Hepatocyte organoid and method for producing the same

Abstract

To provide hepatocyte organoids derived from stem cells such as iPS cells that retain the characteristics of hepatocytes and can be cultured for a long period of time, and to provide methods for producing hepatocyte cell lines containing the same.SOLUTION: The present invention provides a method for producing a hepatocyte cell line that includes a hepatocyte organoid, comprising a step of culturing cells derived from stem cells in a medium containing an intranuclear receptor agonist or a Wnt / β-catenin pathway activator. The present invention also provides a hepatocyte organoid derived from a stem cell, which can be cultured for 21 days or more.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to hepatocyte organoids and a method for producing the same, and hepatocyte cell lines including hepatocyte organoids and a method for producing the same. More specifically, the present invention relates to hepatocyte organoids derived from stem cells such as iPS cells, which retain the traits of hepatocytes and can be cultured for a long period of time, and a method for producing the same, as well as hepatocyte cell lines including hepatocyte organoids and a method for producing the same. [Background technology]

[0002] To elucidate human liver pathology in vitro, human hepatocyte cell lines that can be cultured for a long time while retaining the characteristics of hepatocytes are required, but no cell lines that meet these conditions have been found to date. In liver disease research, hepatoma cell lines have traditionally been used, but they have the problem that they are derived from cancer and have characteristics different from normal cells. One candidate to overcome this problem is hepatocyte lineage cells induced from human iPS cells. However, hepatocyte-like cells induced from human iPS cells in two-dimensional culture using conventional methods have poor proliferation properties similar to primary human hepatocytes, and cannot be cultured for a long time. To perform long-term culture, it is necessary to culture them as immature hepatic progenitor cells at a more differentiated stage, such as iPS-derived hepatic progenitor cells (iPS-HSCs). However, hepatic progenitor cells have the characteristics of bile duct cells and do not have the characteristics acceptable for physiological mature hepatocytes. Therefore, it has been difficult to use them as disease models, reproduce long-term pathology, and apply them to drug screening. In addition, human iPS cell-derived hepatic progenitor cells, which are hepatocyte lineage cells at a more immature differentiation stage, can be subcultured, but they have the problem that they do not have the characteristics required for physiologically mature hepatocytes, such as possessing the characteristics of bile duct cells.

[0003] It has become possible to induce differentiation of human iPS cells into hepatocyte-like cells (iPS-derived hepatocyte-like cells: iPS-Hep), and applications as drug screening, disease models, and alternative cell resources for liver transplantation are expected. However, problems include immaturity of gene expression and function compared to primary human hepatocytes (PHH), poor proliferation similar to PHH, and inability to be cultured for long periods. Regarding the former, it has been reported in recent years that activation of downstream genes of FXR (Non-Patent Document 1) and thyroid hormone (Non-Patent Document 2) are important for the maturation of iPS-Hep. In addition to hepatocyte markers, existing induction methods are accompanied by increased expression of other cell lineage genes such as intestinal epithelial cell markers, but overexpression of FXR and activation of downstream genes by agonists reduced the expression of other cell lineage markers and improved hepatocyte phenotype. It has also been shown that THRB, which is related to thyroid hormone, increases drug metabolism activity such as CYP3A4 in iPS-Hep. Short-term induction of iPS-Hep by plate culture is approaching from fetal hepatocyte to mature hepatocyte, but there are only a limited number of reports of culture systems that can maintain proliferation and culture while maintaining hepatocyte phenotype. In recent years, research has been attempted to overcome this problem by forming a culture by three-dimensional culture, i.e., an organoid, but existing iPS cell-derived hepatocyte-like cell organoids have strong bile duct cell phenotypes and are difficult to culture for a long time while maintaining hepatocyte phenotypes (Non-Patent Document 3), and organoids using human liver tissue are organoids derived from fetal liver cells, which are not easy to obtain (Non-Patent Document 4), and have not yet been put to practical use. In addition, there are reports that liver organoids have been produced from human iPS cells (Non-Patent Document 5), but there are problems such as being dependent on self-organization and being a multicellular culture that also contains other constituent cells such as hepatic stellate cells and Kupffer cells, and an organoid culture method that can be constructed from a single cell type of human hepatocyte and maintained for a long time has not been established until now. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nell et al. J Hepatol 2022 Nov;77(5):1386-1398. [Non-Patent Document 2] Ma et al. Cell Stem Cell 2022 May 5;29(5):795-809. [Non-Patent Document 3] Mun et al. J Hepatol 2019 Nov;71(5):970-985. [Non-Patent Document 4] Hu et al. Cell 2018 Nov 29;175(6):1591-1606. [Non-Patent Document 5] Ouchi et al. Cell Metab. 2019 Aug 6;30(2):374-384. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present invention aims to provide a method for producing hepatocyte organoids derived from stem cells such as iPS cells, which retain the traits of hepatocytes and can be cultured for a long period of time, and a hepatocyte cell line containing the same. [Means for solving the problem]

[0006] Therefore, the present inventors focused on bile acids, which act as agonists of FXR and other receptors and are known to contribute to liver regeneration as well as fat absorption. As mentioned above, it has been shown that bile acid-FXR signaling contributes to hepatocyte maturation in plate culture of iPS-Hep, but the present inventors examined the effects of bile acids in three-dimensional organoid culture of iPS-Hep.

[0007] As a result of this verification, the present inventors invented a method for producing human hepatocyte cell lines, including hepatocyte organoids, from human iPS cells, which can be cultured for a long time while retaining the characteristics of more mature hepatocytes, and developed human iPS cell-derived hepatocyte organoids. Specifically, first, human iPS cells were induced to differentiate into hepatocyte lineages in a two-dimensional culture system via the definitive endoderm lineage and hepatoblast lineage. In the present invention, this was followed by embedding in EHS gel and performing three-dimensional culture. As a result of exploring factors involved in liver regeneration through various condition studies, it was found that it is beneficial to add tauro-obeticolic acid, a strong FXR agonist, and recombinant Wnt3a, etc. as a canonical Wnt agonist, and a new method for producing human hepatocyte cell lines, including human iPS cell-derived hepatocyte organoids, was established.

