Medium for propagating liver organoids

Abstract

To provide a novel medium for propagating liver organoids that achieves cost reduction compared to conventional media.SOLUTION: The present invention provides a medium for propagating human pluripotent stem cell-derived liver organoids, the medium comprising a culture supernatant of modified L cells, which are L cells modified so as to express R-spondin-1, hepatocyte growth factor (HGF), fibroblast growth factor (FGF) 7, and fibroblast growth factor 10.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a medium for growing liver organoids, a method for growing liver organoids, and the grown liver organoids produced by such a method.

Background Art

[0002] Cell aggregates called organoids are used as tools for examining the effects of pharmaceuticals and foods on living organisms because their structures and functions are similar to those of living tissues. Therefore, various methods for forming and growing organoids and the development of media used in those methods are being advanced.

[0003] For example, Patent Document 1 discloses a hepatocyte culture medium containing: i. optionally, a fibroblast growth factor (FGF) of 50 to 500 ng / ml selected from the group consisting of FGF7, FGF10, and combinations thereof; ii. a Wnt agonist containing R-spondin and at least one glycogen synthase kinase 3 (GSK3) inhibitor; iii. an epidermal growth factor (EGF) of 5 to 100 ng / ml; iv. a hepatocyte growth factor (HGF) of 5 to 100 ng / ml; and v. a transforming growth factor beta (TGF-β) inhibitor containing an inhibitor of the activin receptor-like kinase ALK4, ALK5, and / or ALK7 signaling pathway. It is also disclosed that at least a part of the cells isolated from the liver tissue cultured in such a culture medium forms liver organoids, and that the same culture medium is also used for growing liver organoids.

[0004] In addition, Patent Document 2 discloses a medium for the proliferation of liver cells containing any one of R-spondin 1 to 4, noggin, nicotinamide, EGF, FGF10, HGF, gastrin, a TGF-β inhibitor, and PGE2, and it is disclosed that liver organoids can be formed from liver cells or tissue fragments using such a medium. Patent Document 3 discloses a medium containing an Erb3 / 4 ligand such as neuregulin polypeptide, a receptor tyrosine kinase ligand such as FGF-7 and / or 10, a BMP inhibitor such as Noggin, and a Wnt agonist such as R-spondin, and it is disclosed that epithelial stem cells can be proliferated using such a medium to form lung organoids and breast organoids. However, in any of the media for forming and proliferating organoids described in the literature, a large number of expensive recombinant proteins such as growth factors are used, and therefore, the price of the medium is high, and the economic burden on users is large.

[0005] As an example of not using such recombinant proteins, there is a report of culturing small intestinal organoids derived from human induced pluripotent stem cells in a medium prepared using the culture supernatant of cells transfected to express Wnt3a, R-spondin-1, and Noggin in mouse L cells (Non-Patent Document 1). However, although liver organoids are one of the representative organoids, the systematic methodology for handling liver organoids such as culture systems, functional evaluation, and gene transfection is insufficient, and there is still no report of liver organoids cultured without using recombinant proteins.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a new culture medium or the like for growing liver organoids that is lower in cost than conventional culture media.

Means for Solving the Problems

[0009] In the course of intensive research to solve the above problems, the present inventors succeeded in establishing L-RHF2 cells obtained by modifying mouse skin fibroblast cell line L cells to express R-spondin-1, hepatocyte growth factor (HGF), and fibroblast growth factor (FGF). Then, the present inventors obtained the knowledge that by using the culture supernatant of L-RHF2 cells, it is possible to prepare a medium that can grow liver organoids at low cost without using expensive recombinant proteins such as those used in conventional culture media for liver organoids. Based on such findings, further research was conducted, and as a result, the present invention was completed.

[0010] That is, the present invention relates to the following. [1] A culture medium for growing liver organoids, comprising the culture supernatant of modified L cells obtained by modifying L cells to express R-spondin-1, hepatocyte growth factor (HGF), fibroblast growth factor (FGF) 7, and fibroblast growth factor 10. [2] The culture medium for growth according to [1] above, wherein the liver organoid is a pluripotent stem cell-derived liver organoid. [3] The culture medium for growth according to [2] above, wherein the pluripotent stem cell is a human pluripotent stem cell. [4] The growth medium according to any one of [1] to [3] above, wherein the culture supernatant of the modified L cells contains R-spondin-1, HGF, FGF-7 and FGF-10. [5] The growth medium according to any one of [1] to [4] above, wherein the culture supernatant of the modified L cells contains R-spondin-1, HGF, FGF-7 and FGF-10 in a ratio of R-spondin-1:HGF:FGF-7:FGF-10 = 30:1:2:2. [6] The growth medium according to any one of [1] to [5] above, wherein the culture supernatant of the modified L cells is obtained by culturing the modified L cells in a medium containing FBS.

[0011] [7] The growth medium according to any one of [1] to [6] above, wherein the modified L cells are L-RHF2 cells prepared by sequentially infecting L cells with four types of lentiviral vectors each containing the gene sequences of R-spondin-1, HGF, FGF-7 and FGF-10. [8] The growth medium according to any one of [1] to [7] above, wherein the modified L cells are L-RHF2 cells represented by NITE P-04111 (accession number). [9] A method for growing human pluripotent stem cell-derived liver organoids, comprising culturing human pluripotent stem cell-derived liver organoids in the growth medium according to any one of [1] to [8] above.

[10] A method for enhancing the stemness of the grown liver organoids, comprising growing the liver organoids in the growth medium according to any one of [1] to [8] above.

[11] A method for producing proliferated human pluripotent stem cell-derived liver organoids with enhanced stemness, comprising growing the liver organoids in the growth medium according to any one of [1] to [8] above.

[12] Proliferated human pluripotent stem cell-derived liver organoids with enhanced stemness produced by the method of

[11] above.

[13] Modified L cells (ATCC number: CRL-2648) that secrete R-spondin-1, HGF, FGF-7, and FGF-10.

