Preparation of hepatocytes

Abstract

The present invention relates to a method for producing hepatocytes, comprising introducing a set of transcription factors consisting of HNF1A;HNF6;FOXA3;RORc and ERa into an IPSC population and culturing the population, whereby hepatocytes are produced. Provided are methods for producing hepatocytes, hepatocytes produced by the methods, and uses and applications thereof.

Description

[Technical field]

[0001] funding The project leading to this application has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 741707).

[0002] Field The present invention relates to methods for producing hepatocytes, hepatocytes produced by such methods and uses and applications of such hepatocytes. [Background technology]

[0003] Hepatocytes are the major cell type in the liver, accounting for 80% of its volume and performing a myriad of vital functions including lipid metabolism, macronutrient storage, plasma protein secretion, and xenobiotic detoxification (Gordillo et al., 2015; Si-Tayeb et al., 2010; Trefts et al., 2017).

[0004] Diseases affecting these functions are life-threatening and in end-stage forms necessitate liver transplantation, however, due to donor shortages and the side effects of immunosuppression, only a limited number of patients can benefit from this therapy.

[0005] Cell-based therapy using primary hepatocytes has already been found to be an attractive therapeutic option to whole organ transplantation (Dhawan et al., 2020). However, primary human hepatocytes (PHHs) are scarce because they can only be obtained from suboptimal livers that are unsuitable for transplantation. Moreover, PHHs exhibit short life span, lack of proliferation, and rapid loss of function in vitro (Mitry et al., 2002). Similarly, the development of novel platforms for drug discovery and drug screening tests is also greatly affected by the lack of a robust hepatocyte source. For all these reasons, there is an urgent need for alternative hepatocyte sources.

[0006] The generation of hepatocytes from human pluripotent stem cells (hPSCs) using directed differentiation protocols has been shown to be an advantageous alternative to PHHs (Palakkan et al., 2017; Szkolnicka & Hay, 2016; Silier et al., 2015; Hay et al., 2008). These protocols typically follow key hepatic developmental stages in vitro and allow the generation of hepatocyte-like cells (HLCs) that exhibit key hepatic functions including albumin secretion, lipid metabolism, glycogen storage, and urea cycle activity. However, HLCs systematically display an immature / fetal-like phenotype that lacks the full functional repertoire of mature hepatocytes (Baxter et al., 2015; Grandy et al., 2019; Yiangou et al., 2018). The development of fully functional hepatocytes in vitro remains a challenge due to the lack of detailed knowledge regarding the molecular mechanisms that drive functional maturation in vivo. Indeed, this process occurs gradually during fetal life but also after birth, taking almost 12 months for the liver to become functional. Mimicking this timeline and the associated combination of metabolic changes, exposure to oxygen, nutrients and the microbiome represents a major challenge for any directed differentiation protocol (Chen et al., 2011).

[0007] As an alternative, overexpression of transcription factors has been investigated as a way to improve the functionality of in vitro generated hepatocytes (Boon et al., 2020; Nakamori et al., 2016; Zhao et al., 2013). Moreover, transdifferentiation of somatic cells into hepatocytes has been achieved by overexpression of liver-enriched transcription factors (LETFs) in mouse and human fibroblasts (Rombaut et al., 2021). Importantly, these LETFs include the HNF1, HNF3 (FOXA), HNF4 and HNF6 (ONECUT) families, all of which play major roles in coordinating liver development (Gordillo et al., 2015; Lau et al., 2018; Schrem et al., 2002). However, direct cell conversion from somatic cell types has low efficiency / yield due to the intense epigenetic restrictions found in fully differentiated cells. Furthermore, the limited proliferative capacity of somatic cells limits the large-scale production of hepatocytes without oncogene manipulation ( Du et al., 2014 ; Huang et al., 2014 ). Summary of the Invention [Means for solving the problem]

[0008] The present inventors have developed a method for generating functionally mature hepatocytes (referred to herein as FoP-Heps) by forward programming of human pluripotent cells, which may be useful, for example, in the efficient generation of functional hepatocytes for use in modeling liver injury; and in the development of therapeutics for liver injury.

[0009] A first aspect of the present invention is a method for producing hepatocytes, comprising the steps of: (i) providing a population of iPSCs; (ii) introducing a set of transcription factors consisting of HNF1A; HNF6; FOXA3; RORc and ERα into the iPSC population; and (iii) culturing the population, thereby generating hepatocytes. A method is provided.

[0010] A second aspect of the invention is a method for forward programming iPSCs into hepatocytes, comprising the steps of: Introducing a set of transcription factors consisting of HNF1A; HNF6; FOXA3; RORc and ERα into the iPSC population; and and culturing the population, thereby generating hepatocytes. A method is provided.

[0011] A third aspect of the present invention is a method for producing hepatocytes, comprising the steps of: (i) providing a population of iPSCs; (ii) introducing into the iPSC population a set of transcription factors consisting of HNF1A; HNF6; FOXA3; and RORc; and (iii) culturing the population, thereby generating hepatocytes. A method is provided.

[0012] A fourth aspect of the invention is a method for forward programming iPSCs into hepatocytes, comprising the steps of: Introducing a set of transcription factors consisting of HNF1A; HNF6; FOXA3; and RORc into the iPSC population; and and culturing the population, thereby generating hepatocytes. A method is provided.

[0013] A fifth aspect of the invention provides a population of hepatocytes produced by a method according to the first, second, third or fourth aspect.

[0014] A sixth aspect of the invention provides a pharmaceutical composition comprising a population of liver cells according to the fifth aspect and a pharma- ceutically acceptable excipient.

[0015] A seventh aspect of the invention provides a population of liver cells according to the fifth aspect for use in a method of treatment of the human or animal body, such as for use in a method of treatment of liver damage in an individual.

[0016] An eighth aspect of the invention is a method of treating liver damage comprising the steps of: Administering the population of hepatic cells according to the fifth aspect to an individual in need thereof. The present invention provides a method comprising:

[0017] A ninth aspect of the invention provides the use of a population of liver cells according to the fifth aspect in the manufacture of a medicament for use in the treatment of liver damage.

[0018] A tenth aspect of the present invention provides a method of screening for a compound useful in the treatment of liver damage, comprising the steps of: contacting a population of liver cells according to the fifth aspect with a test compound; and Determining the effect of a test compound on said hepatocytes or the effect of hepatocytes on a test compound. The present invention provides a method comprising:

[0019] In some embodiments, hepatocytes for use in the method of the tenth aspect may have a diseased phenotype, such as a liver damaged phenotype.

[0020] An eleventh aspect of the present invention provides a method for determining the hepatotoxicity of a compound, comprising the steps of: contacting an isolated hepatocyte cell according to the fifth aspect with a test compound; and Determining the effect of a test compound on said hepatocytes. The present invention provides a method comprising:

[0021] A twelfth aspect of the present invention provides a method for identifying a transcription factor that promotes hepatocyte maturation, comprising the steps of: To determine the expression of a set of transcription factors in primary human hepatocytes (PHHs) and hepatocyte-like cells (HLCs) generated by in vitro directed differentiation; To identify a set of transcription factors that are upregulated in PHH compared to CLC Including, The identified transcription factor is a candidate transcription factor for promoting hepatocyte maturation. A method is provided.

[0022] A thirteenth aspect of the present invention provides a method for identifying a genetic mutation associated with liver damage, comprising the steps of: providing a test hepatocyte population of the fifth aspect, wherein the hepatocytes in the test population each comprise a genetic mutation; phenotypically comparing the test hepatocyte population with a control hepatocyte population, the control population being free of the genetic mutation; and Identifying hepatocytes that exhibit a disease phenotype, such as a liver injury phenotype, within a study population Including, The identified gene mutations in hepatocytes are candidate gene mutations associated with liver damage, A method is provided.

[0023] A fourteenth aspect of the present invention provides a method for identifying a genetic mutation associated with liver damage, comprising the steps of: Providing a test hepatocyte population of the fifth aspect, wherein hepatocytes in the test population exhibit a disease phenotype, such as a hepatic injury phenotype; and comparing the test hepatocyte population with a control hepatocyte population for genomic sequence, wherein the hepatocytes in the control population do not exhibit a disease phenotype; and Identifying one or more genetic mutations in the genomic sequence of a test population compared to a control population Including, one or more of the identified gene mutations are candidate gene mutations associated with liver damage; A method is provided.

[0024] A fifteenth aspect of the present invention provides a method for identifying a gene associated with liver damage, comprising the steps of: Providing a test hepatocyte population of the fifth aspect, wherein hepatocytes in the test population exhibit a disease phenotype, such as a hepatic injury phenotype; and Comparing the expression of one or more genes in the test hepatocyte population with the expression of one or more genes in a control hepatocyte population. Including, A difference in expression of a gene in the test population compared to the control population indicates that the gene is associated with liver damage. A method is provided.

[0025] Test liver cell populations suitable for use in the methods of the thirteenth to fifteenth aspects may be generated by the methods of the first to fourth aspects from induced pluripotent stem cells (iPSCs) derived from an individual with liver damage.

[0026] A sixteenth aspect of the present invention is a kit for producing hepatocytes, comprising: (i) an agent that activates or increases the expression or amount of an iPSC-source cell and at least three or more transcription factors; and / or (ii) one or more nucleic acids encoding a set of transcription factors consisting of HNF1A; HNF6; FOXA3; RORc, and optionally Erα. A kit comprising:

[0027] The kit of the sixteenth aspect may be useful, for example, in the methods of the first to fourth aspects.

[0028] Other aspects and embodiments of the invention are described in further detail below. [Brief description of the drawings]