[0008] That is, the present invention provides the following. [Aspect 1] A method for producing a hepatocyte line, including a hepatocyte organoid, comprising culturing cells derived from stem cells in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator. [Aspect 2] The method of production according to embodiment 1, wherein the nuclear receptor agonist is an FXR agonist. [Aspect 3] The method of claim 2, wherein the FXR agonist is a bile acid. [Aspect 4] A method according to claim 3, wherein the bile acid is taurobeticholic acid. [Aspect 5] The method of production according to embodiment 1, wherein the Wnt / β-catenin pathway activator is Wnt3a. [Aspect 6] The production method according to embodiment 5, wherein the medium containing the nuclear receptor agonist or Wnt / β-catenin pathway activator contains a Wnt / β-catenin pathway activator that is not Wnt3a. [Aspect 7] The method for production according to embodiment 6, wherein the Wnt / β-catenin pathway activator that is not Wnt3a is R-spondin. [Aspect 8] The method for production according to embodiment 7, wherein the R-spondin is selected from the group consisting of R-spondin1, R-spondin2, R-spondin3 and R-spondin4. [Aspect 9] The production method according to aspect 1, wherein the concentration of CHIR99021 in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is 10 μM or less in the medium. [Aspect 10] The production method according to embodiment 1, wherein the medium containing the nuclear receptor agonist or Wnt / β-catenin pathway activator further contains an ALK inhibitor, a ROCK inhibitor or a growth factor. [Aspect 11] The method of manufacture according to embodiment 10, wherein the ALK inhibitor is A83-01. [Aspect 12] The method of manufacture according to embodiment 10, wherein the ROCK inhibitor is Y-27632. [Aspect 13] 11. The method according to embodiment 10, wherein the growth factor is selected from the group consisting of EGF, HGF, FGF7, FGF10 and TGFα. [Aspect 14] The production method according to embodiment 1, wherein the cells derived from stem cells are cells induced to differentiate from stem cells in two-dimensional culture. [Aspect 15] The production method according to embodiment 1, wherein the cells derived from stem cells are cells induced to differentiate through the definitive endoderm lineage or the hepatoblast lineage. [Aspect 16] The method of production according to aspect 15, wherein the cells induced to differentiate through the definitive endoderm lineage or the hepatoblast lineage are cells induced to differentiate through the definitive endoderm lineage and the hepatoblast lineage. [Aspect 17] The method of production according to embodiment 1, wherein the cells derived from stem cells are of hepatic lineage. [Aspect 18] The production method according to embodiment 1, wherein the step of culturing cells derived from stem cells is a step of three-dimensional culture. [Aspect 19] The method of claim 18, wherein the three-dimensional culture comprises embedding cells derived from stem cells in a gel. [Aspect 20] The method of claim 19, wherein the gel is an EHS gel. [Aspect 21] The method of production according to embodiment 20, wherein the stem cells are iPS cells. [Aspect 22] 22. The method of claim 21, wherein the stem cells are human stem cells. [Aspect 23] The production method according to aspect 1, wherein the hepatic cell line comprises organoids, and among the organoids contained in the hepatic cell line, the number of grape-like organoids is 10% or more. [Aspect 24] A method for producing hepatocyte organoids, comprising a step of identifying grape-like organoids from a hepatocyte cell line comprising the hepatocyte organoids produced by the production method described in embodiment 1. [Aspect 25] 25. The method of claim 24, comprising isolating identified grape bunch-like organoids. [Aspect 26] A hepatocyte organoid derived from a stem cell, which can be cultured for 21 days or more. [Aspect 27] 27. The hepatocyte organoid of embodiment 26, which can be cultured for 180 days or more. [Aspect 28] 27. The hepatocyte organoid of embodiment 26, which is not derived from cancer cells. [Aspect 29] 27. The hepatocyte organoid according to aspect 26, wherein the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatocyte organoid is 30% or less. [Aspect 30] 27. The hepatocyte organoid according to embodiment 26, wherein the expression level of albumin gene in the hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in the hepatocyte organoid. [Aspect 31] 27. The hepatocyte organoid according to embodiment 26, wherein the expression level of HNF4α gene in the hepatocyte organoid is 0.003 times or more than the expression level of GAPDH gene in the hepatocyte organoid. [Aspect 32] 27. The hepatocyte organoid according to embodiment 26, wherein the expression level of AFP gene in the hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in the hepatocyte organoid. [Aspect 33] 27. The hepatocyte organoid according to embodiment 26, wherein the expression level of the EPCAM gene in the hepatocyte organoid is 30 times or less than the expression level of the GAPDH gene in the hepatocyte organoid. [Aspect 34] 27. The hepatocyte organoid of embodiment 26, comprising bile canaliculi or microcilia. [Aspect 35] 35. The hepatocyte organoid according to embodiment 34, in which a bile canalicular structure or microcilia are observed under electron microscopic observation. [Aspect 36] 27. The hepatocyte organoid of embodiment 26, which is comprised in a hepatocyte cell line. [Aspect 37] 37. The hepatocyte organoid of embodiment 36, wherein the hepatocyte cell line comprises organoids, and among the organoids contained in the hepatocyte cell line, the number of grape-like organoids is 10% or more. [Aspect 38] 27. The hepatocyte organoid of embodiment 26, wherein the stem cells are iPS cells. [Aspect 39] 27. The hepatocyte organoid of embodiment 26, wherein the stem cells are human stem cells. Effect of the Invention

[0009] The human iPS cell-derived hepatocyte organoid produced by the method of the present invention in a more specific embodiment can be subcultured for a long period of time of 6 months or more, and the expression of the albumin gene, which is characteristic of mature hepatocytes, can be maintained in a state of expression 5 times higher than that of human iPS cell-derived hepatocyte-like cells produced by conventional two-dimensional culture. In addition, RNA sequencing showed that the human iPS cell-derived hepatocyte-like cells produced by conventional two-dimensional culture and those produced under the culture conditions used in the fetal liver cell-derived organoid culture method were highly expressed in the human iPS cell-derived hepatocyte organoid produced by the method of the present invention in a more specific embodiment, while the more immature progenitor cell-related gene group, such as EpCAM and CK19, was reduced, and it was confirmed that the characteristics of more mature hepatocytes were maintained. Furthermore, in a more specific embodiment of the present invention, the human iPS cell-derived hepatocyte organoid has been confirmed to have the ability to secrete albumin, take up LDL, and store glycogen, and to functionally retain the characteristics of hepatocytes. Furthermore, electron microscopy revealed that the organoids possessed morphologically characteristic fine structures of hepatocytes, such as the presence of bile canalicular structures and microcilia characteristic of hepatocytes.

[0010] Retaining the traits of more mature hepatocytes and enabling long-term culture are mutually exclusive conditions for hepatocytes, and have been considered difficult worldwide up to now. However, the human iPS cell-derived hepatocyte organoids produced by the method of the present invention in a more specific embodiment have the traits of more mature hepatocytes compared to existing cell lines and organoids, and these traits continue to be maintained even after a period of six months or more, so that the two exclusive conditions that have been considered difficult to achieve have been met for the first time, and the present invention is an innovative invention. Human iPS cells have many advantages, such as not only retaining normal human chromosomes and physiological traits, but also being able to be established from any individual, cell lines with various genetic backgrounds are available, quality control as cell lines is becoming standardized, and disease-related gene mutations can be reproduced by genome editing. This hepatocyte organoid production method based on human iPS cells has high industrial applicability, such as being able to be put to practical use in an in vitro liver disease analysis model that enables in vitro elucidation of human liver pathology and being able to be put to practical use in novel drug screening. [Brief description of the drawings]