[14] The modified L cells according to

[13] above, which express R-spondin-1, HGF, FGF-7, and FGF-10 at a ratio of R-spondin-1:HGF:FGF-7:FGF-10 = 30:1:2:2.

[15] Modified L cells represented by NITE P-04111 (deposit number). [Advantages of the Invention]

[0012] By using the growth medium of the present invention, the cost required for culturing liver organoids can be significantly reduced. For example, when using 500 mL of the growth medium, the cost can be reduced to about 1 / 100 or less of the conventional cost. In addition, by using the medium of the present invention, in the preparation of the medium, the step of precisely measuring multiple kinds of proteins and adding them to the medium can be omitted, so that the experimental efficiency of the operator can be increased, and further, the risks of human errors such as contamination during medium preparation, misplacement of proteins by the operator, and error in concentration adjustment can be reduced. The growth medium of the present invention imparts higher growth properties to liver organoids than conventional media. Therefore, by using the growth medium of the present invention, it becomes easy to obtain a large amount of the same lot of liver organoids required for the test. Furthermore, the liver organoids grown using the growth medium of the present invention, after being replaced with a hepatocyte maturation medium and matured, highly express liver function genes, show insulin responsiveness and intracellular fat accumulation, and moreover show very low density lipoprotein secretion that was difficult to reproduce in conventional cell lines and animal models. Therefore, by using human liver organoids grown using the growth medium of the present invention, it becomes possible to apply them to new physiological function evaluation and screening research of hepatocytes. In addition, by using the culture medium for growth of the present invention, it becomes possible to grow human liver organoids derived from pluripotent stem cells, for which the presence or absence of growth ability has not been reported conventionally. Human liver organoids derived from biological tissues have limited proliferative ability, and continuous culture and obtaining a large amount of the same lot are difficult. As a result, when attempting to establish new organoids, it is necessary to collect cells from a living body again. In the case of organoids derived from a living body, there is a risk that their properties may change depending on the living body from which the cells are collected, and the difference between lots can be large. In addition to the fact that biological tissues for establishing human liver organoids derived from biological tissues are not easily available, consideration for ethical aspects such as obtaining informed consent and handling personal information is required. On the other hand, human liver organoids derived from pluripotent stem cells have the advantage of not having these problems in human liver organoids derived from biological tissues. Therefore, the present invention can solve the above problems in human liver organoids derived from biological tissues.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0023] In one aspect, the present invention relates to a culture medium for the growth of liver organoids, comprising modified L cells modified to secrete R-spondin-1, HGF, FGF-7 and FGF-10, and a culture supernatant of such modified L cells. The production of liver organoids is generally divided into a step of proliferation and a step of maturation. When the liver organoids are derived from pluripotent differentiated cells, a step of differentiating pluripotent stem cells is further included before the step of proliferation. In the present invention, the "culture medium for the growth of liver organoids" refers to a medium used in such a step of proliferation for growing liver organoids. The liver organoids grown by the culture medium for growth of the present invention may be liver organoids derived from liver tissue or liver organoids derived from pluripotent stem cells, and preferably are liver organoids derived from pluripotent stem cells. In the present invention, the "pluripotent stem cells" are cells having pluripotency to differentiate into all tissues or cells constituting a living body and self-renewal ability, and examples thereof include ES cells, embryonic germ cells (EG cells), induced pluripotent stem cells (also referred to as iPS cells), and the like. Preferably, the pluripotent stem cells in the present invention are iPS cells. In the present invention, the species from which the pluripotent stem cells are derived is not particularly limited, and examples thereof include humans, monkeys, mice, and the like. Preferably, the pluripotent stem cell-derived liver organoids grown by the culture medium for growth of the present invention are human pluripotent stem cell-derived liver organoids.

[0024] The growth medium in the present invention contains "culture supernatant of modified L cells". In the present invention, "modified L cells" refer to L cells modified to express the R-spondin-1 gene (SEQ ID NO: 1), hepatocyte growth factor (also referred to as HGF) gene (SEQ ID NO: 2), fibroblast growth factor (also referred to as FGF) 7 gene (SEQ ID NO: 3), and fibroblast growth factor 10 gene (SEQ ID NO: 4). L cells can be obtained, for example, from ATCC (ATCC number: CRL-2648 / URL: https: / / www.atcc.org / products / crl-2648). The growth medium in the present invention may contain, instead of the culture supernatant of modified L cells, the culture supernatant of other cells modified to express the R-spondin-1 gene, HGF gene, FGF-7 gene, and FGF-10 gene. Other cells to be modified may be any cells commonly used for protein expression in the art, and for example, CHO cells (Chinese hamster ovary cells), HEK293 cells (human fetal kidney cells), L929 cells (mouse fibroblasts), L929 cell-derived strains, secretory epithelial cells, etc. can be used.

[0025] The lentiviral vector used for the production of the modified L cells of the present invention can be produced using a general gene transfer method. For example, the lentiviral vector used for the production of modified L cells can be obtained by co-transfecting HEK293T cells with a gene expression plasmid incorporating the R-spondin-1 gene, HGF gene, and FGF gene and a packaging plasmid, respectively, by methods such as the calcium phosphate method.

[0026] The modified L cells of the present invention are characterized by over-secreting R-spondin-1, HGF, FGF-7, and FGF-10. In one aspect, the modified L cells of the present invention secrete R-spondin-1 at a concentration of 1500 to 7500 ng / mL, preferably at a concentration of about 3,000 ng / mL. In one aspect, the modified L cells of the present invention secrete HGF at a concentration of 50 ng / mL to 250 ng / mL, preferably at a concentration of about 100 ng / mL. In one aspect, the modified L cells of the present invention secrete FGF-7 at a concentration of 100 ng / mL to 500 ng / mL, preferably at a concentration of about 200 ng / mL. In one aspect, the modified L cells of the present invention secrete FGF-10 at a concentration of 100 ng / mL to 500 ng / mL, preferably at a concentration of about 200 ng / mL. In a preferred aspect, the modified L cells of the present invention are characterized by over-secreting R-spondin-1, HGF, FGF-7, and FGF-10 at a ratio of R-spondin-1:HGF:FGF-7:FGF-10 = 30:1:2:2. In one aspect, the modified L cells of the present invention are L-RHF2 cells established by sequentially infecting L cells with four types of lentiviral vectors each containing the gene sequences of R-spondin-1, HGF, FGF-7, and FGF-10 by the inventors. Such cells were deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation on July 23, 2024 (Reiwa 6), and have been assigned the accession number "NITE P-04111".

