7 pmol / hr / million cells to about 9 pmol / hr / million cells, about 7 pmol / hr / million cells to about 10 pmol / hr / million cells, about 7 pmol / hr / million cells to about 11 pmol / hr / million cells, about 7 pmol / hr / million cells to about 12 pmol / hr / million cells, about 8 pmol / hr / million cells to about 9 pmol / hr / million cells, about 8 pmol / hr / million cells to about 10 pmol / hr / million cells, about 8 pmol / hr / million cells to about 11 pmol / hr / million cells, about 8 pmol / hr / million cells to about 12 pmol / hr / million cells, about 9 pmol / hr / million cells to about 10 pmol / hr / million cells, about 9 pmol / hr / million cells to about 11 pmol / hr / million cells, about 9 pmol / hr / million cells to about 12 pmol / hr / million cells, about 10 pmol / hr / million cells to about 11 pmol / hr / million cells, about 10 pmol / hr / million cells to about 12 pmol / hr / million cells, or about 11 pmol / hr / million cells to about 12 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, about 11pmol / hr / million cells, or about 12 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise at least about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, or about 11 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise at most about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, about 11 pmol / hr / million cells, or about 12 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise about 13 pmol / hr / million cells to about 20 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise about 13 pmol / hr / million cells to about 14 pmol / hr / million cells, about 13 pmol / hr / million cells to about 15 pmol / hr / million cells, about 13 pmol / hr / million cells to about 16 pmol / hr / million cells, about 13 pmol / hr / million cells to about 17 pmol / hr / million cells, about 13 pmol / hr / million cells to about 18 pmol / hr / million cells, about 13 pmol / hr / million cells to about 19 pmol / hr / million cells, about 13 pmol / hr / million cells to about 20 pmol / hr / million cells, about 14 pmol / hr / million cells to about 15 pmol / hr / million cells, about 14 pmol / hr / million cells to about 16 pmol / hr / million cells, about 14 pmol / hr / million cells to about 17 pmol / hr / million cells, about 14 pmol / hr / million cells to about 18 pmol / hr / million cells, about 14 pmol / hr / million cells to about 19 pmol / hr / million cells, about 14 pmol / hr / million cells to about 20 pmol / hr / million cells, about 15 pmol / hr / million cells to about 16 pmol / hr / million cells, about 15 pmol / hr / million cells to about 17 pmol / hr / million cells, about 15 pmol / hr / million cells to about 18 pmol / hr / million cells, about 15 pmol / hr / million cells to about 19 pmol / hr / million cells, about 15 pmol / hr / million cells to about 20 pmol / hr / million cells, about 16 pmol / hr / million cells to about 17 pmol / hr / million cells, about 16 pmol / hr / million cells to about 18 pmol / hr / million cells, about 16 pmol / hr / million cells to about 19 pmol / hr / million cells, about 16 pmol / hr / million cells to about 20 pmol / hr / million cells, about 17 pmol / hr / million cells to about 18 pmol / hr / million cells, about 17 pmol / hr / million cells to about 19 pmol / hr / million cells, about 17 pmol / hr / million cells to about 20 pmol / hr / million cells, about 18 pmol / hr / million cells to about 19 pmol / hr / million cells, about 18 pmol / hr / million cells to about 20 pmol / hr / million cells, or about 19 pmol / hr / million cells to about 20 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise about 13 pmol / hr / million cells, about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, about 19 pmol / hr / million cells, or about 20 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise at least about 13 pmol / hr / million cells, about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16 pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, or about 19 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise at most about 14 pmol / hr / million cells, about 15 pmol / hr / million cells, about 16 pmol / hr / million cells, about 17 pmol / hr / million cells, about 18 pmol / hr / million cells, about 19 pmol / hr / million cells, or about 20 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise at least 40pmol / hr / million cells, 50pmol / hr / million cells, 60pmol / hr / million cells, 70pmol / hr / million cells, 80pmol / hr / million cells, 90pmol / hr / million cells, 100 pmol / hr / million cells, 110 pmol / hr / million cells, 120 pmol / hr / million cells, 130 pmol / hr / million cells, 140 pmol / hr / million cells, or at least 150 pmol / hr / million cells. In some embodiments, the induced expression of CYP3A4 activity can comprise about 40 pmol / hr / million cells to about 150 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 40 pmol / hr / million cells to about 50 pmol / hr / million cells, about 40 pmol / hr / million cells to about 60 pmol / hr / million cells, about 40 pmol / hr / million cells to about 70 pmol / hr / million cells, about 40 pmol / hr / million cells to about 80 pmol / hr / million cells, about 40 pmol / hr / million cells to about 90 pmol / hr / million cells, about 40 pmol / hr / million cells to about 100 pmol / hr / million cells, about 40 pmol / hr / million cells to about 110 pmol / hr / million cells, about 40 pmol / hr / million cells to about 120 pmol / hr / million cells, about 40 pmol / hr / million cells to about 130 pmol / hr / million cells, about 40 pmol / hr / million cells to about 140 pmol / hr / million cells, about 40 pmol / hr / million cells to about 150 pmol / hr / million cells, about 50 pmol / hr / million cells to about 60 pmol / hr / million cells, about 50 pmol / hr / million cells to about 70 pmol / hr / million cells, about 50 pmol / hr / million cells to about 80 pmol / hr / million cells, about 50 pmol / hr / million cells to about 90 pmol / hr / million cells, about 50 pmol / hr / million cells to about 100 pmol / hr / million cells, about 50 pmol / hr / million cells to about 110 pmol / hr / million cells, about 50 pmol / hr / million cells to about 120 pmol / hr / million cells, about 50 pmol / hr / million cells to about 130 pmol / hr / million cells, about 50 pmol / hr / million cells to about 140 pmol / hr / million cells, about 50 pmol / hr / million cells to about 150 pmol / hr / million cells, about 60 pmol / hr / million cells to about 70 pmol / hr / million cells, about 60 pmol / hr / million cells to about 80 pmol / hr / million cells, about 60 pmol / hr / million cells to about 90 pmol / hr / million cells, about 60 pmol / hr / million cells to about 100 pmol / hr / million cells, about 60 pmol / hr / million cells to about 110 pmol / hr / million cells, about 60 pmol / hr / million cells toabout 120 pmol / hr / million cells, about 60 pmol / hr / million cells to about 130 pmol / hr / million cells, about 60 pmol / hr / million cells to about 140 pmol / hr / million cells, about 60 pmol / hr / million cells to about 150 pmol / hr / million cells, about 70 pmol / hr / million cells to about 80 pmol / hr / million cells, about 70 pmol / hr / million cells to about 90 pmol / hr / million cells, about 70 pmol / hr / million cells to about 100 pmol / hr / million cells, about 70 pmol / hr / million cells to about 110 pmol / hr / million cells, about 70 pmol / hr / million cells to about 120 pmol / hr / million cells, about 70 pmol / hr / million cells to about 130 pmol / hr / million cells, about 70 pmol / hr / million cells to about 140 pmol / hr / million cells, about 70 pmol / hr / million cells to about 150 pmol / hr / million cells, about 80 pmol / hr / million cells to about 90 pmol / hr / million cells, about 80 pmol / hr / million cells to about 100 pmol / hr / million cells, about 80 pmol / hr / million cells to about 110 pmol / hr / million cells, about 80 pmol / hr / million cells to about 120 pmol / hr / million cells, about 80 pmol / hr / million cells to about 130 pmol / hr / million cells, about 80 pmol / hr / million cells to about 140 pmol / hr / million cells, about 80 pmol / hr / million cells to about 150 pmol / hr / million cells, about 90 pmol / hr / million cells to about 100 pmol / hr / million cells, about 90 pmol / hr / million cells to about 110 pmol / hr / million cells, about 90 pmol / hr / million cells to about 120 pmol / hr / million cells, about 90 pmol / hr / million cells to about 130 pmol / hr / million cells, about 90 pmol / hr / million cells to about 140 pmol / hr / million cells, about 90 pmol / hr / million cells to about 150 pmol / hr / million cells, about 100 pmol / hr / million cells to about 110 pmol / hr / million cells, about 100 pmol / hr / million cells to about 120 pmol / hr / million cells, about 100 pmol / hr / million cells to about 130 pmol / hr / million cells, about 100 pmol / hr / million cells to about 140 pmol / hr / million cells, about 100 pmol / hr / million cells to about 150 pmol / hr / million cells, about 110 pmol / hr / million cells to about 120 pmol / hr / million cells, about 110 pmol / hr / million cells to about 130 pmol / hr / million cells, about 110 pmol / hr / million cells to about 140 pmol / hr / million cells, about 110 pmol / hr / million cells to about 150 pmol / hr / million cells, about 120 pmol / hr / million cells to about 130 pmol / hr / million cells, about 120 pmol / hr / million cells to about 140 pmol / hr / million cells, about 120 pmol / hr / million cells to about 150 pmol / hr / million cells, about 130 pmol / hr / million cells to about 140 pmol / hr / million cells, about 130 pmol / hr / million cells to about 150 pmol / hr / million cells, or about 140 pmol / hr / million cells to about 150 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 40 pmol / hr / million cells, about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110 pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, about 140 pmol / hr / million cells, or about 150 pmol / hr / million cells.In some embodiments, the induced expression of CYP3 A4 activity can comprise at least about 40 pmol / hr / million cells, about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110 pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, or about 140 pmol / hr / million cells. In some embodiments, the induced expression of CYP3 A4 activity can comprise at most about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110 pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, about 140 pmol / hr / million cells, or about 150 pmol / hr / million cells.