[0011] [Figure 1A] Figure 1A is a schematic diagram showing the process of inducing human iPS cells (iPSCs) to differentiate into hepatocytes in flat plate culture via the definitive endoderm lineage and hepatoblast lineage (2D-iPS-Hep), followed by embedding in EHS gel for three-dimensional culture (3D culture). DE: definitive endoderm. HLC: hepatocyte-like cells. iHO: iPS-Hep organoid. [Figure 1B] Figure 1B shows that iPS-Hep organoids (iHO) induced under culture conditions with the addition of tauro-obeticolic acid and recombinant Wnt3a induced grape-like organoids with a characteristic cluster-like morphology, unlike those induced under culture conditions without bile acid or FO, and were capable of long-term culture for up to 6 months. [Figure 1C]Figure 1C shows the phase contrast of passage 0, showing that iPS-Hep organoids (iHO) induced under culture conditions with the addition of tauro-obeticolic acid and recombinant Wnt3a induced grape-like organoids with a characteristic cluster-of-grapes morphology, unlike those induced under culture conditions without bile acid or FO. [Figure 1D] Figure 1D shows that the overall number of organoids formed in iPS-Hep Organoids (iHO) induced under culture conditions with the addition of tauro-obeticolic acid and recombinant Wnt3a tended to be high. Note that woBA is an abbreviation for Without Bile acids. [Figure 1E] Figure 1E is a graph showing that iHO maintain high expression of hepatocyte marker genes even after subculture. iHO are able to maintain expression of the albumin gene (ALB), which is characteristic of mature hepatocytes, at a level more than five times higher than human iPS-Hep produced by conventional two-dimensional culture. Hepatocyte: liver cell. Fetal hepatocyte: fetal liver cell. Progenitor: precursor cell. [Figure 2A] Figure 2A shows that iHOs have a characteristic Grape-like organoid morphology. When organoids induced using a cell line fluorescently labeled with GFP were observed in whole mounts using a confocal microscope, a Grape-like morphology was observed even in three dimensions. [Figure 2B] Figure 2B is a photograph showing that iHOs exhibit a characteristic Grape-like organoid morphology. This characteristic morphology was confirmed by scanning electron microscopy. [Figure 2C]Figure 2C is an immunostaining photograph showing that iHOs exhibit a characteristic Grape-like organoid morphology. When organoids induced using a cell line fluorescently labeled with GFP were observed in whole mounts using a confocal microscope, a Grape-like morphology was observed even in three dimensions. [Figure 2D] Figure 2D is a photograph of immunostaining showing that iHO express hepatocyte markers. Immunostaining shows strong expression of ALB, a hepatocyte marker, and HNF4α, a marker for hepatocytes and bile duct cells. [Figure 2E] Figure 2E is a photograph of immunostaining showing that some iHO cells co-express AFP and Ki-67. Immunostaining showed strong expression of hepatocyte markers such as AFP, and some cells also expressed Ki-67, suggesting that this is an organoid culture system that maintains proliferation and hepatocyte phenotypes, even at the protein level. [Figure 2F] Figure 2F is an immunostaining photograph showing that the grape-like organoids strongly express ALB, whereas the mixed cystic organoids do not express ALB. Although round and cystic organoids with morphologies other than the grape-like were actually mixed in the iHO, they showed low ALB expression by immunostaining, indicating that the hepatocyte phenotype was relatively weak. [Figure 2G] Figure 2G shows immunostaining results showing that the grape-like organoids do not express cholangiocyte markers, whereas the mixed cystic and round organoids strongly express cholangiocyte markers. Although round and cystic organoids with morphologies other than grape-like were also mixed into the iHO, the expression levels of progenitor cell and cholangiocyte markers such as EPCAM and KRT19 were high, and the hepatocyte phenotype was relatively weak. [Figure 3A]Figure 3A shows that iPS-Hep Organoids (iHOs) induced under culture conditions supplemented with tauro-obeticolic acid and recombinant Wnt3a have abundant mitochondria and glycogen, which are characteristic of hepatocytes, and have a microstructure similar to bile canaliculi and microvilli. When observed under a transmission electron microscope, iHOs have microvilli on the cell surface and a tubular structure backed by tight junctions in some areas, suggesting a bile canaliculi-like structure characteristic of hepatocytes (Figure 3A, left). As with hepatocytes, they have abundant mitochondria and rough endoplasmic reticulum within the cells, and glycogen granules (Figure 3A, right). Mit: mitochondria. RER: rough endoplasmic reticulum. Gly: glycogen. TJ: tight junction. MV: microvilli. Bile canaliculi: Bile canaliculi. [Figure 3B] Figure 3B is a graph showing that iHOs are functionally similar to hepatocytes, and have the ability to produce albumin. [Figure 3C] Figure 3C is a photograph showing that iHOs are functionally similar to hepatocytes, and that iHOs have the glycogen storage ability, a function unique to hepatocytes. [Figure 3D] Figure 3D is a photograph showing that iHOs are functionally similar to hepatocytes, and that iHOs have the ability to uptake LDL, a function unique to hepatocytes. [Figure 4A]Figure 4A shows the results of RNA-seq, which indicates that iHO has an expression profile similar to fetal liver cells (FLC). RNA-seq was performed to investigate the gene expression profile of iHO. In addition to comparison with culture conditions without bile acids and plate culture, comparisons were also made with taurocholic acid in addition to tauroobeticholic acid. In addition, comparisons were made between Passages 1 and 3 to confirm changes over time. Principal component analysis showed that iHO has an expression profile different from existing culture systems and PHH (human primary hepatocytes), and has an expression profile similar to that of fetal liver cells (FLC). [Figure 4B] Figure 4B shows the results of hierarchical clustering (1000 genes with large variance) showing that iHO has a similar mRNA expression profile to FLC and PHH. Hierarchical clustering also suggested that iHO is relatively similar to FLC (fetal liver cells) and PHH (human primary liver cells) compared to other culture conditions and 2D-iPS-Hep. [Figure 4C] Figure 4C is a graph showing that iHO has higher expression of hepatocyte-related genes than those without bile acids. Genes with differential expression were extracted compared to those without bile acids, and enrichment analysis revealed increased expression of gene sets associated with hepatocytes and fetal liver cells. [Figure 4D] Figure 4D is a graph showing that iHO expresses hepatocyte-related genes at higher levels than those in the absence of bile acids. Gene enrichment analysis of differentially expressed genes in iHO compared to those in the absence of bile acids revealed increased expression of gene sets associated with hepatocytes and fetal liver cells. [Figure 4E] Figure 4E is a graph showing that iHO has higher expression of hepatocyte-related genes compared to no bile acid. In terms of functional terms such as GO, increased expression of gene sets related to the coagulation-fibrinolysis system, complement, and lipid metabolism was observed (iHO VS woBA P3). [Figure 4F]Figure 4F shows the results of RNA-seq, showing that iHO strongly expressed hepatocyte markers, but had lower expression of cholangiocyte and other cell lineage markers compared to existing culture systems. When genes related to hepatocytes, fetal liver cells, progenitor cells, or cholangiocytes were specifically extracted and a heat map was created, it was found that iHO had high expression of hepatocyte markers, but lower expression of progenitor cell, cholangiocyte, and intestinal epithelial cell markers compared to other culture systems. Hepatocyte: liver cell. Fetal Hepatocyte: fetal liver cell. Duct Non Liver: bile duct and intestinal epithelial cells other than the liver. [Figure 5A] Figure 5A shows the results of single cell RNA-seq, which shows that a fraction with strong hepatocyte traits (hepatocyte-like fraction) is induced in iHO, while the other cholangiocyte-like fraction expresses cholangiocyte markers and intestinal epithelial cell markers. Single cell RNA-seq analysis showed that iHO had two fractions: a hepatocyte-like fraction that highly expressed hepatocyte lineage genes such as ALB, and a cholangiocyte-like fraction that highly expressed cholangiocyte lineage genes such as KRT19. [Figure 5B] Figure 5B shows the results of single cell RNA-seq, which shows that a hepatocyte-like fraction is induced in iHO, while the cholangiocyte-like fraction expresses cholangiocyte markers and intestinal epithelial cell markers. The hepatocyte-like fraction highly expressed ALB, as well as hepatocyte marker genes such as AHSG, TF, and C3, and also showed high expression of fetal hepatocyte-related genes such as AFP, DLK1, and GPC3. On the other hand, the cholangiocyte-like fraction highly expressed intestinal epithelial cell lineage genes such as CDX2, ISX, and DPP4 in addition to cholangiocyte lineage genes such as KRT19, KRT7, and EPCAM. [Figure 5C]Figure 5C shows the results of single cell RNA-seq, which shows that the hepatocyte-like fraction or iHO contains a fraction that co-expresses hepatocyte-related genes and proliferation-related genes. The hepatocyte-like fraction contains subfractions that highly express proliferation-related genes such as MKI67 and PCNA, suggesting the presence of cells that have both proliferation and hepatocyte characteristics even at the single cell level. [Figure 5D] Figure 5D shows the results of an analysis using single cell RNA-seq results, which shows that iHOs have a fraction similar to human fetal hepatocytes. When combined with previously reported single cell data from 11-19 week human fetal livers (Cao et al. Science, 2020), it was shown that part of iHOs overlaps with fetal hepatocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Manufacturing method: The present invention provides a method for producing a hepatic cell line, including a hepatic cell organoid, comprising a step of culturing cells derived from stem cells in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator.

[0013] Examples of the nuclear receptor agonist include FXR agonists. FXR agonists are molecules that are specific ligands for the farnesoid X receptor (FXR) and have the effect of specifically stimulating ligand-dependent FXR transcriptional activity by binding to FXR. Specific examples include bile acids, INT-767, LY-2562175, Px-104, and LJN-452. Bile acids are a form of cholesterol excreted widely in bile, and are a general term for organic compounds having a cholanic acid skeleton with a steroid skeleton. Examples of the bile acids include primary bile acids, conjugated bile acids, and secondary bile acids. Examples of the primary bile acids include cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, hyocholic acid, and 5α-cyprinol. Examples of the conjugated bile acids include glycocholic acid and taurocholic acid. Examples of secondary bile acids include deoxycholic acid, lithocholic acid, hyodeoxycholic acid, ursodeoxycholic acid, etc. Examples of bile acids include obeticholic acid, tauroobeticholic acid, chenodeoxycholic acid, etc.