[0029] [Figure 1]Forward programming of hPSCs into hepatocytes with four and three liver-enriched transcription factors (LETFs). (A) Schematic representation of the two sequentially targeted loci. Human ROSA26 was targeted with a constitutively expressed reverse tetracycline transactivator (rtTA). The AAVS1 locus was targeted with four LETFs downstream of the Tet response element (TET): HNF1A, HNF6, FOXA3, and HNF4A. (B) mRNA induction levels of the four factors in targeted hESCs (Targ) compared to untargeted (Untarg) hESCs stimulated with dox for 24 h (n=3). Data are shown relative to the untargeted control. (C) Immunofluorescence staining of the four LETFs in targeted and untargeted hESCs after 24 h of inducible overexpression (iOX) with dox confirming transgene induction. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm. (D) Schematic representation of iOX culture conditions for forward programming. Phase contrast images of hESCs targeted with four LETFs after 10 and 15 days of forward programming. (E) mRNA levels of hepatocyte markers (ALB, SERPINA1 and AFP) in hESCs targeted with four LETFs after 10 and 15 days of forward programming. Non-targeted hESCs treated with the same protocol as in (D) were used as controls (n=4). Statistical differences were calculated by unpaired t-test against non-targeted, and p-values ​​are displayed for each comparison. (F) CYP3A4 activity levels normalized to cell number (million) in non-targeted and targeted hESCs (n=5) with four LETFs after 15 days of forward programming and HLCs (n=6) generated by direct differentiation. Statistical differences between targeted and non-targeted cells were calculated by unpaired t-test. (G, H, I) mRNA levels of hepatocyte markers (ALB, SERPINA1 and AFP) in hESCs targeted with four LETFs and a combination of three LETFs (n=4). Factors omitted from each construct are indicated. Expression levels were determined after 10, 15, 20 and 25 days of forward programming. Statistical differences were calculated by one-way ANOVA with correction for multiple comparisons compared to the four LETFs. Significant p-values ​​at each time point are indicated.All mRNA levels were normalized to the average of two housekeeping genes (PBGD and RPLP0). (J) CYP3A4 activity levels (n=3–5) normalized to cell number (million) in hESCs targeted with four LETFs and combinations of three LETFs after 10, 15, 20 and 25 days of forward programming. Statistical differences were calculated by one-way ANOVA with correction for multiple comparisons compared to the four LETFs. Significant p-values ​​at each time point are indicated. In all plots, bars represent the mean and SD, and individual data points are shown for all biological replicates. [Diagram 2]Figure 1 shows that HLCs and PHHs exhibit transcriptomic differences related to their maturation state. (A) Immunofluorescence staining of albumin (yellow) and HNF4A (red) in HLCs differentiated for 30 days. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. (B) CYP3A4 activity levels normalized to cell number (million) in HLCs (n=6) and PHHs (n=4) differentiated for 30 days. Bars represent mean and SD, and individual data points represent different biological replicates. Statistical differences were calculated by unpaired t-test. (C) PCA of HLCs derived from undifferentiated hiPSCs, hESCs (hESC_HLCs) and hiPSCs (hiPSC_HLCs), PHHs immediately after harvest (fPHHs) or after plating (pPHHs). (D) Heatmap showing the proportion of differentially expressed genes in each cell type (cluster 1-PHH, cluster 2-HLC, cluster 4-hiPSC) and Hep (HLC and PHH) versus undifferentiated hiPSC (cluster 3). (E,F) Dot plots showing the top 15 hits in gene ontology enrichment analysis for genes linked to cluster 1 and cluster 3 as shown in (D). The size of each dot represents the number of genes linked to each term and the color represents the adjusted p-value. (G) Heatmap showing differential transcription factor gene expression between PHH (immediately or after plating) and HLC (hESC and hiPSC derived). (H) Reactome pathway enrichment analysis for the transcription factors identified in (G). Differential gene expression was calculated as log2 (fold change) >2 and adjusted p-value <0.05. Hierarchical clustering for samples was created by Euclidean distance. [Diagram 3]We show that the epigenetic state of regulatory regions differs between the mature states of HLCs and PHHs. (A) PCA of global enrichment profiles of H3K27ac, H3K4me1 and H3K27me3 across undifferentiated hiPSCs, hESCs and hiPSC-derived HLCs, and two replicates of PHHs. The average score of 1000 bp genomic regions was calculated for the whole genome. (B) Average density plots and heatmaps showing enrichment levels of H3K27ac, H3K4me1 and H3K27me3 within 10 Kb windows centered on H3K27ac PHH unique regions (blue) or HLC unique regions (green). Scale is adjusted to the maximum peak intensity for each dataset. (C) Enrichment profiles of H3K27ac, H3K4me1 and H3K27me3 across the UGT1A locus. Profiles are shown for undifferentiated hiPSCs, hESCs and hiPSC-derived HLCs, and one replicate of PHHs. Red bars represent H3K27ac peaks unique to PHHs. (D) Nuclear receptor motifs identified as overrepresented binding sites in H3K27ac PHH-unique regions. [Figure 4]Forward programming of hESCs with nuclear receptors into hepatocytes. (A) Phase contrast images and (B) albumin (yellow) and (C) A1AT (green) immunofluorescence staining in hESCs forward programmed with 3TF alone or in combination with nuclear receptors RORc, ERa, and AR for 20 days. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm. (D) mRNA levels of hepatocyte markers (ALB, SERPINA1, and AFP) in FoP-Heps generated with 3TF alone or in combination with nuclear receptors for 20 and 30 days (n=4). Expression data were normalized to the average of two housekeeping genes (PBGD and RPLP0). (E) Protein secretion levels of albumin, A1AT, and AFP in hESC-derived FoP-Heps generated with 3TF alone or in combination with nuclear receptors for 20 days (n=4). Data were normalized to total cell number (million). (F) CYP3A4 activity levels normalized by cell number (million) in FoP-Heps targeted by 3TF with and without nuclear receptors after 20 and 30 days of forward programming (n=3-6). Statistical differences were calculated by one-way ANOVA with correction for multiple comparisons compared to 3TF (day 20). Significant p-values ​​are indicated. (G) CYP3A4 fold induction levels in FoP-Heps treated with 100 nM of ligand as indicated from day 2. Data are normalized to untreated control at day 20 of forward programming (n=3). Significant p-values ​​for paired t-test are indicated. In all plots, bars represent mean and SD, and individual data points are shown for all biological replicates. [Diagram 5]Forward programming of hiPSCs into hepatocytes with 4TF. (A) Phase contrast images and (B) albumin (yellow) and (C) A1AT (green) immunofluorescence staining in hiPSCs forward programmed with 3TF alone or in addition with RORc for 20 days. Nuclei were counterstained with DAPI (blue). Scale bar, 200 μm. (D) mRNA levels of hepatocyte markers (ALB, SERPINA1, and AFP) in hiPSC-derived FoP-Heps generated with 3TF alone or in addition with RORc for 20 and 30 days (n=4). Statistical differences were calculated by unpaired t-test, and significant p-values ​​are indicated. All expression data were normalized to the mean of two housekeeping genes (PBGD and RPLP0). (E) Protein secretion levels of albumin, A1AT and AFP in hiPSC-derived FoP-Heps generated with 3TF alone or in addition with RORc for 20 days (n=4). Data were normalized to total cell number (million). (F) CYP3A4 activity levels normalized to cell number (million) in hiPSC FoP-Heps targeted by 3TF with or without RORc after 20 days of forward programming (n=6). Statistical differences were calculated by unpaired t-test. In all plots, bars represent the mean and SD, and individual data points are shown for all biological replicates. [Figure 6]We show that RORc promotes the functionality of 4TF FoP-Heps. (A) Comparison of CYP3A4 activity levels in FoP-Heps (n=6) versus directly differentiated HLCs (n=6) and PHHs (n=4). hESC (eFoP) and hiPSC (iFoP) derived FoP-Heps were targeted with 4TFs (HNF1A, FOXA3, HNF6 and RORy) and forward programmed for 20 days. Statistical differences between HLCs and FoP-Heps were calculated by unpaired t-test. (B) mRNA levels of phase I (CYP2A6 and CYP2C8) and phase II (UGT1A6) biotransformation enzymes in 4TF FoP-Heps, HLCs and PHHs (n=4). (C) mRNA levels of gluconeogenesis (G6PC and PCK1), lipid (PPARα, PPARy) metabolism, and nuclear receptor RORy in FoP-Heps, HLC, and PHH (n=4). Statistical differences between HLC and other cell types were calculated by unpaired t-test. (D) LDL immunofluorescence staining in FoP-Heps at day 20 of forward programming. Scale bar, 200 μm. Nuclei were counterstained with DAPI (blue). (E) Comparison of mRNA levels of SERPINA1 and UGT1A6 in FoP-Heps cultured in 2D and 3D for up to 20 or 30 days of forward programming (n=4). Statistical differences between 2D and 3D were calculated by unpaired t-test. All expression data were normalized to the mean of two housekeeping genes (PBGD and RPLP0). (F) BODIPY staining of FoP-Heps cultured in 3D from day 20 of forward programming and treated with fatty acids as indicated (oleic acid [OA], palmitic acid [PA] or BSA [Ctr]) for 7 days. Scale bar, 200 μm. Nuclei were counterstained with DAPI (blue). (G) Cell viability in FoP-Heps treated with fatty acids as indicated, normalized to FoP-Heps treated with BSA as control (n=4). (H) Cell viability in FoP-Heps treated with 25 mM acetaminophen (APAP) for 48 h in 3D culture, normalized to untreated FoP-Heps (n=4). Significant differences were determined by paired t-test.p values ​​are indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. In all plots, bars represent the mean and SD, and individual data points are shown for all biological replicates. [Figure 7] Figure 2 shows that ERα promotes the functionality of 5TF FoP-Heps. (A) Schematic representation of factor combinations cloned into the AAVS1 locus. (B) Phase contrast images in hiPSCs forward programmed with 5TF alone or in addition with estrogen (E2) for 20 days. (C) mRNA levels of hepatocyte markers (ALB, SERPINA1 and AFP) in hiPSC-derived FoP-Heps generated with 4TF, 5TF or 5TF+E2 for 20 days (n=4). Statistical differences were calculated by unpaired t-test and significant p-values ​​are indicated. All expression data were normalized to the mean of two housekeeping genes (PBGD and RPLP0). (D) mRNA levels of phase I (CYP2A6 and CYP2C8) and phase II (UGT1A6) biotransformation enzymes and gluconeogenesis enzymes (G6PC and PCK1) in 4TF, 5TF and 5TF+E2 FoP-Heps (n=4). (E) Comparison of CYP3A4 activity levels in 20-day forward-programmed FoP-Heps generated with 4TF, 5TF, or 5TF+E2. (F) BODIPY staining of 5TF-generated FoP-Heps cultured in 3D from day 20 of forward programming and treated with fatty acids as indicated (oleic acid [OA], palmitic acid [PA], or BSA [Ctr]) for 7 days. Scale bar, 200 μm. Nuclei were counterstained with DAPI (blue). (G) Cell viability in 5TF-generated FoP-Heps treated with fatty acids as indicated (n=4), normalized to FoP-Heps treated with BSA as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description The present invention relates to forward programming of pluripotent cells to hepatocytes. A set of transcription factors consisting of HNF1A; HNF6; FOXA3; RORc and optionally ERα is introduced into pluripotent cells. The pluripotent cells are then cultured. The set of transcription factors introduced into the cells confers a mature hepatocyte phenotype to the pluripotent cells, i.e., the pluripotent cell population is forward programmed by the set of transcription factors into functionally mature hepatocytes. Such hepatocytes may be useful, for example, in disease modeling, drug screening and therapeutic methods.

[0031] Forward programming directly confers a more differentiated phenotype on pluripotent stem cells or other progenitor cells, bypassing the normal differentiation pathway; i.e., cells do not pass progressively through intermediate differentiation steps. For example, pluripotent stem cells (PSCs) forward programmed to hepatocytes do not sequentially differentiate through endoderm, foregut, hepatoblast, and fetal liver cell stages before exhibiting a mature hepatocyte phenotype. Forward programming by direct overexpression of transcription factors in PSCs has been successfully used to generate neurons, skeletal muscle cells, and oligodendrocytes (Pawlowski et al., 2017).

[0032] Pluripotent stem cells (PSCs) have the ability to self-renew in vitro, exhibit an undifferentiated phenotype, and have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, and endoderm) of any fetal or adult cell type. Pluripotent stem cells are distinct from totipotent stem cells and cannot give rise to extraembryonic cell lineages. A population of PSCs may be clonal, i.e., genetically identical cells that are descendants of a single common ancestral cell. PSCs may express one or more of the following pluripotency-associated markers: Oct4, Sox2, alkaline phosphatase, POU5f1, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4, and c-myc, preferably one or more of POU5f1, NANOG, and SOX2. PSCs may lack markers associated with specific differentiation fates, such as Bra, Sox17, FoxA2, ​​αFP, Sox1, NCAM, GATA6, GATA4, Hand1, and CDX2. In particular, PSCs may lack markers associated with an endoderm fate.

[0033] Preferably, the PSCs are human PSCs (hPSCs).

[0034] PSCs can include embryonic stem cells (ESCs) and non-embryonic stem cells, such as fetal stem cells, adult stem cells, amniotic stem cells, umbilical cord stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the PSCs are not human embryonic stem cells. In some embodiments, the PSCs are not human embryonic cells. Suitable techniques for generating PSCs are well known in the art.

[0035] Preferably, the PSCs are iPSCs, more preferably human iPSCs (hiPSCs).

[0036] iPSCs are pluripotent cells derived from non-pluripotent fully differentiated progenitor or ancestor cells. Suitable progenitor cells include somatic cells, such as adult fibroblasts and peripheral blood cells. Progenitor cells are typically reprogrammed by introducing pluripotency genes or proteins, such as Oct4, Sox2 and Sox1, into the cells. Such genes or proteins can be introduced into differentiated cells by any suitable technique, including plasmid or, more preferably, viral transfection or direct delivery of proteins. To increase induction efficiency, other genes can also be introduced into cells, such as Klf genes, such as Klf-1, -2, -4 and -5; Myc genes, such as C-myc, L-myc and N-myc; nanog; and Lin28. After introduction of pluripotency genes or proteins, the progenitor cells can be cultured. Cells expressing pluripotency markers can be isolated and / or purified to generate iPSC populations. Techniques for producing iPSCs are well known in the art (Yamanaka et al Nature 2007;448:313-7; Yamanaka 6 2007 Jun 7;1(1):39-49; Kim et al Nature.2008 Jul 31;454(7204):646-50; Takahashi Cell.2007 Nov 30;131(5):861-72. Park et al Nature.2008 Jan 10;451(7175):141-6; Kimet et al Cell Stem Cell.2009 Jun 5;4(6):472-6; Vallier, L., et al. Stem Cells,2009.9999(999A):pN / A).

[0037] iPSCs for use in the present methods may be derived from somatic cells, such as fibroblasts or blood cells, having a normal (i.e., non-disease-associated) genotype, e.g., cells obtained from an individual of normal genetic background, e.g., an individual without a genetic disorder. Such iPSCs may be used to generate hepatocytes of normal (i.e., non-disease-associated) genotype, for use in, for example, therapeutic, modeling, screening, or other applications.

[0038] iPSCs for use in some embodiments of the method may be derived from somatic cells or other progenitor cells obtained from individuals with distinctively different genetic backgrounds. For example, iPSCs may be generated from cells from individuals with disease states, individuals with high risk of disease states, and / or individuals with low risk of disease states. Disease states may include liver disorders, such as hepatopathy or other liver-related disorders. iPSCs generated from cells obtained from individuals with distinctively different genetic backgrounds may be used to generate hepatocytes with that genetic background, which may be useful for studying the mechanisms of disease states, such as liver disorders, and for identifying therapeutic targets.

[0039] Conventional techniques may be used for the culture and maintenance of PSCs (Vallier, L. et al Dev. Biol. 275, 403-421 (2004); Cowan, CA et al. N. Engl. J. Med. 350, 1353-1356 (2004); Joannides, A. et al. Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005); Ludwig, TE et al. Nat. Biotechnol. 24, 185-187 (2006)). PSCs for use in the present methods may be grown under defined conditions or on feeder cells. For example, PSCs are conventionally grown at an appropriate density (e.g. 10 5 ~10 6 Pluripotent cells for use in the present method may be cultured on a layer of feeder cells such as irradiated mouse embryonic fibroblasts (MEFs) (1000 cells / 60 mm culture dish) or on a suitable substrate containing feeder-conditioned or defined medium. Pluripotent cells for use in the present method may be passaged by enzymatic or mechanical means.

[0040] In a preferred embodiment, PSCs for use in the present methods may be cultured in a chemically defined culture medium.

[0041] A chemically defined medium is a nutrient solution for culturing cells that contains only specified components, preferably components of known chemical structure. A chemically defined medium lacks undefined or undefined components, such as feeder cells, stromal cells, serum, serum albumin, and complex extracellular matrices, such as Matrigel™. In some embodiments, a chemically defined medium is humanized. A humanized chemically defined medium lacks components or supplements derived from or isolated from non-human animals, such as fetal bovine serum (FBS) and bovine serum albumin (BSA), and mouse or other feeder cells. Proteins in humanized CDM may be recombinant human proteins. Conditioned media contain undefined components from cultured cells and are not chemically defined. Suitable chemically defined media are well known in the art and are described in more detail below. Culture media and its ingredients may be obtained from commercial sources (e.g., Gibco, Roche, Sigma, Europabioproducts, Cellgenix, Life Sciences).

[0042] In some embodiments, the chemically defined medium may include a chemically defined basal medium supplemented with serum-free media supplements and / or one or more additional components, for example, transferrin, 1-thioglycerol, defined lipids, substitutes such as L-glutamine or GlutaMAX-1™, nicotinamide, dexamethasone, selenium, pyruvate, buffers such as HEPES, sodium bicarbonate, glucose, and antibiotics such as penicillin and streptomycin, and optionally polyvinyl alcohol; polyvinyl alcohol and insulin; serum albumin; or serum albumin and insulin. Suitable chemically defined basal media, such as improved Dulbecco's Modified Eagle's Medium (DMEM) (Price et al Focus (2003) 25 3-6), Iscove's Modified Dulbecco's Medium (IMDM), William's E medium and RPMI-1640 (Moore, GE and Woods LK, (1976) Tissue Culture Association Manual. 3, 503-508; see Table 3), are known in the art and available from commercial sources (e.g., Sigma-Aldrich MI USA; Life Technologies USA). Other suitable chemically defined basal media are also known in the art and available from commercial sources (e.g., Sigma-Aldrich MI USA; Life Technologies USA). Suitable serum-free media supplements include B27 (Brewer et al Brain Res (1989) 494 65-74; Brewer et al J. Neurosci Res 35 567-576 (1993); Brewer et al Focus 16 1 6-9; Brewer et al (1995) J. Neurosci. Res. 42:674-683; Roth et al J Trace Elem Med Biol (2010) 24 130-137) and NS21 (Chen et al J. Neurosci Meths (2008) 171 239-247).Serum-free media supplements, such as B27 and N21, are well known in the art and widely available commercially (eg, Invitrogen; Sigma Aldrich Inc.).