[0082] In some embodiments, the engineered cell that may further be engineered to express one or more functional polypeptide, which is CYP3 A4 may comprise a CYP3 A4 that may not be a recombinant, exogenous or transgenic polypeptide. In some embodiments, CYP3 A4 can be a copy of a wild-type CYP3 A4 polypeptide. Such an engineered cell may be an iPSC or a stem cell that may be induced to express the one or more functional polypeptide. For example, the iPSC may be induced to express CYP3 A4. In some embodiments, the induced expression of CYP3A4 activity can comprise at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800% 900%, 1000%, 1200%, 1400%, 1600%, 1800%, 2000%, 2200%, 2600% or at least 3000% higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise about 100 % to about 1,400 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise about 100 % to about 200 %, about 100 % to about 300 %, about 100 % to about 400 %, about 100 % to about 500 %, about 100 % to about 600 %, about 100 % to about 700 %, about 100 % to about 800 %, about 100 % to about 900 %, about 100 % to about 1,000 %, about 100 % to about 1,200 %, about 100 % to about 1,400 %, about 200 % to about 300 %, about 200 % to about 400 %, about 200 % to about 500 %, about 200 % to about 600 %, about 200 % to about 700 %, about 200 % to about 800 %, about 200 % to about 900 %, about 200 % to about 1,000 %, about 200 % to about 1,200 %, about 200 % to about 1,400 %, about 300 % to about 400 %, about 300 % to about 500 %, about 300 % to about 600 %, about 300 % to about 700 %, about 300 % to about 800 %, about 300 % to about 900 %, about 300 % to about 1,000 %, about 300 % to about 1,200 %, about 300 % to about 1,400 %, about 400 % to about 500 %, about 400 % to about 600 %, about 400 % to about 700 %, about 400 % to about 800 %, about 400 % to about 900 %, about 400 % to about1,000 %, about 400 % to about 1,200 %, about 400 % to about 1,400 %, about 500 % to about600 %, about 500 % to about 700 %, about 500 % to about 800 %, about 500 % to about 900 %, about 500 % to about 1,000 %, about 500 % to about 1,200 %, about 500 % to about 1,400 %, about 600 % to about 700 %, about 600 % to about 800 %, about 600 % to about 900 %, about 600 % to about 1,000 %, about 600 % to about 1,200 %, about 600 % to about 1,400 %, about700 % to about 800 %, about 700 % to about 900 %, about 700 % to about 1,000 %, about700 % to about 1,200 %, about 700 % to about 1,400 %, about 800 % to about 900 %, about800 % to about 1,000 %, about 800 % to about 1,200 %, about 800 % to about 1,400 %, about900 % to about 1,000 %, about 900 % to about 1,200 %, about 900 % to about 1,400 %, about1,000 % to about 1,200 %, about 1,000 % to about 1,400 %, or about 1,200 % to about 1,400 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise about 100 %, about 200 %, about 300 %, about 400 %, about 500 %, about 600 %, about 700 %, about 800 %, about 900 %, about 1,000 %, about 1,200 %, or about 1,400 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3 A4 activity can comprise at least about 100 %, about 200 %, about 300 %, about 400 %, about 500 %, about 600 %, about 700 %, about 800 %, about 900 %, about 1,000 %, or about 1,200 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise at most about 200 %, about 300 %, about 400 %, about 500 %, about 600 %, about 700 %, about 800 %, about 900 %, about 1,000 %, about1.200 %, or about 1,400 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1,600 % to about 3,000 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1,600 % to about 1,800 %, about 1,600 % to about 2,000 %, about 1,600 % to about2.200 %, about 1,600 % to about 2,600 %, about 1,600 % to about 3,000 %, about 1,800 % to about 2,000 %, about 1,800 % to about 2,200 %, about 1,800 % to about 2,600 %, about 1,800 % to about 3,000 %, about 2,000 % to about 2,200 %, about 2,000 % to about 2,600 %, about 2,000 % to about 3,000 %, about 2,200 % to about 2,600 %, about 2,200 % to about 3,000 %, or about 2,600 % to about 3,000 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3 A4 activity can comprise about 1,600 %, about 1,800 %, about 2,000 %, about 2,200 %, about 2,600 %, or about 3,000 % higher in the engineered cell than in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise at least about 1,600 %, about 1,800 %, about 2,000 %, about 2,200 %, or about 2,600 % higher in the engineered cellthan in a primary or a wild-type hepatocyte. In some embodiments, the induced expression of CYP3A4 activity can comprise at most about 1,800 %, about 2,000 %, about 2,200 %, about 2,600 %, or about 3,000 % higher in the engineered cell than in a primary or a wild-type hepatocyte.

[0083] In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a hepatocyte or hepatocyte-like cell. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a hepatocyte or hepatocyte-like cell is not derived from a patient. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a hepatocyte or hepatocyte-like cell may be derived from a patient. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a hepatocyte or hepatocyte-like cell derived from a human induced pluripotent stem cell. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, or an embryonic stem cell. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be the human stem cell, the human induced pluripotent stem cell, the human progenitor cell, the human fetal stem cell, the hepatic stem cell, or the embryonic stem cell is not derived from a patient. In some embodiments, the engineered cell further comprising at least one expression cassette encoding one or more functional polypeptide may be a human stem cell, the human induced pluripotent stem cell, the human progenitor cell, the human fetal stem cell, the hepatic stem cell, or the embryonic stem cell may be derived from a patient. In some embodiments, the primary hepatocyte or the wild-type hepatocyte may be a human hepatocyte cell, wherein the human hepatocyte cell is not derived from a patient. In some embodiments, the primary hepatocyte or the wild-type hepatocyte may be a human hepatocyte cell, wherein the human hepatocyte cell may be derived from a patient. In some cases, the engineered cell may not comprise a human cell, e.g., may not be a human hepatocyte or human hepatocyte-like cell. In some embodiments, the primary hepatocyte or the wild-type hepatocyte used herein may not comprise a human primary hepatocyte or may not comprise a wild-type hepatocyte.

[0084] Provided herein in some embodiments is a composition comprising a modified hepatocyte or hepatocyte-like cell comprising at least one functional polypeptide whose activity may bedifferent from activity of a similar polypeptide wild-type hepatocytes when the function is assessed in vivo or in vitro by one or more functional assay. In some embodiments, the function of the polypeptide in the modified hepatocyte or hepatocyte-like cell may be exhibit reduced activity from that in wild-type hepatocyte, e.g., the polypeptide of the modified hepatocyte or hepatocyte-like cell may comprise 90%, 85%, 80%, 75%, 70%, all the way to 0.1% less than the activity of the polypeptide of the wild-type hepatocyte. In some embodiments, the function of the polypeptide in the modified hepatocyte or hepatocyte-like cell may be exhibit greater activity than the activity of a similar polypeptide in the wild-type hepatocyte. In some embodiments, the polypeptide of the modified hepatocyte or hepatocyte-like cell may comprise 90%, 85%, 80%, 75%, 70%, all the way to 0.1% more than the activity of the polypeptide of the wild-type hepatocyte.

[0085] Methods of generating the engineered or modified cell: The present disclosure includes methods of generating, producing, engineered or genetically modified cells cell configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wildtype hepatocyte, the method comprising administering at least one expression cassette comprising one or more transcription factors to a cell, thereby generating the engineered cell.

[0086] Provided herein in some embodiments are methods of generating an engineered cell comprising a cell modified to express one or more functional recombinant polypeptide. In some embodiments, the engineered cell can further be generated by contacting the cell with an expression cassette comprising a nucleic acid molecule that encodes the one or more functional recombinant polypeptide. In some embodiments, the engineered cell expressing the one or more functional recombinant polypeptide may be configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

[0087] The engineered cell or population of engineered cells disclosed in the composition, formulation, vectors, pharmaceuticals section of this disclosure may be generated by any number of ways. The methods of generating the engineered cell or modified cell is written in reference to the compositions, or therapeutic compositions or formulations presently disclosed (see section on compositions, pharmaceuticals, vectors, formulations, kits of the present disclosure etcetera). In some embodiments, the engineered cell can be created by modifying the cell to comprise one or more transcription factors, one or more transcription factor members or one or more enhancers of transcription. The engineered cell can comprise at least one exogenous cassette comprising the one or more transcription factors, one or more transcription factor family members, or one or more enhancers of transcription. In addition, the engineered cell can comprise any number ofrecombinant constructs required for the specific function of the engineered cell. The constructs required to generate a functional expression cassette are known by a skilled artisan. For example, the engineered cell can comprise at least one promoter region comprising all the standard regulatory molecules to drive transcription, translation, and production of a functional polypeptide in the cell. In some embodiments, a promoter constructed in the engineered cell can comprise an inducible promoter, a transient promoter, or a constitutively expressed promoter. In some embodiments, the promoter region may comprise or express at least one reporter gene. In other cases, the promoter region in the exogenous expression cassette may not comprise a reporter gene. The exogenous expression cassette may comprise any of the selectable markers disclosed herein or those known to a person of skill in the art. In some embodiments, the engineered cell can be a stem cell. In some embodiments, the engineered cell can be an embryonic stem cell or adult stem cell an induced pluripotent stem cell (IPSC), a mesenchymal stem cell (MSC). In some cases, the engineered cell can be differentiated from a non-hepatic stem cell (non-liver stem cell). In some embodiments, the engineered cell is a pre-existing hepatocyte cell such as a mammalian preexisting hepatocyte cell. In other embodiments, the engineered cell is generated from a human pre-existing hepatocyte cell. The engineered cell can be derived from a subject or a patient with a liver disease. In other cases, depending on the use, the engineered cell may not be derived from a subject or a patient with a liver disease. In some embodiments, the engineered cell may comprise a hepatocyte cell. In some embodiments, the engineered cell may not comprise a hepatocyte. The hepatocyte may comprise a hepatocyte stem cell. In some embodiment, the hepatocyte stem cell may be a human hepatocyte. In some embodiments, the engineered cell comprises a stem cell, an induced pluripotent stem cell (iPSCs) or any suitable cell disclosed herein. In some embodiments, the engineered cell comprises an iPSC. In some embodiments, the iPSC can be induced to differentiate into one or more hepatocytes or hepatocyte-like cells. The iPSC may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells through various methods, including being induced to express one or more transcription factors, being contacted with one or more transcription factor proteins or nucleic acids encoding transcription factor proteins, or through other means as disclosed herein. The PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in less than 12 hours, less than to about 20 hours, or between at least about 1, 2, 3, 4 (96 hours) days or less. In some embodiments, the PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in 30 days or less. The PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in at most about 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. The PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in at most about 1 day. The PSCs may beinduced to differentiate into one or more hepatocytes or hepatocyte-like cells in at least most 4 days. The PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in at most about 96 hours. The PSCs may be induced to differentiate into one or more hepatocytes or hepatocyte-like cells in at most about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10 hours or less. The one or more nucleic acids may be expressed in one or more exogenous expression cassettes. The one or more exogenous expression cassettes may comprise one or more transcription factors. The one or more transcription factors may induce differentiation of the PSC. The one or more transcription factors may induce differentiation of the PSC into a hepatocyte or a hepatocyte-like cell with improved efficiency of differentiation. In some embodiments, the improved efficiency is much higher relative to a control cell. The improved efficiency may be characterized by any method known to one of skill in the art. Non-limiting examples of characteristics that can be used to detect improved efficiency relative to a control include for example, increased secretion of markers, higher percentage of engineered cells expressing a gene, RNA, protein, or protein activity, morphological features (of the differentiated cell relative to a wild-type cell), or by enzymatic activity levels of the differentiated cell relative to a control cell. The control cell may be an uninduced differentiated cell, a stem cell, a native cell, a primary or wild-type cell or any control, positive or negative deemed sufficient. In the present disclosure, the control cell may serve to confirm that the engineered cell has differentiated to e.g., a hepatocyte or hepatocyte-like cell. As such, the control cell may comprise a primary hepatocyte, or a wildtype hepatocyte, or an undifferentiated stem cell, or an uninduced iPSC or any other control as disclosed here. A skilled artisan would be able to determine the appropriate control for the experiments disclosed herein. The methods of generating the engineered cell or population of engineered cell disclosed can be used to assess the characteristics of a differentiated engineered cell disclosed. The engineered cell or engineered population of cells disclosed can comprise one or more exogenous expression cassettes engineered stem cells, embryonic stem cells, mesenchymal stem cell or iPSC. For example, the iPSC may comprise one or more exogenous expression cassettes. The one or more exogenous expression cassettes can comprise any number of recombinant constructs to generate the engineered cell. For example, the one or more exogenous cassettes may comprise one or more transcription factors that induce differentiation of the PSC into a hepatocyte, hepatocyte-like cell, or a cell that expresses albumin (ALB), cell that expresses alpha- 1 antitrypsin (Al AT), or a cell that expresses any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated markers disclosed herein. In some cases, the differentiated iPSC may express any other markers disclosed herein, that are associated with the (TF cocktail or TF recipe) non-natural, exogenous induction of iPSC differentiation along thedifferentiation trajectory. The exogenous expression cassettes may induce the differentiation of the PSC into the hepatocyte, hepatocyte-like cell, cell that expresses albumin (ALB), cell that expresses alpha-1 antitrypsin (Al AT), cell that does not express albumin (ALB), or cell that does not express alpha-1 antitrypsin (Al AT), in 30 days or less. The exogenous expression cassettes may induce the differentiation of the PSC into the hepatocyte, hepatocyte-like cell, cell that expresses detectable levels of CYP3A4. The exogenous expression cassettes can induce the differentiation of the PSC into the hepatocyte, hepatocyte-like cell, cell that does not express high levels of CYP3A4, but expresses another enzymatic activity or cellular lineage marker. The exogenous expression cassettes can induce the differentiation of the PSC into any cell lineage. In a non-limiting example, the exogenous expression cassette in the engineered cell or population of engineered cells can induce differentiation of the pluripotent stem cell (PSC) into a hepatocyte, hepatocyte-like cell, cell that expresses albumin (ALB), cell that expresses alpha-1 antitrypsin (Al AT), cell that does not express albumin (ALB), or cell that does not express alpha- 1 antitrypsin (Al AT), in 96 hours or less. In a non-limiting example, the exogenous expression cassette in the engineered cell or population of engineered cells can induce differentiation of the iPSC into a hepatocyte, hepatocyte-like cell, cell that expresses albumin (ALB), cell that expresses alpha- 1 antitrypsin (Al AT), cell that does not express albumin (ALB), or cell that does not express alpha- 1 antitrypsin (Al AT), in 30 hours or less. The PSC may be provided in a media. The media may not have to be altered during the differentiation of the PSC into the hepatocyte, hepatocyte-like cell, cell that expresses albumin (ALB), cell that expresses alpha-1 antitrypsin (Al AT), cell that does not express albumin (ALB), or cell that does not express alpha- 1 antitrypsin (Al AT).