[0014] The content of the nuclear receptor agonist in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 0.3 to 100 μM, more preferably 1 to 30 μM, and even more preferably 3 to 10 μM, relative to the medium.

[0015] The Wnt / β-catenin pathway activator is a molecule that activates the β-catenin pathway that controls gene expression via β-catenin in the Wnt signal pathway, which is an intracellular signal transduction mechanism activated by the action of Wnt on cells, and examples of such activators include R-spondin1, R-spondin2, R-spondin3, R-spondin4, Wnt3a, CHIR99021, and various GSK3b inhibitors. The Wnt / β-catenin pathway activator, such as Wnt3a, may be a complex with another protein, such as Afamin. The complex of Wnt3a and Afamin is called Wnt3a-AFM, etc. Therefore, examples of Wnt3a include Wnt3a-AFM, in addition to Wnt3a. Examples of Wnt / β-catenin pathway activators include Wnt3a, and in this case, the medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator may contain a Wnt / β-catenin pathway activator other than Wnt3a. Examples of Wnt / β-catenin pathway activators other than Wnt3a include R-spondin. Examples of R-spondin include R-spondin1, R-spondin2, R-spondin3, and R-spondin4.

[0016] The content of the Wnt / β-catenin pathway activator in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 10 to 3000 ng / ml, more preferably 30 to 1000 ng / ml, and even more preferably 100 to 300 ng / ml, relative to the medium.

[0017] The concentration of CHIR99021 in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 10 μM or less, more preferably 3 μM or less, even more preferably 1 μM or less, even more preferably 0.3 μM or less, and even more preferably 0.1 μM or less. A medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably substantially free of CHIR99021, more preferably does not contain CHIR99021. An example of a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator being substantially free of CHIR99021 is when CHIR99021 is not detected in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator. CHIR99021 is an inhibitor of the GSK-3 enzyme, and is a C 22 H 18 It is represented by the chemical formula Cl2N8.

[0018] The medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator may contain an ALK inhibitor, a ROCK inhibitor or a growth factor, preferably contains two or more selected from the group consisting of an ALK inhibitor, a ROCK inhibitor and a growth factor, more preferably contains an ALK inhibitor, a ROCK inhibitor and a growth factor. Here, containing two or more selected from the group consisting of an ALK inhibitor, a ROCK inhibitor and a growth factor means, for example, containing an ALK inhibitor and a ROCK inhibitor, containing an ALK inhibitor and a growth factor, containing a ROCK inhibitor and a growth factor, or containing an ALK inhibitor, a ROCK inhibitor and a growth factor.

[0019] ALK inhibitors are molecules that inhibit the function of the ALK (Activin receptor-Like Kinase) family, and are, for example, inhibitors of ALK4, ALK5, or ALK7. Examples of ALK inhibitors include Lefty-1 (NCBI accession numbers include mouse: NP_034224.1 and human: NP_066277.1), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline) and derivatives thereof. 25 H 19 It has the chemical formula N5S. A83-01 is used as an inhibitor of transforming growth factor beta kinase type 1 receptor. A83-01 is an inhibitor of TGF beta kinase / activin receptor-like kinase (ALK5) that blocks phosphorylation of Smad2 / 3, inhibiting TGF beta-induced proliferation. A83-01 blocks phosphorylation of Smad2 and inhibits epithelial-mesenchymal transition induced by TGF-beta. A83-01 also inhibits transcriptional activity induced by TGF beta type I receptor ALK-5, activin type IB receptor ALK-4, and nodal type I receptor ALK-7.

[0020] The content of the ALK inhibitor in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 0.1 to 100 μM, more preferably 0.3 to 30 μM, and even more preferably 1 to 10 μM, relative to the medium.

[0021] ROCK inhibitors are molecules that inhibit the function of Rho-kinase (ROCK), and examples thereof include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)) and derivatives thereof. Y-27632 is also known as (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.2HCl, and is 14 H 21 Its chemical formula is NO.2HCl. Y-27632 is a cell-permeable, potent, ATP-antagonistic, specific ROCK inhibitor that inhibits Ca2+ phosphorylation of myosin and smooth muscle contraction. 2+ Y-27632 acts as a potent inhibitor of receptor agonists, inhibiting cell spreading and suppressing Rho-A-induced stress fiber formation in hepatic stellate cells. Y-27632 also reduces the increase in inflammatory cytokines after reperfusion and prevents the onset of acute renal failure.

[0022] The content of the ROCK inhibitor in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 0.3 to 100 μM, more preferably 1 to 30 μM, and even more preferably 3 to 10 μM, relative to the medium.

[0023] Growth factors are molecules that have the effect of promoting cell division, and specific examples include fibroblast growth factors (FGFs) such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), FGF4, FGF7, and FGF10, as well as TGFα.

[0024] The content of the growth factor in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is preferably 1 to 1000 ng / ml, more preferably 3 to 300 ng / ml, and even more preferably 10 to 100 ng / ml, relative to the medium.

[0025] The medium in the medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator may be, for example, DMEM, DMEM / F12, or DME culture medium containing 10 to 15% FBS, or these culture media may appropriately contain HEPES, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, B27 supplement, N-acetylcysteine, nicotinamide, Gastrin, etc. Furthermore, the medium in the medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator may be a commercially available culture medium such as a culture medium for primate ES / iPS cells (ReproCell), a serum-free medium (mTESR, Stemcell Technology), a medium for proliferation of iPS / ES cells / medium for regenerative medicine (StemFit (registered trademark) AK02N, Ajinomoto Healthy Supply Co., Inc.), a feeder-free medium for iPS cell culture (Essential 8, Gibco), or Cellartis DEF-CS (Takara Bio Inc.), or may be a serum-free medium (e.g., Sun N, et al. (2009), Proc Natl Acad Sci USA. 106:15720-15725).

[0026] Examples of cells derived from stem cells include cells induced to differentiate from stem cells in two-dimensional culture, as well as cells induced to differentiate from stem cells in three-dimensional culture, and the cells derived from stem cells are preferably cells induced to differentiate from stem cells in two-dimensional culture. Examples of cells derived from stem cells include cells induced to differentiate through the definitive endoderm lineage or the hepatoblast lineage, and the cells derived from stem cells are preferably cells induced to differentiate through the definitive endoderm lineage and the hepatoblast lineage. The cells derived from stem cells are preferably hepatocyte lineage.

[0027] The step of culturing the cells derived from stem cells is preferably a step of three-dimensional culture. The three-dimensional culture includes, for example, a step of embedding the cells derived from stem cells in a gel. For example, the gel includes EHS gel.

[0028] An example of the stem cells is an iPS cell. The stem cells are preferably mammalian stem cells, and more preferably human stem cells.

[0029] The hepatocyte cell line produced by the production method of the present invention is preferably that the hepatocyte cell line comprises organoid, and the number of grape bunch-shaped organoids in the organoids contained in the hepatocyte cell line is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, or 99.99% or more.Here, grape bunch-shaped organoids are distinguished from circular organoids.

[0030] The organoid of grape bunch in the hepatocyte line comprising the hepatocyte organoid produced by the above-mentioned production method of the present invention has the phenotype of hepatocyte organoid.Therefore, the present invention provides a method for producing hepatocyte organoid, comprising the step of identifying the organoid of grape bunch from the hepatocyte line comprising the hepatocyte organoid produced by the above-mentioned production method.

[0031] The method for producing hepatocyte organoids provided by the present invention may, for example, include the step of isolating the organoids of the specified bunch of grapes.That is, the hepatocyte organoids produced by the method for producing hepatocyte organoids provided by the present invention may be isolated from other organoids or cells, or may be integrated with other organoids or cells without being isolated from other organoids or cells, such as being contained in a liquid containing other organoids or cells.

