Hepatic organoid maturation medium

Abstract

To provide a new hepatic organoid maturation medium for induction or promotion of hepatocyte maturation within a hepatic organoid.SOLUTION: A hepatic organoid maturation medium comprises at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention relates to a liver organoid maturation medium for inducing or promoting the maturation of hepatocytes in liver organoids. [Background technology]

[0002] Human organoids are becoming increasingly important as a reliable tool for evaluating the physiological functions of the human liver, replacing animal experiments (Non-Patent Document 1). In particular, human liver organoids (HLOs) are expected to have physiological properties.

[0003] Human liver organoids reported to date include human tissue-derived liver organoids (Non-Patent Document 2) and human iPS cell-derived liver organoids. Human iPS cell-derived liver organoids further include organoids produced by artificially mixing three types of cells (Non-Patent Document 3) and organoids produced by the spontaneous generation of multiple types of cells during differentiation (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Journal of Clinical Investigation 124, 328-337, 2014 [Non-patent document 2] Cell 175, 1591-1606, 2018 [Non-patent document 3] Nature 499, 7459, 481-484, 2013 [Non-patent document 4] Cell Metabolism 30(2) 378-384, 2019 Summary of the Invention [Problem to be solved by the invention]

[0005] In the production of human iPS cell-derived liver organoids, including the proliferation and maturation process of human liver organoids, there was a problem in that the maturity of hepatocytes in liver organoids was insufficient when conventional culture media were used.

[0006] The problem to be solved by the present invention is to provide a liver organoid maturation medium for inducing or promoting the maturation of hepatocytes in liver organoids, a method for producing liver organoids, and a method for inducing or promoting the maturation of hepatocytes in liver organoids. [Means for solving the problem]

[0007] The present invention encompasses the embodiments described below. Section 1. A liver organoid maturation medium containing at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin. Section 2. The liver organoid maturation medium according to paragraph 1, further containing amino acids and inorganic salts. Section 3. The liver organoid maturation medium described in paragraph 1, containing a basal medium. Section 4. The liver organoid maturation medium according to paragraph 1, which does not contain growth factors or contains a total of 1000 ng / mL or less of growth factors. Section 5. A method for producing liver organoids, comprising: A method comprising culturing liver organoids in the liver organoid maturation medium according to any one of items 1 to 4 for a sufficient time to induce or promote the maturation of hepatocytes in the liver organoids. Section 6. Item 6. The method according to item 5, wherein after the step of culturing, the expression level of the gene that is related to metabolism in at least one hepatocyte in liver organoid or that is expressed in liver-specific manner increases compared with the expression level of the gene that is related to metabolism in at least one hepatocyte or that is expressed in liver-specific manner before the step of culturing. Section 7. the hepatocyte maturation medium contains diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, and the at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner includes at least one gene selected from the group consisting of albumin, CYP7A1, ARG1, ABCG5, ABCG8, and UGT2B2; or Item 6. The method according to Item 5, wherein the hepatocyte maturation medium contains calcipotriene, and the at least one gene involved in metabolism in hepatocytes or expressed in a liver-specific manner includes at least one gene selected from the group consisting of genes encoding ALB, CYP2C8, CYP2C9, CYP3A4, CYP23A1, UGT1A3, UGT1A4, SLC30A10, and ceruloplasmin. Section 8. A liver organoid produced by the method for producing a liver organoid described in paragraph 5. Section 9. A liver organoid, wherein the liver organoid is cultured in a liver organoid maturation medium containing at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin, and the expression level of at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner is increased by three times or more compared to the expression level of the at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner in the liver organoid cultured in a medium that does not contain at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin. Section 10. A method for inducing or promoting hepatocyte maturation in liver organoids, comprising: A method comprising culturing liver organoids in the liver organoid maturation medium according to any one of items 1 to 4 for a sufficient time to induce or promote the maturation of hepatocytes in liver organoids. Section 11. Fe 3+ and Zn 2+ Item 11. The method according to item 10, further comprising the step of counteracting the maturation of hepatocytes in liver organoids by at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin, by adding at least one of the following to the liver organoid maturation medium. Section 12. A method for identifying a molecule that controls the maturation of hepatocytes in liver organoids, comprising: Item 1 to 4. The method comprises contacting the liver organoid cultured in the liver organoid maturation medium with a candidate molecule, and the change in the maturation of hepatocytes in the liver organoid indicates that the candidate molecule is a molecule that controls the maturation of hepatocytes in the liver organoid. [Brief explanation of the drawings]