[0088] The engineered cell provided, or the population of engineered cells herein can comprise adherent cells. The engineered cell or population of engineered cells can comprise suspension cells. The engineered cell or population of engineered cells can comprise adherent cells and suspension cells. The engineered cell or population of engineered cells can be provided in a media. In some embodiments, soluble versions of the TF can be added to culture media of the stem cell or PSC. Addition of the soluble TFs can lead to induction of stem cells or PSCs to differentiate to a different cell lineage or phenotype. For example, the induced stem cells or iPSC can differentiate to one or more hepatocyte or hepatocyte-like cell. The media may not have to be altered during the differentiation of the PSCs into hepatocytes, hepatocyte-like cells, cells that express albumin (ALB), cells that express A1AT, or cells that express or any other hepatocyte-like, hepatocyte lineage or hepatocyte-associated markers disclosed herein, or any other markers disclosed herein. In some embodiments, the any other markers can be associated with a TF recipe or a non-natural, exogenous induction of iPSC differentiation along a differentiation cell trajectory. The engineeredcell or the population of engineered cells may be provided in a media. The media may not need any nutrients, growth factors, or microenvironmental or matrix optimizations. At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 95%of the cells may express albumin (ALB). The media may not need any nutrients, growth factors, or microenvironmental or matrix optimizations. At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 95%of the cells may express Al AT. At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 95%of the cells may express measurable levels of CYP3A4 activity in a standard enzymatic assay. The engineered cell disclosed can comprise one or more exogenous expressions cassettes. The engineered cell can comprise a population of cells. The one or more exogenous cassettes may comprise one or more transcription factors (TFs) that induce differentiation of the engineered cell. In some cases, the engineered cell can be a stem cell, a progenitor cell, an induced pluripotent cell, or any other cell type as disclosed herein or known in the art. For example, the engineered cell can be an iPSC which can differentiate into a different cell lineage of choice. In some embodiment, the iPSC can differentiate into a hepatocyte, hepatocyte-like cell that expresses albumin, cell that expresses alpha- 1 -anti -trypsin (A1AT), cell that expresses cytochrome p450 (CYP3A4) activity or a cell that expresses any other hepatocyte or hepatocyte-like cell markers. In some cases, the differentiated engineered cell can express or secrete any other hepatic markers, hepatocyte or hepatocyte-like cell markers disclosed herein and / or known in the art. The differentiation of the engineered cell may be associated with any disclosed TF cocktail or TF recipe. In some embodiments, the hepatic, hepatocyte, or hepatocyte-like cell markers can comprise at least one marker associated with unnatural, exogenous induction of iPSC differentiation along the differentiation trajectory. The present disclosure provides an engineered cell or a population of cells comprising one or more hepatic markers, hepatocyte, or hepatocyte-like cell markers. In some embodiments, the engineered cell or population of engineered cells can secrete albumin. The amount or quantity of secreted albumin can be higher in the engineered cell relative to the amount or quantity secreted by an uninduced control cell. The uninduced control cell may be an uninduced engineered cell, a stem cell or iPSC. A control cell may be a primary or a wild-type cell. In some embodiments, the engineered cell or population of engineered cells can secrete Al AT. The amount or quantity of secreted albumin can be higher in the engineered cell relative to the amount or quantity secreted by an uninduced control cell. The uninduced control cell may be an uninduced engineered cell, a stem cell or iPSC. A control cell may be a primary or a wildtype cell. The engineered cell may also express a greater amount of one or more hepatocyte geneproduct relative to the expression detected in an uninduced control cell. In some embodiments, the control uninduced cells do not comprise or express any of the differentiation markers disclosed herein. In some embodiments, the engineered cells that are induced to differentiate can comprise a hepatocyte or hepatocyte-like cell morphology. In some cases, the hepatocyte or hepatocyte-like cell morphology can comprise a large cytoplasm and polygonal cell shapes indicative that the engineered cell has differentiated.

[0089] In some embodiments, the genetic cargo can be a therapeutic cargo. The therapeutic cargo can be at least one agent, at least one inhibitor, at least one small molecule, at least one polypeptide or any therapeutically effective molecule deliverable by human and non-human cell utilizing the present disclosure. In some embodiments, the genetic cargo can be overexpressed in any cell. In some embodiments, the overexpressed genetic cargo can be overexpressed in a control cell, a stem cell, an iPSC, a hepatocyte, a stem cell, a pluripotent stem cell, a progenitor cell, a fetal stem cell, an induced pluripotent stem cell, a hepatic stem cell, an adult stem cell, a hepatocyte, or any type of cell disclosed herein and / or known in the art. The cell can be a mammalian cell or a nonmammalian cell. In some cases, the wild-type or native cell disclosed herein as a reference cell can comprise or a non-human cell.

[0090] Transcription factor family members or recipes: In some embodiments, the at least one expression cassette may comprise one or more transcription factors or at least one transcription factor family member or a combination thereof. The TFs, TF recipes or cocktails used to generate different cell lines of the present disclosure are provided throughout the present application e.g., TFs may be accessed in Table 1, 2, 3, 5 in this disclosure. Table 2 show non-limiting example of the functionality of the engineered cells disclosed. In some embodiments, the engineered cell comprising the at least one or more exogenous expression cassette can comprise one or more transcription factor (TF) cocktail or TF recipe (various TF recipes are shown in Table 1 and elsewhere throughout). TF recipes can be designated in long form or short form, for instance, the TF recipe, H0036A refers to TF recipe H36A, and e.g., TF recipe H0062A refers to the same TF recipe H62A and so forth, such that these designations are used interchangeably for all TF recipes in any section of the present disclosure. In some embodiments, the at least one exogenous expression cassette can comprise one or more transcription factors (any TFs shown in Table 1 or elsewhere herein). Non-limiting examples can include at least one exogenous expression cassette comprising the one or more transcription factors in any combination. In some embodiments, in non-limiting examples, the at least one exogenous expression cassette can consist of one or more transcription factor recipes disclosed. For each of the TFs disclosed, or TF recipes disclosed herein,different amounts of one or more transcription factors may be required or introduced into an PSC. In some embodiments, groups of TFs or TFs and TF family can be combined. In some embodiments, at least one TF may be expressed in the iPSC or in a stem cell. The introduction of the TF can be by any methods disclosed, e.g., via an expression cassette in an engineered cell, via delivery systems, or introduction of soluble TFs into the media. Non-limiting example of amounts of individual TFs or groups of TFs can vary but may include at least about 5, about 10, about 15, about 20, about 25, or about 50 copies of the open reading frame (ORF) for each TF per cell may be introduced. Alternatively, or in addition to, at most about 50, about 25, about 20, about 15, about 10, or about 5 copies of the open reading frame (ORF) for each TF per cell may be introduced. Increased levels of expression 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.