[0027] In the present invention, the "culture supernatant of modified L cells" refers to the culture supernatant obtained when modified L cells are cultured. The medium used for culturing the modified L cells is not particularly limited as long as the modified L cells can survive. Examples include those obtained by adding penicillin / streptomycin, fetal bovine serum (also referred to as FBS), etc. to a basal medium. In the present invention, known basal media can be used and are not limited thereto. Examples include DMEM (Dulbecco's Modified Eagle Medium), DMEM high glucose, DMEM low glucose, Advanced DMEM, MEM (Minimum Essential Medium), Advanced MEM, α-MEM (α-modified Minimum Essential Medium), GMEM (Glasgow Minimum Essential Medium), Ham's F-12 (Nutrient Mixture F-12 Ham), DMEM / Ham, IMDM (Iscove's Modified Dulbecco's Medium), DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 Ham), Advanced DMEM / F-12, RPMI (Roswell Park Memorial Institute) 1640, etc. Preferably, the basal medium is DMEM high glucose. The culture of the modified L cells can be carried out under conditions commonly known in the art. In one embodiment, the culture supernatant is recovered after culturing the modified L cells for 24 to 96 hours. Preferably, the culture supernatant is recovered after culturing the modified L cells for about 72 hours.

[0028] The culture supernatant of the modified L cells in the present invention contains R-spondin-1, HGF, FGF-7 and FGF-10. In one aspect, the culture supernatant of the modified L cells of the present invention contains R-spondin-1 at a concentration of 1500 to 7500 ng / mL, preferably at a concentration of about 3,000 ng / mL. In one aspect, the culture supernatant of the modified L cells in the present invention contains HGF at a concentration of 50 to 250 ng / mL, preferably at a concentration of about 100 ng / mL. In one aspect, the culture supernatant of the modified L cells in the present invention contains FGF-7 at a concentration of 100 to 500 ng / mL, preferably at a concentration of about 200 ng / mL. In one aspect, the culture supernatant of the modified L cells in the present invention contains FGF-10 at a concentration of 100 to 500 ng / mL, preferably at a concentration of about 200 ng / mL. In a preferred aspect, the culture supernatant of the modified L cells in the present invention contains R-spondin-1, HGF, FGF-7 and FGF-10 in a ratio of R-spondin-1:HGF:FGF-7:FGF-10 = 30:1:2:2.

[0029] Without being bound by theory, the culture supernatant of the modified L cells in the present invention also contains components other than proteins secreted by the modified L cells such as FBS, and it is considered that they can contribute to the stabilization of R-spondin-1, HGF, FGF-7 and FGF-10, so that a state with higher activity than that of commercially available recombinant proteins used in conventional media can be maintained.

[0030] The culture medium for the growth of the human pluripotent stem cell-derived liver organoids of the present invention is not particularly limited as long as it contains the culture supernatant of the modified L cells. For example, it can be based on a basal medium. The basal medium may be the same as or different from that used for the culture of the modified L cells. Preferably, the basal medium as the base of the culture medium for the growth of liver organoids is Advanced DMEM / F-12. The culture medium for the growth of the liver organoids of the present invention may contain known additives as required. The additives may be those that do not inhibit the growth of liver organoids. For example, they may include inorganic salts, carbohydrates, amino acids, vitamins, lipids, buffers, GSK3β (glycogen synthase kinase 3β) inhibitors, TGFβ inhibitors, ROCK (Rho-associated protein kinase) inhibitors, antibiotics, serum substitutes, antioxidants, hormones, growth factors, and the like. Suitable antibiotics for use in the culture medium for the growth of the liver organoids of the present invention include, for example, penicillin / streptomycin. Suitable buffers for use in the culture medium for the growth of the liver organoids of the present invention include, for example, HEPES. Suitable serum substitutes for use in the culture medium for the growth of the liver organoids of the present invention include, for example, B-27 Supplement (minus Vitamin A) or bovine serum albumin (BSA). Suitable GSK3β inhibitors for use in the culture medium for the growth of the liver organoids of the present invention include, for example, CHIR99021. Suitable antioxidants for use in the culture medium for the growth of the liver organoids of the present invention include N-acetylcysteine. Suitable hormones for use in the culture medium for the growth of the liver organoids of the present invention include, for example, gastrin. Suitable growth factors for use in the culture medium for the growth of the liver organoids of the present invention include, for example, TGFα, EGF. Suitable TGFβ inhibitors for use in the culture medium for the growth of the liver organoids of the present invention include, for example, A83-01. Suitable ROCK inhibitors for use in the culture medium for the growth of the liver organoids of the present invention include, for example, Y-27632. In one aspect, the culture medium for the growth of the liver organoids of the present invention may contain components useful for cell growth such as nicotinamide and L-alanyl-L-glutamine. In some aspects, the culture supernatant of the L-RHF2 cells in the present invention is added to the basal medium so as to be diluted 2-fold to 10-fold. In a preferred aspect, the culture supernatant of the L-RHF2 cells in the present invention is added to the basal medium so as to be diluted 4-fold.