[0008] [Figure 1] iHLO passage. Biotechnol.J.2024;19:2300365 [Figure 2] Microscopic image of iHLO. AD in Figure 2 corresponds to each stage of AD in Figure 1. AC in Figure 2 is taken from Biotechnol.J.2024;19:2300365 [Figure 3] Changes in the expression levels of various markers when iHLOs were cultured in EM medium for 6 days after differentiation induction in HDM medium. Quoted from Biotechnol.J.2024;19:2300365 [Figure 4] Changes in the expression levels of various markers when iHLOs were grown in EM medium and then cultured in EM or HDM medium for 3 days. Quoted from Biotechnol.J.2024;19:2300365 [Figure 5] Overview of iHLO differentiation and culture methods. See Biotechnol.J.2024;19:2300365. [Figure 6A] Albumin (ALB) mRNA levels in various hepatocytes and liver organoids. Mean ± SEM. [Figure 6B] Graph showing the amount of albumin secreted into the medium by iHLO cultured in EM medium and iHLO cultured in HDM, when the amount of albumin secreted into the medium by HepG2 cells is set to 1. Mean ± SEM. [Figure 7] Graph showing the concentration-dependent albumin protein secretion amount of each screening hit compound in iHLO (mean ± SEM). [Figure 8] Graph showing changes in the expression of various genes when iHLO cells were treated with DMSO, 10 μM each of DTPA, calcipotriene, and bleomycin. [Figure 9] Graph showing changes in the expression of various genes when iHLO cells were treated with DMSO, 10 μM each of DTPA, calcipotriene, and bleomycin. [Figure 10] Signaling pathways (left) extracted from the RNA sequencing results using Ingenuity pathway analysis, and a list of upstream factors (right) whose activity is predicted to have changed. [Figure 11] Bright-field microscope image of pHLO. [Figure 12] Graph showing changes in the expression of various genes upon treatment with 10 μM DTPA in pHLO. [Figure 13] Graph showing changes in the expression of various genes upon treatment with 3 μM calcipotriene in pHLO. [Figure 14] Graph showing changes in the expression of various genes when HepG2 cells and HuH-7 cells were treated with 10 μM each of DTPA, calcipotriene, and bleomycin. [Figure 15] Time course of the experiment. [Figure 16]Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 17] Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 18] Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 19] Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 20] Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 21] Graph showing the difference in mRNA expression induction of various genes when the basal medium is changed. [Figure 22] Graph showing the inhibitory effect of adding Fe3+ and Zn2+ on the induction of ALB expression when it was induced by DTPA treatment. [Figure 23] (A) Candidates for upstream factors predicted to be suppressed by the addition of Fe3+ and Zn2+ when hepatocyte maturation was induced by DTPA treatment. (B) Changes in the expression of various proteins when hepatocyte maturation was induced by DTPA treatment. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments for carrying out the present invention will be described. Note that the embodiment described below shows an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0010] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0011] In the numerical ranges described in stages in this specification, the upper or lower limit of a certain numerical range can be arbitrarily combined with the upper or lower limit of another numerical range. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in an example or a value that can be unambiguously derived from an example. Furthermore, in this specification, a numerical value connected with "~" means a numerical range that includes the numbers before and after "~" as the upper and lower limits.

[0012] As used herein, organoid refers to the miniaturized organ that is generated in vitro in three dimensions.Generally, organoid comprises multiple types of organ-specific cells.

[0013] As used herein, liver organoids refer to organoids containing hepatocytes.

[0014] Herein, human tissue-derived liver organoids are referred to as pHLO( P rimary H uman L iver O It is sometimes called rganoid.

[0015] As used herein, human iPS cell-derived liver organoids are referred to as iHLO( i PSC-derived H uman L iver O It is sometimes called rganoid.

[0016] As used herein, "genes related to metabolism in hepatocytes" refers to genes involved in metabolism, including nutrient metabolism and drug metabolism, in hepatocytes, including genes encoding proteins that control the metabolism of at least one nutrient selected from the group consisting of sugars, lipids, proteins, drugs, and vitamins in hepatocytes.

[0017] As used herein, a liver-specifically expressed gene is a gene that encodes a protein whose expression in hepatocytes increases as hepatocyte function matures, and includes genes that serve as hepatocyte markers.

[0018] Culture media for human liver organoids include, for example, EM medium (Expansion Medium) as a proliferation medium (described in Hu et al., Cell 175, 1591-1606, 2018), and HDM medium (Hepatocyte Differentiation Medium) as a differentiation and maturation medium (Ouchi et al., Cell Metabolism 30(2) 378-384, 2019) (Table 1).

[0019] [Table 1]

[0020] For example, iHLOs are produced from human iPS cells according to the procedure described in Cell Metabolism 30(2) 378-384, 2019, and are cultured in HDM medium from day 10 to day 20 after differentiation induction.

[0021] As shown in Figures 1 and 2, when iHLOs were subcultured on the 20th day after the start of iPS cell culture, proliferation began and they could be subcultured when they were transferred to EM medium, but they could not proliferate if they were maintained in HDM.

[0022] Furthermore, as shown in Figure 3, when iHLOs were differentiated in HDM medium and then cultured in EM medium for 6 days, the mRNA expression levels of the hepatocyte marker LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5) increased, the mRNA expression levels of the fetal liver cell marker AFP (alpha-fetoprotein) decreased, and the mRNA expression levels of the hepatocyte marker albumin (ALB) decreased. LGR5 is an indicator of proliferation, while AFP and ALB are indicators of maturation. ALB, in particular, is the most commonly used marker for hepatocyte maturation. Furthermore, as shown in Figure 4, when iHLOs were grown in EM medium and then cultured in EM or HDM medium for 3 days, the mRNA expression levels of LGR5 decreased and the mRNA expression levels of AFP and ALB increased when cultured in HDM medium.

[0023] Based on this, we developed a method for producing iPS cell-derived liver organoids and subsequent culture, as shown in Figure 5. iHLOs were prepared, for example, as described in Cell Metabolism 30(2) 378-384, 2019, then passaged in EM medium and matured in HDM medium. For details of the experiments in Figures 1-5, please refer to Biotechnol.J.2024;19:2300365; https: / / doi.org / 10.1002 / biot.202300365.