[0091] In some embodiments, the number of copies of the ORF for one or more TF, one or more groups of TFs or one or more TF family member or members (note any plural and any singular are used interchangeably in the present application) introduced per cell can be about 1 to about 90. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be at least about 1. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be at most about 90. In a non-limiting example, the number of copies of an ORF of interest e.g., for FoxAl, that may be introduced per cell can be about 1 to about 2, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 90, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 90, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about 25, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 4 to about 90, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 5 to about 90, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 90, about 15 to about 20, about 15 to about 25, about 15 to about 40, about 15 to about 50, about 15 to about 60, about 15 to about 90, about 20 to about 25, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 90, about 25 to about 40, about 25 to about 50, about 25 to about 60, about 25 to about 90, about 40 to about 50, about 40 to about 60, about 40 to about 90, about 50 to about 60, about 50 to about 90, or about 60 to about 90. In some embodiments, the number of copies of the ORF for each TF introduced per cell can be about 1, about 2, about 4, about 5, about 10, about 15, about20, about 25, about 40, about 50, about 60, or about 90. In some embodiments, new cell lines can be engineered with novel combinations of transcription factors (TFs). Following induction and differentiation of engineered cells (e.g., engineered iPSCs) for 7 days, the differentiated cells, e.g., differentiated hepatocyte or hepatocyte-like cells can be evaluated to assess cell functionality. In some cases, the cell functionality assessment can comprise evaluating function relative to a mature cell functionality. For example, the cell functionality of a differentiated engineered cell can be evaluated in light of functionality or functional pathways that may be expressed by a mature cell or a cell that may have differentiated into a mature-cell-like phenotype. In some cases, the cell functionality assessment can comprise testing functionality of another stage, e.g., an intermediary differentiation stage which can be anywhere on the differentiation trajectory. In some embodiments, the cell functionality assessment can comprise evaluating function of known physiological cell functionality such as for example, functionality relative to a control cell or e.g., functionality relative to a hepatocyte or a primary hepatocyte or a wild-type hepatocyte, uninduced engineered cell etcetera. In some embodiments, cell functionality of the new engineered cells can be conducted relative to cell functionality of a control cell or a suitable comparator cell. Exemplary cell functionality assays can be conducted to determine functional performance of the differentiated or engineered cell. For illustrative purposes, exemplary cell functionality can be determined for various functional pathways, genes, and / or gene products e.g., as shown in Table 2 and in various figures disclosed herein. Cell functionality can examine expression levels or activity levels e.g., an enzyme activity. For illustrative purposes only, the functionality can comprise assessing activity of CYP3A4 enzyme in the engineered new cell lines disclosed herein. Various TF recipes (one or more for example) can be engineered into the cell line e.g., stem cells, expanded and induced to differentiated for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the cells may be induced to differentiated for less than 24 hours all the way to less than 1 hour. In some embodiments, induction of engineered cell to allow differentiation can start for about 0.5 hours or less, all the way monitoring by hour or less than an hour up to 7 days, 8 days, 9 days, 10 days, 11 days and in between all the way to at least 30 days or less. In some embodiments, the engineered cell can be differentiated for the time desired then assessed for expression activity of one or more gene or gene product (exemplary data is provided here in and for example in Table 2 or figures herein). In some cases, the TF recipes can provide improved CYP3A4 activity to at least about 2-8 pmol / hr / million cells compared to Ipmol / hr / million cells of the comparator cell line. In some embodiments, the comparator cell line can be another engineered cell line or not. In some embodiments, one or more TF recipes of the presentdisclosure can improve expression of CYP3 A4 activity in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be superior to CYP3 A4 activity of primary cells e.g., primary hepatocyte or wild-type cells. The improved TF-associated CYP3A4 activity can be from 0.5 to at least about 12 pmol / hr / million cells. In some embodiments, the control cell e.g., primary hepatocyte can produce CYP3 A4 activity that is much less e.g., about 1 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity in the engineered cell disclosed can be about 0.5 pmol / hr / million cells to about 12 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be about 0.5 pmol / hr / million cells to about 1 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 2 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 3 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 4 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 5 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 6 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 7 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 8 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 9 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 10 pmol / hr / million cells, about 0.5 pmol / hr / million cells to about 12 pmol / hr / million cells, about 1 pmol / hr / million cells to about 2 pmol / hr / million cells, about 1 pmol / hr / million cells to about 3 pmol / hr / million cells, about 1 pmol / hr / million cells to about 4 pmol / hr / million cells, about 1 pmol / hr / million cells to about 5 pmol / hr / million cells, about 1 pmol / hr / million cells to about 6 pmol / hr / million cells, about 1 pmol / hr / million cells to about 7 pmol / hr / million cells, about 1 pmol / hr / million cells to about 8 pmol / hr / million cells, about 1 pmol / hr / million cells to about 9 pmol / hr / million cells, about 1 pmol / hr / million cells to about 10 pmol / hr / million cells, about 1 pmol / hr / million cells to about 12 pmol / hr / million cells, about 2 pmol / hr / million cells to about 3 pmol / hr / million cells, about 2 pmol / hr / million cells to about 4 pmol / hr / million cells, about 2 pmol / hr / million cells to about 5 pmol / hr / million cells, about 2 pmol / hr / million cells to about 6 pmol / hr / million cells, about 2 pmol / hr / million cells to about 7 pmol / hr / million cells, about 2 pmol / hr / million cells to about 8 pmol / hr / million cells, about 2 pmol / hr / million cells to about 9 pmol / hr / million cells, about 2 pmol / hr / million cells to about 10 pmol / hr / million cells, about 2 pmol / hr / million cells to about 12 pmol / hr / million cells, about 3 pmol / hr / million cells to about 4 pmol / hr / million cells, about 3 pmol / hr / million cells to about 5 pmol / hr / million cells, about 3 pmol / hr / million cells to about 6 pmol / hr / million cells, about 3 pmol / hr / million cells to about 7 pmol / hr / million cells, about 3 pmol / hr / million cells to about 8 pmol / hr / million cells, about 3 pmol / hr / million cells to about 9 pmol / hr / million cells, about 3 pmol / hr / million cells to about 10 pmol / hr / million cells, about 3 pmol / hr / million cells to about 12 pmol / hr / million cells, about 4 pmol / hr / million cells to about 5 pmol / hr / million cells, about 4pmol / hr / million cells to about 6 pmol / hr / million cells, about 4 pmol / hr / million cells to about 7 pmol / hr / million cells, about 4 pmol / hr / million cells to about 8 pmol / hr / million cells, about 4 pmol / hr / million cells to about 9 pmol / hr / million cells, about 4 pmol / hr / million cells to about 10 pmol / hr / million cells, about 4 pmol / hr / million cells to about 12 pmol / hr / million cells, about 5 pmol / hr / million cells to about 6 pmol / hr / million cells, about 5 pmol / hr / million cells to about 7 pmol / hr / million cells, about 5 pmol / hr / million cells to about 8 pmol / hr / million cells, about 5 pmol / hr / million cells to about 9 pmol / hr / million cells, about 5 pmol / hr / million cells to about 10 pmol / hr / million cells, about 5 pmol / hr / million cells to about 12 pmol / hr / million cells, about 6 pmol / hr / million cells to about 7 pmol / hr / million cells, about 6 pmol / hr / million cells to about 8 pmol / hr / million cells, about 6 pmol / hr / million cells to about 9 pmol / hr / million cells, about 6 pmol / hr / million cells to about 10 pmol / hr / million cells, about 6 pmol / hr / million cells to about 12 pmol / hr / million cells, about 7 pmol / hr / million cells to about 8 pmol / hr / million cells, about 7 pmol / hr / million cells to about 9 pmol / hr / million cells, about 7 pmol / hr / million cells to about 10 pmol / hr / million cells, about 7 pmol / hr / million cells to about 12 pmol / hr / million cells, about 8 pmol / hr / million cells to about 9 pmol / hr / million cells, about 8 pmol / hr / million cells to about 10 pmol / hr / million cells, about 8 pmol / hr / million cells to about 12 pmol / hr / million cells, about 9 pmol / hr / million cells to about 10 pmol / hr / million cells, about 9 pmol / hr / million cells to about 12 pmol / hr / million cells, or about 10 pmol / hr / million cells to about 12 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be about 0.5 pmol / hr / million cells, about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, or about 12 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be at least about 0.5 pmol / hr / million cells, about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, or about 10 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3 A4 activity can be at most about 1 pmol / hr / million cells, about 2 pmol / hr / million cells, about 3 pmol / hr / million cells, about 4 pmol / hr / million cells, about 5 pmol / hr / million cells, about 6 pmol / hr / million cells, about 7 pmol / hr / million cells, about 8 pmol / hr / million cells, about 9 pmol / hr / million cells, about 10 pmol / hr / million cells, or about 12 pmol / hr / million cells.

[0092] In other embodiments, one or more TF recipes of the present disclosure can provide improved expression of CYP3 A4 activity in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be superior to CYP3 A4 activity of primary cells e.g., primary hepatocyte or wild-type cells. The improved TF-associated CYP3 A4 activity can be at least about 100 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be about 40 pmol / hr / million cells to about 150 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be about 40 pmol / hr / million cells to about 50 pmol / hr / million cells, about 40 pmol / hr / million cells to about 60 pmol / hr / million cells, about 40 pmol / hr / million cells to about 70 pmol / hr / million cells, about 40 pmol / hr / million cells to about 80 pmol / hr / million cells, about 40 pmol / hr / million cells to about 90 pmol / hr / million cells, about 40 pmol / hr / million cells to about 100 pmol / hr / million cells, about 40 pmol / hr / million cells to about 110 pmol / hr / million cells, about 40 pmol / hr / million cells to about 120 pmol / hr / million cells, about 40 pmol / hr / million cells to about 130 pmol / hr / million cells, about 40 pmol / hr / million cells to about 140 pmol / hr / million cells, about 40 pmol / hr / million cells to about 150 pmol / hr / million cells, about 50 pmol / hr / million cells to about 60 pmol / hr / million cells, about 50 pmol / hr / million cells to about 70 pmol / hr / million cells, about 50 pmol / hr / million cells to about 80 pmol / hr / million cells, about 50 pmol / hr / million cells to about 90 pmol / hr / million cells, about 50 pmol / hr / million cells to about 100 pmol / hr / million cells, about 50 pmol / hr / million cells to about 110 pmol / hr / million cells, about 50 pmol / hr / million cells to about 120 pmol / hr / million cells, about 50 pmol / hr / million cells to about 130 pmol / hr / million cells, about 50 pmol / hr / million cells to about 140 pmol / hr / million cells, about 50 pmol / hr / million cells to about 150 pmol / hr / million cells, about 60 pmol / hr / million cells to about 70 pmol / hr / million cells, about 60 pmol / hr / million cells to about 80 pmol / hr / million cells, about 60 pmol / hr / million cells to about 90 pmol / hr / million cells, about 60 pmol / hr / million cells to about 100 pmol / hr / million cells, about 60 pmol / hr / million cells to about 110 pmol / hr / million cells, about 60 pmol / hr / million cells to about 120 pmol / hr / million cells, about 60 pmol / hr / million cells to about 130 pmol / hr / million cells, about 60 pmol / hr / million cells to about 140 pmol / hr / million cells, about 60 pmol / hr / million cells to about 150 pmol / hr / million cells, about 70 pmol / hr / million cells to about 80 pmol / hr / million cells, about 70 pmol / hr / million cells to about 90 pmol / hr / million cells, about 70 pmol / hr / million cells to about 100 pmol / hr / million cells, about 70 pmol / hr / million cells to about 110 pmol / hr / million cells, about 70 pmol / hr / million cells to about 120 pmol / hr / million cells, about 70 pmol / hr / million cells to about 130 pmol / hr / million cells, about 70 pmol / hr / million cells to about 140 pmol / hr / million cells, about 70 pmol / hr / million cells to about 150 pmol / hr / million cells,about 80 pmol / hr / million cells to about 90 pmol / hr / million cells, about 80 pmol / hr / million cells to about 100 pmol / hr / million cells, about 80 pmol / hr / million cells to about 110 pmol / hr / million cells, about 80 pmol / hr / million cells to about 120 pmol / hr / million cells, about 80 pmol / hr / million cells to about 130 pmol / hr / million cells, about 80 pmol / hr / million cells to about 140 pmol / hr / million cells, about 80 pmol / hr / million cells to about 150 pmol / hr / million cells, about 90 pmol / hr / million cells to about 100 pmol / hr / million cells, about 90 pmol / hr / million cells to about 110 pmol / hr / million cells, about 90 pmol / hr / million cells to about 120 pmol / hr / million cells, about 90 pmol / hr / million cells to about 130 pmol / hr / million cells, about 90 pmol / hr / million cells to about 140 pmol / hr / million cells, about 90 pmol / hr / million cells to about 150 pmol / hr / million cells, about 100 pmol / hr / million cells to about 110 pmol / hr / million cells, about 100 pmol / hr / million cells to about 120 pmol / hr / million cells, about 100 pmol / hr / million cells to about 130 pmol / hr / million cells, about 100 pmol / hr / million cells to about 140 pmol / hr / million cells, about 100 pmol / hr / million cells to about 150 pmol / hr / million cells, about 110 pmol / hr / million cells to about 120 pmol / hr / million cells, about 110 pmol / hr / million cells to about 130 pmol / hr / million cells, about 110 pmol / hr / million cells to about 140 pmol / hr / million cells, about 110 pmol / hr / million cells to about 150 pmol / hr / million cells, about 120 pmol / hr / million cells to about 130 pmol / hr / million cells, about 120 pmol / hr / million cells to about 140 pmol / hr / million cells, about 120 pmol / hr / million cells to about 150 pmol / hr / million cells, about 130 pmol / hr / million cells to about 140 pmol / hr / million cells, about 130 pmol / hr / million cells to about 150 pmol / hr / million cells, or about 140 pmol / hr / million cells to about 150 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be about 40 pmol / hr / million cells, about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110 pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, about 140 pmol / hr / million cells, or about 150 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be at least about 40 pmol / hr / million cells, about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110 pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, or about 140 pmol / hr / million cells. In some embodiments, the improved TF-associated CYP3A4 activity can be at most about 50 pmol / hr / million cells, about 60 pmol / hr / million cells, about 70 pmol / hr / million cells, about 80 pmol / hr / million cells, about 90 pmol / hr / million cells, about 100 pmol / hr / million cells, about 110pmol / hr / million cells, about 120 pmol / hr / million cells, about 130 pmol / hr / million cells, about 140 pmol / hr / million cells, or about 150 pmol / hr / million cells.