[0031] In another aspect, the present invention relates to a method for enhancing the stem cell property of proliferated liver organoids, which includes proliferating liver organoids in a growth medium containing the culture supernatant of modified L cells, a method for producing proliferated liver organoids with maintained stem cell property, which includes proliferating liver organoids in a growth medium containing the culture supernatant of modified L cells, and proliferated liver organoids with enhanced stem cell property produced by such a method. The growth medium containing the culture supernatant of modified L cells in the present invention is as described above. In one aspect, proliferating liver organoids in a growth medium containing the culture supernatant of modified L cells refers to culturing liver organoids in a growth medium containing the culture supernatant of modified L cells. In such an aspect, culturing liver organoids may be, for example, contacting the liver organoids or a suspension obtained by crushing the liver organoids with a substrate such as an extracellular matrix, and embedding and culturing them in the extracellular matrix. Examples of the extracellular matrix include Matrigel and Cultrex UltiMatrix Reduced Growth Factor (RGF) Basement Membrane Extract (BME). In addition to Matrigel, gels containing collagen, laminin, fibronectin, etc. can be used as the extracellular matrix. Also, instead of the extracellular matrix, gels not containing extracellular matrix constituent proteins can be used, and examples of such gels include 4-PEG and alginate hydrogel.

[0032] The proliferated liver organoids produced by the production method of the present invention have enhanced stem cell properties compared to the proliferated liver organoids produced by conventional media. In the present invention, the proliferated liver organoids refer to liver organoids before transitioning to the maturation process after completing the proliferation process using a proliferation medium. In the present invention, stem cell properties refer to the fact that at least some of the cells constituting the liver organoids have stem cell-like properties such that they can proliferate while maintaining the ability to differentiate into other cells. Stem cell properties can be measured, for example, by the expression levels of stem cell marker genes or marker genes of differentiated somatic cells, such as liver cell marker genes. For example, high stem cell properties mean that the expression level of the stem cell marker gene is high and the expression level of a marker gene of a differentiated somatic cell, such as a liver cell marker gene, is low. The human pluripotent stem cell-derived liver organoids produced by the production method of the present invention have a higher expression level of the stem cell marker gene and a lower expression level of the liver cell marker gene compared to the liver organoids produced using a conventional proliferation medium. In one aspect, the stem cell marker gene is LGR5. In one aspect, the liver cell marker gene is AFP and / or ALB. The expression level of LGR5 in the proliferated liver organoids produced by the production method of the present invention is, for example, 1.5 times or more, 2.0 times or more, or 2.5 times or more the expression level in the proliferated liver organoids produced by a medium using a recombinant protein. Preferably, the expression level of LGR5 in the proliferated liver organoids produced by the production method of the present invention is 2.0 times or more the expression level in the proliferated liver organoids produced by a conventional medium. The expression level of AFP and / or ALB in the proliferated liver organoids produced by the production method of the present invention is, for example, 0.8 times or less, 0.5 times or less, or 0.3 times or less the expression level in the proliferated liver organoids produced by a conventional medium. Preferably, the expression level of AFP and / or ALB in the proliferated liver organoids produced by the production method of the present invention is 0.5 times or less the expression level in the proliferated liver organoids produced by a conventional medium. Although not bound by theory, it is considered that the high stemness of the grown liver organoids produced by the production method of the present invention leads to the high proliferative ability of the liver organoids.

[0033] In addition, the liver organoids produced by the production method of the present invention can exhibit an anabolic effect in response to insulin. The liver organoids produced by the production method of the present invention express nuclear receptors involved in lipid and drug metabolism, such as liver X receptor (LXR), farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR) α, and pregnane X receptor (PXR). The liver organoids produced by the production method of the present invention can reproduce the physiological metabolism and functions of hepatocytes, such as the accumulation of lipid droplets. The liver organoids produced by the production method of the present invention have an expression level of lipid metabolism-related genes equal to or higher than that of primary human hepatocytes and established cell lines (such as HepaRG, HepG2, and Huh-7 cells). Furthermore, the liver organoids produced by the production method of the present invention can secrete mature VLDL.

Examples

[0034] Hereinafter, the present invention will be described in more detail based on examples. Needless to say, the present invention is not limited to these examples.

[0035] Example 1. Establishment of L-RHF2 cells (1) Preparation of lentiviral vectors Four types of lentiviral vectors having the gene sequences of the respective target proteins were prepared by co-transfecting HEK293T cells with a plasmid for virus particle construction and a plasmid containing the genes of the respective target proteins, that is, R-spondin-1 (RSPO1), HGF, FGF7, and FGF10, which are exogenous factors essential for the growth of human liver organoids. Specifically, HEK293T cells (obtained from ATCC) were used at 3.3×10 6Cells were seeded in dishes at a density of 100 cells / mm², and transfected with two packaging plasmids (pCAG-HIVgp; 8 μg / dish, pCMV-VSV-G-RSV-Rev; 8 μg / dish) and a lentiviral expression plasmid (20 μg / dish) using the calcium phosphate method. The calcium phosphate-plasmid DNA complex was prepared by mixing 36 μL of plasmid DNA (1 μg / μL), 60 μL of 2.5 M CaCl 2 , 504 μL of distilled water, and 600 μL of 2×HEPES-buffered saline (pH 7.05) in a 15 mL tube. The mixture was bubbled 20 - 30 times using a pipette, vortexed for 10 seconds, and incubated in a 37°C water bath for 30 minutes. The complex was uniformly dropped onto the cells in each 100 mm dish. After 12 hours, the medium was replaced with fresh medium containing 10 μM forskolin. After 24 hours, the medium containing the lentiviral vector was collected and filtered through a 0.45 μm filter.