[0024] However, both liver organoids grown using this culture method (iHLO EM, pHLO EM) and those transferred to conventional HLO medium for maturation and differentiation (iHLO HDM, pHLO HDM) showed lower expression levels of the maturation marker albumin, both in mRNA and protein, compared to primary human tissue-derived liver organoids (PHH, PHH4h) and established hepatocyte cell lines (HepG2, HuH-7) (Figures 6A, 6B). This means that the use of HDM medium resulted in insufficient hepatocyte maturation in liver organoids.

[0025] Therefore, we investigated the improvement of the medium composition for the maturation of hepatocytes in liver organoids.First, we found that among 21 factors reported to have the potential to enhance hepatocyte function, no factor promoting the maturation of HLOs was identified (Table 2). For details on the 21 factors, see Huch, M. et al. Cell 160, 299-312; Lee, JM et al. Nature 474, 506-510 (2011); Xiang, C. et al. Science 364, 399-402; Xiang, C. et al. Science 364, 399-402 (2019); Kotaka, M. et al. Sci. Rep. 7, 16734 (2017); Hu, H. et al. Cell 175, 1591-1606.e19 (2018); Miyazaki, M. et al. Exp. Cell Res. 159, 176-190 (1985); Xiang, C. et al. Science 364, 399-402 (2019); Boon, R. et al. Nat. Commun. 11, 1-16 (2020); Yuan, F. et al. J. Steroid Biochem. Mol. Biol. 211, 105881 (2021); Sun, P. et al. Cell Rep. 29, 3212-3222.e4 (2019); Wu, A.-L. et al. PLoS One 6, e17868 (2011); Kojima, M., et al., Biol. Pharm. Bull. 34, 1644-1647 (2011); Lammel Lindemann, JA et al., Mol. Cell. Endocrinol. 388, 32-40 (2014); Yang, C. et al. Redox Biol. 32, 101445 (2020); Lam, N. V et al. Amino Acids 30, 43-48 (2006); Wang, Y. et al. Nat. Commun. 11, 3612 (2020).Therefore, we aimed to identify novel factors that promote the maturation of HLOs using the albumin protein secretion level of hepatocytes as an indicator from the Validated Compound Library (approximately 3,500 compounds with known pharmacological activity, https: / / www.ddi.fu-tokyo.ac.jp / chemical_library / #1) maintained by the University of Tokyo Drug Discovery Institute. As a result, we identified the following compounds that significantly induce the expression of hepatocyte markers, including albumin: the chelator DTPA (diethylenetriamine-N,N,N',N'',N''-pentaacetic acid, CAS Registry Number 67-43-6), calcipotriene (CAS Registry Number 112965-21-6), which exhibits vitamin D activity, and the anticancer drug bleomycin (CAS Registry Number 11056-06-7). Comprehensive gene expression analysis indicated that these compounds independently enhance a wide range of hepatocyte functions. Furthermore, DTPA and calcipotriene have shown similar effects not only on HLOs differentiated from iPS cells but also on HLOs established from human liver tissue. The present invention relates to a method for inducing and / or promoting HLO maturation, which comprises adding at least one of these compounds to a culture medium, and a liver organoid maturation medium.

[0026] [ka]

[0027] [Table 2] LRH-1: Liver receptor homolog-1 ALK: activin receptor-like kinase MEM: Minimum essential medium PI3K: phosphatidylinositol-3 kinase TFEB: transcription factor EB MAPK: MAP kinase (mitogen-activated protein kinase)

[0028] In one embodiment, the present invention provides a liver organoid maturation medium, comprising at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid (DTPA), calcipotriene, and bleomycin.

[0029] Liver organoid maturation medium is suitable for the maturation of hepatocytes in liver organoid.Liver organoid preferably comprises human liver organoid, and human liver organoid comprises human tissue-derived liver organoid (pHLO) and human iPS cell-derived liver organoid (iHLO).pHLO is the organoid made from the liver tissue isolated from human.iHLO can be made by artificially mixing multiple kinds of cells, including hepatic endoderm, which are made by differentiating human iPS cells, and the organoid made by spontaneously generating multiple kinds of cells in the differentiation process, including primitive intestinal spheroids, which are made by differentiating human iPS cells.

[0030] Liver organoid maturation medium can be cell culture medium and at least one selected from the group consisting of DTPA, calcipotriene and bleomycin.Cell culture medium can be commercially available or the cell culture medium described in the literature.

[0031] The cell culture medium is preferably a cell culture medium containing nutrients necessary for cell survival in order to induce or promote cell maturation. Examples of such cell culture media include cell culture media containing one or more amino acids and one or more inorganic salts. Alternatively, a basal medium may be used.

[0032] In a preferred embodiment, liver organoid maturation medium comprises one or more amino acids, one or more inorganic salts, and at least one selected from the group consisting of DTPA, calcipotriene and bleomycin.In another preferred embodiment, liver organoid maturation medium further comprises glucose.Optionally, liver organoid maturation medium can also comprise vitamin, organic acid and / or buffering agent.Liver organoid maturation medium can also comprise pH indicator such as phenol red.

[0033] In another preferred embodiment, the liver organoid maturation medium comprises a basal medium and at least one selected from the group consisting of DTPA, calcipotriene, and bleomycin.