[0043] Suitable chemically defined media for use in culturing PSCs include E8 medium comprising DMEM / F12, pH adjusted with NaHCO3, supplemented with insulin, selenium, transferrin, L-ascorbic acid, FGF2, and TGFβ (or NODAL or activin) (Chen et al 2011 Nat Methods 8(5)424-U76); and E6 medium comprising DMEM / F12, pH adjusted with NaHCO3, supplemented with insulin, e.g., at a concentration of 0.5 μg / ml to 70 μg / ml, transferrin, e.g., at a concentration of 1.5 μg / ml to 150 μg / ml, L-ascorbic acid, e.g., at a concentration of 30 μg / ml to 120 μg / ml, FGF2 (Chen et al 2011 Nat Methods 8(5)424-U76).

[0044] Other suitable chemically defined media include CDM-PVA (Johansson and Wiles (1995) Mol Cell Biol 15, 141-151), which comprises a basal medium supplemented with polyvinyl alcohol, insulin, transferrin and defined lipids. For example, CDM-PVA medium can consist of 50% Iscove's Modified Dulbecco's Medium (IMDM) + 50% Ham's F12 with GlutaMAX-1™ or 50% F12 NUT-MIX (Gibco) supplemented with 1% chemically defined lipid concentrate, 450 μM 1-thiolglycerol, 15 μg / ml transferrin, 1 mg / ml polyvinyl alcohol, 7 μg / ml insulin. Other suitable chemically defined nutrient media include hESC Maintenance Medium (CDMA) identical to CDM-PVA described above, except that PVA is replaced with 5 mg / ml BSA; and RPMI basal medium supplemented with B27 and activin (e.g., at least 50 ng / ml). CDM-PVA medium is described in Vallier et al 2009 PLoS ONE 4:e6082.doi:10.1371; Vallier et al 2009 Stem Cells 27:2655-2666, Touboul 2010 51:1754-1765. Teo et al 2011 Genes & Dev. (2011) 25:238-250 and Peterson & Loring Human Stem Cell Manual: A Laboratory Guide (2012) Academic Press.

[0045] The PSC population may be cultured in the seeding medium for 12-36 hours, preferably about 24 hours, in the methods described herein. Suitable seeding medium includes E8 medium. The seeding medium may be supplemented with a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, for example, 1-100 μM ROCK inhibitor, such as 10 μM Y-27632.

[0046] In the methods described herein, PSCs are forward programmed to become hepatocytes through the introduction of a set of transcription factors into the PSCs, which increases the intracellular levels of the set of transcription factors in the PSCs and induces conversion of the PSCs in the population into hepatocytes.

[0047] Transcription factors are DNA-binding proteins that regulate the expression of genes in cells.Preferably, the transcription factors introduced into PSCs are human transcription factors.The set of transcription factors used in the method described herein for forward programming of PSCs to hepatocytes consists of HNF1A;HNF6;FOXA3;RORc and optionally ERα.For example, the set of transcription factors may consist of HNF1A;HNF6;FOXA3;and RORc;or the set of transcription factors may consist of HNF1A;HNF6;FOXA3;RORc and ERα.

[0048] Hepatocyte nuclear factor 1 homeobox A (HNF1A; Gene ID number 6927) is a liver-enriched transcription factor (LETF). HNF1A may have a reference amino acid sequence of NP_00536.6 or NP_001293108.2, and may be encoded by a reference nucleotide amino acid sequence of NM_00545.8 or NM_001306179.2.

[0049] Hepatocyte nuclear factor 6 (HNF6; gene ID 3175; also known as one cut homeobox 1; ONECUT1) is a liver-enriched transcription factor (LETF). HNF6 may have a reference amino acid sequence of NP_004489.1 and may be encoded by a reference nucleotide amino acid sequence of NM_004498.4.

[0050] Forkhead box A3 (FOXA3; Gene ID: 3171; also known as hepatocyte nuclear factor 3-gamma HNF3G) is a liver-enriched transcription factor (LETF). FOXA3 may have a reference amino acid sequence of NP_004488.2 and may be encoded by a reference nucleotide amino acid sequence of NM_004497.3.

[0051] RAR-related orphan receptor C (RORc; gene ID: 6097) is a nuclear transcription factor that is mainly expressed in immune cells. RORc may have a reference amino acid sequence of NP_001001523.1 or NP_005051.2, and may be encoded by a reference nucleotide amino acid sequence of NM_001001523.2 or NM_005060.4.

[0052] Estrogen receptor alpha (ESR1, Era, ERα or NR3A1; Gene ID: 2099) is a nuclear receptor that is activated by estrogen. ERα may have a reference amino acid sequence of NP_000116.2 or NP_001116212.1 and may be encoded by a reference nucleotide amino acid sequence of NM_000125.4 or NM_001122740.2.

[0053] Transcription factors suitable for use as described herein may include reference database amino acid sequences or variants thereof. Suitable variants may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with the reference sequence. Amino acid sequence identity is generally defined according to the algorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego CA). GAP uses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) to align two full-length sequences in a way that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters of gap creation penalty = 12 and gap extension penalty = 4 are used. Although the use of GAP may be preferred, other algorithms may be used, such as BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147:195-197), generally with default parameters. Detailed sequence variants may differ from the reference sequence by insertions, additions, substitutions or deletions of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids.

[0054] Suitable transcription factor nucleic acids and proteins may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g., R&D Systems, Minneapolis, MN, USA; Cellgenix, Germany; Life Technologies, USA).

[0055] In some preferred embodiments, the defined set of transcription factors are the only transcription factors that are introduced into the PSCs. No other transcription factors, such as HNF4A, are introduced into the PSCs.

[0056] In other embodiments, one or more additional transcription factors selected from the group consisting of NR1, CUX2, AR, ZNF558, TSHZ2, TBX15, NF1X, NF1B, ATOH8, ZMAT1, ONECUT2, ZNF3858, FOS, FOSB, NR113, NPAS2, L3MBTL4, JAZF1, NF1A, ZNF680, HNF4G, CREBL2, DMRTA1, IRF6, ARID5A, SOX5, ZBTB20, ZNF704, ZEB1, ZNF367, NR1H4, KLF15, HLF, and NR4A2 may also be introduced into the PSCs in addition to the set of transcription factors.

[0057] A set of transcription factors may be introduced into the PSCs in the form of nucleic acids (Warren L et al. Cell Stem Cell. 2010 Nov 5;7(5):618-30) or proteins (Zhou H, et al Cell Stem Cell. 2009 May 8;4(5):381-4). After introduction of the reprogramming nucleic acids or proteins, the treated population of cells may be cultured.

[0058] In some embodiments, the set of transcription factors may be introduced into the PSC by expressing a heterologous nucleic acid encoding the set of transcription factors in the PSC. The amount of the transcription factors in the set is then increased in the PSC. The amount of the transcription factors in the set may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control (e.g., a PSC in which the set of transcription factors has not been introduced).

[0059] The nucleic acid may be operably linked to an inducible or non-inducible regulatory element in a vector suitable for expression in a cell, for example, a plasmid or a viral vector such as a retroviral or lentiviral vector. The vector containing the nucleic acid is then transfected into the PSC. Any convenient transfection technique may be used. In some embodiments, the set of transcription factors may be introduced into the PSC by a method comprising: (a) inserting a nucleic acid encoding a transcription regulator protein into a first genetic safe harbor site of the PSC; and (b) inserting one or more nucleic acids encoding a set of transcription factors consisting of a set of transcription factors consisting of HNF1A; HNF6; FOXA3; RORc and optionally ERα into a second genetic safe harbor site of the PSC, the one or more nucleic acids being operably linked to an inducible promoter regulated by the transcription regulator protein; and (c) culturing the PSC, thereby generating a hepatocyte. Safe harbor loci are well known in the art and include the hROSA26 locus and the AAVS1 locus.

[0060] After transfection, a set of transcription factors are expressed in the PSCs, thereby programming the PSCs to become hepatocytes. The transcription factors may be overexpressed in the PSCs. For example, the set of transcription factors may be expressed at a higher level, such as at least two-fold higher, at least three-fold higher, at least two-fold higher, or at least five-fold higher, than the endogenous expression level of the set of transcription factors in the cell. In some embodiments, transposon-mediated or other random integration transgenesis techniques may be used. Reprogramming cells through expression of nucleic acids encoding one or more transcription factors is well known in the art (Takahashi et al. 2007; Takahashi et al. 2007; Seki et al. 2010; Loh et al. 2010; Staerk et al. 2010).

[0061] The expression of transcription factors from the encoding nucleic acid in iPSCs may be inducible. For example, the encoding nucleic acid may be operably linked to one or more inducible regulatory elements in a suitable vector. Inducible regulatory elements may include tetracycline (Tc) or doxycycline (dox) inducible regulatory elements. Suitable methods for inducible expression of transcription factors in PSCs are established in the art (see, for example, Pawlowski et al (2017); WO2018096343A1).

[0062] In some preferred embodiments, PSCs can be programmed to become hepatocytes with minimal or no genetic modification to the cells.Suitable techniques are known in the art and include the use of excisable lentivirus and transposon vectors; repeated application of transient plasmids, episomes and adenovirus or adeno-associated vectors; or the use of small molecules, synthetic mRNA and / or microRNA (Sidhu KS.Expert Opin Biol Ther.(2011)May;11(5):569-79;Woltjen K et al(2009)Nature 458(7239):766-70;Chou BK et al.Cell Res.2011 21(3):518-29).

[0063] In other embodiments, a set of transcription factors may be introduced into the PSCs by contacting a population of PSCs with transcription factor nucleic acids, such as transcription factor proteins or mRNAs encoding the transcription factors. Programming of cells through direct delivery of transcription factor nucleic acids (Warren L et al. Cell Stem Cell. 2010 Nov 5; 7(5): 618-30) or proteins (Zhou H, et al Cell Stem Cell. 2009 May 8; 4(5): 381-4) is well known in the art and any suitable technique may be used. For example, a combination of transcription factor proteins or nucleic acids may be cultured in the presence of PSCs under conditions that allow the proteins or nucleic acids to enter the cells. In some embodiments, entry of the transcription factor proteins into the cells may be facilitated by a membrane-permeable peptide that may be linked or attached to the transcription factor proteins. The transcription factor protein or nucleic acid combination may be introduced into the PSC by conventional methods such as lipofection, electroporation, calcium phosphate precipitation, particle bombardment and / or microinjection, or may be delivered to the cell by a protein delivery agent. For example, the transcription factor protein or nucleic acid combination may be introduced into the cell by covalently or non-covalently attached lipids, such as myristoyl groups.

[0064] Transcription factor nucleic acids delivered directly to PSCs may be translatable by endogenous translation factors within the cells. Suitable synthetic mRNAs may be modified, for example, cytidine may be replaced with 5-methylcytidine and uridine with pseudouridine, followed by phosphatase treatment to generate transcription factor nucleic acids (Zhou H, et al 2009).

[0065] In other embodiments, a set of transcription factors may be introduced into the PSCs by activating expression of endogenous nucleic acid sequences encoding those transcription factors in the PSC population. Suitable techniques for activating endogenous genes include zinc finger or Transcription like Activator (TAL) techniques, which are well established in the art (see, e.g., Hum Gene Ther. 2012 May 15; Zhang P et al. Hum Gene Ther. 2012 Nov; 23(11):1186-99).

[0066] Once the set of transcription factors has been introduced, the PSCs can be cultured in a programming medium.

[0067] Preferably, the programming medium is a chemically defined medium. A suitable programming medium may include a basal culture medium, such as DMEM / F12, supplemented with, for example, 0.5 μg / ml to 70 μg / ml insulin, for example, 1.5 μg / ml to 150 μg / ml transferrin, for example, 30 μg / ml to 120 μg / ml L-ascorbic acid, for example, 0.05 μg / ml to 0.2 μg / ml FGF2, and for example, 0.05 μg / ml to 0.2 μg / ml TGFβ (or NODAL). For example, a suitable chemically defined programming medium may include a basal culture medium, such as DMEM / F12, supplemented with, for example, 0.5 μg / ml to 70 μg / ml insulin, for example, 1.5 μg / ml to 150 μg / ml transferrin, and L-ascorbic acid. In some preferred embodiments, the programming medium is E6 medium. E6 medium is described in detail above.

[0068] In embodiments in which the nucleic acid encoding a set of transcription factors is operably linked to one or more inducible regulatory elements, the programming medium may be supplemented with one or more agents that induce expression of the set of transcription factors, for example, when the encoding nucleic acid is operably linked to one or more doxycycline-inducible regulatory elements, the programming medium may be supplemented with doxycycline.

[0069] Following introduction of the set of transcription factors into the PSCs, the cells may be cultured in programming medium for 12 hours or more, 1 day or more, 2 days or more, or 3 days or more, preferably about 1 day.

[0070] Methods for culturing mammalian cells, such as PSCs, are well known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. US (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., US (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293; Ho WY et al J Immunol Methods. (2006) 310: 40-52; Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430). Culture media and its ingredients may be obtained from commercial sources (e.g., Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be used, e.g., 37°C, 21% oxygen, 5% carbon dioxide. Culture medium is preferably changed every 2 days and cells are allowed to settle by gravity.

[0071] Following culture in the programming medium, the cells may then be cultured in hepatocyte medium.