[0032] Hepatocyte Organoids: The present invention provides hepatocyte organoids derived from stem cells, which can be cultured for 21 days or more. Hepatocyte organoids derived from stem cells, which can be cultured for 21 days or more, are preferably hepatocyte organoids that can be cultured for 30 days or more, more preferably hepatocyte organoids that can be cultured for 60 days or more, and even more preferably hepatocyte organoids that can be cultured for 180 days or more. More specifically, the hepatocyte organoids provided by the present invention are hepatocyte organoids derived from stem cells, which can be cultured for 21 days or more, preferably 30 days or more, more preferably 60 days or more, and even more preferably 180 days or more after differentiation induction from stem cells, while maintaining the properties of a hepatocyte line.

[0033] Here, the hepatocyte organoids that can be cultured for a predetermined time or more are hepatocyte organoids that can be cultured as hepatocyte organoids for a period of time that is equal to or longer than the predetermined time, and the predetermined time does not include the period of culturing as stem cells or the period from the start of differentiation induction from stem cells to the time of becoming hepatocyte organoids. Therefore, the hepatocyte organoids that can be cultured for a predetermined time or more are, for example, hepatocyte organoids that can be cultured for a predetermined time or more after starting the culture of cells that are derived from stem cells and are hepatocyte lineage, and for example, hepatocyte organoids that can be cultured for a predetermined time or more after starting the culture in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator. However, this does not mean that the hepatocyte organoids that are derived from stem cells and can be cultured for 21 days or more provided by the present invention are limited to those produced by culturing in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator. In addition, even if the start time of the predetermined time is not specified, the predetermined hepatocyte organoid can be cultured for a predetermined time or more from any start time, and therefore the predetermined hepatocyte organoid can be said to be a hepatocyte organoid that can be cultured for a predetermined time or more. However, this does not mean that the predetermined hepatocyte organoid is not a hepatocyte organoid that can be cultured for a predetermined time or more just because it cannot be cultured for a predetermined time or more from any start time such as the time of obtaining the predetermined hepatocyte organoid. For example, when determining whether the obtained predetermined hepatocyte organoid is a hepatocyte organoid that can be cultured for a predetermined time or more, the culture period of the predetermined hepatocyte organoid before the time of obtaining the predetermined hepatocyte organoid can also be taken into consideration.

[0034] Hepatocyte organoids derived from stem cells and capable of being cultured for 21 days or more are preferably not derived from cancer cells.

[0035] Hepatic cell organoids derived from stem cells and capable of being cultured for 21 days or more are preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 30% or less, more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 20% or less, more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 10% or less, more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 5% or less, even more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 3% or less, even more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 1% or less, and even more preferably those in which the number of hepatic stellate cells and Kupffer cells among the cells constituting the hepatic cell organoids is 1% or less, Of the cells, hepatic stellate cells and Kupffer cells are 0.5% or less in terms of the number of cells, and more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells and Kupffer cells are 0.3% or less in terms of the number of cells, and more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells and Kupffer cells are 0.1% or less in terms of the number of cells, and more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells and Kupffer cells are 0.05% or less in terms of the number of cells, and more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells and Kupffer cells are 0.03% or less in terms of the number of cells, and more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells and Kupffer cells are 0.01% or less in terms of the number of cells, and more preferably, substantially free of hepatic stellate cells and Kupffer cells, and more preferably, hepatic stellate cells and Kupffer cells are not detected, and more preferably, hepatic stellate cells and Kupffer cells are not included. Here, the fact that the number of hepatic stellate cells and Kupffer cells is less than a certain numerical percentage does not mean that the number of hepatic stellate cells and Kupffer cells are each less than a certain numerical percentage, but rather that the sum of the number of hepatic stellate cells and the number of Kupffer cells is less than a certain numerical percentage.Moreover, in the hepatocyte organoid derived from stem cells and capable of being cultured for 21 days or more, preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 30% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 20% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 10% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 5% or less in terms of the number of cells, even more preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 3% or less in terms of the number of cells, even more preferably, among the cells constituting the hepatocyte organoid, hepatic stellate cells are 1% or less in terms of the number of cells, and even more preferably, Of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.5% or less in terms of the number of cells, more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.3% or less in terms of the number of cells, more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.1% or less in terms of the number of cells, more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.05% or less in terms of the number of cells, more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.03% or less in terms of the number of cells, more preferably, of the cells constituting the hepatic organoid, the hepatic stellate cells are 0.01% or less in terms of the number of cells, more preferably, substantially free of hepatic stellate cells, more preferably, hepatic stellate cells are not detected, more preferably, free of hepatic stellate cells.Furthermore, in the hepatocyte organoid derived from stem cells and capable of being cultured for 21 days or more, preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 30% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 20% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 10% or less in terms of the number of cells, more preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 5% or less in terms of the number of cells, even more preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 3% or less in terms of the number of cells, even more preferably, among the cells constituting the hepatocyte organoid, Kupffer cells account for 1% or less in terms of the number of cells, and even more preferably, Of the cells constituting the ganoids, Kupffer cells are 0.5% or less in terms of the number of cells, more preferably, of the cells constituting the hepatocyte organoids, Kupffer cells are 0.3% or less in terms of the number of cells, more preferably, of the cells constituting the hepatocyte organoids, Kupffer cells are 0.1% or less in terms of the number of cells, more preferably, of the cells constituting the hepatocyte organoids, Kupffer cells are 0.05% or less in terms of the number of cells, more preferably, of the cells constituting the hepatocyte organoids, Kupffer cells are 0.03% or less in terms of the number of cells, more preferably, of the cells constituting the hepatocyte organoids, Kupffer cells are 0.01% or less in terms of the number of cells, more preferably, substantially free of Kupffer cells, more preferably, Kupffer cells are not detected, and more preferably, Kupffer cells are not included.

[0036] The hepatocyte organoid derived from stem cells and can be cultured for 21 days or more is preferably such that the expression level of albumin gene in hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of albumin gene in hepatocyte organoid is 0.3 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of albumin gene in hepatocyte organoid is 1 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of albumin gene in hepatocyte organoid is 3 times or more than the expression level of GAPDH gene in hepatocyte organoid. The expression level of albumin gene in hepatocyte organoid is measured, for example, by RT-PCR. The expression level of GAPDH gene in hepatocyte organoid is measured, for example, by RT-PCR. The comparison of the expression level of albumin gene in hepatocyte organoid and the expression level of GAPDH gene in hepatocyte organoid is performed, for example, by ΔCT method.

[0037] In this specification, when the predetermined gene is a collective term for a plurality of genes, if it can be said that the expression level of at least one gene among the plurality of genes is a predetermined numerical value multiple or more of the expression level of a specific gene, even if the expression levels of other genes among the plurality of genes are not the predetermined numerical value multiple or more of the expression level of the specific gene, it can be said that the expression level of the predetermined gene is the predetermined numerical value multiple or more of the expression level of the specific gene. In other words, when the predetermined gene is a collective term for a plurality of genes, it cannot be said that the expression level of the predetermined gene is not the predetermined numerical value multiple or more of the expression level of the specific gene, even if the expression level of at least one gene among the plurality of genes is not the predetermined numerical value multiple or more of the expression level of the specific gene. The same applies to the case where the expression level of the specific gene is not the predetermined numerical value multiple or more, but is the predetermined numerical value multiple or less.