[0034] Examples of basal media include, but are not limited to, media selected from Dulbecco's Modified Eagle Medium (DMEM medium), DMEM / F12 medium, MEM medium, opti-MEM (registered trademark) medium, IMDM medium, RPMI1640 medium, Leivovitz L15 medium, MCDB medium, 199 medium, Ham's F10 medium, Ham's F12 medium, Ham's F14 medium, GMEM medium, Ames' medium, Eagle's Basal Medium (BME medium), CMRL1066 medium, Claycomb medium, Crick medium, Glasgow Minimum Essential Medium (GMEM), MegaCell medium, McCoy's Modified 5A Medium, NCTC medium, Williams' Medium E, Weymouth's Medium, TC-10 medium, and IPL-10 medium.

[0035] When the liver organoid maturation medium contains a basal medium, the proportion of the basal medium in the liver organoid maturation medium is preferably 50% by mass or more, and may be 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more.

[0036] In order to suppress or stop the proliferation of hepatocytes in liver organoids, liver organoid maturation medium preferably does not contain the growth factor that causes hepatocytes to proliferate, or if it does contain, the total amount of the growth factor that causes hepatocytes to proliferate is preferably 1000ng / mL or less, preferably 100ng / mL or less, more preferably 10ng / mL or less.Growth factor includes but is not limited to hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) etc.

[0037] In some embodiments, the liver organoid maturation medium does not contain insulin.

[0038] The liver organoid maturation medium is preferably a serum-free medium in terms of suppressing or stopping the growth of liver organoids, but may contain serum.

[0039] In another aspect, the present invention provides a method for producing liver organoid, comprising culturing liver organoid in any of the liver organoid maturation mediums described above for a sufficient time to induce or promote the maturation of hepatocytes in liver organoid.

[0040] The method for producing liver organoids using the above-mentioned liver organoid maturation medium can enhance the function of hepatocytes that constitute liver organoids.

[0041] The improvement of the function of the hepatocytes that constitute liver organoid can be evaluated based on the expression level of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver.The expression level of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver includes the expression level of the mRNA of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver, and the expression level of the protein that is the translation product of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver.The expression level of the protein that is the translation product of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver can be measured by known immunological methods such as Western blotting.The expression level of the RNA of the gene that is related to metabolism in hepatocytes or that is specifically expressed in liver can be measured by known methods such as quantitative PCR, RNA sequencing.

[0042] Genes related to hepatocyte metabolism or expressed in a liver-specific manner include, but are not limited to, the genes listed in Tables 4 and 5.

[0043] In some embodiments, the hepatocyte metabolism-related or liver-specifically expressed gene is a hepatocyte metabolism-related gene.

[0044] In some embodiments, the hepatocyte metabolism-related or liver-specifically expressed gene is a liver-specifically expressed gene.

[0045] In a preferred embodiment, when liver organoids are cultured in a liver organoid maturation medium containing DTPA, the expression level of at least one selected from the group consisting of alpha-1 microglobulin / bikunin precursor (AMBP), GC vitamin D binding protein (GC), fibrinogen-like 1 (FGL1), pyruvate kinase L / R (PKLR), aldo-keto reductase family 1 member C4 (AKR1C4), bile acid-CoA: amino acid N-acyltransferase (BAAT), serpin family C member 1 (SERPINC1), and vitronectin (VTN) in the liver organoids is increased.

[0046] In a preferred embodiment, when hepatic organoid is cultured in the hepatic organoid maturation medium that contains DTPA, the expression level of at least one selected from the group consisting of albumin (ALB), CYP7A1, arginase 1 (ARG1), ABCG5 and ABCG8 and UGT2B2 in hepatic organoid increases.In this case, hepatic organoid can be pHLO or iHLO, and preferably pHLO.

[0047] In preferred embodiment, when hepatic organoid is cultured in the hepatic organoid maturation medium that contains DTPA, the expression level of at least one selected from the group consisting of AMBP, GC vitamin D binding protein (GC), PKLP, CYP7A1, BAAT, ABCG5, ABCG8, ARG1, and AKR1C4, CYP3A4, CYP2C8, CYP24A1, SLC30A10, ceruloplasmin (CP) coding gene increases.In this case, hepatic organoid can be pHLO or iHLO, and preferably iHLO.

[0048] In a preferred embodiment, culturing liver organoids in a liver organoid maturation medium containing DTPA increases the expression level of apolipoproteins in the liver organoids.

[0049] In a preferred embodiment, culturing liver organoids in a liver organoid maturation medium containing calcipotriene increases the expression level of at least one selected from ceruloplasmin, GC, solute carrier family 30 member 10 (SLC30A10), mannan-binding lectin serine peptidase 1 (MASP1), vanin 1 (VNN1), and alpha-1 microglobulin bikunin precursor (AMBP) in the liver organoids.

[0050] In a preferred embodiment, culturing liver organoids in a liver organoid maturation medium containing DTPA and / or calcipotriene increases the expression levels of UDP glucuronosyltransferase family (UGT), cytochrome P450 family (CYP), or both.

[0051] In preferred embodiments, when hepatic organoid is cultured in the hepatic organoid maturation medium that contains calcipotriene, the expression level of at least one selected from the group consisting of ALB, CYP2C8, CYP2C9, CYP3A4, CYP23A1, UGT1A3, UGT1A4, SLC30A10 and CP in hepatic organoid increases.In this case, hepatic organoid can be pHLO or iHLO, and preferably pHLO.