[0072] A hepatocyte culture medium is a culture medium that supports the maintenance of the hepatocyte phenotype in cultured cells. The hepatocyte culture medium is preferably a chemically defined medium.Suitable media include Hepatozyme (ThermoFisher Scientific; Jasmund et al (2007) Biomol En 24(1) 59-69), Hepatocyte Culture Medium (HCM; Lonza), Power Primary HEP Medium (Cellartis), DMEM / F12 (ThermoFisher Scientific), and William E Medium (WEM) (ThermoFisher Scientific) (Toda et al (2020) PLos One 15(2) e0229654; Jasmund et al (2007) Biomol En 24(1) 59-69; De Bartolo et al Biomaterials. 2006; 27: 4794-4803; Herrera et al Stem Cells. 2006; 24: 2840-2850. pmid: 16945998; Ammerschlaeger et al Toxicol Sci.2004;78:229-24;Yu et al Stem Cell Res.2012;9:196-207;Mallanna et al Curr Protoc Stem Cell Biol.2013;26:1G.4.1-1G.4.13;Cameron et al Stem Cell Reports.2015;5:1250-1262;Gieseck et al PLoS One.2014;9:e86372;Carpentier et al Stem Cell Res.2016;16:640-650;Leibovitch L-15 medium (Sigma-Aldrich; Leibovitch(1963) Amer J.Hyg 78 173-180);Waymouth MB 752 / 1 medium (Sigma-Aldrich; Waymouth,Tca Manual 3,521-525(1977)); and SF3 (Peng et.al, IOVS 44:808-17,2003; Jasmund et al (2007) Biomol En 24(1)59-69) and Chee's medium (Zirvi et al Cancer Biochem Biophys. 1991 Aug;12(2):137-51).In some preferred embodiments, the cell population may be cultured with Hepatozyme.

[0073] In some preferred embodiments, when the set of transcription factors includes ERα, the hepatocyte culture medium may be supplemented with β-estradiol (E2).

[0074] The cell population may be cultured in hepatocyte medium under suitable conditions for a time sufficient to allow one or more cells in the population to assume a hepatocyte phenotype following introduction of the set of transcription factors, for example, the cells may be cultured for 10 to 40 days, preferably 20 to 30 days, for example about 20 days.

[0075] In some embodiments, the hepatocyte culture medium may be supplemented with one or more agents that induce expression of the set of transcription factors from the encoding nucleic acid. For example, the set of transcription factors may be operably linked to one or more doxycycline-inducible regulatory elements, and the hepatocyte culture medium may be supplemented with doxycycline. The cell population may be cultured in the hepatocyte culture medium supplemented with the one or more agents for 7-9 days, preferably 8 days. The cell population may then be cultured in the hepatocyte culture medium without the one or more agents for an additional 8-12 days, preferably 10 days.

[0076] In some embodiments, cells in hepatocyte culture medium can be further cultured in 3D. For example, cells can be embedded in a scaffold such as Matrigel growth factor basement membrane matrix and cultured in hepatocyte culture medium for, for example, 5 days or more, or 10 days or more. This can be useful for promoting hepatocyte functionality, such as sensitivity to cytotoxic drugs.

[0077] A set of transcription factors introduced into a cell population confers a mature hepatocyte phenotype, ie, the cells in the population are forward programmed into hepatocytes by the set of transcription factors.

[0078] During cell culture, expression of one or more hepatocyte markers and / or one or more pluripotent cell markers may be monitored or detected in the cells in the population. This allows one to determine the degree of forward programming in the population as the population is cultured. In some embodiments, the method may include identifying or confirming the identity of the hepatocytes in the culture. Hepatocytes may be identified in the cell culture after, for example, at least 15 days.

[0079] After programming, the hepatocyte cell population may be cultured, expanded, and optionally stored, for example by cryopreservation.

[0080] The methods described above may result in the generation of hepatocyte populations that are substantially free of other cell types. For example, the populations generated by the methods described herein may contain 80% or more, 85% or more, 90% or more, or 95% or more hepatocytes after culturing. The presence or proportion of hepatocytes in the population may be determined through the expression of albumin and / or alpha 1-antitrypsin, as described above. Preferably, the hepatocyte population is sufficiently free of other cell types that purification is not necessary. If necessary, the hepatocyte population may be purified by any convenient technique, including FACS.

[0081] Also provided is a population of hepatocytes generated from PSCs by the methods described herein, wherein at least 90%, at least 95%, at least 98%, or 100% of the PSC population are capable of becoming hepatocytes following forward programming by the methods described herein.

[0082] In some embodiments, the hepatocytes may contain heterologous nucleic acid encoding a set of transcription factors.

[0083] The hepatocytes in the population may be functionally mature. Functionally mature hepatocytes may exhibit a mature hepatocyte phenotype.

[0084] Hepatocytes produced by the methods described herein may express hepatocyte markers; albumin (ALB), alpha 1-antitrypsin (AAT, A1AT or SERPINA1), CYP2A6, CYP3A4, CYP2C8, CYP2C9, UGT1A1, ApoA1, FASN, NR1H4, G6PC, UGT1A6, PCK1, PPRa / g and RORg.

[0085] Other hepatocyte markers include fumarylacetoacetase (FAH), cytokeratin 8 (CK8), cytokeratin 18 (CK18), asialoglycoprotein receptor (ASGR), alcohol dehydrogenase 1, arginase type I, and liver-specific organic anion transporter (LST-1).

[0086] The hepatocytes may express hepatocyte markers at the same or substantially the same levels as primary adult human hepatocytes, for example, the expression level in the hepatocytes may be the same or no more than 20%, no more than 10%, or no more than 5% higher or lower than the expression level in primary adult human hepatocytes.

[0087] The hepatocytes may express hepatocyte markers at levels higher than those in hepatocyte-like cells (HLCs) generated by directed differentiation, such as HLCs generated by the methods described below or in Palakkan et al., 2017; Szkolnicka & Hay, 2016; Silier et al., 2015; Hay et al., 2008; Baxter et al., 2015; Grandy et al., 2019; or Yiangou et al., 2018. For example, the expression levels in the hepatocytes may be 10% or more, 20% or more, 30% or more, or 50% or more higher than those in HLCs.

[0088] The hepatocytes may not express progenitor cell markers or may express them at low levels, such as AFP, CK18 and Sox17. For example, the expression level of the progenitor cell markers in the hepatocytes may be less than 20%, less than 10%, less than 5% or less than 1% of the expression level of the above hepatocyte markers.

[0089] Preferably, the hepatocytes do not express or exhibit reduced expression compared to PSCs of pluripotency-associated markers expressed by PSCs, such as Oct4, Sox2, alkaline phosphatase, SSEA-3, Nanog, SSEA-4 and Tra-1-60.

[0090] Expression of cell markers may be monitored and / or detected in the cell population. For example, expression or production of albumin (ALB), alpha 1-antitrypsin (AAT) or other hepatocyte markers by the hepatocyte population may be determined. This allows one to determine and / or monitor the degree of differentiation in the cultured population. Expression of cell markers may be determined by any suitable technique, including immunocytochemistry, immunofluorescence, RT-PCR, fluorescence activated cell sorting (FACS), and enzymatic analysis.

[0091] The hepatocytes produced by the methods described herein may have the ability to perform the functions of primary adult human hepatocytes. For example, hepatocytes may be capable of storing glycogen and LDL, synthesizing and secreting AAT and / or albumin (ALB), taking up LDL and fatty acids, and detoxifying xenobiotics via the CytP450 pathway. Hepatocytes may be capable of producing bile, thrombopoietin, angiotensinogen, urea, and cholesterol; and performing glycogenolysis, gluconeogenesis, glycogenogenesis, and lipogenesis.

[0092] The methods described herein may further include monitoring and / or determining whether the cells in the population are capable of performing one or more of the above hepatocyte functions.

[0093] Hepatocyte functions may be performed by hepatocytes produced by the methods described herein with the same or substantially the same activity as primary adult human hepatocytes. For example, the amount of activity in the hepatocytes may be the same as, or no more than 20%, no more than 10%, or no more than 5% higher or lower than the amount of activity in primary adult human hepatocytes.

[0094] Hepatocytes produced by the methods described herein may perform hepatocyte functions with greater activity than that of hepatocyte-like cells (HLCs) produced by directed differentiation, e.g., HLCs produced by the methods disclosed in Palakkan et al., 2017; Szkolnicka & Hay, 2016; Silier et al., 2015; Hay et al., 2008; Baxter et al., 2015; Grandy et al., 2019; or Yiangou et al., 2018. For example, the activity in hepatocytes may be 10% or more, 20% or more, 30% or more, or 50% or more higher than that in HLCs.

[0095] Hepatocytes generated by the methods described herein may have the capacity for in vivo engraftment and liver colonization in model systems, e.g., murine mouse models such as humanized FRG mice (Strom et al Methods Mol Biol 2010 640 491-509).

[0096] Hepatocytes produced by the methods described herein can exhibit the same, or substantially the same, gene expression profile of mature primary human hepatocytes (PHH).

[0097] Hepatocytes produced by this method may exhibit one or more of the following hepatocyte morphological characteristics: cobblestone morphology, occasional binucleation; glycogen deposits; apical microspikes; rough and smooth endoplasmic reticulum (ER) and prominent Golgi apparatus.

[0098] In some embodiments, the hepatocytes administered to an individual may be genetically engineered to produce therapeutic molecules, such as drugs or growth factors (Behrstock S et al, Gene Ther 2006 Mar;13(5):379-88, Klein SM et al, Hum Gene Ther 2005 Apr;16(4):509-21).

[0099] The hepatocyte populations produced by the methods described herein may be used in methods of treatment of the human or animal body, for example, treating an individual with liver damage, liver injury, and / or damaged or dysfunctional liver tissue. The populations may also be used in the manufacture of medicaments for use in treating an individual's liver damage, liver injury, and / or damaged or dysfunctional liver tissue. Suitable individuals may have acute liver injury, e.g., drug-induced liver injury; chronic liver disease, e.g., hepatitis (e.g., hepatitis A, B, C, D, E, G, or K), cirrhosis of the liver, hepatocellular carcinoma, nonalcoholic fatty liver disease, alcoholic liver disease, autoimmune liver disease, or a genetic metabolic disorder, e.g., alpha 1 antitrypsin deficiency, glycogen storage disease, e.g., glycogen storage disease type 1a, familial hypercholesterolemia, hereditary tyrosinemia, Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, or factor IX deficiency or other hemophilias, hemochromatosis, Wilson's disease, Dubin-Johnson syndrome, familial amyloidosis, or Refsum's disease.

[0100] For therapeutic applications, the hepatocytes are preferably clinical grade hepatocytes.

[0101] Aspects of the invention also relate to pharmaceutical compositions, medicaments, drugs or other compositions comprising hepatocytes produced as described herein, methods comprising administration of such hepatocytes to an individual in need thereof, for example for treatment (which may include prophylactic treatment) of liver damage or damaged or dysfunctional liver tissue as described above, and methods of making a pharmaceutical composition comprising admixing such hepatocytes with a pharma- ceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients.

[0102] The pharmaceutical composition may contain hepatocytes prepared as described herein and one or more additional components. In addition to hepatocytes, the pharmaceutical composition may contain pharma- ceutical acceptable excipients, carriers, buffers, preservatives, stabilizers, antioxidants, and / or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the activity of hepatocytes. The exact nature of the carriers or other materials will depend on the route of administration.

[0103] Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, tissue or cell culture medium, dextrose or other saccharide solution, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may also be included. The composition may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and exhibits suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions, for example, using isotonic vehicles such as sodium chloride, Ringer's solution, or lactated Ringer's solution. The composition may also be prepared using artificial cerebrospinal fluid.

[0104] Hepatocytes may be transplanted into a patient by any technique known in the art (e.g., Lindvall, O. (1998) Mov. Disord. 13, Suppl. 1:83-7; Freed, CR, et al., (1997) Cell Transplant, 6, 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332, 1118-1124; Freed, CR, (1992) New England Journal of Medicine, 327, 1549-1555; Le Blanc et al, Lancet 2004 May 1; 363(9419):1439-41). In particular, a cell suspension may be injected into the patient's portal vein. In some embodiments, hepatocytes may be encapsulated in alginate and transplanted as microbeads (Dhawan et al J. Hepatology 2020 72 5 877-884).

[0105] Administration of the pharmaceutical composition is preferably in a "therapeutically effective amount" (although in some cases prevention may be considered treatment), which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time-course of administration, will depend on the nature and severity of what is being treated. Decisions regarding prescription of treatment, e.g. dosage, etc., are within the responsibility of general practitioners and other physicians. The composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0106] In some embodiments, the hepatocytes in the population produced as described herein may exhibit a normal phenotype. For example, cells may be obtained from an individual with liver damage or liver injury or liver dysfunction and used to produce iPS cells. In some embodiments, the iPS cells may contain a mutation or genetic defect, which may be corrected using conventional recombinant techniques to produce iPS cells of normal phenotype. Hepatocytes of normal phenotype may be produced from these iPS cells as described herein and transplanted into a patient to repair or ameliorate liver injury or liver dysfunction.

[0107] In other embodiments, the hepatocytes in the population generated as described herein may exhibit a disease phenotype. For example, cells may be obtained from an individual with liver injury or liver damage or liver dysfunction and used to generate disease-specific iPS (ds-IPS) cells. Disease phenotype hepatocytes may then be generated from these iPS cells as described herein. These cells may then be treated to restore normal phenotype. For example, genetic mutations or defects that cause disease phenotypes may be corrected in vitro. Various techniques are available for correcting genetic mutations or defects in isolated mammalian cells. Once the defects or mutations are corrected and the normal phenotype is restored, the hepatocytes may be transplanted into a patient to repair or ameliorate liver injury or liver dysfunction.

[0108] Hepatocyte populations generated as described above may be useful in modeling the interaction of test compounds with hepatocytes, for example in toxicity screening, modeling liver damage and screening for compounds with potential therapeutic effects.

[0109] For example, a method for screening for a compound useful in treating liver damage includes: contacting isolated hepatocyte cells produced by the methods described herein with a test compound; and determining the effect of the test compound on said hepatocyte cells; may include.