[0093] In yet another embodiment, one or more TF recipes of the present disclosure can provide improved expression of CYP3A4 activity in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) than that can be superior to CYP3 A4 activity of primary cells e.g., primary hepatocyte or wild-type cells. The improved the TF-associated CYP3 A4 activity can be at least about 100 to 500% greater than primary hepatocytes. In some embodiments, the improved TF-associated CYP3A4 activity can be about 100 % to about 600 %. In some embodiments, the improved TF-associated CYP3A4 activity can be about 100 % to about 150 %, about 100 % to about 200 %, about 100 % to about 250 %, about 100 % to about 300 %, about 100 % to about 350 %, about 100 % to about 400 %, about 100 % to about 450 %, about 100 % to about 500 %, about 100 % to about 550 %, about 100 % to about 600 %, about 150 % to about 200 %, about 150 % to about 250 %, about 150 % to about 300 %, about 150 % to about 350 %, about 150 % to about 400 %, about 150 % to about 450 %, about 150 % to about 500 %, about 150 % to about 550 %, about 150 % to about 600 %, about 200 % to about 250 %, about 200 % to about 300 %, about 200 % to about 350 %, about 200 % to about 400 %, about 200 % to about 450 %, about 200 % to about 500 %, about 200 % to about 550 %, about 200 % to about 600 %, about 250 % to about 300 %, about 250 % to about 350 %, about 250 % to about 400 %, about 250 % to about 450 %, about 250 % to about 500 %, about 250 % to about 550 %, about 250 % to about 600 %, about 300 % to about 350 %, about 300 % to about 400 %, about 300 % to about 450 %, about 300 % to about 500 %, about 300 % to about 550 %, about 300 % to about 600 %, about 350 % to about 400 %, about 350 % to about 450 %, about 350 % to about 500 %, about 350 % to about 550 %, about 350 % to about 600 %, about 400 % to about 450 %, about 400 % to about 500 %, about 400 % to about 550 %, about 400 % to about 600 %, about 450 % to about 500 %, about 450 % to about 550 %, about 450 % to about 600 %, about 500 % to about 550 %, about 500 % to about 600 %, or about 550 % to about 600 %. In some embodiments, the improved TF-associated CYP3A4 activity can be about 100 %, about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 %. In some embodiments, the improved TF-associated CYP3A4 activity can be at least about 100 %, about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, or about 550 %. In some embodiments, the improved TF-associated CYP3A4 activity can be at most about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 %.

[0094] In other embodiments, one or more TF recipes of the present disclosure can provide improved secretion of albumin levels in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be superior to the albumin secreted levels by primary cells e.g., primary hepatocyte or wild-type cells. The improved TF-associated secreted albumin levels can be at least about 5 to 60 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin levels can be about 5 pg / day / million cells to about 60 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin levels can be about 5 pg / day / million cells to about 10 pg / day / million cells, about 5 pg / day / million cells to about 15 pg / day / million cells, about 5 pg / day / million cells to about 20 pg / day / million cells, about 5 pg / day / million cells to about 25 pg / day / million cells, about 5 pg / day / million cells to about 30 pg / day / million cells, about 5 pg / day / million cells to about 35 pg / day / million cells, about 5 pg / day / million cells to about 40 pg / day / million cells, about 5 pg / day / million cells to about 45 pg / day / million cells, about 5 pg / day / million cells to about 50 pg / day / million cells, about 5 pg / day / million cells to about 55 pg / day / million cells, about 5 pg / day / million cells to about 60 pg / day / million cells, about 10 pg / day / million cells to about 15 pg / day / million cells, about 10 pg / day / million cells to about 20 pg / day / million cells, about 10 pg / day / million cells to about 25 pg / day / million cells, about 10 pg / day / million cells to about 30 pg / day / million cells, about 10 pg / day / million cells to about 35 pg / day / million cells, about 10 pg / day / million cells to about 40 pg / day / million cells, about 10 pg / day / million cells to about 45 pg / day / million cells, about 10 pg / day / million cells to about 50 pg / day / million cells, about 10 pg / day / million cells to about 55 pg / day / million cells, about 10 pg / day / million cells to about 60 pg / day / million cells, about 15 pg / day / million cells to about 20 pg / day / million cells, about 15 pg / day / million cells to about 25 pg / day / million cells, about 15 pg / day / million cells to about 30 pg / day / million cells, about 15 pg / day / million cells to about 35 pg / day / million cells, about 15 pg / day / million cells to about 40 pg / day / million cells, about 15 pg / day / million cells to about 45 pg / day / million cells, about 15 pg / day / million cells to about 50 pg / day / million cells, about 15 pg / day / million cells to about 55 pg / day / million cells, about 15 pg / day / million cells to about 60 pg / day / million cells, about 20 pg / day / million cells to about 25 pg / day / million cells, about 20 pg / day / million cells to about 30 pg / day / million cells, about 20 pg / day / million cells to about 35 pg / day / million cells, about 20 pg / day / million cells to about 40 pg / day / million cells, about 20 pg / day / million cells to about 45 pg / day / million cells, about 20 pg / day / million cells to about 50 pg / day / million cells, about 20 pg / day / million cells to about 55 pg / day / million cells, about 20 pg / day / million cells to about 60 pg / day / million cells, about 25 pg / day / million cells to about 30 pg / day / million cells, about 25 pg / day / million cells to about 35 pg / day / million cells, about 25 pg / day / million cells toabout 40 pg / day / million cells, about 25 pg / day / million cells to about 45 pg / day / million cells, about 25 pg / day / million cells to about 50 pg / day / million cells, about 25 pg / day / million cells to about 55 pg / day / million cells, about 25 pg / day / million cells to about 60 pg / day / million cells, about 30 pg / day / million cells to about 35 pg / day / million cells, about 30 pg / day / million cells to about 40 pg / day / million cells, about 30 pg / day / million cells to about 45 pg / day / million cells, about 30 pg / day / million cells to about 50 pg / day / million cells, about 30 pg / day / million cells to about 55 pg / day / million cells, about 30 pg / day / million cells to about 60 pg / day / million cells, about 35 pg / day / million cells to about 40 pg / day / million cells, about 35 pg / day / million cells to about 45 pg / day / million cells, about 35 pg / day / million cells to about 50 pg / day / million cells, about 35 pg / day / million cells to about 55 pg / day / million cells, about 35 pg / day / million cells to about 60 pg / day / million cells, about 40 pg / day / million cells to about 45 pg / day / million cells, about 40 pg / day / million cells to about 50 pg / day / million cells, about 40 pg / day / million cells to about 55 pg / day / million cells, about 40 pg / day / million cells to about 60 pg / day / million cells, about 45 pg / day / million cells to about 50 pg / day / million cells, about 45 pg / day / million cells to about 55 pg / day / million cells, about 45 pg / day / million cells to about 60 pg / day / million cells, about 50 pg / day / million cells to about 55 pg / day / million cells, about 50 pg / day / million cells to about 60 pg / day / million cells, or about 55 pg / day / million cells to about 60 pg / day / million cells. In some embodiments, the improved TF -associated secreted albumin can be about 5 pg / day / million cells, about 10 pg / day / million cells, about 15 pg / day / million cells, about 20 pg / day / million cells, about 25 pg / day / million cells, about 30 pg / day / million cells, about 35 pg / day / million cells, about 40 pg / day / million cells, about 45 pg / day / million cells, about 50 pg / day / million cells, about 55 pg / day / million cells, or about 60 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin levels can be at least about 5 pg / day / million cells, about 10 pg / day / million cells, about 15 pg / day / million cells, about 20 pg / day / million cells, about 25 pg / day / million cells, about 30 pg / day / million cells, about 35 pg / day / million cells, about 40 pg / day / million cells, about 45 pg / day / million cells, about 50 pg / day / million cells, or about 55 pg / day / million cells. In some embodiments, the improved TF- associated secreted albumin levels can be at most about 10 pg / day / million cells, about 15 pg / day / million cells, about 20 pg / day / million cells, about 25 pg / day / million cells, about 30 pg / day / million cells, about 35 pg / day / million cells, about 40 pg / day / million cells, about 45 pg / day / million cells, about 50 pg / day / million cells, about 55 pg / day / million cells, or about 60 pg / day / million cells.

[0095] In another embodiment, one or more TF recipes of the present disclosure can provide improved secretion of albumin levels in the differentiated engineered cell (e.g., engineeredhepatocyte or hepatocyte-like cell) that can be superior to that secreted by primary cells e.g., primary hepatocyte or wild-type cells. In some embodiments, the TF-associated improved secretion of albumin levels can reach 100% - 600% albumin secretion compared to primary hepatocyte. In some embodiments, the improved TF-associated secreted albumin can be about 100 % to about 600 %. In some embodiments, the improved TF-associated secreted albumin can be about 100 % to about 150 %, about 100 % to about 200 %, about 100 % to about 250 %, about 100 % to about 300 %, about 100 % to about 350 %, about 100 % to about 400 %, about 100 % to about 450 %, about 100 % to about 500 %, about 100 % to about 550 %, about 100 % to about 600 %, about 150 % to about 200 %, about 150 % to about 250 %, about 150 % to about 300 %, about 150 % to about 350 %, about 150 % to about 400 %, about 150 % to about 450 %, about 150 % to about 500 %, about 150 % to about 550 %, about 150 % to about 600 %, about 200 % to about 250 %, about 200 % to about 300 %, about 200 % to about 350 %, about 200 % to about 400 %, about 200 % to about 450 %, about 200 % to about 500 %, about 200 % to about 550 %, about 200 % to about 600 %, about 250 % to about 300 %, about 250 % to about 350 %, about 250 % to about 400 %, about 250 % to about 450 %, about 250 % to about 500 %, about 250 % to about 550 %, about 250 % to about 600 %, about 300 % to about 350 %, about 300 % to about 400 %, about 300 % to about 450 %, about 300 % to about 500 %, about 300 % to about 550 %, about 300 % to about 600 %, about 350 % to about 400 %, about 350 % to about 450 %, about 350 % to about 500 %, about 350 % to about 550 %, about 350 % to about 600 %, about 400 % to about 450 %, about 400 % to about 500 %, about 400 % to about 550 %, about 400 % to about 600 %, about 450 % to about 500 %, about 450 % to about 550 %, about 450 % to about 600 %, about 500 % to about 550 %, about 500 % to about 600 %, or about 550 % to about 600 %. In some embodiments, the improved TF-associated secreted albumin can be about 100 %, about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 %. In some embodiments, the improved TF-associated secreted albumin can be at least about 100 %, about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, or about 550 %. In some embodiments, the improved TF-associated secreted albumin can be at most about 150 %, about 200 %, about 250 %, about 300 %, about 350 %, about 400 %, about 450 %, about 500 %, about 550 %, or about 600 %.