[0038] In this specification, the expression level of a given gene in hepatocyte organoids is a given numerical value times or more of the expression level of another gene in hepatocyte organoids, for example, the expression level of the transcription product of a given gene in hepatocyte organoids is a given numerical value times or more of the expression level of another gene in hepatocyte organoids, and the expression level of the translation product of the transcription product of a given gene in hepatocyte organoids is a given numerical value times or more of the expression level of the translation product of the transcription product of another gene in hepatocyte organoids. Therefore, if the expression level of the transcription product of a given gene in hepatocyte organoids is a given numerical value times or more of the expression level of the transcription product of another gene in hepatocyte organoids, regardless of the expression level of the translation product of the transcription product of a given gene in hepatocyte organoids, it can be said that the expression level of a given gene in hepatocyte organoids is a given numerical value times or more of the expression level of another gene in hepatocyte organoids. Furthermore, if the expression level of a translation product of a transcription product of a given gene in a hepatocyte organoid is a given numerical value or more or a given numerical value or less than the expression level of a translation product of a transcription product of another gene in a hepatocyte organoid, then, regardless of the expression level of the transcription product of the given gene in the hepatocyte organoid, it can be said that the expression level of the given gene in the hepatocyte organoid is a given numerical value or more or a given numerical value or less than the expression level of another gene in the hepatocyte organoid.

[0039] The hepatocyte organoid derived from stem cells, which can be cultured for 21 days or more, preferably, the expression level of HNF4α gene in hepatocyte organoid is 0.003 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of HNF4α gene in hepatocyte organoid is 0.01 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of HNF4α gene in hepatocyte organoid is 0.03 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of HNF4α gene in hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in hepatocyte organoid. The expression level of HNF4α gene in hepatocyte organoid is measured, for example, by RT-PCR. The expression level of GAPDH gene in hepatocyte organoid is measured, for example, by RT-PCR. The expression level of the HNF4α gene in the hepatocyte organoid and the expression level of the GAPDH gene in the hepatocyte organoid are compared, for example, by the ΔCT method.

[0040] The hepatocyte organoid derived from stem cells and can be cultured for 21 days or more is preferably such that the expression level of AFP gene in hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of AFP gene in hepatocyte organoid is 0.3 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of AFP gene in hepatocyte organoid is 1 times or more than the expression level of GAPDH gene in hepatocyte organoid, more preferably, the expression level of AFP gene in hepatocyte organoid is 5 times or more than the expression level of GAPDH gene in hepatocyte organoid. The expression level of AFP gene in hepatocyte organoid is measured, for example, by RT-PCR. The expression level of GAPDH gene in hepatocyte organoid is measured, for example, by RT-PCR. The comparison of the expression level of AFP gene in hepatocyte organoid and the expression level of GAPDH gene in hepatocyte organoid is performed, for example, by ΔCT method.

[0041] The hepatocyte organoid derived from stem cells and can be cultured for 21 days or more is preferably such that the expression level of the EPCAM gene in the hepatocyte organoid is 30 times or less than the expression level of the GAPDH gene in the hepatocyte organoid, more preferably, the expression level of the EPCAM gene in the hepatocyte organoid is 10 times or less than the expression level of the GAPDH gene in the hepatocyte organoid, more preferably, the expression level of the EPCAM gene in the hepatocyte organoid is 5 times or less than the expression level of the GAPDH gene in the hepatocyte organoid, and more preferably, the expression level of the EPCAM gene in the hepatocyte organoid is 2 times or less than the expression level of the GAPDH gene in the hepatocyte organoid. The expression level of the EPCAM gene in the hepatocyte organoid is measured, for example, by RT-PCR. The expression level of the GAPDH gene in the hepatocyte organoid is measured, for example, by RT-PCR. The comparison of the expression level of the EPCAM gene in the hepatocyte organoid and the expression level of the GAPDH gene in the hepatocyte organoid is performed, for example, by the ΔCT method.

[0042] The hepatocyte organoid derived from stem cells and can be cultured for 21 days or more preferably has bile canalicular structure or microcilia.The hepatocyte organoid derived from stem cells and can be cultured for 21 days or more has bile canalicular structure or microcilia, for example, the hepatocyte organoid derived from stem cells and can be cultured for 21 days or more can be observed by observing bile canalicular structure or microcilia under electron microscope.

[0043] The hepatocyte organoid of the present invention may be isolated, but may also be contained in hepatocyte line.When hepatocyte organoid is contained in hepatocyte line, for example, hepatocyte line contains organoid, and the number of grape bunch-shaped organoids in the organoids contained in hepatocyte line is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, or 99.99% or more.Here, grape bunch-shaped organoid is distinguished from circular organoid.

[0044] In the hepatocyte organoid derived from stem cell, and can be cultured for more than 21 days, the stem cell is preferably iPS cell.In addition, in the hepatocyte organoid derived from stem cell, and can be cultured for more than 21 days, the stem cell is preferably mammalian stem cell, more preferably human stem cell. EXAMPLES

[0045] Cell line: Human iPS cell lines (P11025, RIKEN-2F, PB001) were maintained and cultured in a 37°C incubator with 5% CO2 in air by a Cellartis® DEF-CS™ 500 culture system (Takara Bio) according to the manufacturer's protocol. Human iPS cell line P11025 was established from male fibroblasts reprogrammed by overexpression of Oct3 / 4, Sox2, Klf4, and c-Myc (OKSM) transduced by episomal vectors. Human iPS cell line RIKEN2F was established from umbilical cord fibroblasts of a Japanese male and reprogrammed by retroviral expression of OKSM. Human iPS cell line PB001 was established from peripheral blood cells, reprogrammed using a Sendai virus vector expressing OKSM, and transduced by a lentiviral vector expressing tdTOMATO. P11025 was purchased from Takara Bio. RIKEN2F and PB001 were kindly provided by Dr. Y. Nakamura and Dr. Masaki H., respectively. 2F-GFP was established from RIKEN2F by overexpression of GFP using the piggyBac transposon system.

[0046] Human 2D iPSC-derived hepatocyte-like cell culture: iPSCs were differentiated into hepatocytes using the Cellartis® Definitive Endoderm Differentiation Kit and the Cellartis® Hepatocyte Differentiation Kit (Takara Bio) according to the manufacturer's protocol. During hepatic differentiation, iPSCs differentiated into hepatocyte-like cells after day 21, so iPSC-derived hepatocytes (iPS-Heps) were used as differentiated cells on day 36. iPSCs were also differentiated into hepatocytes using the protocol described by Ang et al. with some modifications. Briefly, iPSCs were plated at approximately 2x10 4 cells / cm 2(day 0). For differentiation into definitive endoderm (day 1 to 2), iPSCs were cultured in IMDM / F12 (1:1) supplemented with 1x insulin-serine-transferrin (ITS), 1x lipid concentrate (LC), 1x penicillin / streptomycin (PS), 0.1% polyvinyl alcohol (PVA), 100 ng / mL activin A (Nacalai Tesque), 2 μM CHIR99021 (day 1), 250 nM LDN193189 (day 2), and 50 μM PI-103. For differentiation towards hepatic lineages (days 3 to 6), cells were cultured in IMDM / F12 (1:1) supplemented with 10% knockout serum (KOSR), 1xLC, 1xPS, 0.1% PVA, basic FGF 10 ng / mL (day 3), 10 ng / mL activin A (days 4 to 6), 30 ng / mL BMP4, 1 μM A83-01 (day 3), 2 μM all-trans retinoic acid (day 3), 1 μM forskolin (days 4 to 6), 1 μM C59 (days 4 to 5), and 1 μM CHIR99021 (day 6). For differentiation into hepatocytes, cells were cultured for the next 6 days (days 7 to 12) in IMDM / F12 (1:1) supplemented with 1x ITS, 1x Glutamax™, 1x Non-Essential Amino Acids (NEAA), 1xLC, 1xPS, 10ng / mL BMP4, 10ng / mL Oncostatin M, 10μM Forskolin, 10μM DAPT, 10μM Dexamethasone, 200μg / mL Ascorbic Acid-2-phosphate (AAP), and 1μM SB431252 or SB505124 (days 7 to 8). For further differentiation, cells were cultured in IMDM / F12 (1:1) supplemented with 10% KOSR, 1xITS, 1xGlutamax™, 1xNEAA, 1xLC, 1xPS, 10 μM forskolin, 10 μM DAPT, 10 μM dexamethasone, 200 μg / mL AAP (days 13 to 18). After 18 days, iPSCs were differentiated into iPS-Heps.