[0110] In some embodiments, liver cells may be generated from iPSCs derived from a sample of cells having a disease-associated phenotype or genotype, and the effect of a test compound on the cells may be determined, e.g., the effect on one or more disease-associated pathologies may be determined.

[0111] Toxicity screening methods include: contacting isolated hepatocyte cells produced by the methods described herein with a test compound; and determining the effect of the test compound on said hepatocyte cells or the effect of the hepatocyte cells on the test compound. may include.

[0112] The effect of the test compound on the growth or viability of hepatocytes; gene expression; or function is determined. The growth or viability of hepatocytes may be determined in the presence of the test compound compared to its absence. A decrease in growth or viability is indicative of the compound having a hepatotoxic effect. Gene expression may be determined in the presence of the test compound compared to its absence. For example, the expression of albumin, alpha 1-antitrypsin (AAT), cytochrome p450 enzymes, such as CYP3A4, CYP1A2, CYP2E1, CYP2C19, CYP2C9, and CYP2D6, factor IX, apolipoprotein A2, CEBPα, and / or transthyretin may be determined. A decrease in expression is indicative of the compound having a hepatotoxic effect. Gene expression may be determined at the nucleic acid level, for example by RT-PCR, or at the protein level, for example by immunological techniques such as ELISA, or by activity assays. Cytochrome p450 assays, such as luminescence, fluorescence or colorimetric assays, are well known in the art and are available from commercial suppliers. One or more functions of hepatocytes may be determined and / or measured in the presence of test compound compared to the absence of test compound. For example, the ability of hepatocytes to perform one or more of the following functions may be determined and / or measured: detoxification of organic compounds, glycogen storage, secretion of AAT or albumin, bile production, thrombopoietin production, angiotensinogen production, conversion of ammonia to urea, cholesterol synthesis, glycogenolysis, glycogenogenesis and lipogenesis. The ability of hepatocytes to perform one or more of these functions is reduced in the presence of test compound compared to the absence of test compound, which indicates that the compound has hepatotoxic effect.

[0113] In some embodiments, the metabolism, degradation, or destruction of the test compound by liver cells may be determined. For example, the change in the amount or concentration of the test compound and / or metabolites of said test compound over time may be determined or measured, either continuously or at one or more time points. The decrease in the amount or concentration of the test compound and / or the increase in the amount or concentration of metabolites of said test compound may be determined or measured. In some embodiments, the rate at which the amount or concentration of the test compound and / or metabolites changes may be determined. Suitable techniques for measuring the amount of the test compound or metabolite include mass spectrometry. This may be useful in determining the in vivo half-life, toxicity, efficacy, or other in vivo properties of the test compound.

[0114] The hepatocyte suitable for use in the method for screening compounds useful in the treatment of liver injury or liver damage or liver dysfunction can exhibit disease phenotype.The effect of test compound on one or more disease pathologies in hepatocytes can be determined.For example, the effect of test compound on one or more of cell growth, gene expression, protein aggregation or polymerization; protein trapping in ER; cholesterol uptake; lipid and / or glycogen accumulation; and lactate production can be determined.Suitable techniques are well known in the art and include immunostaining, mass spectrometry, Western blot, and enzyme assay.

[0115] A reduction or amelioration of one or more disease pathologies in liver cells in the presence of the test compound compared to its absence may be an indication that the compound may be useful in treating liver damage or liver injury or dysfunction.

[0116] The methods as described herein may include identifying a test compound that reduces or ameliorates one or more disease pathologies in hepatocytes. Compounds that reduce disease pathologies may be useful in the development of therapeutics for the treatment of liver damage.

[0117] Other liver cells suitable for use in the method of screening for compounds useful in the treatment of liver damage or liver injury or liver dysfunction may exhibit normal phenotype, for example, may be derived from individuals at high risk or susceptible to liver damage compared to the general population. The effect of the test compound on cell growth, or on one or more of gene expression, for example, expression of cytochrome p450 (CYP), such as CYP3A4, CYP1A2, CYP2E1, CYP2C19, CYP2C9, and CYP2D6, may be determined. The effect of the test compound on one or more of the functions of the liver cells may be determined. For example, the ability of the liver cells to perform one or more of the following functions may be determined and / or measured in the presence of the test compound compared to its absence: detoxification of organic compounds, glycogen storage, secretion of AAT or albumin, bile production, thrombopoietin production, angiotensinogen production, conversion of ammonia to urea, cholesterol synthesis, glycogenolysis, glycogen production, and lipogenesis.

[0118] An increase in gene expression, growth and / or one or more functions in the presence of the test compound compared to its absence may indicate that the compound may be useful in treating liver damage or liver injury or dysfunction, such as hepatitis (e.g., hepatitis A, B, C, D, E, G or K), cirrhosis, hepatocellular carcinoma, non-alcoholic fatty liver disease, drug-induced liver injury, alcoholic liver disease, or autoimmune liver disease.

[0119] Following identification of a compound that reduces or ameliorates one or more disease pathologies in hepatocytes, the compound may be modified to optimize its pharmaceutical properties, which may be done using modeling techniques that are well known in the art.

[0120] Test compounds identified as having the ability to reduce or ameliorate one or more disease pathologies in hepatocytes using one or more initial screens may be further evaluated using one or more secondary screens.

[0121] Secondary screening may involve testing biological function or activity in vitro and / or in vivo, e.g., in an animal model. For example, the ability of a test compound to reduce or ameliorate one or more symptoms or pathologies associated with liver damage in an animal model of the disease may be determined.

[0122] After identifying a test compound that reduces or ameliorates one or more disease pathologies in liver cells, the compound may be isolated and / or purified, or alternatively, it may be synthesized using conventional techniques of recombinant expression or chemical synthesis.Furthermore, it may be manufactured and / or used in the preparation, i.e., manufacture or formulation, of compositions such as pharmaceuticals, pharmaceutical compositions or drugs.They may be administered to individuals for the treatment of liver disorders as described herein.

[0123] Hepatocytes generated as described above may be useful in disease modeling and identifying drug targets for liver disorders.

[0124] In some embodiments, the effect of the genetic mutation on the phenotype of the hepatocytes generated as described above may be determined. For example, a method for identifying a genetic mutation associated with liver damage may include: providing a test hepatocyte population of the fifth aspect, wherein the hepatocytes in the test population each comprise a genetic mutation; and phenotypically comparing the test hepatocyte population with a control hepatocyte population, the control population being free of the genetic mutation; and Identifying hepatocytes that exhibit a disease phenotype, such as a liver injury phenotype, within a study population It may include, Here, the disease phenotype indicates that the identified genetic mutations in hepatocytes are associated with liver damage.

[0125] Disease phenotype is the hepatocyte phenotype associated with disease, such as liver damage.Disease phenotype hepatocytes can exhibit abnormal function compared with normal phenotype hepatocytes.For example, disease phenotype hepatocytes can exhibit reduced or absent one or more of the hepatocyte functions described above.

[0126] In other embodiments, causative genetic mutations may be identified by examining liver cells for a disease phenotype. For example, a method for identifying genetic mutations associated with liver damage may include: Providing a test hepatocyte population of the fifth aspect, wherein hepatocytes in the test population exhibit a disease phenotype, such as a hepatic injury phenotype; and comparing the test hepatocyte population with a control hepatocyte population for genomic sequence, wherein the hepatocytes in the control population do not exhibit a disease phenotype; and Identifying one or more genetic mutations in the genomic sequence of a test population compared to a control population It may include, Here, the presence of a genetic mutation in a test population compared to a control population indicates that the mutation is associated with a disease phenotype.

[0127] In other embodiments, mutant gene expression may be identified by examining liver cells for a disease phenotype. For example, a method for identifying genes associated with liver damage may include: Providing a test hepatocyte population of the fifth aspect, wherein hepatocytes in the test population exhibit a disease phenotype, such as a hepatic injury phenotype; and Comparing the expression of one or more genes in the hepatocyte population with the expression of one or more genes in a control hepatocyte population. It may include, Here, a difference in expression of a gene in a population compared to a control population indicates that the gene is associated with liver damage.

[0128] Test liver cell populations suitable for use in these methods may be generated by the methods of the first to fourth aspects from induced pluripotent stem cells (iPSCs) derived from individuals with liver damage.

[0129] Also provided are methods for identifying transcription factors useful for forward programming of PSCs to hepatocytes. For example, a method for identifying transcription factors that promote hepatocyte maturation includes: To determine the expression of a set of transcription factors in primary human hepatocytes (PHHs) and hepatocyte-like cells (HLCs) generated by in vitro directed differentiation; and To identify transcription factors within that set that are upregulated in PHH compared to CLC. It may include, The identified transcription factors are candidate transcription factors that promote hepatocyte maturation.

[0130] Suitable hepatocyte-like cells (HLCs) may be generated by established methods (see below or, for example, Palakkan et al., 2017; Szkolnicka & Hay, 2016; Silier et al. 2015; Hay et al. 2008; Baxter et al., 2015; Grandy et al., 2019; or Yiangou et al., 2018).

[0131] For example, expression of one or more additional transcription factors selected from the group consisting of NR1, CUX2, AR, ZNF558, TSHZ2, TBX15, NF1X, NF1B, ATOH8, ZMAT1, ONECUT2, ZNF3858, FOS, FOSB, NR113, NPAS2, L3MBTL4, JAZF1, NF1A, ZNF680, HNF4G, CREBL2, DMRTA1, IRF6, ARID5A, SOX5, ZBTB20, ZNF704, ZEB1, ZNF367, NR1H4, KLF15, HLF, and NR4A2 may be determined.

[0132] Other aspects and embodiments of the invention provide those aspects and embodiments described above in which the term "comprising" is replaced with the term "consisting of" and those aspects and embodiments described above in which the term "comprising" is replaced with the term "consisting essentially of".

[0133] It should be understood that the present application discloses any combination of any of the above aspects and embodiments described above with each other, unless the context requires otherwise.Similarly, the present application discloses any combination of preferred and / or optional features, either alone or together with any of the other aspects, unless the context requires otherwise.

[0134] Variations of the above embodiments, further embodiments and variations thereof will be apparent to those of skill in the art upon reading this disclosure, and therefore are within the scope of the invention.

[0135] All documents and sequence database entries mentioned herein are hereby incorporated by reference in their entirety for all purposes.

[0136] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. EXAMPLES

[0137] Experimental Example To generate hepatocytes by forward programming, we first tested different combinations of LETFs and identified a cocktail of three factors sufficient to drive the conversion to immature hepatocytes (FoP-Heps). We then performed a transcriptomic and epigenetic comparison between HLCs and PHHs to identify additional transcription factors that may further enhance the functional maturation of hepatocytes. This comparison revealed that a number of nuclear receptors are expressed in adult hepatocytes and thus likely inducers of functionality and maturation in vivo. Combining a selection of these factors with LETFs, we identified the 4TFs HNF1A-HNF6-FOXA3-RORc as the most efficient cocktail to generate FoP-Heps that exhibit features of mature hepatocytes, including CYP3A4 activity, protein secretion, and hepatotoxicity response. Thus, forward programming offers an alternative to direct differentiation by avoiding the need for complex culture conditions and long timelines. Moreover, FoP-Heps exhibit a level of functional relevance for regenerative medicine, as well as disease modeling or drug screening.

[0138] Materials and Methods hPSC culture The human ESC H9 (WiCell) and iPSC A1ATDR / R, FS13B and NIH Lrg1 (Yusa et al., 2011) lines were used in this project. Following approval by the local research ethics committee (REC 08 / H0311 / 201), human iPSC lines were derived as previously described. Both types of hPSCs were cultured on plates coated with Vitronectin XFTM (10 μg / mL, StemCell Technologies) in Essential 8 (E8) chemically defined medium (Chen et al., 2011) consisting of freshly prepared DMEM / F12 (Gibco), L-ascorbic acid 2-phosphate (1%), insulin-transferrin-selenium solution (2%, Life Technologies), sodium bicarbonate (0.7%), and penicillin / streptomycin (1%) supplemented with TGFB (10 ng / ml, R&D) and FGF2 (12 ng / ml, Qkine). For routine dissociation, cells seeded in small clumps were incubated with 0.5 μM EDTA (ThermoFisher Scientific) for 3 min at 37°C. Cells were maintained at 37°C in 20% O2 and 5% CO2, and medium was changed every 24 h.

[0139] Gene targeting Inducible hESC and hiPSC lines were generated using the OPTi-OX system as previously described (Bertero et al., 2016; Pawlowski et al., 2017). Briefly, two gene safe harbors were targeted (GSH). The hROSA26 locus was targeted with a constitutively expressed transactivator (rtTA) and the AAVS1 locus was targeted with the transgene of interest under a TET-responsive element (TRE). Different combinations of transcription factors and / or nuclear receptors were cloned as described throughout this manuscript. Template cDNA sequences were either obtained from Dharmacon: HNF6 (MHS6278-213244170), HNF1A (MHS6278-202857902), RORy (MHS6278-202800991) and ESR1 (MHS6278-211691051); or amplified from human primary liver cDNA: HNF4A, FOXA3 and AR. Sequences were amplified using KAPA HiFi HotStart ReadyMix (Roche). Primers used for amplification and cloning of sequences into backbone vectors contained upstream and downstream overhangs to generate GSG (Gly-Ser-Gly) linkers and different 2A peptides as listed in Table 1. These different vectors were constructed by Gibson Assembly (New England Biolabs) using a vector to insert ratio of 1:3 pmol. For targeting, hPSCs were dissociated into single cells with STEMpro accutase (Thermo Fisher) for 5 min, and 1 million cells were transfected with 2 μg of donor vector and 2 μg of each AAVS1 ZFN expression plasmid using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza). Cells were plated in E8 medium supplemented with 10 μM ROCK inhibitor Y-27632 (Selleckchem).After 5–7 days, colonies were selected with 1 μg / ml puromycin (Sigma Aldrich) for at least 2 days, after which they were individually picked and genotyped as previously described ( Bertero et al., 2016 ; Pawlowski et al., 2017 ).