[0096] In another embodiment, one or more TF recipes of the present disclosure can provide improved secretion of albumin levels in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be superior to that secreted by primary cells e.g., primary hepatocyte or wild-type cells. In some embodiments, the TF-associated improvedsecretion of albumin levels can be at least about 1 to 15 pg / day / million cells, which can be greater than albumin secreted in a control cell. The control cell can comprise a primary hepatocyte. In some embodiments, the improved TF-associated secreted albumin can be about 1 pg / day / million cells to about 15 pg / day / million cells. In some embodiments, the improved TF- associated secreted albumin can be about 1 pg / day / million cells to about 2 pg / day / million cells, about 1 pg / day / million cells to about 3 pg / day / million cells, about 1 pg / day / million cells to about 4 pg / day / million cells, about 1 pg / day / million cells to about 5 pg / day / million cells, about 1 pg / day / million cells to about 10 pg / day / million cells, about 1 pg / day / million cells to about 11 pg / day / million cells, about 1 pg / day / million cells to about 12 pg / day / million cells, about 1 pg / day / million cells to about 13 pg / day / million cells, about 1 pg / day / million cells to about 14 pg / day / million cells, about 1 pg / day / million cells to about 15 pg / day / million cells, about 2 pg / day / million cells to about 3 pg / day / million cells, about 2 pg / day / million cells to about 4 pg / day / million cells, about 2 pg / day / million cells to about 5 pg / day / million cells, about 2 pg / day / million cells to about 10 pg / day / million cells, about 2 pg / day / million cells to about 11 pg / day / million cells, about 2 pg / day / million cells to about 12 pg / day / million cells, about 2 pg / day / million cells to about 13 pg / day / million cells, about 2 pg / day / million cells to about 14 pg / day / million cells, about 2 pg / day / million cells to about 15 pg / day / million cells, about 3 pg / day / million cells to about 4 pg / day / million cells, about 3 pg / day / million cells to about 5 pg / day / million cells, about 3 pg / day / million cells to about 10 pg / day / million cells, about 3 pg / day / million cells to about 11 pg / day / million cells, about 3 pg / day / million cells to about 12 pg / day / million cells, about 3 pg / day / million cells to about 13 pg / day / million cells, about 3 pg / day / million cells to about 14 pg / day / million cells, about 3 pg / day / million cells to about 15 pg / day / million cells, about 4 pg / day / million cells to about 5 pg / day / million cells, about 4 pg / day / million cells to about 10 pg / day / million cells, about 4 pg / day / million cells to about 11 pg / day / million cells, about 4 pg / day / million cells to about 12 pg / day / million cells, about 4 pg / day / million cells to about 13 pg / day / million cells, about 4 pg / day / million cells to about 14 pg / day / million cells, about 4 pg / day / million cells to about 15 pg / day / million cells, about 5 pg / day / million cells to about 10 pg / day / million cells, about 5 pg / day / million cells to about 11 pg / day / million cells, about 5 pg / day / million cells to about 12 pg / day / million cells, about 5 pg / day / million cells to about 13 pg / day / million cells, about 5 pg / day / million cells to about 14 pg / day / million cells, about 5 pg / day / million cells to about 15 pg / day / million cells, about 10 pg / day / million cells to about 11 pg / day / million cells, about 10 pg / day / million cells to about 12 pg / day / million cells, about 10 pg / day / million cells to about 13 pg / day / million cells, about 10 pg / day / million cells to about 14 pg / day / million cells, about 10 pg / day / million cells to about 15pg / day / million cells, about 11 pg / day / million cells to about 12 pg / day / million cells, about 11 pg / day / million cells to about 13 pg / day / million cells, about 11 pg / day / million cells to about 14 pg / day / million cells, about 11 pg / day / million cells to about 15 pg / day / million cells, about 12 pg / day / million cells to about 13 pg / day / million cells, about 12 pg / day / million cells to about 14 pg / day / million cells, about 12 pg / day / million cells to about 15 pg / day / million cells, about 13 pg / day / million cells to about 14 pg / day / million cells, about 13 pg / day / million cells to about 15 pg / day / million cells, or about 14 pg / day / million cells to about 15 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin can be about 1 pg / day / million cells, about 2 pg / day / million cells, about 3 pg / day / million cells, about 4 pg / day / million cells, about 5 pg / day / million cells, about 10 pg / day / million cells, about 11 pg / day / million cells, about 12 pg / day / million cells, about 13 pg / day / million cells, about 14 pg / day / million cells, or about 15 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin can be at least about 1 pg / day / million cells, about 2 pg / day / million cells, about 3 pg / day / million cells, about 4 pg / day / million cells, about 5 pg / day / million cells, about 10 pg / day / million cells, about 11 pg / day / million cells, about 12 pg / day / million cells, about 13 pg / day / million cells, or about 14 pg / day / million cells. In some embodiments, the improved TF-associated secreted albumin can be at most about 2 pg / day / million cells, about 3 pg / day / million cells, about 4 pg / day / million cells, about 5 pg / day / million cells, about 10 pg / day / million cells, about 11 pg / day / million cells, about 12 pg / day / million cells, about 13 pg / day / million cells, about 14 pg / day / million cells, or about 15 pg / day / million cells.

[0097] In another embodiment, one or more TF recipes of the present disclosure can provide improved activity levels of alpha- 1 -anti -trypsin (A1AT) in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be higher or superior to the Al AT activity levels produced by a primary hepatocyte or a control cells. In some embodiments, the TF-associated improved levels of Al AT can be at least about 1,000 to 12,000 ng / day / million cells, which can be greater than Al AT levels in a primary hepatocyte or a control cell. For example, the control cell can comprise a primary hepatocyte or non-hepatocyte e.g., uninduced cells. In some embodiments, the activity levels of Al AT produced by a control cell or a primary hepatocyte can be at least about lx to lOx less than that of the engineered cell, e.g., an engineered differentiated hepatocyte or hepatocyte-like cell. In some embodiments, the primary hepatocyte or control cell can produce at least lOx or 1,000 ng / day / million cells less than the engineered cell. In some embodiments, the improved TF-associated levels of Al AT produced in the engineered cell can be about 1,000 ng / day / million cells to about 15,000 ng / day / million cells. In some embodiments, the improved TF-associated levels of Al AT produced in the engineeredcell can be about 1,000 ng / day / million cells to about 2,000 ng / day / million cells, about 1,000 ng / day / million cells to about 3,000 ng / day / million cells, about 1,000 ng / day / million cells to about 4,000 ng / day / million cells, about 1,000 ng / day / million cells to about 7,000 ng / day / million cells, about 1,000 ng / day / million cells to about 8,000 ng / day / million cells, about 1,000 ng / day / million cells to about 9,000 ng / day / million cells, about 1,000 ng / day / million cells to about 10,000 ng / day / million cells, about 1,000 ng / day / million cells to about 11,000 ng / day / million cells, about 1,000 ng / day / million cells to about 12,000 ng / day / million cells, about 1,000 ng / day / million cells to about 13,000 ng / day / million cells, about 1,000 ng / day / million cells to about 15,000 ng / day / million cells, about 2,000 ng / day / million cells to about 3,000 ng / day / million cells, about 2,000 ng / day / million cells to about 4,000 ng / day / million cells, about 2,000 ng / day / million cells to about 7,000 ng / day / million cells, about 2,000 ng / day / million cells to about 8,000 ng / day / million cells, about 2,000 ng / day / million cells to about 9,000 ng / day / million cells, about 2,000 ng / day / million cells to about 10,000 ng / day / million cells, about 2,000 ng / day / million cells to about 11,000 ng / day / million cells, about 2,000 ng / day / million cells to about 12,000 ng / day / million cells, about 2,000 ng / day / million cells to about 13,000 ng / day / million cells, about 2,000 ng / day / million cells to about 15,000 ng / day / million cells, about 3,000 ng / day / million cells to about 4,000 ng / day / million cells, about 3,000 ng / day / million cells to about 7,000 ng / day / million cells, about 3,000 ng / day / million cells to about 8,000 ng / day / million cells, about 3,000 ng / day / million cells to about 9,000 ng / day / million cells, about 3,000 ng / day / million cells to about 10,000 ng / day / million cells, about 3,000 ng / day / million cells to about 11,000 ng / day / million cells, about 3,000 ng / day / million cells to about 12,000 ng / day / million cells, about 3,000 ng / day / million cells to about 13,000 ng / day / million cells, about 3,000 ng / day / million cells to about 15,000 ng / day / million cells, about 4,000 ng / day / million cells to about 7,000 ng / day / million cells, about 4,000 ng / day / million cells to about 8,000 ng / day / million cells, about 4,000 ng / day / million cells to about 9,000 ng / day / million cells, about 4,000 ng / day / million cells to about 10,000 ng / day / million cells, about 4,000 ng / day / million cells to about 11,000 ng / day / million cells, about 4,000 ng / day / million cells to about 12,000 ng / day / million cells, about 4,000 ng / day / million cells to about 13,000 ng / day / million cells, about 4,000 ng / day / million cells to about 15,000 ng / day / million cells, about 7,000 ng / day / million cells to about 8,000 ng / day / million cells, about 7,000 ng / day / million cells to about 9,000 ng / day / million cells, about 7,000 ng / day / million cells to about 10,000 ng / day / million cells, about 7,000 ng / day / million cells to about 11,000 ng / day / million cells, about 7,000 ng / day / million cells to about 12,000 ng / day / million cells, about 7,000 ng / day / million cells to about 13,000ng / day / million cells, about 7,000 ng / day / million cells to about 15,000 ng / day / million cells, about 8,000 ng / day / million cells to about 9,000 ng / day / million cells, about 8,000 ng / day / million cells to about 10,000 ng / day / million cells, about 8,000 ng / day / million cells to about 11,000 ng / day / million cells, about 8,000 ng / day / million cells to about 12,000 ng / day / million cells, about 8,000 ng / day / million cells to about 13,000 ng / day / million cells, about 8,000 ng / day / million cells to about 15,000 ng / day / million cells, about 9,000 ng / day / million cells to about 10,000 ng / day / million cells, about 9,000 ng / day / million cells to about 11,000 ng / day / million cells, about 9,000 ng / day / million cells to about 12,000 ng / day / million cells, about 9,000 ng / day / million cells to about 13,000 ng / day / million cells, about 9,000 ng / day / million cells to about 15,000 ng / day / million cells, about 10,000 ng / day / million cells to about 11,000 ng / day / million cells, about 10,000 ng / day / million cells to about 12,000 ng / day / million cells, about 10,000 ng / day / million cells to about 13,000 ng / day / million cells, about 10,000 ng / day / million cells to about 15,000 ng / day / million cells, about 11,000 ng / day / million cells to about 12,000 ng / day / million cells, about 11,000 ng / day / million cells to about 13,000 ng / day / million cells, about 11,000 ng / day / million cells to about 15,000 ng / day / million cells, about 12,000 ng / day / million cells to about 13,000 ng / day / million cells, about 12,000 ng / day / million cells to about 15,000 ng / day / million cells, or about 13,000 ng / day / million cells to about 15,000 ng / day / million cells. In some embodiments, the improved TF-associated levels of Al AT produced in the engineered cell can be about 1,000 ng / day / million cells, about 2,000 ng / day / million cells, about 3,000 ng / day / million cells, about 4,000 ng / day / million cells, about 7,000 ng / day / million cells, about 8,000 ng / day / million cells, about 9,000 ng / day / million cells, about 10,000 ng / day / million cells, about 11,000 ng / day / million cells, about 12,000 ng / day / million cells, about 13,000 ng / day / million cells, or about 15,000 ng / day / million cells. In some embodiments, the improved TF-associated levels of A1AT produced in the engineered cell can be at least about 1,000 ng / day / million cells, about 2,000 ng / day / million cells, about 3,000 ng / day / million cells, about 4,000 ng / day / million cells, about 7,000 ng / day / million cells, about 8,000 ng / day / million cells, about 9,000 ng / day / million cells, about 10,000 ng / day / million cells, about 11,000 ng / day / million cells, about 12,000 ng / day / million cells, or about 13,000 ng / day / million cells. In some embodiments, the improved TF-associated levels of Al AT produced in the engineered cell can be at most about 2,000 ng / day / million cells, about 3,000 ng / day / million cells, about 4,000 ng / day / million cells, about 7,000 ng / day / million cells, about 8,000 ng / day / million cells, about 9,000 ng / day / million cells, about 10,000 ng / day / million cells, about 11,000 ng / day / million cells, about 12,000 ng / day / million cells, about 13,000 ng / day / million cells, or about 15,000 ng / day / million cells.