[0036] Schematic diagrams of the packaging plasmids (pCAG-HIVgp, pCMV-VSV-G-RSV-Rev) and the lentiviral expression plasmid (CSII-EF-MCS-IRES2-Venus) used are shown in Figures 1 - 3, respectively. Note that the symbols in the figures have the following meanings. [Figure 1] CAG: Human cytomegalovirus enhancer and chicken β-actin promoter HIV-1 gp: HIV gag and pol genes RRE: Rev response element SV40pA: SV40 polyadenylation signal Amp pro: Ampicillin resistance (β-lactamase) promoter SV40 pro&ori: SV40 early promoter and origin Kan / Neo; Kanamycin / neomycin resistance gene HSV TK pA; Herpes simplex virus thymidine kinase polyadenylation signal [Figure 2] CMV: Human Cytomegalovirus Immediate Early Promoter 5’SD: 5’ Splicing Donor Site 3’SA: 3’ Splicing Acceptor Site VSV-G: Vesicular Stomatitis Virus G Glycoprotein Gene BGH pA: Bovine Growth Hormone Polyadenylation Signal RSV LTR: Rous Sarcoma Virus LTR Rev: HIV-1 rev Gene R, U5: HIV-1 LTR and U5 Amp: Ampicillin Resistance Gene [Figure 3] CMV: Human Cytomegalovirus Immediate Early Promoter y: Packaging Signal 5’SD: 5’ Splicing Donor Site 3’SA: 3’ Splicing Acceptor Site RRE: Rev Response Element cPPT: Central Polymeric Purine Tract CTS: Central Termination Sequence EF-1: Human Elongation Factor 1α Subunit Promoter MCS: Multiple Cloning Site IRES2: Encephalomyocarditis Virus Internal Ribosome Entry Site Venus: Variant of Yellow Fluorescent Protein (YFP) Gene PRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element del U3: Deletion of Enhancer and Promoter Sequences in the U3 Region BGH pA: Bovine Growth Hormone Polyadenylation Signal SV40 pro&ori: SV40 Immediate Early Promoter and Origin Zeo: Zeocin Resistance Gene SV40 pA: SV40 Polyadenylation Signal Amp: Ampicillin Resistance Gene

[0037] (2) Establishment of L-RHF2 Cells 1. Detach and collect L cells (ATCC number: CRL-2648) cultured to 80% confluence in a 100 mm dish, and suspend them in 20 mL of subculture medium (DMEM high Glucose + 10% FBS, penicillin / streptomycin). Seed the cell suspension into a 6-well plate at 2 mL / well for 2 wells, and incubate overnight at 37°C, 5% CO 2 2. The next day, remove the medium, and add the RSPO1 lentiviral vector solution diluted to the optimal concentration in subculture medium and a 10 μg / mL polybrene solution. 2. Centrifuge the plate at 2,500 rpm for 90 minutes at room temperature. 3. After centrifugation, remove all of the viral vector solution, and wash twice with 1 mL / well of subculture medium. 4. Add subculture medium at 2 mL / well, and incubate overnight at 37°C, 5% CO 5. The next day, after removing the medium, wash with 2 mL of PBS(-), and detach the cells using 0.5 mL of trypsin-EDTA solution. Add 2 mL of subculture medium to collect the cells into a 15 mL tube, and centrifuge at 800 rpm for 5 minutes at room temperature. 2 6. After removing the supernatant, suspend in 4 mL of subculture medium, and seed the cells at 4 mL / 60 mm dish (passage number 1). 7. Approximately 2 days later, after removing the medium, wash with 4 mL of PBS(-), and detach the cells using 1 mL of trypsin-EDTA solution. Add 4 mL of subculture medium to collect the cells into a 15 mL tube, and centrifuge at 800 rpm for 5 minutes at room temperature. 8. After removing the supernatant, suspend in 10 mL of subculture medium, and seed the cells at 10 mL / 100 mm dish (passage number 2). 9. Approximately 3 days later, after removing the medium, wash with 8 mL of PBS(-), and detach the cells using 2 mL of trypsin-EDTA solution. Add 6 mL of subculture medium to collect the cells into a 15 mL tube, and centrifuge at 800 rpm for 5 minutes at room temperature. 10. After removing the supernatant, suspend in 30 mL of subculture medium, and seed the cells at 10 mL / 100 mm dish (passage number 3). 11. Approximately 3 days later, after removing the medium, wash with 8 mL of PBS(-), and detach the cells using 2 mL of trypsin-EDTA solution. Add 6 mL of subculture medium to collect the cells into a 15 mL tube, and centrifuge at 800 rpm for 5 minutes at room temperature. 12. After removing the supernatant, suspend in 30 mL of subculture medium, and seed the cells at 10 mL / 100 mm dish (passage number 3). 12. After confirming the secretion of RSPO1 by Western blotting, the same viral infection procedure was performed for each of the proteins of HGF, FGF-7, and FGF-10. 13. After infection with all lentiviral vectors, the established L-RHF2 cells were repeated through 9 - 11 steps and passaged up to passage 5. 14. Cells at passage 5 were collected, suspended in passage medium + 10% DMSO, and a frozen stock was prepared.

[0038] Example 2. Preparation of the culture supernatant of L-RHF2 cells (1) Collection of the culture supernatant of L-RHF2 cells The culture medium for L-RHF2 cells was prepared as shown in Table 1. L-RHF2 cells suspended in this medium were seeded at 5.0×10 6 cells / 12 mL / 100 mm dish and incubated at 37 °C, 5% CO 2 for 72 hours. Finally, the culture supernatant was collected, filtered using a 0.2 μm filter, and stored at -80 °C until use.

[0039] Table 1. Composition of the culture medium for L-RHF2 cells

Table 1

[0040] (2) Confirmation of proteins secreted from L-RHF2 cells (Western blotting) After thawing the culture supernatant, Western blotting was performed using antibodies against R-spondin-1, HGF, FGF-7, and FGF-10 respectively. Recombinant proteins with known concentrations were used as standard substances to calculate the amount of each protein in the culture supernatant. Furthermore, the culture supernatant was collected after subculturing L-RHF2 cells every 3 days, and this was continued for 20 passages.