[0140] Direct Hepatocyte Differentiation hPSCs were dissociated into single cells after incubation with StemPro Accutase (Thermo Fisher) for 5 min at 37°C and seeded at a density of 50,000 cells / cm2 in E8 medium supplemented with 10 μM ROCK inhibitor Y-27632 (Selleckchem). Hepatocytes were differentiated for 48 h after seeding as previously described (Hannan et al., 2013), with minor modifications. Following endoderm differentiation, anterior foregut specification was achieved in RPMI-B27 differentiation medium supplemented with 50 ng / ml activin A (R&D) for 5 days. These foregut stage cells were further differentiated into hepatocytes in Hepatozyme complete medium (HepatoZYME-SFM) (Thermo Fisher) supplemented with 2 mM L-glutamine (Thermo Fisher), 1% penicillin-streptomycin (Thermo Fisher), 2% non-essential amino acids (Thermo Fisher), 2% chemically defined lipids (Thermo Fisher), 14 μg / ml insulin (Roche), 30 μg / ml transferrin (Roche), 50 ng / ml hepatocyte growth factor (R&D), and 20 ng / ml oncostatin M (R&D) for up to 27 days.

[0141] Forward programming of hepatocytes hPSCs were dissociated into single cells after 5 min incubation at 37 °C with StemPro Accutase (Thermo Fisher) and seeded at a density of 40–50.000 cells / cm2 in E8 medium supplemented with 10 μM ROCK inhibitor Y-27632 (Selleckchem). The next day, E8 medium was replenished. After 48 h, initial induction of the transgene was achieved by incubation for 24 h in E6 medium (E8 without growth factors) supplemented with 1 mg / ml doxycycline (dox). Cells were then maintained in Hepatozyme complete medium supplemented with 1 mg / ml dox for the remainder of the protocol. Medium was replenished daily for the next 4 days and every other day thereafter. For special experiments, cell lines were treated with 100 nM desmosterol, testosterone or β-estradiol (E2) from the second day of forward programming. All ligands were purchased from Sigma-Aldrich and reconstituted in ethanol. For 3D culture, on days 15 or 20, forward-programmed cells were embedded in Matrigel low growth factor basement membrane matrix, phenol red free (Corning) and cultured for 5 or 10 days, respectively. Cells were dissociated with Hank's Balanced Salt Solution-based cell dissociation buffer (Gibco) for 20 min at 37°C, resuspended in Matrigel, and plated in 40-50 μL domes in Hepatozyme complete medium supplemented with 1 mg / ml dox.

[0142] Primary Human Hepatocytes Fresh primary hepatocytes used for RNA-seq were obtained as previously described (Segeritz et al., 2018). Primary plated hepatocytes from four donors (three males and one female) that met the manufacturer's quality control requirements were purchased from Biopredic International (Rennes, France). Cells were maintained in short-term monolayer culture in William E (Gibco) supplemented with 1% glutamine (Gibco), 1% penicillin-streptomycin (Gibco), 700 nM insulin (Sigma-Aldrich), and 50 μM hydrocortisone (Sigma). Functional assays, including CYP3A4 activity measurements, were performed in Hepatozyme complete medium within 8 hours of receipt.

[0143] CYP3A4 assay Measurement of CYP3A4 enzyme activity was performed using the P450 Glo kit (Promega). Cells were incubated with luciferin-IPA 1:1000 in Hepatozyme complete for 1 h at 37°C. Supernatants were mixed with detection reagent in a 1:1 ratio and incubated for 20 min at room temperature in a Greiner white 96-well microplate (Sigma Aldrich). Luminescence was measured in triplicate on a GloMax plate reader. Hepatozyme complete medium was used as background control. Relative luminescence units were normalized to background, volume and mean total cell number obtained after differentiation.

[0144] LDL uptake assay LDL uptake capacity was measured with an LDL uptake assay kit (Abcam). Cells were incubated with 1:100 human LDL conjugated to DyLight™ 550 in Hepatozyme complete medium for 3 hours at 37° C. Cells were then washed and fixed with 4% PFA for 20 minutes at 4° C.

[0145] Fatty Acid Processing Forward-programmed cells were embedded in 3D from day 20 and cultured for 7 days in Hepatozyme complete medium supplemented with either BSA (control) or oleic acid (0.25 mM) or palmitic acid (0.25 mM) conjugated with BSA. Intracellular lipid accumulation was detected by incubating cells with 1 μl / ml Bodipy (Thermo scientific) for 30 min, followed by DAPI (Hoechst) diluted 1:10,000 in PBS for 30 min, and imaged with a Zeiss LSM 700 confocal microscope.

[0146] APAP toxicity Forward programmed cells cultured in 3D from day 15 were incubated in Hepatozyme complete medium supplemented with 25 mM acetaminophen (R&D) for 48 h (days 18-20) and then tested for hepatotoxicity of acetaminophen (APAP) by determining cell viability.

[0147] Cell viability Cell viability was determined by incubating cells with 1:10 Presto Blue reagent (Invitrogen) in Hepatozyme complete medium for 4 h at 37° C. Fluorescence was measured at excitation / emission of 560 nm / 590 nm using an EnVision plate reader.

[0148] RT-qPCR RNA was extracted from either cells or tissues using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer's instructions. 500 ng of RNA was reverse transcribed into cDNA using random primers and SuperScript II (Invitrogen) according to the manufacturer's instructions. qPCR was performed using the KAPA SYBR FAST qPCR Kit Low ROX (Sigma-Aldrich) with 200 nM forward and reverse primers (Sigma-Aldrich; primers are listed in Table 2) in a QuantStudio 5 (Applied Biosystems). qPCR was performed in technical duplicates and normalized to the average of two housekeeping genes (RPLP0 and PBGD) using the 2-ΔCt method.

[0149] Immunofluorescence staining Monolayers of cells were fixed in 4% PFA for 20 min at 4°C and blocked with 10% donkey serum (BioRad) and 0.1% Triton X-100 (Sigma-Aldrich) for 30 min. Fixed cells were incubated overnight at 4°C with the primary antibodies listed in Table 3 in 1% donkey serum and 0.01% Triton X-100. After washing, cells were incubated with AlexaFluor 488, 568 or 647-conjugated secondary antibodies (Life Technologies) diluted in 1% donkey serum and 0.01% Triton X-100 for 1 h at room temperature. To visualize nuclei, cells were incubated with DAPI / Hoechst 33258 (Bisbenzimide H, Sigma-Aldrich) diluted 1:10,000 in PBS for 10 min at room temperature. Cells were imaged on either a Zeiss Axiovert 200M or a Zeiss LSM 700 confocal microscope.

[0150] Secreted protein quantification Albumin, α-fetoprotein and α1-antitrypsin were measured in cell culture supernatants from monolayer cultures supplemented with fresh Hepatozyme complete medium 24 h before harvesting. Concentrations were detected by ELISA (performed by the core biomedical assay laboratory, Cambridge University Hospitals) and normalized to cell number.

[0151] RNA-seq analysis RNA-seq datasets were generated for undifferentiated hiPSCs (n=3), hESC-derived HLCs (n=2), hiPSC-derived HLCs (n=6), freshly harvested PHHs (fPHHs, n=3) and commercially purchased PHHs (pPHHs, n=2). RNA was extracted from either cells or tissues using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer's instructions. PolyA library preparation and sequencing were performed by Cambridge Genomic Services (hESC_HLCs; pPHHs) and the Wellcome Trust Sanger Institute (hiPSCs, hiPSC_HLCs, fPHHs). Read quality was assessed with FastQC. For consistency, fastq reads were split into single-end reads and trimmed to the same length (40bp) using cutadapt version 2.10. Single-end fastq files were mapped and quantified using salmon version 1.2.1 with the following parameters: -l A, -GCbias, -posbias, -validatemappings (Patro et al., 2017). The index used was previously generated from the human GRCh38 cDNA reference sequence from Ensembl (refgenomes.databio.org). Differential gene expression was calculated using DESeq2 (Love et al., 2014) with the following parameters: padj>0.05, basemean>100, and between-group log2 fold change>2 or <-2 as illustrated in each figure. Gene ontology enrichment was calculated with the clusterProfiler package (Yu et al., 2012). Pathway analysis of significantly misregulated transcription factors was determined using ReactomePA (Yu & He, 2016). Mouse liver polyA+RNA-seq was downloaded from ENCODE (Consortium, 2012). Single-end fastq reads in both replicates from each dataset were trimmed to 70 bp using cutadapt version 2.10.Fastq were mapped and quantified using a pregenerated mm10 cDNA reference genome using salmon version 1.2.1 with the following parameters: -l A, -Gbias, -seqbias, -validatemappings. DeSeq2 was used to generate all plots for visualization.

[0152] Chromatin immunoprecipitation (ChIP) ChIP was performed as previously described (Brown et al., 2011). Briefly, chromatin was crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature and quenched with 0.125 M glycine (Sigma-Aldrich). Cells and nuclei were then lysed, and chromatin was sonicated in a Bioruptor Pico sonicator (Diagenode) to fragment DNA to approximately 200–500 bp. Sonicated chromatin was precleared with same host IgG and Protein G Dynabeads (Thermo Fisher), and 100 μg of cleared chromatin (protein) was incubated with 2 μg of the following antibodies: H3K27ac (Abcam, ab4729), H3K4me1 (Abcam, ab8895), H3K27me3 (active motif, 39155) and H3K4me3 (Merk, 05-745R) at 4°C overnight, after which the complexes were captured with 30 μl Protein G Dynabeads (Thermo Fisher). The complexes were washed and treated with RNase A (Thermo Fisher) and Proteinase K (Sigma-Aldrich), and DNA was purified by phenol-chloroform extraction and precipitated with GlycoBlue (Thermo Fisher), sodium acetate (Thermo Fisher) and ethanol (Sigma-Aldrich). Sonicated chromatin samples (1%) were also collected as input for normalization, and 10 ng of DNA was used for ChIP-sequencing library preparation.

[0153] ChIP-seq analysis Library preparation as well as sequencing and alignment were performed by the Wellcome Trust Sanger Institute DNA Sequencing Facility (Hinxton, UK). Sequencing was performed on an Illumina HiSeq v4, obtaining paired-end reads of 75 bp length. ChIP-seq reads were mapped to the human genome assembly GRCh38 with bwa. Alignment data in BAM format were sorted and indexed with samtools. Coverage files were generated using deeptools bamCoverage with a bin size of 10 bp and normalized as RPKM for visualization in IGV and heatmap representation in deeptools. To plot principal component analysis (PCA), the average score of 1000 bp bins was calculated. For peak calling, BAM files were converted to SAM and peaks were called using homer (Heinz et al., 2010). Both replicates were used for peak calling on the input, where local filtering was disabled for H3K27ac by invoking the following flag: -region-L 0. To identify regulatory regions showing specific activity in PHH or HLC, differentially bound peaks were determined using the PHH dataset as target against all HLC datasets as background, and vice versa, with an enrichment fold over background of 4. For motif enrichment, peak calling was performed by invoking flag -L 1 -nfr on nucleosome-free regions, thereby determining "dips" within H3K27ac-rich regions. PHH or HLC-specific motif enrichment was performed by overlaying these sets of regions with the differentially bound peaks as described above. Peak annotation and gene ontology enrichment were determined with the clusterProfiler R package (Yu et al., 2012). Undifferentiated hiPSC ChIP-seq reads aligned to the same genome assembly were downloaded from ENCODE (Consortium, 2012) and processed as described above.

[0154] Data availability The RNA-seq dataset used in this study is accessible on Array Express under the accession number E-MTAB-10634. In addition, three of the hiPSC_HLC datasets have been previously deposited under the accession number E-MTAB-6781 (Segeritz et al., 2018). The Mus musculus C57BL / 6 liver embryo RNA-seq datasets were obtained from the ENCODE database (Nakamori et al., 2016) (https: / / www.encodeproject.org / ) under the following accession numbers: ENCSR216KLZ (E12.5 liver), ENCSR826HIQ (E16.5 liver), ENCSR096STK (P0 liver), ENCSR000BYS (8-week-old mixed-sex adult liver) and ENCSR216KLZ (10-week-old adult liver). The ChIP-seq dataset generated in this study has been deposited in Array Express under the accession number E-MTAB-10637, and publicly available datasets of hiPSCs were used from the ENCODE database under the following accession numbers: ENCSR729ENO (H3K27ac), ENCSR249YGG (H3K4me1), ENCSR386RIJ (H3K27me3), ENCSR657DYL (H3K4me3), and ENCSR773IYZ (input).

[0155] statistical analysis Statistical analyses were performed using GraphPad 9.0.0, and specific tests are indicated in the figure legends. For each figure, the sample size n indicates the number of independent experiments or biological replicates, and individual values ​​are represented per graph. Tests between groups were performed on at least n>3 independent experiments, and when significant, exact p values ​​are indicated in the figures.