[0047] Human iPSC-derived hepatocyte organoid culture: Differentiated iPSC-derived hepatocytes were dissociated with collagenase and TrypLE select (Thermofisher), washed twice with PBS, mixed with Matrigel, and seeded per 24-well plate. After Matrigel solidification, medium was added to each well. Human fetal hepatocyte organoid medium (FO) was prepared as previously described. iPSC-derived hepatocyte organoid medium (iHO medium) consisted of AdDMEM / F12 (Thermofisher) supplemented with HEPES, Glutamax, penicillin-streptomycin, B27, 1.25 mM N-acetylcysteine ​​(Sigma), 10 mM nicotinamide (Sigma), 15% RSPO conditioned medium (homemade; contains R-spondin1), 300 ng / mL mouse Wnt3a (R&D), 10 ng / mL Gastrin, 2 μM A83-01, 10 μM Y-27632, 50 ng / mL EGF, 50 ng / mL HGF, 100 ng / mL FGF7, 100 ng / mL FGF10, 20 ng / mL TGFα, and 10 μM taurobeticholic acid. Medium was changed every 2–3 days (3 times a week). iHOs were routinely passaged after 2 weeks of culture, with a passage ratio of 1:3.

[0048] RNA isolation and qRT-PCR analysis: Total RNA was extracted using RNeasy Mini and Micro Kit (Qiagen) according to the manufacturer's protocol. First-strand cDNA was synthesized using PrimeScript™ IV 1st Strand cDNA Synthesis Mix (Takara Bio) and used as a template for PCR. Quantitative RT-PCR analysis was performed using QuantiTect SYBR Green PCR Kit (Qiagen). All data were normalized to GAPDH. For quantification of GAPDH mRNA, expression levels were quantified using TaqMan™ GAPDH Control Reagents (human) (Thermo Fisher Scientific).

[0049] RNA-seq and analysis: Total RNA samples were extracted using RNeasy Micro kit and DNase I treatment. The quality check of RNA samples was confirmed by measuring RNA Integrity Number (RIN). Sequencing libraries were prepared by Dynabeads mRNA Purification Kit and RNA Directional Library Prep Set according to the manufacturer's protocol. Sequence data were obtained using DNBSEQ-G400. We combined our own RNA-seq data with published data from GEO (GSM3067802, GSM3067803 for fetal hepatocytes, GSM3090157, GSM3090158 for adult hepatocytes) to compare liver phenotypes. Transcript level counts and TPM were quantified by salmon package using gencode v38 transcriptome reference (see Nat Methods. 2017 Apr;14(4):417-419.). They were aggregated to gene level for gene-level differential expression analysis using the tximport package.

[0050] Single-cell RNA sequencing and analysis: Organoids embedded in Matrigel (iHO condition and passage 1) were washed with ice-cold PBS and dissociated into single cells using trypsin / EDTA (0.05%) for 10–20 min. Trypan blue staining confirmed that cell viability was 80% or higher. For 5000 viable cells, scRNA-seq library preparation was performed and sequenced on the 10x Genomics Chronium platform according to the manufacturer's protocol. The resulting libraries were sequenced with DNBSEQ (MGI). Sequenced reads were processed using the Cell Ranger gene expression pipeline mkfastq and count, starting with demultiplexing and conversion of barcode and read data into fastq files. Raw reads were aligned to the GRCh38 genome, filtered, and a gene barcode matrix was created. Cell and gene filtering, data scaling, clustering, and visualization were performed with the Seurat v3 toolkit (https: / / github.com / satijalab / seurat). Cells with fewer than 200 unique features or more than 25% mitochondrial counts were removed. Data were normalized using the default global scaling method.

[0051] Immunofluorescence: Immunostaining of organoids was performed similarly to previous methods. Briefly, organoids were fixed with 4% paraformaldehyde (PFA) for approximately 2 h, washed with PBS, soaked in 30% sucrose buffer overnight, and embedded in OCT compound. Organoid sections were cut at 10 μm, washed with PBS, permeabilized with 0.05% Tween20 / PBS (PBS-T), and blocked with Blocking One (Nacalai Tesque) for at least 2 h. Subsequently, sections were incubated with primary antibodies overnight, washed with PBS-T, and incubated with secondary antibodies for 1 h. Stained sections were washed and mounted using VECTASHIELD mounting medium containing DAPI (VECTOR Laboratories). Images were scanned and captured using a KEYENCE BZ-X710 and FLUOVIEW FV3000.

[0052] Immunostaining of whole-mount organoids: Immunostaining of whole-tissue organoids was performed according to a previously reported protocol for whole-tissue immunostaining. Organoids were fixed overnight in 4% PFA and permeabilized with 1% Triton X-100 / PBS for at least 6 h. Organoids were incubated with primary antibodies for 2 days, washed twice with 0.1% Triton X-100 / 0.2% BSA / PBS, and then incubated with secondary antibodies overnight. Organoids were then washed three times and incubated in fructose-glycerol clearing solution (60% (vol / vol) glycerol and 2.5 M fructose) for 20 min at room temperature. Confocal images were captured using an FV3000, and Z-stacks of organoid images were analyzed and assembled using FV3000 software (OLYMPUS).

[0053] Example 1 Bile acids induce 3D proliferation and hepatocyte phenotypes of iPS-Hep: Human iPS cells were induced to differentiate into hepatocytes through the definitive endoderm lineage and hepatoblast lineage in plate culture (2D-iPS-Hep), and then embedded in EHS gel for 3D culture (Figure 1A). Based on the previously reported medium composition of human fetal hepatocyte organoids (FO), we explored factors involved in liver regeneration through various condition studies and found that adding tauro-obeticolic acid, a strong FXR agonist, and recombinant Wnt3a as a canonical Wnt agonist was effective. iPS-Hep Organoids (iHO) induced under these culture conditions were different from those induced under bile acid-free or FO culture conditions. Grape-like organoids with a characteristic bunch-of-grapes morphology were induced (Fig. 1B and 1C), and the overall number of organoids formed tended to be higher (Fig. 1D). iHOs were able to maintain expression of the albumin gene, which is characteristic of mature hepatocytes, at a level more than five times higher than that of human iPS-Hep produced by conventional two-dimensional culture (Fig. 1E). iHOs could be cultured for sustained proliferation, with 1:2 subcultures continued for about two weeks, allowing for culture for up to six months. During this time, iHOs continued to proliferate while maintaining the morphology of grape-like organoids. When whole mount observations of organoids induced using cell lines fluorescently labeled with GFP were performed using a confocal microscope, a grape-like morphology was observed even in three dimensions (Fig. 2A and 2C). The characteristic morphology was also confirmed by scanning electron microscopy (Fig. 2B). Immunostaining also showed strong expression of hepatocyte markers such as ALB and AFP, and some also showed expression of Ki-67, suggesting that this is an organoid culture system that maintains proliferation and hepatocyte phenotypes at the protein level (Figures 2D and 2E).However, iHO actually contained round organoids and cystic organoids that exhibited morphologies other than grape-like.However, immunohistochemistry revealed that the expression of ALB was low, and the expression levels of progenitor cell and bile duct cell markers such as EPCAM and KRT19 were high, indicating that the hepatocyte phenotype was relatively weak (Figures 2F and 2G). These results suggest that organoids with strong hepatocyte phenotypes, which characterize iHO, exhibit a grape-like morphology.

[0054] Example 2 iHOs have similar microstructure and functions to hepatocytes: When iHO were observed under a transmission electron microscope, they had microvilli on the cell surface and some tubular structures lined with tight junctions, suggesting a bile canaliculi-like structure characteristic of hepatocytes (Fig. 3A, left). Like hepatocytes, they contained abundant mitochondria and rough endoplasmic reticulum, as well as glycogen granules (Fig. 3A, right). In addition to producing albumin, iHO also had the ability to take up LDL and store glycogen, which are functions unique to hepatocytes (Fig. 3B-D).