[0041] The results are shown in Figure 4. Bands were detected at the positions of the expected molecular weights for all proteins, indicating that each protein was secreted into the culture supernatant as expected. For FGF-7 and FGF-10, the molecular weights of the proteins in the culture supernatant were higher than those of the standard proteins. However, treatment of the obtained FGF-7 with glycopeptidase F resulted in a molecular weight comparable to that of the standard protein. Since FGF-7 and FGF-10 have high sequence similarity and both have glycosylation sites, it was suggested that these proteins were N-glycosylated in host cells before being secreted into the medium. Finally, a culture supernatant containing approximately 2,400 ng / mL of R-spondin-1, approximately 80 ng / mL of HGF, approximately 160 ng / mL of FGF-7, and approximately 150 ng / mL of FGF-10 was obtained. Also, the bands of each protein remained constant from P6 to P26, indicating that the secretion ability of each protein in L-RHF2 cells was maintained constantly for at least two months.

[0042] Example 3. Comparison between the growth medium of the present invention and the medium prepared using recombinant proteins (1) Preparation of media The growth medium of the present invention was prepared with the composition as shown in Table 2. As a control, a growth medium using commercially available recombinant proteins instead of the culture supernatant of L-RHF2 cells in the present invention was prepared with the composition as shown in Table 3. Also, the DE1 medium, DE2 medium, DE3 medium, primitive gut spheroid differentiation medium, posterior foregut spheroid medium, and maturation medium (HDM) used when producing liver organoids from iPS cells were each prepared with the composition as shown in Tables 4 to 9.

[0043] Table 2. Composition of the growth medium of the present invention

Table 2

[0044] Table 3. Composition of the control growth medium

Table 3

[0045] Table 4. Composition of DE1 Medium

Table 4

[0046] Table 5. Composition of DE2 Medium

Table 5

[0047] Table 6. Composition of DE3 Medium

Table 6

[0048] Table 7. Composition of Gastrula Spheroid Differentiation Medium

Table 7

[0049] Table 8. Composition of Posterior Foregut Spheroid Medium

Table 8

[0050] Table 9. Composition of Maturation Medium (HDM)

Table 9

[0051] (2) Differentiation of iPS Cells into Human Pluripotent Stem Cell-Derived Liver Organoids Human iPS cells were differentiated into liver organoids derived from human pluripotent stem cells by modifying the differentiation process into definitive endoderm (DE) according to a known protocol. Briefly, iPS cells at 80-90% confluence were treated with DE1 medium for 24 hours, then with DE2 medium for 24 hours, and further with DE3 medium for 24 hours to differentiate them into DE. Subsequently, the DE was cultured in primitive gut spheroid differentiation medium for 3 days to recover free-floating spheroids, which were embedded in Matrigel on a Nunc Multidish 4-well plate (Thermo Fisher Scientific), and then supplemented with posterior foregut spheroid medium and exchanged every 2 days for 4 days of culture. Thereafter, the medium was changed to HDM and cultured for 10 days while changing the medium every 3 days.

[0052] (3) Preparation of liver organoids derived from human biological samples For comparison with liver organoids derived from human pluripotent stem cells, liver organoids derived from human biological samples were established by modifying the two-step collagenase perfusion method. Healthy liver tissue from surgical specimens was washed with cold EBSS (Gibco), cut into approximately 3-mm fragments in 5 mL of EBSS supplemented with 2.5 mg / mL collagenase D (Roche) and 0.1 mg / mL DNase I (Sigma). The fragmented tissue was transferred to a 15-mL tube and incubated in a 37°C water bath with shaking for 40 minutes. After adding 5 mL of basal medium, the tissue fragments were filtered through a 70-μm nylon mesh, 10 mL of basal medium was added, and the mixture was left standing on ice. The tissue remaining on the mesh was further digested with 5 mL of Accutase (Gibco) in a 37°C water bath with shaking for 10 minutes, 5 mL of basal medium was added, filtered through a 70-μm nylon mesh, and an additional 10 mL of basal medium was added. After combining the digested fractions, the mixture was centrifuged at 100×g for 5 minutes to recover the pellet. This pellet was resuspended in 10 mL of basal medium and centrifuged at 100×g for 5 minutes to recover again. After resuspension in 10 mL of basal medium, the cell density was measured using a hemocytometer. Cells (10,000 - 20,000) embedded in Matrigel were dispensed into each well of a Nunc Multidish 4-well plate. 5% CO 2After solidifying Matrigel at 37°C for 15 minutes in an incubator, 500 μL of the growth medium (EM) of the present invention was added to each well. The entire medium was replaced every three days.

[0053] (4) Subculture and culture of human liver organoids Human liver organoids embedded in Matrigel were washed with phosphate-buffered saline (PBS) and treated with TrypLE Express solution (Thermo Fisher Scientific) at 37°C in a water bath for 10 minutes. The collected organoids were disrupted by pipetting 30 - 40 times, 10 mL of basal medium was added, and centrifuged at 440×g for 3 minutes. After removing the supernatant, the cells were suspended in the growth medium. 5 μL of the cell suspension was mixed with 40 μL of Matrigel on ice, and 40 μL of the mixture was added to the wells of a Nunc Multidish 4-well plate, and 5% CO 2 It was solidified at 37°C for 15 minutes in an incubator. Then, 500 μL of the growth medium was added to each well. The total amount of the medium was replaced every 3 - 4 days. Subculture of human liver organoids was performed every 6 - 7 days. The subculture ratio was 1:20 - 1:24. The cells were cultured in an incubator at 37°C, 5% CO 2 The bright-field image or fluorescence image of the organoids was obtained by generating a focus-stacked image from a series of Z-stack images using an all-in-one fluorescence microscope (Keyence, Japan).