[0098] In another embodiment, one or more TF recipes of the present disclosure can provide improved levels of cholesterol production in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be higher or superior to levels of cholesterol production detected in a primary hepatocyte or a control cells. In some embodiments, the TF- associated improved levels of cholesterol production can be from ~1 nmol / day / million cells to 40 nmol / day / million cells. In some embodiments, the improved TF-associated levels of cholesterol produced in the engineered cell can be about 1 nmol / day / million cells to about 40 nmol / day / million cells. In some embodiments, the improved TF-associated levels of cholesterol produced in the engineered cell can be about 1 nmol / day / million cells to about 2 nmol / day / million cells, about 1 nmol / day / million cells to about 3 nmol / day / million cells, about 1 nmol / day / million cells to about 4 nmol / day / million cells, about 1 nmol / day / million cells to about 5 nmol / day / million cells, about 1 nmol / day / million cells to about 10 nmol / day / million cells, about 1 nmol / day / million cells to about 15 nmol / day / million cells, about 1 nmol / day / million cells to about 20 nmol / day / million cells, about 1 nmol / day / million cells to about 25 nmol / day / million cells, about 1 nmol / day / million cells to about 30 nmol / day / million cells, about1 nmol / day / million cells to about 35 nmol / day / million cells, about 1 nmol / day / million cells to about 40 nmol / day / million cells, about 2 nmol / day / million cells to about 3 nmol / day / million cells, about 2 nmol / day / million cells to about 4 nmol / day / million cells, about 2 nmol / day / million cells to about 5 nmol / day / million cells, about 2 nmol / day / million cells to about 10 nmol / day / million cells, about 2 nmol / day / million cells to about 15 nmol / day / million cells, about2 nmol / day / million cells to about 20 nmol / day / million cells, about 2 nmol / day / million cells to about 25 nmol / day / million cells, about 2 nmol / day / million cells to about 30 nmol / day / million cells, about 2 nmol / day / million cells to about 35 nmol / day / million cells, about 2 nmol / day / million cells to about 40 nmol / day / million cells, about 3 nmol / day / million cells to about 4 nmol / day / million cells, about 3 nmol / day / million cells to about 5 nmol / day / million cells, about 3 nmol / day / million cells to about 10 nmol / day / million cells, about 3 nmol / day / million cells to about 15 nmol / day / million cells, about 3 nmol / day / million cells to about 20 nmol / day / million cells, about 3 nmol / day / million cells to about 25 nmol / day / million cells, about3 nmol / day / million cells to about 30 nmol / day / million cells, about 3 nmol / day / million cells to about 35 nmol / day / million cells, about 3 nmol / day / million cells to about 40 nmol / day / million cells, about 4 nmol / day / million cells to about 5 nmol / day / million cells, about 4 nmol / day / million cells to about 10 nmol / day / million cells, about 4 nmol / day / million cells to about 15 nmol / day / million cells, about 4 nmol / day / million cells to about 20 nmol / day / million cells, about4 nmol / day / million cells to about 25 nmol / day / million cells, about 4 nmol / day / million cells toabout 30 nmol / day / million cells, about 4 nmol / day / million cells to about 35 nmol / day / million cells, about 4 nmol / day / million cells to about 40 nmol / day / million cells, about 5 nmol / day / million cells to about 10 nmol / day / million cells, about 5 nmol / day / million cells to about 15 nmol / day / million cells, about 5 nmol / day / million cells to about 20 nmol / day / million cells, about 5 nmol / day / million cells to about 25 nmol / day / million cells, about 5 nmol / day / million cells to about 30 nmol / day / million cells, about 5 nmol / day / million cells to about 35 nmol / day / million cells, about 5 nmol / day / million cells to about 40 nmol / day / million cells, about 10 nmol / day / million cells to about 15 nmol / day / million cells, about 10 nmol / day / million cells to about 20 nmol / day / million cells, about 10 nmol / day / million cells to about 25 nmol / day / million cells, about 10 nmol / day / million cells to about 30 nmol / day / million cells, about 10 nmol / day / million cells to about 35 nmol / day / million cells, about 10 nmol / day / million cells to about 40 nmol / day / million cells, about 15 nmol / day / million cells to about 20 nmol / day / million cells, about 15 nmol / day / million cells to about 25 nmol / day / million cells, about 15 nmol / day / million cells to about 30 nmol / day / million cells, about 15 nmol / day / million cells to about 35 nmol / day / million cells, about 15 nmol / day / million cells to about 40 nmol / day / million cells, about 20 nmol / day / million cells to about 25 nmol / day / million cells, about 20 nmol / day / million cells to about 30 nmol / day / million cells, about 20 nmol / day / million cells to about 35 nmol / day / million cells, about 20 nmol / day / million cells to about 40 nmol / day / million cells, about 25 nmol / day / million cells to about 30 nmol / day / million cells, about 25 nmol / day / million cells to about 35 nmol / day / million cells, about 25 nmol / day / million cells to about 40 nmol / day / million cells, about 30 nmol / day / million cells to about 35 nmol / day / million cells, about 30 nmol / day / million cells to about 40 nmol / day / million cells, or about 35 nmol / day / million cells to about 40 nmol / day / million cells. In some embodiments, the improved TF-associated levels of cholesterol produced in the engineered cell can be about 1 nmol / day / million cells, about 2 nmol / day / million cells, about 3 nmol / day / million cells, about 4 nmol / day / million cells, about 5 nmol / day / million cells, about 10 nmol / day / million cells, about 15 nmol / day / million cells, about 20 nmol / day / million cells, about 25 nmol / day / million cells, about 30 nmol / day / million cells, about 35 nmol / day / million cells, or about 40 nmol / day / million cells. In some embodiments, the improved TF-associated levels of cholesterol produced in the engineered cell can be at least about 1 nmol / day / million cells, about 2 nmol / day / million cells, about 3 nmol / day / million cells, about 4 nmol / day / million cells, about 5 nmol / day / million cells, about 10 nmol / day / million cells, about 15 nmol / day / million cells, about 20 nmol / day / million cells, about 25 nmol / day / million cells, about 30 nmol / day / million cells, or about 35 nmol / day / million cells. In some embodiments, the improved TF-associated levels ofcholesterol produced in the engineered cell can be at most about 2 nmol / day / million cells, about 3 nmol / day / million cells, about 4 nmol / day / million cells, about 5 nmol / day / million cells, about 10 nmol / day / million cells, about 15 nmol / day / million cells, about 20 nmol / day / million cells, about 25 nmol / day / million cells, about 30 nmol / day / million cells, about 35 nmol / day / million cells, or about 40 nmol / day / million cells.

[0099] In another embodiment, one or more TF recipes of the present disclosure can provide improved levels of triglycerides secretion in the differentiated engineered cell (e.g., engineered hepatocyte or hepatocyte-like cell) that can be higher or superior to levels of triglycerides secretion produced by a primary hepatocyte or a control cells. In some embodiments, the TF- associated improved triglycerides secretion can be between ~20 to -500 pg / day / million cells. In some embodiments, the improved TF-associated levels of triglycerides secretion produced in the engineered cell can be about 20 pg / day / million cells to about 130 pg / day / million cells. In some embodiments, the improved TF-associated levels of triglycerides secretion produced in the engineered cell can be about 20 pg / day / million cells to about 30 pg / day / million cells, about 20 pg / day / million cells to about 40 pg / day / million cells, about 20 pg / day / million cells to about 50 pg / day / million cells, about 20 pg / day / million cells to about 60 pg / day / million cells, about 20 pg / day / million cells to about 70 pg / day / million cells, about 20 pg / day / million cells to about 80 pg / day / million cells, about 20 pg / day / million cells to about 90 pg / day / million cells, about 20 pg / day / million cells to about 100 pg / day / million c...

Claims

CLAIMSWhat is claimed:

1. An engineered cell engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise HNF1 A, HNF4A, and F0XA1, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte.

2. The engineered cell of claim 1, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

3. The engineered cell of claim 1 or 2, wherein the engineered cell comprises a pluripotent stem cell (PSC).

4. The engineered cell of any one of the preceding claims, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell (iPSC), a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

5. The engineered cell of any one of the preceding claims, wherein the rate is at least 30% greater than a primary hepatocyte or a wild-type hepatocyte.

6. The engineered cell of any one of the preceding claims, wherein one or more hepatocyte functions comprise: coagulation, copper transport, ion transport, retinol transport, protection against neutrophil elastase, inflammatory response, regulation of urea cycle, tyrosine metabolism, histidine metabolism, alanine metabolism, alcohol metabolism, transporting lipoproteins, fatty acid biosynthesis, albumin production, Al AT production, CYP3A4 activity, or any combination thereof.

7. The engineered cell of any one of the preceding claims, wherein the engineered cell comprises increased CYP3 A4 activity relative to the primary hepatocyte or the wild-type hepatocyte.

8. The engineered cell of any one of the preceding claims, wherein one or more hepatocyte markers comprise: SLC40A1, ATP7B, ARG1, HAL, ALDH6A1, APOM, Al AT, albumin or any combination thereof.

9. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform ion transport at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the engineered cell expresses an equal to or greater amount ofSLC40A1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

10. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform copper transport at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the cell expresses an equal to or greater amount of ATP7B relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

11. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform urea cycle at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the cell expresses an equal to or greater amount of ARG1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

12. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform histidine metabolism at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal to or greater amount of HAL relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

13. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform alcohol metabolism at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the engineered cell expresses an equal to or greater amount of ALDH6A1 relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

14. The engineered cell of any one of the preceding claims, wherein the engineered cell is configured to perform transporting lipoproteins at a rate equal to or greater than the primary hepatocyte or the wild-type hepatocyte and the engineered cell expresses an equal or greater amount of APOM relative to an amount of the hepatocyte marker expressed by a primary hepatocyte or a wild-type hepatocyte.

15. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses ATP7B at 4x greater than a primary cell or a wild-type hepatocyte.

16. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses SLC40A1 at least 2x greater than a primary cell or a wild-type hepatocyte.

17. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses HAL at least 2.7x greater than a primary cell or a wild-type hepatocyte.

18. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses ALDH6A1 at least 40% greater than a primary cell or a wild-type hepatocyte.

19. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses APOM at least 2x greater than a primary cell or a wild-type hepatocyte.

20. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses ARG1 at least 33% greater than a primary cell or a wild-type hepatocyte.

21. The engineered cell of any one of the preceding claims, wherein the engineered cell comprises at least one expression cassette comprising the one or more transcription factors.

22. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors further comprise one or more additional transcription factors comprising: a FOS, JUN, PROX, GATA, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

23. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors further comprise GATA6.

24. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors further comprise FOXA2.

25. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors further comprise CEBP A.

26. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

27. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more HNF family members.

28. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more FOX family members.

29. The engineered cell of any one of the preceding claims, further comprising at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

30. The engineered cell of any one of the preceding claims, further comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF,HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

31. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors further comprise one or more transcription factors from Table 3.

32. The engineered cell of any one of the preceding claims, wherein the activity of CYP3 A4 in the cell is at least 40% greater than in a primary hepatocyte or a wild-type hepatocyte when the engineered cell is cultured in the presence of rifampicin.

33. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses at least 2x greater CYP3 A4 than a primary cell or a wild-type hepatocyte.

34. The engineered cell of any one of the preceding claims, wherein the engineered cell comprises a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, or an embryonic stem cell.

35. The engineered cell of any one of the preceding claims, wherein the engineered cell is further not derived from a patient.

36. The engineered cell of any one of the preceding claims, wherein a primary hepatocyte or a wild-type hepatocyte comprises a human hepatocyte cell.

37. The engineered cell of any one of the preceding claims, wherein the engineered cell secretes at least 4x more albumin than a primary cell or a wild-type hepatocyte.

38. The engineered cell of any one of the preceding claims, wherein the engineered cell secretes at least 5x more Al AT than in a primary cell or a wild-type hepatocyte.

39. The engineered cell of any one of the preceding claims, wherein the engineered cell further comprises an expression cassette comprising a nucleic acid molecule encoding one or more functional polypeptides.