[0055] Example 3: RNA-seq analysis reveals that iHOs have an expression profile similar to that of fetal liver cells: RNA-seq was performed to investigate the gene expression profile of iHO. In addition to comparison with culture conditions without bile acids and plate culture, we also compared with taurocholic acid in addition to taurobeticholic acid. In addition, we compared passages 1 and 3 to confirm changes over time. Principal component analysis showed that iHO has an expression profile different from existing culture systems and PHH, and has an expression profile similar to fetal liver cells (FLC) (Figure 4A). Hierarchical clustering also suggested that iHO is relatively similar to FLC and PHH compared with other culture conditions and 2D-iPS-Hep (Figure 4B). When genes with different expression levels were extracted in comparison with culture conditions without bile acids and enrichment analysis was performed, the expression of gene sets related to hepatocytes and fetal liver cells was increased (Figure 4C and Figure 4D). In addition, in terms of functional terms such as GO, the expression of gene sets related to the coagulation-fibrinolysis system, complement, and lipid metabolism was increased (Figure 4E). When genes related to hepatocytes, fetal liver cells, progenitor cells, and bile duct cells were specifically extracted and a heat map was created, it was found that iHO had high expression of hepatocyte markers, while the expression of progenitor cell, bile duct cell markers, and intestinal epithelial cell markers was low compared to other culture systems (Figure 4F). The difference with PHH was that the expression of CYPs such as CYP3A4 was low, and fetal liver cell markers such as AFP and DLK1 were high. iHO had an expression profile close to fetal liver cells, and was shown to have a relatively similar expression profile to PHH compared to 2D-iPS-Hep.

[0056] Example 4 Single cell analysis shows that iHOs have hepatocyte-like and cholangiocyte-like fractions: Single-cell RNA-seq analysis showed that iHO cells were divided into two fractions: a hepatocyte-like fraction that highly expressed hepatocyte lineage genes such as ALB, and a cholangiocyte-like fraction that highly expressed bile duct lineage genes such as KRT19 (Figure 5A). The hepatocyte-like fraction highly expressed ALB as well as hepatocyte marker genes such as AHSG, TF, and C3, and also showed high expression of fetal hepatocyte-related genes such as AFP, DLK1, and GPC3 (Figure 5B). On the other hand, the cholangiocyte-like fraction highly expressed intestinal epithelial cell lineage genes such as CDX2, ISX, and DPP4 in addition to bile duct lineage genes such as KRT19, KRT7, and EPCAM (Figure 5B). It has been reported that iPSC-derived hepatocytes in two-dimensional culture co-express intestinal epithelial cell lineage genes, which are a part of other organs. In iHOs cultured in three dimensions, this characteristic remained in the bile duct cell-like fraction, but was thought to have almost disappeared in the hepatocyte-like fraction. In addition, the hepatocyte-like fraction contained a subfraction that highly expressed the proliferation-related genes MKI67 and PCNA (Figure 5C), suggesting the presence of cells that have both proliferation and hepatocyte characteristics even at the single cell level. In addition, when combined with previously reported single cell data on human fetal liver (Cao et al. Science, 2020), it was shown that part of the iHO overlapped with fetal hepatocytes (Figure 5D). In combination with the results of immunostaining and other methods, it was suggested that the grape-like organoids in iHOs in particular were almost entirely composed of cells from the hepatocyte-like fraction, and that they could be considered as organoids consisting of only hepatocytes.

Claims

1. A method for producing a hepatocyte line, including a hepatocyte organoid, comprising culturing cells derived from stem cells in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator.

2. The method of claim 1, wherein the nuclear receptor agonist is an FXR agonist.

3. The method of claim 2, wherein the FXR agonist is a bile acid.

4. The method according to claim 3, wherein the bile acid is taurobeticholic acid.

5. The method of claim 1, wherein the Wnt / β-catenin pathway activator is Wnt3a.

6. The production method according to claim 5, wherein the medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator contains a Wnt / β-catenin pathway activator other than Wnt3a.

7. The method of claim 6, wherein the Wnt / β-catenin pathway activator is not Wnt3a but is R-spondin.

8. The method of claim 7, wherein the R-spondin is selected from the group consisting of R-spondin1, R-spondin2, R-spondin3 and R-spondin4.

9. The manufacturing method according to claim 1, wherein the concentration of CHIR99021 in a medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator is 10 μM or less in said medium.

10. The production method according to claim 1 , wherein the medium containing a nuclear receptor agonist or a Wnt / β-catenin pathway activator contains an ALK inhibitor, a ROCK inhibitor or a growth factor.

11. The method of claim 10, wherein the ALK inhibitor is A83-01.

12. The method according to claim 10, wherein the ROCK inhibitor is Y-27632.

13. The method according to claim 10, wherein the growth factor is selected from the group consisting of EGF, HGF, FGF7, FGF10 and TGFα.

14. The method according to claim 1, wherein the cells derived from stem cells are cells induced to differentiate from stem cells in two-dimensional culture.

15. The method according to claim 1, wherein the cells derived from stem cells are cells induced to differentiate through a definitive endoderm lineage or a hepatoblast lineage.

16. The method of claim 15, wherein the cells induced to differentiate through the definitive endoderm lineage or the hepatoblast lineage are cells induced to differentiate through the definitive endoderm lineage and the hepatoblast lineage.

17. The method of claim 1, wherein the cells derived from stem cells are of hepatic cell lineage.

18. The method according to claim 1, wherein the step of culturing cells derived from stem cells is a step of three-dimensional culture.

19. The method of claim 18, wherein the three-dimensional culture comprises embedding cells derived from stem cells in a gel.

20. 20. The method of claim 19, wherein the gel is an EHS gel.

21. The method of claim 20, wherein the stem cells are iPS cells.

22. The method of claim 21 , wherein the stem cells are human stem cells.

23. The method of claim 1, wherein the hepatic cell line comprises organoids, and among the organoids contained in the hepatic cell line, the number of grape-like organoids is 10% or more.

24. A method for producing hepatocyte organoids, comprising a step of identifying grape-like organoids from a hepatocyte cell line containing the hepatocyte organoids produced by the production method described in claim 1.

25. The method of claim 24, comprising isolating the identified grape bunch-like organoids.

26. A hepatocyte organoid derived from a stem cell, which can be cultured for 21 days or more.

27. 27. The hepatocyte organoid of claim 26, which can be cultured for 180 days or more.

28. 27. The hepatocyte organoid of claim 26, which is not derived from a cancer cell.

29. The hepatocyte organoid according to claim 26, wherein the number of hepatic stellate cells and Kupffer cells in the cells constituting the hepatocyte organoid is 30% or less.

30. The hepatocyte organoid of claim 26, wherein the expression level of the albumin gene in the hepatocyte organoid is 0.1 times or more than the expression level of the GAPDH gene in the hepatocyte organoid.

31. The hepatocyte organoid of claim 26, wherein the expression level of HNF4α gene in the hepatocyte organoid is 0.003 times or more than the expression level of GAPDH gene in the hepatocyte organoid.

32. The hepatocyte organoid of claim 26, wherein the expression level of AFP gene in hepatocyte organoid is 0.1 times or more than the expression level of GAPDH gene in hepatocyte organoid.

33. The hepatocyte organoid of claim 26, wherein the expression level of the EPCAM gene in the hepatocyte organoid is 30 times or less than the expression level of the GAPDH gene in the hepatocyte organoid.

34. 27. The hepatocyte organoid of claim 26, having a bile canalicular structure or microcilia.

35. 35. The hepatocyte organoid of claim 34, wherein a bile canalicular structure or microcilia is observed under an electron microscope.

36. The hepatocyte organoid of claim 26, which is contained in a hepatocyte cell line.

37. The hepatocyte organoid of claim 36, wherein the hepatocyte cell line comprises organoids, and the number of grape-like organoids in the organoids contained in the hepatocyte cell line is 10% or more.

38. 27. The hepatocyte organoid of claim 26, wherein the stem cell is an iPS cell.

39. 27. The hepatocyte organoid of claim 26, wherein the stem cells are human stem cells.

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