[0054] (5) Evaluation of cell morphology and proliferative ability Human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention and the control growth medium were passaged at a constant dilution ratio from P1 to P3 and observed under a bright-field microscope. The proliferative ability of human pluripotent stem cell-derived liver organoids was evaluated by an ATP assay using the CellTiter-Glo 3D reagent. Specifically, on the passage day, the cells were collected at 1 well / tube, washed twice with DMEM, and then suspended in 100 μL of DMEM. Next, 100 μL of the CellTiter-Glo 3D reagent was added to each, mixed, and incubated at room temperature for 30 minutes. Thereafter, 2 μL of the sample was aliquoted and mixed with 60 μL of the CellTiter-Glo 3D reagent, and luminescence was measured with a luminometer. The luminescence signal was measured using Lumat3 LB9508 (Berthold) according to the manufacturer's protocol. Bright-field full-focus images of human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention and the control growth medium at each time point from P1 to P3 are shown in Fig. 5 (scale bar: 500 μm). Also, the relative ATP amounts of human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention and the control growth medium at each time point from P1 to P3 are shown in Fig. 6 (n = 4, mean ± standard deviation, ** p < 0.01: compared to the control). Although there was no significant difference in the concentration of each protein, human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention showed higher proliferative ability compared to those cultured in the control growth medium.

[0055] Also, as shown in Fig. 7, human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention had a higher expression level of LGR5, a stem cell marker, and lower expression levels of AFP and ALB, stem cell markers, at P3 compared to those cultured in the control growth medium. Therefore, it was shown that the growth medium of the present invention can promote the proliferation of LGR5+ hepatic stem cells compared to the control growth medium. In addition, it was confirmed that human body-derived liver organoids can also be proliferated by the growth medium of the present invention. Furthermore, by switching the medium for culturing human pluripotent stem cell-derived liver organoids from the growth medium of the present invention to HDM, the expression of LGR5 decreased, and the expressions of AFP and ALB increased. The results are shown in Fig. 8. Therefore, while the stemness of human pluripotent stem cell-derived liver organoids is maintained by the growth medium, it was shown that the maturation of human pluripotent stem cell-derived liver organoids can be promoted by switching the medium to HDM.

[0056] Furthermore, growth media were similarly prepared using culture supernatants stored at -80°C for 1 month or culture supernatants stored at -80°C for 1 year, and the cell morphology and proliferative ability of human pluripotent stem cell-derived liver organoids cultured in each were confirmed. Bright-field full-focus images of human pluripotent stem cell-derived liver organoids cultured in the medium using the 1-month stored culture supernatant (New) and the medium using the 1-year stored culture supernatant (Old), respectively, obtained at each time point from P1 to P3 are shown in Fig. 9 (scale bar: 500 μm). Also, the relative ATP amounts of human pluripotent stem cell-derived liver organoids cultured in the medium using the 1-month stored culture supernatant (New) and the medium using the 1-year stored culture supernatant (Old), respectively, obtained at each time point from P1 to P3 are shown in Fig. 10 (n = 4, mean ± standard deviation, ** p < 0.01: compared to the control). From these results, it was shown that even when using the culture supernatant stored at -80°C for 1 year, human pluripotent stem cell-derived liver organoids can be proliferated in the same manner as with freshly prepared culture supernatant.

[0057] Example 4. Properties of human pluripotent stem cell-derived liver organoids cultured in the growth medium of the present invention Since the liver is responsible for the upregulation of anabolic pathways in response to insulin, activation of insulin signaling was focused on. Because HDM contains insulin, William's E medium was used as the basal medium. As shown in Fig. 11, when cultured in William's E medium for 18 hours and then treated with 100 μM insulin for 6 hours after serum deprivation, the phosphorylation levels of Akt and its downstream proteins, mTOR and S6K, were increased in human pluripotent stem cell-derived liver organoids. Furthermore, as shown in Fig. 12, the expression of SREBP-1 target genes, namely acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, which mediate fatty acid synthesis, was also increased. On the other hand, the expression of gluconeogenic genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1 was decreased by insulin administration. These results indicate that insulin exerts an anabolic effect in human pluripotent stem cell-derived liver organoids by controlling the expression of gluconeogenic and lipid synthesis genes.

[0058] Nuclear receptors such as liver X receptor (LXR), farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR) α, and pregnane X receptor (PXR) are involved in glucose, lipid, and drug metabolism in the liver. Whether these transcription factors function in human pluripotent stem cell-derived liver organoids matured by HDM was confirmed. As shown in Fig. 13, after treatment with each ligand (LXR agonist: 1 μM of T0901317; FXR agonist: 1 μM of GW4064; PPARα agonist: 1 μM of GW7647; PXR agonist: 20 μM of rifampicin), the expression of target genes of each nuclear receptor was significantly induced. As shown in Fig. 14, after treatment with inducers of each CYP450 molecular species (1000 μM of phenobarbital, 10 μM of rifampicin), the expression of CYP450 with PXR as an inducer was significantly induced. From these results, it was shown that these receptors expressed in human pluripotent stem cell-derived liver organoids are activated at the protein level.

[0059] Hepatocytes accumulate excess energy as lipid droplets composed of neutral lipids including triacylglycerol. Since hepatocytes have a high ability to synthesize triacylglycerol from free fatty acids, we examined whether lipid droplets accumulate intracellularly when human pluripotent stem cell-derived liver organoids are loaded with oleic acid. After culturing with HDM for 3 days, human pluripotent stem cell-derived liver organoids were loaded with 500 μM oleic acid and cultured for 2, 4, and 6 days. The results are shown in Fig. 15. The amount of intracellular lipid droplet accumulation stained with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493 / 503) increased in proportion to the culture period. The results of quantitative analysis of triacylglycerol are shown in Fig. 16. The amount of intracellular triacylglycerol increased by treatment with 500 μM oleic acid. Furthermore, as shown in Fig. 17, the expression of tumor necrosis factor (TNF), a marker of inflammation, increased by oleic acid treatment. From the above results, it was suggested that human pluripotent stem cell-derived liver organoids maintained with HDM after being grown in the growth medium of the present invention reproduce the physiological metabolism and functions of hepatocytes.