40. The engineered cell of any one of the preceding claims, wherein activity of the one or more functional polypeptides is equal to or greater in the engineered cell than in a primary hepatocyte or a wild-type hepatocyte.

41. The engineered cell of any one of the preceding claims, wherein the one or more functional polypeptides comprise green fluorescent protein (GFP).

42. The engineered cell of any one of the preceding claims, wherein the one or more functional polypeptides comprise an enzyme.

43. The engineered cell of any one of the preceding claims, wherein the one or more functional polypeptides improves functionality of the engineered cell.

44. The engineered cell of any one of the preceding claims, wherein the improved functionality of the engineered cell involves an enzymatic cellular activity.

45. The engineered cell of any one of the preceding claims, wherein the enzymatic cellular activity comprises metabolizing xenobiotics.

46. The engineered cell of any one of the preceding claims, wherein the one or more functional polypeptides comprises CYP3A4.

47. The engineered cell of any one of the preceding claims, wherein the engineered cell expresses one or more hepatocyte or hepatocyte-like markers comprising SLC40A1, ARG1, HAL, ALDH6A1, APOM, ATP7B, CYP3A4 activity, AIAT production, or any combination thereof.

48. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBPA, F0XA1, and GATA6.

49. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors consists of HNF4A, HNF1A, CEBPA, F0XA1, and GATA6.

50. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1 A, CEBPA, FOXA1, GATA6, and FOXA2.

51. The engineered cell of any one of the preceding claims, wherein the one or more transcription factors consists of HNF4A, HNF1A, CEBPA, FOXA1, GATA6, and FOXA2.

52. A pharmaceutical composition comprising the engineered cell of any one of the preceding claims.

53. The pharmaceutical composition of claim 52, further comprising an excipient.

54. A method of treating a liver condition in a subject in need thereof, the method comprising administering the engineered cell of any one of the preceding claims to the subject in need thereof, thereby treating the liver condition.

55. A method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise HNF1A, HNF4A, and FOXA1;inducing expression of the one or more transcription factors, thereby generating the engineered cell, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte.

56. The method of claim 55, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

57. The method of claim 55 or 56, wherein the engineered cell comprises a pluripotent stem cell (PSC).

58. The method of any one of the preceding claims, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, or a hepatocyte or hepatocytelike cell derived from a human stem cell.

59. The method of any one of the preceding claims, wherein the engineered cell is configured to perform ion transport at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses a greater amount of SLC40A1 relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

60. The method of any one of the preceding claims, wherein the engineered cell is configured to perform copper transport at the rate equal to or greater than a primary hepatocyte or a wildtype hepatocyte and the cell expresses an equal or greater amount of ATP7B relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

61. The method of any one of the preceding claims, wherein the engineered cell is configured to perform urea cycle at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ARG1 relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

62. The method of any one of the preceding claims, wherein the engineered cell is configured to perform histidine metabolism at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of HAL relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

63. The method of any one of the preceding claims, wherein the engineered cell is configured to perform alcohol metabolism at the rate equal to or greater than a primary hepatocyte or a wildtype hepatocyte and the cell expresses an equal or greater amount of ALDH6A1 relative to an amount of expressed by the primary hepatocyte or the wild-type hepatocyte.

64. The method of any one of the preceding claims, wherein the engineered cell is configured to perform transporting lipoproteins at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of APOM relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

65. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise GATA6.

66. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise F0XA2.

67. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise CEBPA.

68. The method of any one of the preceding claims, wherein the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

69. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more HNF family members.

70. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more FOX family members.

71. The method of any one of the preceding claims, further comprising at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

72. The method of any one of the preceding claims, further comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

73. The method of any one of the preceding claims, wherein the one or more additional transcription factors further comprise one or more transcription factors from Table 3.

74. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBPA, FOXA1, and GATA6.

75. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1 A, CEBPA, FOXA1, and GATA6.

76. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2.

77. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1A, CEBPA, F0XA1, GATA6, and F0XA2.

78. A method of treating a disease characterized by a liver dysfunction in a subject in need thereof, the method comprising: administering a pluripotent stem cell (PSC) engineered to express one or more nucleic acid molecules comprising an open reading frames encoding one or more transcription factors, one or more transcription factors, or activator of transcription of the open reading frame encoding the one or more transcription factors, to the subject in need thereof, wherein the one or more transcription factors comprise HNF1 A, HNF4A, and F0XA1.

79. The method of claim 78, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell (iPSC), a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, an engineered cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

80. The method of any one of the preceding claims, wherein the PSC improves oxygen rate consumption compared to an uninduced cell type.

81. The method of any one of the preceding claims, wherein the PSC improves maximal oxygen rate consumption compared to an uninduced cell type.

82. The method of any one of the preceding claims, wherein the PSC improves spare respiratory capacity (SRC) compared to an uninduced cell type.

83. The method of any one of the preceding claims, wherein the liver dysfunction comprises: a urea cycle disorders, a methylmalonic acidemia, a propionic acidemia, a glycogen storge disease type 1, a familiar hypercholesterolemia, or any combination thereof.

84. The method of any one of the preceding claims, wherein the PSC increases expression of urea cycle enzymes.

85. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise GATA6.

86. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise FOXA2.

87. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise CEBPA.

88. The method of any one of the preceding claims, wherein the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

89. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more HNF family members.

90. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more FOX family members.

91. The method of any one of the preceding claims, further comprising at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

92. The method of any one of the preceding claims, further comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

93. The method of any one of the preceding claims, wherein the one or more additional transcription factors further comprise one or more transcription factors from Table 3.

94. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBP A, FOXA1, and GATA6.

95. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1 A, CEBP A, FOXA1, and GATA6.

96. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBP A, FOXA1, GATA6, and FOXA2.

97. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1A, CEBP A, FOXA1, GATA6, and FOXA2.

98. The method of any one of the preceding claims, wherein the PSC is a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

99. The method of any one of the preceding claims, wherein the PSC is not derived from the subject in need thereof.

100. The method of any one of the preceding claims, wherein the PSC is derived from the subject in need thereof.

101. A method for preparing a population of cells for infusion into a subject in need thereof, wherein the population of cells comprises pluripotent stem cells (PSCs), wherein the PSCs are engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprising HNF1 A, HNF4A, and FOXA1, wherein the method comprises: a) at least a first passage of the PSCs; b) maintaining the PSCs on ice or at 0° C, and further passaging the PSCs at least one time; or c) cryopreserving the PSCs at less than 0° C and thawing the PSCs; d) wherein after (a), (b), or (c), the PSCs secrete albumin at a rate of at least 4pg / day / million cells, wherein the PSCs produce hepatocytes or hepatocyte-like cells.

102. The method of claim 101, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, an engineered cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

103. The method of any one of the preceding claims, wherein the PSCs are maintained on ice or at 0°C for at least 6 hours prior to the first passage of the PSCs.

104. The method of any one of the preceding claims, wherein the PSCs are plated or cultured for 24 hours after (a), (b), or (c).

105. The method of any one of the preceding claims, wherein the PSCs comprise distinct hepatocyte or hepatocyte-like morphology.

106. The method of any one of the preceding claims, wherein the PSCs comprise at least 80% viable cells after (a), (b), (c), or any combination thereof.

107. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise GATA6.

108. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise FOXA2.

109. The method of any one of the preceding claims, wherein the one or more transcription factors further comprise CEBPA.

110. The method of any one of the preceding claims, wherein the one or more transcription factors comprise one or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

111. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more HNF family members.

112. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more FOX family members.

113. The method of any one of the preceding claims, further comprising at least 2 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

114. The method of any one of the preceding claims, further comprising at least 3 or more additional transcription factors comprising: a FOS, JUN, FOX, PROX, GATA, HHEX, HIF, HNF, CEBP, MYC, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member.

115. The method of any one of the preceding claims, wherein the one or more additional transcription factors further comprise one or more transcription factors from Table 3.

116. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBPA, FOXA1, and GATA6.

117. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1 A, CEBPA, FOXA1, and GATA6.

118. The method of any one of the preceding claims, wherein the one or more transcription factors comprise HNF4A, HNF1A, CEBPA, FOXA1, GATA6, and FOXA2.

119. The method of any one of the preceding claims, wherein the one or more transcription factors consist of HNF4A, HNF1 A, CEBPA, FOXA1, GATA6, and FOXA2.

120. A method of generating an engineered cell, wherein the method comprises: contacting a pluripotent stem cell (PSC) with an expression cassette comprising one or more nucleic acidmolecules encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member; inducing expression of the expression cassette, thereby generating an engineered cell, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte.

121. The method of claim 120, wherein the engineered cell is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

122. The method of any one of the preceding claims, wherein the engineered cell is configured to perform ion transport at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of SLC40A1 relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

123. The method of any one of the preceding claims, wherein the engineered cell is configured to perform copper transport at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ATP7B relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

124. The method of any one of the preceding claims, wherein the engineered cell is configured to perform urea cycle at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ARG1 relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

125. The method of any one of the preceding claims, wherein the engineered cell is configured to perform histidine metabolism at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of HAL relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

126. The method of any one of the preceding claims, wherein the engineered cell is configured to perform alcohol metabolism at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of ALDH6A1 relative to an amount of expressed by the primary hepatocyte or the wild-type hepatocyte.

127. The method of any one of the preceding claims, wherein the engineered cell is configured to perform transporting lipoproteins at the rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte and the cell expresses an equal or greater amount of APOM relative to an amount expressed by the primary hepatocyte or the wild-type hepatocyte.

128. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors further comprise 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.

129. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more HNF family members.

130. The engineered cell of any one of the preceding claims, wherein the one or more additional transcription factors comprise 2 or more FOX family members.

131. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise FOXA1.

132. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise FOXA2.

133. The method of any one of the preceding claims, wherein the one or more additional transcription factors comprise CEBP A.

134. A pluripotent stem cell (PSC), wherein the PSC is engineered to express one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame encoding the one or more transcription factors, wherein the one or more transcription factors comprise: GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member, wherein the PSC is configured to perform one or more hepatocyte functions at a rate equal to or greater than a primary hepatocyte or a wild-type hepatocyte.

135. The PSC of claim 134, wherein the PSC is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

136. The method of claim 134 or 135, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetalstem cell, a hepatic stem cell, an embryonic stem cell, an engineered cell or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

137. A method of treating a disease characterized by a liver dysfunction in a subject in need thereof, the method comprising: administering a pluripotent stem cell (PSC) comprising one or more nucleic acid molecules comprising an open reading frame encoding one or more transcription factors, one or more transcription factors, or an activator of transcription of the open reading frame, to the subject in need thereof, wherein the one or more transcription factors comprise: GATA6 and additional one or more transcription factors selected from: a FOS, JUN, FOX, PROX, GATA, HNF, HHEX, HIF, MYC, CEBP, NFE, NFI, NR, RBPJ, RXR, SMAD, SOX, SPI, TBX, TP, PPAR, SALL, EGR, ONECUT, SKI or HLF family member; and wherein the PSC is configured to perform one or more hepatocyte functions at a rate greater than or equal to a primary hepatocyte or a wild-type hepatocyte.

138. The method of claim 137, wherein the PSC is configured to perform one or more hepatocyte functions at a rate greater than a primary hepatocyte or a wild-type hepatocyte.

139. The method of claim 137 or 138, wherein the PSC comprises a mammalian stem cell, a human stem cell, a human induced pluripotent stem cell, a human progenitor cell, a human fetal stem cell, a hepatic stem cell, an embryonic stem cell, an engineered cell, or a hepatocyte or hepatocyte-like cell derived from a human stem cell.

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