[0156] result Liver-enriched transcription factors enable cells to be forward programmed for hepatocyte identity The first step in the development of a forward programming method is to identify a cocktail of transcription factors capable of recapitulating the transcriptional network that characterizes the target cell type. However, for hepatocytes, this step is challenging, as liver development is not initiated by a single specific master regulator, and the factors that drive the functional maturation of hepatocytes are not fully understood. To circumvent these limitations, we decided to focus on LETFs that are known to control the induction of the liver program during fetal development and have been tested in somatic cell conversion (Rombaut et al., 2021). The coding sequences of four LETFs (HNF4A, HNF1A, HNF6 and FOXA3) were cloned into the OPTi-OX system (Figure 1A) and the resulting inducible cassettes were targeted to the AVSS1 gene safe harbor (Bertero et al., 2016; Pawlowski et al., 2017). After selection, individual sub-lineages were picked, expanded and genotyped before further characterization. Addition of doxycycline (dox) for 24 hours was sufficient to induce uniform and robust expression of each LETF in selected hESCs (Figure 1B, 1C), confirming the efficacy of this OPTi-OX system in inducing transgene expression. Importantly, this induction was not associated with differentiation into hepatocytes, suggesting that LETFs alone are not sufficient to confer hepatocyte identity. Thus, we decided to screen culture conditions that could sustain both hepatocyte survival and differentiation (data not shown) and found that cells acquired hepatocyte-like morphology when cultured for 14 days in Hepatozyme complete medium after the first 24 hours in E6 medium (Figure 1D). Interestingly, the resulting cells expressed hepatocyte markers such as albumin (ALB), α1-antitrypsin (A1AT or SERPINA1), and α-fetoprotein (AFP) (Figure 1E), and exhibited CYP3A4 activity levels comparable to those of HLCs generated by direct differentiation (Figure 1F). We next asked whether all four of these LETFs were essential to achieve this hepatocyte-like phenotype.Therefore, we generated hESC sublines expressing combinations of the three factors by removing each factor (Figure 1). Again, robust and uniform expression was observed at the protein level after 24 hours of dox induction (Figure 1). Induction of each of the three LETF combinations in the culture conditions identified above showed that HNF1A, HNF6, or FOXA3 were essential to generate cells expressing hepatocyte markers such as ALB (Figure 1G). Overexpression of HNF4A appeared to be dispensable, as cells generated by overexpression of the remaining three LETFs (HNF1A, HNF6, FOXA3) acquired a cobblestone-like morphology and expressed high levels of ALB, SERPINA1, and AFP (Figure 1G, H, I). Notably, hepatocytes generated using these three TFs (3TF FoP-Heps) achieved the highest level of CYP3A4 activity, suggesting that overexpression of HNF4A may have prevented the acquisition of functional properties (Figure 1J). Collectively, these results demonstrated that overexpression of HNF1A, HNF6, and FOXA3 was sufficient for forward programming of hPSCs into hepatocyte-like cells.

[0157] HLCs generated by directed differentiation lack expression of specific nuclear receptors Given these promising results, we aimed to enhance the functionality of 3TF FoP-Heps by adding TFs that could play a role in promoting liver maturation and function. However, identifying these factors proved to be a challenge, as there is little information on the mechanisms driving functional maturation of hepatocytes, especially around the neonatal period when adult liver function is established. To circumvent this limitation, we decided to compare the transcriptome profile of adult PHHs with that of HLCs generated by direct differentiation from hPSCs. Indeed, HLCs represent a fetal state that has been extensively characterized (Baxter et al., 2015), whereas 3TF FoP-Heps seem less relevant to natural development. For this comparison, we used state-of-the-art protocols (Hannan et al., 2013; Touboul et al., 2010) that have been used to model liver injury (Rashid et al., 2010; Segeritz et al., 2018) and as proof-of-concept for the application of cell-based therapies (Yusa et al., 2011). The protocol begins with the generation of endodermal cells expressing SOX17, followed by the specification of foregut expressing HHEX, after which the cells transition through a hepatoblast-like state characterized by TBX3 (Figure 2). Interestingly, LETF is expressed at levels comparable to PHH during this differentiation (Figure 2), confirming that these initial steps follow a natural developmental pathway. The resulting progenitor cells undergo terminal differentiation steps to become HLCs expressing functional markers such as ALB and SERPINA1 (Figure 2A, Figure 1A, Figure 1D). Despite exhibiting major hepatic functions ( Baxter et al., 2015 ; Grandy et al., 2019 ; Yiangou et al., 2018 ), HLCs represent a “fetal” state, as indicated by expression of AFP ( Figure 2 ) or limited activity / expression of CYP3A4, CYP2A6 or CYP2C9 ( Figure 1B ).RNA sequencing (RNA-seq) performed on HLCs generated from either human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) and on PHHs freshly harvested (fPHHs) or cultured in vitro as monolayers (pPHHs) reinforces these observations. Principal component analysis (PCA) of the 500 most variable genes showed clear differences between these three cell types, with HLCs clustering with undifferentiated hiPSCs and PHHs confirming their intermediate differentiation state (PC1: 52%, Figure 2C). To further explore the differences between HLCs and PHHs, we combined differential gene expression (DGE) and gene ontology (GO) analysis to identify genes and biological functions specific to each cell type (Figure 2D). Genes uniquely expressed in PHHs (cluster 1) were associated with adult liver functions, including response to and metabolism of xenobiotics, inflammatory responses, and complement activation (Figure 2E). Genes expressed in both HLCs and PHHs (Heps; cluster 3) were involved in liver development, fatty acid metabolism or a wide range of cellular functions (Figure 1F). Notably, genes specifically upregulated in HLCs were associated with various developmental functions that may have arisen from the organization of the extracellular matrix and its in vitro environment (Figure 2-Supplementary Figure 1F). Using a previously curated list (Lambert et al., 2018), we next decided to focus specifically on transcription factors (TFs) (Figure 2G, Figure 2H), and identified 36 TFs highly expressed (p<0.05, log2 fold change>2) in PHHs relative to HLCs. Interestingly, in reactome pathway analysis, these TFs were grouped into two major pathways: the NFI family and a cohort of eight nuclear receptors (Figure 2H), which are known to play a role in hepatic metabolic activity. Taken together, these observations indicate that the "fetal" state of HLCs is associated with the absence of several nuclear receptors, thus suggesting that these factors may be essential to drive the functional maturation of hepatocytes.

[0158] Epigenetic characterization of HLCs suggests roles for the nuclear receptors RORc, AR, and ERαTo further refine the list of TFs identified by our transcriptome analysis, we decided to compare the epigenetic landscape between HLCs and PHHs. Indeed, we hypothesized that the regulatory regions bound by TFs in PHHs may play key functions in maturation. ChIP sequencing (ChIP-seq) was performed for histone marks including H3K27ac (active regulatory regions), H3K4me1 (active or primed regulatory regions), and H3K27me3 (silenced genes) (Creyghton et al., 2010; Wang et al., 2015). These marks were profiled in HLCs derived from both hiPSCs and hESCs, as well as in PHHs, while undifferentiated hiPSCs were used as controls. As expected, PCA analysis showed striking differences in the epigenetic profiles between HLCs and hiPSCs, independent of the marks analyzed (Figure 3A). Interestingly, HLCs and PHHs were grouped into close clusters, suggesting that these cell types share important parts of their epigenetic profiles despite differences in their transcriptomes. Analysis of H3K27ac yielded the strongest signature difference between HLCs and their native counterparts, confirming that this mark is important for establishing cell identity (Figure 3A) and suggesting that H3K27ac may be the most informative mark for understanding the differences between HLCs and PHHs. We then performed differential peak calling to identify active regulatory regions that were uniquely enriched in PHHs compared to HLCs ("PHH-specific") and vice versa ("HLC-specific") (Figure 3). We profiled H3K4me1 and H3K27me3 in specific regions of either PHHs or HLCs. This analysis revealed that H3K4me1 was absent from “PHH-specific” regions of HLCs and instead appeared to be extensively replaced by spreading H3K27me3 deposition ( Fig. 3B ).Interestingly, in genes downregulated in HLC, individual portions of the regulatory regions were lacking H3K27ac (e.g., CYP3A4 and UGT1A, see Fig. 3C). According to the gene ontology associated with each set of regions, in agreement with the transcriptome analysis, several adult hepatic metabolic processes in the "PHH-specific" set, such as steroid, lipid, and xenobiotic metabolism, and a range of developmental functions for the "HLC-unique" regions emerged (Fig. 3). Overall, these results suggest that a subset of genes involved in adult liver function lacks H3K4me1 priming as well as global H3K27ac deposition in HLC.

[0159] These genes may also exhibit repressive marks, such as H3K27me3. In addition, HLCs exhibit active histone marks in regions containing genes not associated with hepatic differentiation, confirming that cells generated from hPSCs also display an epigenetic signature specific to their in vitro state (Figure 3). Collectively, these observations suggest that HLCs and PHHs share the same broad epigenetic identity. However, activation of a limited set of specific regulatory regions was absent in HLCs, explaining their lack of functional maturation. To identify nuclear receptors that may be involved in regulating these regions, we performed motif enrichment analysis in "PHH-specific" regions characterized by H3K27ac. Interestingly, this analysis identified significant enrichment of androgen (AR) and estrogen (ERα) response elements, as well as RORc motifs (Figure 3D), which were among the top differentially expressed nuclear receptors in our transcriptome analysis. We next decided to further investigate the importance of these nuclear receptors throughout development using mouse RNA-seq datasets obtained at different stages of liver organogenesis (E12.5, E16.5, P0, 8 weeks and 10 weeks adult) (Figure 3). Interestingly, the expression of these three nuclear receptors was found to be specifically upregulated in adult liver (Figure 3). Collectively, these observations suggested that the nuclear receptors AR, ERα and RORc may play a role in the establishment or maintenance of the transcriptional network that characterizes mature hepatocytes.

[0160] Overexpression of RORc enhances the functionality of hepatocytes generated by forward programming We next tested the ability of RORc (RORy), AR, and ERα (ESR1) to further improve the functionality of FoP-Heps generated using the three LETFs. To this end, we generated hESC lines inducible for expression of HNF1A, HNF6, and FOXA3 (3TFs) in combination with each of the nuclear receptors identified above. The uniformity of induction of the 4TFs was verified using immunostaining. Interestingly, these analyses showed that the overexpressed nuclear receptors were localized to the nucleus, suggesting that their overexpression may circumvent the need for ligands to promote their activity. We then induced forward programming using the culture conditions identified above and observed the production of polyploid cells with cobblestone morphology (Figure 4A). The hepatocyte identity of these cells was confirmed by the expression of albumin, SERPINA1 / A1AT, and AFP in all lineages (Figure 4B, Figure 4C, Figure 4D, Figure 4E). Notably, overexpression of RORc resulted in cells with the highest albumin protein levels (Figure 4B, 4D, 4E), and AFP expression levels were lower, especially after 30 days of differentiation (Figure 4D, 4E). CYP3A4 activity levels were also significantly higher in cells generated in the presence of RORc when compared to cells reprogrammed with 3TF alone (Figure 4F). We next tested whether stimulation with exogenous ligands specific for each nuclear receptor could further induce functional maturation as measured by CYP3A4 activity (desmosterol for RORc, β-estradiol for ERα, and testosterone for AR). Interestingly, only β-estradiol treatment resulted in a three-fold increase in CYP3A4 activity, while testosterone treatment significantly reduced CYP3A4 activity, and desmosterol had no effect (Figure 4G). This increase was not observed in 3TF Fop-Heps, thus suggesting that the effect of these ligands was related to overexpression of their receptors.

[0161] FoP-Heps generated with RORc appeared to have the highest level of functionality, therefore we decided to test the potential of this combination of factors in alternative pluripotent stem cell lines. We generated Opti-OX hiPSCs with 3TF or 3TF+RORc FoP systems and tested the upregulation of these factors following 24 hours of dox treatment, followed by induced differentiation, according to the protocol established above. When RORc was overexpressed, FoP-Heps derived from hiPSCs also exhibited a cobblestone morphology (Figure 5A) and expressed higher levels of albumin, AFP and SERPINA1 / A1AT (Figure 5B, Figure 5C, Figure 5D, Figure 5E). In addition, the presence of RORc significantly increased basal CYP3A4 activity, thereby confirming the positive effect of this factor on the functional maturity of FoP-Heps (Figure 5F). Overall, these results demonstrated that overexpression of specific nuclear receptors was equivalent to generating FoP hepatocytes. In particular, overexpression of RORc was able to improve the functionality of hepatocytes generated by overexpression of the three LETFs, confirming the role of this nuclear receptor in hepatocyte maturation.

[0162] 4TF FoP-Heps exhibit functional characteristics in vitro Next, we sought to further characterize the functionality of 4TF (HNF1A, HNF6, FOXA3, and RORc) FoP-Heps derived from either hESCs (eFoP-Heps) or hiPSCs (iFoP-Heps) in comparison with HLCs and PHHs generated by directed differentiation. CYP3A4 activity was significantly higher in forward-programmed 4TF FoP-Heps after 20 days compared with that achieved by HLCs after 30 days of directed differentiation (Figure 6A). In addition, we analyzed the expression of markers related to hepatic metabolic functions, such as phase I (cytochrome P450 enzymes) and phase II (UGT) biotransformation, gluconeogenesis (G6PC and PCK1), and lipid (PPAR, PPARy, FASN, and APOA1) and bile acid (NR1H4) metabolism. FoP-Heps expressed a range of these functional markers, confirming the acquisition of hepatic functionality (Figure 6B, Figure 6C). Overall, the expression levels achieved by forward programming were equivalent to those achieved by direct differentiation, except for gluconeogenic genes, which were increased in FoP-Heps (Figure 6C). Interestingly, the expression of gluconeogenic and lipid metabolism genes was comparable between FoP-Heps and PHH. However, induction of cytochrome P450 genes remains a challenge, indicating that further refinement of our protocol may be required to obtain this specific liver function (Figure 6B). Notably, in FoP-Heps, RORc remained expressed at physiological levels even at the end of our protocol (Figure 6B). In addition to expressing mature hepatocyte markers, eFoP-Heps and iFoP-Heps were also able to uptake LDL from the culture medium, confirming their ability to transport lipids (Figure 6D). To further investigate their ability to metabolize lipids, FoP-Heps were grown in 3D for an additional 5 days (D20) or 10 days (D30), as we recently observed that such culture conditions promote lipid accumulation in HLCs (Carola Morell, personal communication).We first confirmed that FoP-Heps grown in 3D retained the expression of hepatocyte markers (Figure 6E). Interestingly, the expression of SERPINA1 or UGT1A6 was increased in these conditions, suggesting that increased functional maturation was promoted in 3D (Figure 6E). We next tested the ability of both eFoP and iFoP-Heps to respond to fatty acids by treating these cells with both oleic acid (OA) and palmitic acid (PA), which are known to induce steatosis and lipotoxicity, respectively (Ricchi et al., 2009). Consistent with its known effects on hepatocytes, OA treatment induced a strong accumulation of lipids as shown by BODIPY staining (Figure 6F), while PA treatment induced a decrease in cell viability consistent with lipotoxicity (Figure 6G). Thus, FoP-Heps appear to respond to fatty acids similarly to their primary counterparts. Finally, we investigated the benefit of FoP-Heps in modeling the hepatotoxic effects of paracetamol / acetaminophen. To this end, FoP-Heps were grown in the presence of a dose of acetaminophen (APAP) known to induce liver failure. This treatment reduced cell viability by 50% (Figure 6H), suggesting that FoP-Heps may be used for cytotoxicity studies. In summary, these results show that 4TF FoP-Heps derived from either hESCs or hiPSCs exhibit features of functional hepatocytes, including expression of genes involved in drug, lipid, glucose and bile acid metabolism, the ability to take up LDL and fatty acids from the culture medium, and response to hepatotoxic agents, demonstrating their potential benefit in modeling liver injury and toxicity screening in vitro.