[0052] In preferred embodiment, when hepatic organoid is cultured in the hepatic organoid maturation medium that contains DTPA and / or calcipotriene, the expression level of at least one selected from the group consisting of ALB, AMBP, GC, PKRL, ARG1, BAAT, ABCG5, ABCG8, AKR1C4, SLC30A10, CP, VNN1, CYP3A4, CYP2C8, CYP2C9, CYP7A1 and CYP24 increases.In this case, hepatic organoid can be pHLO or iHLO, and preferably iHLO.

[0053] In some embodiments, after the process of culturing in the liver organoid maturation medium described above, the expression level of at least one kind of hepatocyte metabolism-related or liver-specifically expressed gene in liver organoid is increased compared with the expression level of the same at least one kind of hepatocyte metabolism-related or liver-specifically expressed gene when the culture medium is containing DMSO instead of containing at least one kind selected from the group consisting of DTPA, calcipotriene and bleomycin.Expression level includes protein expression level and mRNA expression level.Preferably, the process of culturing in the liver organoid maturation medium and the process of culturing in the culture medium containing DMSO are the same except for the conditions of the culture medium.

[0054] In some embodiments, after being cultured in any of the above-mentioned liver organoid maturation medium, the expression level of the gene that is related to metabolism in at least one hepatocyte in liver organoid or that is specifically expressed in liver increases compared with the expression level of the gene that is related to metabolism in at least one hepatocyte before being cultured or that is specifically expressed in liver.Expression level includes the expression level of protein and the expression level of mRNA.

[0055] In some embodiments, after being cultured in any of the above-mentioned liver organoid maturation medium, the expression level of at least one gene that is related to metabolism in liver cell or that is specifically expressed in liver in liver organoid increases compared with the expression level of the same at least one gene that is cultured in the medium that does not contain at least one selected from the group consisting of DTPA, calcipotriene and bleomycin.Expression level includes the expression level of protein and the expression level of mRNA.

[0056] The medium that does not contain at least one selected from the group consisting of DTPA, calcipotriene, and bleomycin is not limited to a medium having the same composition as the liver organoid maturation medium except that it does not contain at least one selected from the group consisting of DTPA, calcipotriene, and bleomycin, and may be a commercially available basal medium.

[0057] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in liver in the liver organoid after the process of culturing in the liver organoid maturation medium that contains DTPA is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more, when the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in liver is set as 1, when the comparison medium that contains DMSO instead of DTPA is used for culturing.

[0058] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte in the liver organoid after the process of culturing in the liver organoid maturation medium that contains DTPA or that is expressed in liver-specific manner is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more, as compared with the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte before the process of culturing.

[0059] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one kind of hepatocyte or that is expressed in liver-specific manner in the liver organoid after the process of culturing in the liver organoid maturation medium that contains DTPA is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more, when the expression level of the mRNA of the gene that is related to metabolism in at least one kind of hepatocyte or that is expressed in liver-specific manner in the liver organoid is set as 1 when the culture medium that does not contain DTPA is used.

[0060] In some embodiments, the expression level of mRNA of at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner in the liver organoids after culturing in a liver organoid maturation medium containing calcipotriene is increased by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 40-fold or more, or 50-fold or more compared to the expression level of mRNA of the same at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner when cultured in a comparative medium containing DMSO instead of calcipotriene.

[0061] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in the liver in the liver organoid after the step of culturing in a liver organoid maturation medium containing calcipotriene is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more compared to the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in the liver before the step of culturing.

[0062] In some embodiments, the expression level of mRNA of at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner in the liver organoids after culturing in a liver organoid maturation medium containing calcipotriene is increased by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 40-fold or more, or 50-fold or more, relative to the expression level of mRNA of the at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner when cultured in a culture medium that does not contain calcipotriene.

[0063] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in the liver in the liver organoid after the process of culturing in the liver organoid maturation medium containing bleomycin is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more, when the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is specifically expressed in the liver is set as 1 in the comparison medium that contains DMSO instead of bleomycin.

[0064] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte in the liver organoid after the step of culturing in the liver organoid maturation medium containing bleomycin or that is expressed in a liver-specific manner is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more, as compared with the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is expressed in a liver-specific manner before the step of culturing.

[0065] In some embodiments, the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte in the liver organoid after the process of culturing in the liver organoid maturation medium containing bleomycin or that is expressed in a liver-specific manner is set as 1, and the expression level of the mRNA of the gene that is related to metabolism in at least one hepatocyte or that is expressed in a liver-specific manner is set as 1, and is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more.

[0066] Unless there are special circumstances, it is preferable that the conditions for the process of culturing in a liver organoid maturation medium and the process of culturing in any of the above-mentioned comparative culture solutions or media are the same except for the difference in the culture solution.

[0067] In another aspect, the present invention provides the liver organoid that is produced by the method for producing above-mentioned liver organoid.This liver organoid is a novel liver organoid, but the analysis of the change in cell structure or characteristics compared with the prior art requires a huge number of trials, and at the time of filing, it is not practical to directly identify liver organoid by its structure or characteristics, so it is identified by the method for producing liver organoid.

[0068] In another aspect, the present invention provides liver organoid, wherein the liver organoid is comprised of at least one selected from the group consisting of DTPA, calcipotriene and bleomycin, and the expression level of the metabolism-related or liver-specifically expressed gene in at least one hepatocyte after culturing in the liver organoid maturation medium is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more compared with the expression level of the metabolism-related or liver-specifically expressed gene in the same at least one hepatocyte before culturing in the liver organoid maturation medium.

