Method for producing human liver organoid

Abstract

This method for producing a genetically modified human liver organoid comprises: a step for introducing a construct for genetic modification into the genome of a human liver organoid in the presence of a HIPPO signaling pathway inhibitor; and a step for culturing, in the presence of a human platelet lysate, the human liver organoid into which the construct has been introduced.

Description

Method for producing human liver organoids

[0001] The present invention relates to a method for producing human liver organoids. More specifically, the present invention relates to a method for producing genetically modified human liver organoids, human liver organoids and genetically modified human liver organoids, and a method for producing primary hepatocytes with excellent organoid-forming ability. This application claims priority based on US 63 / 745,798, which was provisionally filed in the United States on January 16, 2025, and the contents thereof are incorporated herein by reference.

[0002] The liver performs extremely important metabolic functions such as gluconeogenesis, glycogenesis, urea cycle, lipid production, bile acid synthesis, protein production, drug metabolism, etc. When these functions are severely impaired, liver failure occurs, threatening life and requiring transplantation.

[0003] With the recent progress of antiviral therapies, the causes of liver failure have changed from viral hepatitis to metabolic liver diseases associated with modern lifestyles, particularly metabolic dysfunction-associated steatotic liver disease (MASLD). If metabolic liver diseases are left untreated, they progress to metabolic dysfunction-associated steatohepatitis, cirrhosis, and liver failure.

[0004] The development of technologies for preventing or treating metabolic liver diseases requires a human hepatocyte model that faithfully reproduces metabolic functions. For example, Patent Document 1 describes a method for producing metabolically activated liver organoids with activated metabolic functions.

[0005] International Publication No. 2024 / 225466

[0006] However, in Patent Document 1, no consideration has been given to the genetic modification of human liver organoids. The present invention aims to provide a technique for genetically modifying human liver organoids.

[0007] The present invention includes the following aspects: [1] A method for producing a genetically modified human liver organoid, comprising the steps of: introducing a construct for genetic modification into the genome of a human liver organoid in the presence of a HIPPO signaling pathway inhibitor; and culturing the human liver organoid after the introduction of the construct in the presence of human platelet lysate. [2] The method according to [1], wherein the step of introducing the construct for genetic modification is performed by electroporation. [3] The method according to [1] or [2], wherein the construct for genetic modification is a CRISPR Cas protein or its expression vector, and a gRNA or its expression vector, or an expression vector. [4] The method for producing a human liver organoid according to any one of [1] to [3], wherein the human liver organoid is a proliferative liver organoid or a metabolically activated liver organoid, the proliferative liver organoid is obtained by culturing human primary hepatocytes in a growth medium, the metabolically activated liver organoid is obtained by culturing the proliferative liver organoid in a differentiation medium, the growth medium is a medium containing a Wnt agonist, a bone morphogenetic protein inhibitor, a growth factor, a TGF-β inhibitor, forskolin and a STAT3 activating molecule, and the differentiation medium is a medium substantially free of a STAT3 activating molecule and containing a growth factor, a TGF-β inhibitor, forskolin, growth hormone, prolactin, glucocorticoids and a γ-secretase inhibitor. [5] The method for producing a human liver organoid according to [4], wherein the STAT3 activating molecule is IL-6 or oncostatin M. [6] A human liver organoid produced by the method for producing a human liver organoid according to any one of [1] to [5]. [7] A human liver gene disease model produced by any of the manufacturing methods described in [1] to [5]. [8] The human liver gene disease model described in [7], wherein the G6PC gene or the OTC gene is disrupted. [9] A method for producing primary hepatocytes with excellent organoid formation ability, comprising the step of subjecting human primary hepatocytes dissociated into single cells to flow cytometry and recovering cells whose lateral scattering values ​​are in the lower 30% or less of the population of human primary hepatocytes, wherein the recovered cells are primary hepatocytes with excellent organoid formation ability.

[0008] The present invention can also be said to include the following embodiments. [P1] A method for gene editing human liver organoids, comprising the following steps: i) culturing proliferative human liver organoids in a second growth medium for 3 to 5 days before gene introduction; ii) adding DMSO to the second growth medium the day before gene introduction and culturing; iii) separating the human liver organoids into single cells, washing with gene introduction medium, and then introducing the gene; iv) culturing in a second growth medium containing DMSO at 30°C for 24 hours after gene introduction; v) culturing in the second growth medium with DMSO removed at 30°C for a further 2 days; vi) culturing in the second growth medium at 37°C for a further 4 to 7 days; vii) selecting the gene-introduced cells; viiii) culturing in a medium containing human-platelet lysate, wherein the second growth medium is a medium containing a HIPPO signaling pathway inhibitor, and the human liver organoids are organoids derived from adult human tissue. [P2] The method according to [P1], wherein the human liver organoid is produced from primary hepatocytes derived from human adult tissue or human adult hepatocytes using a growth medium and further differentiated using a differentiation medium to have normal liver function. [P3] The method according to [P2], wherein the growth medium is a medium containing a Wnt agonist, a TGF-β inhibitor, a STAT3 activating molecule, EGF, HGF, and FGF-10. [P4] The method according to [P3], wherein the STAT3 activating molecule is IL-6 or oncostatin. [P5] The method according to [P2], wherein the differentiation medium is substantially free of EGF, HGF, and FGF-10 and contains growth hormone, prolactin, glucocorticoids, and a γ-secretase inhibitor. [P6] A disease model liver organoid having the function of a human liver and serving as a disease model, produced by the method according to [P1]. [P7] A method for separating human adult hepatocytes by flow cytometry, wherein the SSC / BSC (lateral-backscattering) values ​​are in the lower 30% or less of the cell population.