[0163] 5TF FoP-Heps exhibit functional characteristics in vitro Next, we sought to further characterize the functionality of hiPSC-derived 5TF (HNF1A, HNF6, FOXA3, RORc, ERα) FoP-Heps (iFoP-Heps) in comparison with 4TF iFoP-Heps. Overexpression of 5TF in the presence or absence of eostrogen allowed the generation of hepatocyte-like cells (Figure 7B). These cells were shown to express key hepatocyte markers (Figures 7C and 7D). 5FT-generated iFop-Heps were also found to exhibit high levels of CYP3A4 activity (Figure 7E), lipid transport / accumulation (Figure 7F) and lipotoxicity (Figure 7G).

[0164] In this study, we established a method to forward program hPSCs into hepatocytes. The success of this approach relies on the selection of TFs that combine factors controlling early liver development with regulators of adult liver function. Nevertheless, most forward programming methods rely on master regulators to convert hPSCs into specific cell types. As an example, neurons and muscle cells can be generated by simple overexpression of NGN2 and MYOD, respectively (Pawlowski et al., 2017). Our results show that the generation of hepatocytes requires a more complex process involving three transcription factors, but also a culture medium that supports primary hepatocytes. Moreover, our best LETF combination did not include HNF4A, which is known to be a master regulator of hepatocyte function in the adult liver. On the contrary, removing HNF4A significantly improved the identity of the generated hepatocytes. Similar observations have been recently reported for the direct reprogramming of human umbilical vein endothelial cells into bipotential hepatocyte progenitors, where HNF4A was found to be deleterious (Inada et al., 2020). HNF4A is essential not only in the adult liver but also during development, especially in the establishment of the liver bud (Gordillo et al., 2015). Thus, HNF4A may also have a role in the maintenance of fetal liver cells, such as hepatoblasts, and its overexpression during forward programming may prevent the acquisition of adult hepatocyte identity. This example illustrates the challenge of identifying factors uniquely expressed in the adult liver. Importantly, FoP-Heps created by LETF overexpression acquired a hepatocyte identity with reduced adult function, suggesting that this transcriptional cocktail may only convert hiPSCs into fetal-like cells. Thus, we decided to add factors that can direct the functional maturation of the liver. Factors in this last category were identified by performing transcriptomic and epigenetic comparisons of PHH and HLCs generated by directed differentiation.Our focus on HLCs was based on their well-characterized fetal state and also on our extensive experience with these cells. From these analyses, a subset of nuclear receptors was identified that are expressed exclusively in PHH and adult liver, thereby validating our approach. Of particular interest, RORc, ERα, and AR were identified as key candidates that control functional maturation in hepatocytes. Importantly, nuclear receptors are well known to control diverse liver functions, including lipid and glucose homeostasis, bile acid clearance, xenobiotic sensing, and regeneration (Rudraiah et al., 2016). Both steroid hormone receptors ER and AR have been shown to play a role in regulating hepatic energy homeostasis (Shen & Shi, 2015). Moreover, ER is involved in cholesterol clearance (Zhu et al., 2018), and has also been linked to liver regeneration (Kao et al., 2018) and bilirubin metabolism through CYP2A6 (Kao et al., 2017). RORc is a nuclear receptor expressed in peripheral tissues, including liver, muscle, and adipose tissue, and has been proposed to function as an intermediary between the circadian clock and glucose / lipid metabolism (Cook et al., 2015). Moreover, RORy-deficient mice exhibit reduced expression of a subset of phase I and phase II enzymes involved in insulin sensitivity, gluconeogenesis, lipid metabolism markers, and bile acid synthesis (Kang et al., 2007; Takeda, Kang, Freudenberg, et al., 2014; Takeda, Kang, Lih, et al., 2014). Based on these previous reports, we propose that overexpression of RORc and other nuclear receptors may improve certain functions of FoP-Heps by activating a subset of target genes in the hepatic context induced by LETF overexpression. Importantly, hepatocyte functionality varies spatially across the liver lobule and is influenced by oxygen gradients, nutrients and signaling ( Trefts et al., 2017 ).This separation of liver segments drives distinct metabolic processes for glucose, lipids, iron, or even xenobiotics under the control of distinct transcriptomic programs (Halpern et al., 2017).We therefore predict that different combinations of background nuclear factors induced by LETF overexpression may result in the generation of hepatocytes with distinct and distinct functional repertoires.

[0165] FoP-Heps generated with overexpression of 4 TFs (HNF1A, HNF6, FOXA3 and RORc) exhibited functional characteristics of adult hepatocytes, including albumin and A1AT secretion, basal CYP3A4 activity, expression of phase I / phase II enzymes, gluconeogenesis and lipid metabolism markers, ability to take up LDL and fatty acids, and response to toxic compounds. Nevertheless, CYP3A4 expression remains limited, and this gene remains difficult to induce in vitro. Thus, additional TFs may be essential to generate FoP-Heps that exhibit the full range of functional activities exhibited by PHHs. Similarly, culture conditions may further improve support of key liver functions. Indeed, the basal medium used in our protocol does not prevent dedifferentiation of PHHs and may therefore be incompatible with the generation of fully functional cells by forward programming. Nevertheless, the forward programming method established here presents several advantages over conventional directed differentiation protocols. This is a robust two-step method, avoiding the need for multi-step differentiation, which is often associated with batch-to-batch variability. Moreover, forward programming is faster, generating functional cells in 20 days, as opposed to 30-35 days for direct differentiation. Finally, the cell yield appears to be good and amenable to large-scale production. Indeed, we observed that forward programming was accompanied by a 6-8-fold increase in cell number upon differentiation, whereas the direct differentiation yield was lower. Taken together, our results represent the first method to generate hepatocytes using forward programming. This approach represents a first step towards high-throughput, large-scale production of specialized hepatocytes that exhibit a variety of functions relevant for various applications in disease modeling and drug screening.

[0166] array

[0167] [Table 1] TIFF2024531593000002.tif96170

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Table 2

[0169] References Baxter et al(2015)J Hepatol 62(3)581 - 589. Bertero,A.et al(2016,Dec 1).Development,143(23),4405 - 4418. Boon,R et al.(2020,Mar 13).Nat Commun,11(1),1393. Brown,S.et al Stem Cells,29(8),1176 - 1185. Chen,G.et al(2011,May)Nat Methods,8(5),424 - 429. Consortium,E.P.(2012,Sep 6).Nature,489(7414),57 - 74. Cook,D.N.,et al.(2015).Nucl Receptor Res,2.101185 Creyghton,M.P.et al(2010,Dec 14).Proc Natl Acad Sci U S A,107(50)21931 - 21936 Dhawan,A.et al(2020,May)J Hepatol,72(5),877 - 884 Du,Y.et al(2014 Mar 6).Cell Stem Cell 14(3)394 - 403. Gordillo,M et al(2015,Jun 15).Development,142(12),2094 - 2108. Grandy,R.et al(2019,Jan 24).Annu Rev Pathol 14,449 - 468. Halpern,K.B.et al(2017,Feb 16).Nature,542(7641),352 - 356 Hannan,N.R.et al(2013,Feb)Nat Protoc,8(2),430-437. Hay et al Stem Cells(2008)Apr;26(4):894-902 Heinz,S.,et al.(2010,May 28).Mol Cell 38(4)576-589 Huang,P.et al(2014,Mar 6).Cell Stem Cell 14(3)370-384. Inada,H.,et al(2020,Oct 21).Nat Commun,11(1)5292. Kang,H.S.et al(2007,Oct 22).Physiol Genomics 31(2)281-294. Kao,T.L.,et al(2017,Nov).Cell Transplant 26(11)1822-1829. Kao,T.L.et al(2018).Theranostics,8(10),2672-2682. Lambert,S.A.et al(2018,Oct 4).Cell,175(2),598-599. Lau,H.H et al(2018,May).J Hepatol,68(5),1033-1048. Love,M.I.et al(2014)Genome Biol 15(12)550. Mitry,R.R.et al(2002,Dec).Semin Cell Dev Biol, 13(6),463-467. Nakamori,D.et al(2016, Jan 15).Biochem Biophys Res Commun,469(3),424-429. Palakkan,A.A.et al(2017,Apr)..Biomed Rep,6(4),367-373. Patro,R.,et al(2017,Apr).Nat Methods,14(4),417-419 Pawlowski,M.et al(2017,Apr 11).Stem Cell Reports 8(4)803-812. Rashid,STet al(2010,Sep 1).J Clin Invest,120(9),3127-3136. Ricchi,M.et al(2009,May)J Gastroenterol Hepatol 24(5)830-840. Rombaut,M.et al(2021,May 11).J Hepatol.75 690-705 Rudraiah,S.et al(2016).Annu Rev Pharmacol Toxicol,56,605-626. Schrem,H.et al(2002,Mar).Pharmacol Rev,54(1),129-158. Segeritz,CPet al(2018,Oct).J Hepatol,69(4),851-860. Shen,M.,& Shi,H.(2015).Int J Endocrinol,2015,294278. Silier et al Stem Cell Reports 2015 4 5 939-952 Si-Tayeb,K.et al(2010,Feb 16)Dev Cell,18(2)175-189. Szkolnicka,D.,&Hay,DC(2016,Jun).Stem Cells,813 34(6),1421-1426. Takeda,Y.,et al(2014)PLoS Genet, Takeda,Y.et al(2014).Nucleic Acids Res,42(16),10448- Touboul,T.et al(2010,May)Hepatology, Trefts,E.et al(2017,Nov 6).Curr Biol,27(21), Wang,A.et al(2015,Apr)Cell Stem Cell,16(4),386-399. Yiangou,L.et al(2018,Apr 5).Cell Stem Cell,22(4),485-499. Yu,G.,&He,Q.Y.(2016,Feb).Mol Biosyst,12(2),477-479. Yu,G.et al(2012,May) OMICS, 16(5), 284-287. Yusa,K.et al(2011,Oct 20).Nature,478(7369),391-394. Zhao,D.et al(2013,Jan).Cell Res,23(1),157-161. Zhu,L.et al(2018,Feb).Mol Metab,8,106-116.

Claims

1. 1. A method for producing hepatocytes, comprising: (i) Providing a population of iPSCs (ii) introducing into the iPSC population a set of transcription factors comprising HNF1A, HNF6, FOXA3, RORc, and optionally ERα and / or NR1I3; and (iii) culturing the population such that one or more cells in the population are programmed to become hepatocytes.

2. 2. The method of claim 1, wherein the set of transcription factors consists of HNF1A, HNF6, FOXA3, RORc, and ERα.

3. 2. The method of claim 1, wherein the set of transcription factors consists of HNF1A, HNF6, FOXA3, and RORc.

4. 2. The method of claim 1, wherein the set of transcription factors consists of HNF1A, HNF6, FOXA3, RORc, and NR1I3.

5. The method of claim 1, wherein the hepatocytes are functionally mature.

6. 2. The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells (hPSCs).

7. 2. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

8. The method of claim 1 , wherein the transcription factor is a human transcription factor.

9. 10. The method of claim 1, wherein the cell population is cultured under suitable conditions for a time sufficient to allow one or more cells to assume a hepatocyte phenotype.

10. 2. The method of claim 1, wherein during step (iii), the cells in the population do not exhibit an endodermal and hepatic progenitor cell phenotype.

11. the set of transcription factors (a) expressing in the PSCs nucleic acids encoding the combination of transcription factors; (b) contacting the PSCs with the combination of transcription factor mRNA or protein; or (c) activating the endogenous expression of genes encoding the combination of transcription factors in the PSCs. The method of claim 1, wherein the PSC is introduced by

12. 12. The method of claim 11, wherein the nucleic acid encoding the set of transcription factors is expressed in the PSC by transfecting the PSC with one or more vectors comprising the nucleic acid operably linked to one or more regulatory elements for expression of the nucleic acid in the PSC.

13. 13. The method of claim 12, wherein the one or more regulatory elements are inducible regulatory elements.

14. The method of claim 12 or 13, wherein the vector is a lentiviral vector.

15. 10. The method of claim 1, wherein the PSC population is cultured for at least 7 days.

16. 2. The method of claim 1, wherein after step (iii), at least 5% of the population is forward programmed to become hepatocytes.

17. 10. The method of claim 1, comprising isolating and / or purifying hepatocytes.

18. The method of claim 1, comprising expanding the population of hepatocytes.

19. The method of claim 1 , comprising storing the population of hepatocytes.

20. A hepatocyte population produced by the method of claim 1.

21. 21. A pharmaceutical composition comprising the hepatocyte population of claim 20 and a pharmaceutically acceptable excipient.

22. 21. The hepatocyte cell population of claim 20 for use in a method of treating the human or animal body.

23. 21. The hepatocyte cell population of claim 20 for use in a method for treating liver damage in an individual.

24. 21. A pharmaceutical composition comprising the population of liver cells described in claim 20 for use in the treatment of liver damage.

25. 21. Use of the hepatocyte population of claim 20 in the manufacture of a medicament for use in the treatment of liver damage.

26. 10. Use of a set of transcription factors comprising HNF1A, HNF6, FOXA3, RORc, and optionally ERα and / or NR1I3, or one or more nucleic acids encoding said set of transcription factors, for generating hepatocytes.

27. 1. A method for screening for a compound useful in the treatment of liver damage, comprising: contacting the population of liver cells of claim 20 with a test compound; and determining the effect of the test compound on the hepatocytes or the effect of the hepatocytes on the test compound.

28. 1. A method for determining the hepatotoxicity of a compound, comprising: contacting the isolated hepatocyte of claim 20 with a test compound; and determining the effect of said test compound on said hepatocytes.

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