[0060] Example 5. Expression of lipid metabolism-related genes in human liver organoids As shown in Example 4, mature human liver organoids exhibited multiple liver-specific functions. Next, to further elucidate the physiological characteristics of human liver organoids, extensive mRNA expression analysis was performed. Focusing on the pathways related to nutrient metabolism and drug metabolism, representative gene expression profiles of primary human hepatocytes, established cell lines (HepaRG, HepG2, and Huh-7 cells), and human liver organoids (human pluripotent stem cell-derived liver organoids and human cadaver-derived liver organoids) were compared. The results are shown in Fig. 18. Human pluripotent stem cell liver organoids and cadaver-derived liver organoids showed similar gene expression patterns, and the expression levels of many genes related to lipid metabolism, drug metabolism, glycolysis, gluconeogenesis, and the urea cycle increased when the culture medium was changed from the growth medium to HDM. Among them, the expression levels of lipid metabolism-related genes mediating fatty acid, triacylglycerol, cholesterol synthesis, fatty acid β-oxidation, LDL uptake, and VLDL secretion in human liver organoids were generally equivalent to or higher than those of primary human hepatocytes and established cell lines. These results suggest that human liver organoids are useful particularly for the evaluation of lipid metabolism-related events.

[0061] Example 6. Secretion of VLDL at the physiological level in human liver organoids The metabolic mechanism of VLDL differs between humans and rodents such as mice and rats. In addition, it is secreted from HepG2 cells and HuH-7 cells, which are human liver cancer-derived cells, but contains far less lipid than VLDL in vivo. We confirmed whether human pluripotent stem cell-derived liver organoids could secrete mature VLDL. The results are shown in Fig. 22. When the medium was changed from the growth medium to HDM in human pluripotent stem cell-derived liver organoids, it was confirmed that ApoB secretion increased significantly. Considering that VLDL and LDL contain one molecule of ApoB, especially ApoB-100 per particle, this result indicates that culturing human pluripotent stem cell-derived liver organoids in HDM enhanced the liver function of VLDL secretion. Subsequently, iodixanol (OptiPrep) density gradient ultracentrifugation was performed to separate each lipoprotein fraction, and apoB-100 in each fraction was analyzed to monitor VLDL and LDL levels. As shown in Fig. 20, it was confirmed that HepG2 cells hardly secreted ApoB-100 into the VLDL fraction (0.950 - 1.006 g / mL), accounting for only 1.9% of the total ApoB-100 protein.

[0062] Interestingly, as shown in Figure 21, human pluripotent stem cell-derived liver organoids were able to secrete a large amount of ApoB-100 into the VLDL fraction, accounting for 54.9% of the total ApoB-100 protein. This ratio is close to the ratio secreted from primary human hepatocytes. It is known that VLDL secretion deficiency in HepG2 cells is caused by the overactivation of the mitogen-activated protein kinase (MEK) / extracellular signal-regulated kinase (ERK) signaling pathway. As shown in Figures 20 and 21, the ratio of ApoB-100 in the VLDL fraction of HepG2 cells increased to 27.6% when treated with 30 μM of PD98059, a cell-permeable MEK inhibitor, but did not increase after PD98059 treatment in human pluripotent stem cell-derived liver organoids. As shown in Figure 22, since PD98059 effectively inhibited MEK phosphorylation in the whole cell lysate of human pluripotent stem cell liver organoids, activated MEK, which is expressed in HepG2 cells but not in human pluripotent stem cell-derived liver organoids, is considered to be the cause of the physiological secretion disorder of VLDL. Notably, as shown in Figure 23, PD98059 did not affect the secretion amount of ApoB-100 in either HepG2 cells or human liver organoids. Taken together, human pluripotent stem cell-derived liver organoids can secrete VLDL in a physiological manner similar to primary human hepatocytes and are considered a reliable model for elucidating the molecular mechanism of VLDL secretion in humans.

Claims

1. A culture medium for growing liver organoids, comprising a culture supernatant of modified L cells obtained by modifying L cells to express R-spondin-1, hepatocyte growth factor (HGF), fibroblast growth factor (FGF) 7, and fibroblast growth factor 10.

2. The proliferation medium according to claim 1, wherein the liver organoid is a liver organoid derived from a pluripotent stem cell.

3. The proliferation medium according to claim 2 , wherein the pluripotent stem cells are human pluripotent stem cells.

4. The growth medium according to any one of claims 1 to 3, wherein the culture supernatant of the modified L cells contains R-spondin-1, HGF, FGF-7 and FGF-10.

5. The growth medium according to any one of claims 1 to 3, wherein the culture supernatant of the modified L cells contains R-spondin-1, HGF, FGF-7 and FGF-10 in a ratio of R-spondin-1:HGF:FGF-7:FGF-10=30:1:2:

2.

6. The growth medium according to any one of claims 1 to 3, wherein the culture supernatant of the modified L cells is obtained by culturing the modified L cells in a medium containing FBS.

7. The growth medium according to any one of claims 1 to 3, wherein the modified L cells are L-RHF2 cells prepared by sequentially infecting L cells with four types of lentivirus vectors each containing the gene sequences of R-spondin-1, HGF, FGF-7, and FGF-10.

8. The growth medium according to any one of claims 1 to 3, wherein the modified L cells are L-RHF2 cells represented by NITE P-04111 (accession number).

9. A method for growing human pluripotent stem cell derived liver organoids, comprising culturing human pluripotent stem cell derived liver organoids in a growth medium according to any one of claims 1 to 3.

10. A method for enhancing stemness of a grown liver organoid, comprising growing the liver organoid in a growth medium according to any one of claims 1 to 3.

11. A method for producing a hepatic organoid derived from a proliferated human pluripotent stem cell with enhanced stemness, comprising growing the hepatic organoid in a growth medium according to any one of claims 1 to 3.

12. A proliferated human pluripotent stem cell-derived liver organoid with enhanced stemness produced by the method of claim 11.

13. The modified L cells (ATCC number: CRL-2648) are modified to secrete R-spondin-1, HGF, FGF-7, and FGF-10.

14. The modified L cell of claim 13, which expresses R-spondin-1, HGF, FGF-7 and FGF-10 in a ratio of R-spondin-1:HGF:FGF-7:FGF-10=30:1:2:

2.

15. Modified L cells, designated NITE P-04111 (accession number).

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