[0069] The present invention also encompasses liver organoids, which are described in relation to each embodiment of the method for producing the above-mentioned liver organoids, and the expression level of at least one of the genes related to metabolism in at least one liver cell or expressed in a liver-specific manner after culturing in a liver organoid maturation medium containing at least one selected from the group consisting of DTPA, calcipotriene, and bleomycin, is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 40 times or more, or 50 times or more.

[0070] In another aspect, the present invention provides a method for inducing or promoting hepatocyte maturation in liver organoids, comprising culturing liver organoids in any of the liver organoid maturation media described above for a sufficient time to induce or promote hepatocyte maturation in the liver organoids.

[0071] In some embodiments, the method further comprises: 3+ and Zn 2+ The method further comprises the step of adding at least one of the following to the liver organoid maturation medium to counteract the maturation of hepatocytes in the liver organoid caused by at least one selected from the group consisting of DTPA, calcipotriene, and bleomycin.

[0072] In another aspect, the present invention provides a method for identifying the molecule that controls the maturation of hepatocytes in liver organoid, comprising the step of contacting the liver organoid that is cultured in the above-mentioned liver organoid maturation medium of the present invention with a candidate molecule, and the change in the maturation of hepatocytes in liver organoid indicates that the candidate molecule is the molecule that controls the maturation of hepatocytes in liver organoid.

[0073] For example, if the maturation of hepatocytes in liver organoids cultured in liver organoid maturation medium is delayed by contact with a candidate molecule compared to when not contacted with the candidate molecule, the candidate molecule can be determined and selected as a molecule that suppresses or inhibits the maturation of hepatocytes in liver organoids.If the maturation of hepatocytes in liver organoids cultured in liver organoid maturation medium is faster by contact with a candidate molecule compared to when not contacted with the candidate molecule, the candidate molecule can be determined and selected as a molecule that promotes or enhances the maturation of hepatocytes in liver organoids.

[0074] The maturation of hepatocytes in liver organoids can be confirmed by an increase or decrease in the gene expression levels of hepatocyte maturation markers, such as albumin (ALB), AFP, and HNF4A.

[0075] In another aspect, the present invention provides a method for identifying the molecule that controls the expression of the gene that is related to metabolism in hepatocyte of liver organoid or that is specifically expressed in liver, comprising the step of contacting the liver organoid that is cultured in the above-mentioned hepatocyte maturation medium of the present invention embodiment with candidate molecule; and the change in the expression of the gene that is related to metabolism in hepatocyte of liver organoid or that is specifically expressed in liver indicates that candidate molecule is the molecule that controls the expression of the gene that is related to metabolism in hepatocyte of liver organoid or that is specifically expressed in liver.

[0076] For example, when the expression level of the gene that is related to metabolism in hepatocytes in the liver organoid that is cultured in hepatocyte maturation medium is lower by contacting with candidate molecule compared to when it is not contacted with candidate molecule, the candidate molecule can be determined to be the molecule that suppresses or inhibits the expression of the gene that is related to metabolism in hepatocytes in the liver organoid or is specifically expressed in the liver.When the expression level of the gene that is related to metabolism in hepatocytes in the liver organoid that is cultured in hepatocyte maturation medium is higher by contacting with candidate molecule compared to when it is not contacted with candidate molecule, the candidate molecule can be determined to be the molecule that promotes or enhances the expression of the gene that is related to metabolism in hepatocytes in the liver organoid or is specifically expressed in the liver.

[0077] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0078] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or literature procedures in the art. [Example]

[0079] Example 1 Frozen primary human hepatocytes (PHH) purchased from Ronza before culture, PHH harvested after 4 hours of culture (PHH 4h), HepaRG cells purchased from KAC, HepG2 and HuH-7 cells, which are established hepatic cancer cells, iHLO cultured in EM (iHLO EM), iHLO cultured for 3 days in HDM (iHLO HDM), pHLO cultured in EM (pHLO EM), and pHLO cultured for 3 days in HDM (pHLO HDM) were harvested and the mRNA expression level of albumin, a marker of maturity of cultured cells, was quantified by quantitative PCR. Mean ± SEM. The results are shown in Figure 6A. Compared to PHH isolated from living organisms, the expression level of albumin mRNA in human liver organoids cultured in HDM (iHLO EM and pHLO EM) was several tens to 100 times higher than when cultured in EM. However, this was still only a few tens of times lower than that of PHH and HepG2 cells, suggesting that sufficient maturation had not occurred. Various cells were cultured for 3 days, and the culture supernatants were collected. The amount of albumin secreted was quantified by ELISA. The values ​​are mean ± SEM. The amount of albumin secreted into the medium by HepG2 cells was set at 1, and the values ​​are expressed as relative values. The results are shown in Figure 6B. Compared to HepG2 cells, both iHLOs cultured in EM medium and iHLOs cultured in HDM medium secreted less albumin protein into the medium, at only a few tenths the amount.

[0080] Example 2 From the results of Example 1, it was thought that the maturation of hepatocytes in liver organoids required improvement of the medium composition, and therefore a screening for compounds that induce hepatocyte maturation in liver organoids was carried out. From the Validated Compound Library of the University of Tokyo Drug Discovery Institute, a screening for compounds that increase the amount of albumin secreted, a hepatocyte maturation marker, into the iHLO culture supernatant was carried out by ELISA using a 384-well plate. As a result, six compounds were selected: diethylenetriamine-N',N',N',N'',N''-pentaacetic acid (DTPA), BU99006, calcipotriene, bleomycin, trovafloxacin mesylate, and dolutegravir. 7 is a graph showing that the amount of albumin secreted into the medium increases in a compound concentration-dependent manner (mean ± SEM). Calcipotriene was shown to secrete a large amount of human albumin even at low concentrations.

[0081] Example 3 iHLOs were treated with 10 μM of each of the six compounds shown in Figure 7 for three days by culturing them in medium. The expression levels of representative genes known to be important for hepatocyte function were quantified by qPCR. Mean ± SEM. Significant differences were tested using Dunnett's test, with **p<0.01 and *p<0.05 (vs. DMSO). The results of the expression levels of representative genes known to be important for hepatocyte function are shown in Figures 8 and 9. DTPA, calcipotriene, and bleomycin, which clearly affected gene expression levels, were selected as final hit compounds.

[0082] Example 4 RNA was extracted from iHLOs treated by culturing them for 3 days in medium containing 10 μM each of DTPA, calcipotriene, and bleomycin. RNA sequencing analysis was then performed using the NovaSeq6000 system, and pathway analysis was performed using the Ingenuity pathway analysis software provided by QIAGEN. Figure 10 shows a list of signal pathways (left) important for hepatocyte metabolism and response that were determined to be activated by compound treatment, as well as upstream factors (right) whose activity is predicted to have changed. The upstream factors include transcription factors such as HNF1, HNF4A, and p53 that are thought to be important for hepatocyte maturation, and it was confirmed that the expression of their target genes was uniformly increased. The above results suggest that calcipotriene improves various hepatocyte functions. Calcipotriene is a derivative of vitamin D and is the active form of vitamin D3. Therefore, like calcitriene and calcitriol, calcipotriene has physiological functions mediated by the vitamin D receptor (VDR). However, the hepatocyte function-improving effect of calcipotriene cannot be fully explained solely by its activation of the existing vitamin D receptor, suggesting that the maturation effect of calcipotriene differs from that of calcitriol and is not mediated by VDR activation.

[0083] [ka]

[0084] Example 5 pHLO was established from human liver tissue obtained from surgical specimens (see Nat. Protoc. 16: 182-217, 2021). Figure 11 shows images of pHLO cells observed under a bright-field microscope during growth in EM. Six days after subculture, pHLO cells were cultured for 3 days in a medium containing 10 μM DTPA or 3 μM calcipotriene in a basal medium of Williams' medium E+0.1% BSA. The mRNA expression levels of various genes important for hepatocyte function, ALB, CYP7A1, ARG1, ABCG5, and UGT2B4, whose expression is enhanced by DTPA, were quantified by quantitative PCR (Figure 12). Mean ± SEM. Similar to iHLO, pHLO also demonstrated an increase in the expression of liver-specific genes by DTPA. The mRNA expression levels of various genes important for hepatocellular function, including ALB, CYP2C8, CYP2C9, CYP3A4, CYP23A1, UGT1A3, UGT1A4, SLC30A10, and CP, whose expression is upregulated by calcipotriene, were quantified by quantitative PCR (Figure 13). Mean ± SEM. Similar to iHLO, pHLO also demonstrated increased expression of liver-specific genes.

[0085] Example 6 HepG2 and HuH-7 cells, established hepatocyte cell lines, were cultured for three days in medium supplemented with 10 μM each of DTPA, calcipotriene, and bleomycin. The mRNA expression levels of various liver-specific genes, ALB, AMBP, CYP7A1, CYP3A4, ARG1, and ABCG5, were quantified by quantitative PCR (Figure 14). Figures are mean ± SEM. It was confirmed that the maturation medium of this example promotes the maturation of HLOs but has no effect on conventional cancer-derived hepatocyte cell lines such as HepG2 cells.

[0086] Example 7 Based on the results of Examples 3 and 4, DTPA and bleomycin are expected to have similar modes of action. However, bleomycin is not a very stable compound. Therefore, in this example, DTPA and calcipotriene were used as the basic components of the maturation medium. Additives to the basal medium containing 10 μM DTPA and 3 μM calcipotriene were investigated. Williams Medium E, which is rich in amino acids and considered suitable for hepatocyte culture, was supplemented with serum substitutes 1xB27 (insulin-free) or 0.1% BSA (fatty acid-free) on day 6 after passage and EM culture, as shown in Figure 15. After culturing iHLOs in these media for 3 days, we evaluated the effects of these supplements on the upregulation of mRNA expression of various maturation indicator genes: ALB, AMBP, GC (vitamin D-binding protein (GC)), PKRL, ARG1, BAAT, ABCG5, ABCG8, AKR1C4, SLC30A10, CP, VNN1, CYP3A4, CYP2C8, CYP2C9, CYP7A1, and CYP24 (Figures 16-21). Under all conditions, upregulation of all genes was observed, indicating that Williams Medium + 0.1% BSA is a suitable basal medium for maturation.

[0087] Example 8 iHLOs were treated with Williams' medium E + 0.1% BSA basal medium supplemented with 10 μM DTPA for 3 days to induce ALB expression. 3+ (iron citrate), Zn 2+ The addition of Fe (ZnCl) and DTPA inhibited ALB mRNA expression in a concentration-dependent manner (Figure 22, Table 3). 3+ and Zn 2+ When a chelator with a weak chelating effect of DTPA was used, no maturation effect was observed unless the concentration of the chelator was increased, suggesting that the maturation effect of DTPA is mediated by the chelating effect of these ions.

[0088] [Table 3]

[0089] Example 9 After culturing iHLO for 3 days in a medium supplemented with 10 μM DTPA and 10 μM iron citrate or 3 μM zinc chloride, the cells were harvested and RNA samples were prepared. RNA sequencing analysis was performed using the NovaSeq6000 system, and the results were analyzed for pathways using Ingenuity pathway analysis software (Figure 23(A)). 3+ Or Zn 2+ The following shows candidates for upstream factors whose activity was predicted to be suppressed by the addition of DTPA. Protein samples were collected from iHLO treated in the same way, and changes in the expression of albumin, HNF1, and p53 proteins were analyzed by Western blotting using antibodies against each protein (β-actin was used as an internal standard) (Figure 23(B)). The induction of albumin and p53 protein expression by DTPA and the effect of Fe 3+ , Zn 2+ The inhibitory effect of DTPA on hepatocyte maturation was also demonstrated at the protein level.

[0090] Example 10 After culturing in EM for 6 days, iHLOs were treated by culturing in medium containing 10 μM each of DTPA, calcipotriene, and bleomycin for 3 days. RNA was extracted from the treated iHLOs and subjected to RNA sequencing analysis using the NovaSeq6000 system. Table 4 shows genes whose TPM values, which are an indicator of expression level, exceeded a certain level (TPM>10) and whose RNA expression levels were increased by more than three times by DTPA treatment compared to untreated (DMSO) (multiplications are shown in the rightmost column of the table). Table 5 shows genes whose TPM values, which are an indicator of expression level, exceeded a certain level (TMP>10) and whose RNA expression levels were increased by more than three times by treatment with calcipotriene compared to untreated (DMSO) (multiplication factors are shown in the far right column of the table). TPM (Transcrips per million) is the gene expression level calculated by correcting the number of reads for each transcript to make the gene length 1000 bp, and then adjusting the total number of reads for each sample to 1 million (see, for example, Bioinformatics 26: 493-500, 2010).

[0091] [Table 4]

[0092] [Table 4]

[0093] [Table 4]

[0094] [Table 4]

[0095] [Table 5]

[0096] [Table 5]

[0097] [Table 5]

Claims

1. A liver organoid maturation medium containing at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin.

2. The liver organoid maturation medium of claim 1, further comprising an amino acid and an inorganic salt.

3. 2. The liver organoid maturation medium of claim 1, comprising a basal medium.

4. The liver organoid maturation medium of claim 1, which does not contain growth factors or contains a total of 1000 ng / mL or less of growth factors.

5. A method for producing liver organoids, comprising: A method comprising culturing liver organoids in a liver organoid maturation medium according to any one of claims 1 to 4 for a period of time sufficient to induce or promote maturation of hepatocytes in the liver organoids.

6. The method according to claim 5, wherein after the step of culturing, the expression level of the gene that is related to metabolism in at least one hepatocyte in liver organoid or that is expressed in liver-specific manner increases compared with the expression level of the gene that is related to metabolism in at least one hepatocyte or that is expressed in liver-specific manner before the step of culturing.

7. the hepatocyte maturation medium contains diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, and the at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner includes at least one gene selected from the group consisting of albumin, CYP7A1, ARG1, ABCG5, ABCG8, and UGT2B2; or 6. The method of claim 5, wherein the hepatocyte maturation medium contains calcipotriene, and the gene involved in metabolism in at least one hepatocyte or expressed in a liver-specific manner comprises at least one gene selected from the group consisting of genes encoding ALB, CYP2C8, CYP2C9, CYP3A4, CYP23A1, UGT1A3, UGT1A4, SLC30A10, and ceruloplasmin.

8. A liver organoid produced by the method for producing a liver organoid according to claim 5.

9. A liver organoid, wherein the liver organoid is cultured in a liver organoid maturation medium containing at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin, and the expression level of at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner is increased by three times or more compared to the expression level of the at least one gene related to metabolism in hepatocytes or expressed in a liver-specific manner in the liver organoid cultured in a medium that does not contain at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin.

10. A method for inducing or promoting hepatocyte maturation in liver organoids, comprising: A method comprising culturing liver organoids in a liver organoid maturation medium according to any one of claims 1 to 4 for a period of time sufficient to induce or promote maturation of hepatocytes in the liver organoids.

11. Fe 3+ and Zn 2+ The method of claim 10, further comprising the step of counteracting the maturation of hepatocytes in liver organoids caused by at least one selected from the group consisting of diethylenetriamine-N',N',N',N'',N''-pentaacetic acid, calcipotriene, and bleomycin, by adding at least one of the following to the liver organoid maturation medium.

12. A method for identifying a molecule that controls the maturation of hepatocytes in liver organoids, comprising: A method comprising contacting liver organoids cultured in a liver organoid maturation medium according to any one of claims 1 to 4 with a candidate molecule, wherein a change in the maturation of hepatocytes in the liver organoid indicates that the candidate molecule is a molecule that controls the maturation of hepatocytes in the liver organoid.

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