[0009] According to the present invention, a technology for genetically modifying human liver organoids can be provided.

[0010] Figure 1 is a graph showing the results of measuring the percentage of proliferating cells by 5-ethynyl-2'-deoxyuridine (EDU) staining in Experimental Example 2. Figure 2 is a graph showing the results of measuring albumin secretion, glucose production, and urea synthesis from TP53 knockout human liver organoids (HHO) in Experimental Example 2. Figure 3 left is an image showing the results of confirming the expression of G6PC protein in G6PC-knockout (KO) HHO by Western blotting in Experimental Example 3. Figure 3 right is a representative image of a G6PC-KO HHO clone. Figure 4 left is an image showing the results of confirming the expression of OTC protein in OTC-KO HHO by Western blotting in Experimental Example 3. Figure 4 right is a representative image of an OTC-KO HHO clone. Figure 5 is a graph showing the results of measuring albumin production by G6PC-KO HHO and OTC-KO HHO in Experimental Example 3. Figure 6 is a graph showing the results of measuring glucose production by G6PC-KO HHO in Experimental Example 3. Figure 7 is a graph showing the results of measuring citrulline production by OTC-KO HHO in Experimental Example 3. Figure 8 is a graph showing the results of measuring the percentage of proliferating cells by EDU staining for OTC-KO HHO in Experimental Example 3. Figure 9 is an image showing a time-series snapshot of organoid growth using oncostatin M (OSM) derived from a single primary human hepatocyte (PHH) in Experimental Example 4. Figure 10 is a graph showing the results of classifying the lateral scattering values ​​of DAPI-surviving cells into three levels and an image of sorted PHH in Experimental Example 4. Figure 11 is a graph showing the results of measuring the organoid formation efficiency when each PHH with side scattering values ​​of Low, Middle, and High was cultured in growth medium (EM medium) in Experimental Example 4.

[0011] [Method for Producing Human Liver Organoids] In one embodiment, the present invention provides a method for producing genetically modified human liver organoids (HHO), comprising the steps of: introducing a construct for genetic modification into the genome of human liver organoids in the presence of a HIPPO signaling pathway inhibitor; and culturing the human liver organoids after the introduction of the construct in the presence of human platelet lysates. The method for producing human liver organoids in this embodiment can also be described as a method for producing electroporated human liver organoids, comprising the steps of: performing electroporation on human liver organoids in the presence of a HIPPO signaling pathway inhibitor; and culturing the human liver organoids after electroporation in the presence of human platelet lysates.

[0012] As described later in the examples, genetic modification of HHO by electroporation leads to poor proliferation due to stress on the cells. Furthermore, in order to clone the genetically modified cells, it is necessary to stably proliferate the HHO cells that have been dissociated into single cells, but conventionally, HHO cells that have been dissociated into single cells sometimes suffer from poor proliferation.

[0013] In response, the inventors found that electroporation of HHO in the presence of a HIPPO signaling pathway inhibitor improved the proliferative capacity of HHO after electroporation. The inventors also found that by culturing the human liver organoids after electroporation in the presence of human platelet lysates, the HHO dissociated into single cells could be stably proliferated and cloned.

[0014] The electroporation conditions can be any conditions typically used for gene transfer into cells. For example, when using the NEPAGENE electroporator (NEPAGENE Co., Ltd.), the conditions are: Poring Pulse (Voltage = 150V, Pulse Length = 10ms, Pulse Interval = 50ms, Number of Pulses = 2, Decay Rate: 10%, Polarity: +), and Transfer Pulse (Voltage = 20V, Pulse Length = 50ms, Pulse Interval = 50ms, Number of Pulses = 5, Decay The conditions are: Rate: 40%, Polarity: + / -).

[0015] The HIPPO signaling pathway is a signaling pathway known to be involved in cell proliferation, apoptosis, and stem cell self-renewal, and is known to be an evolutionarily conserved pathway.

[0016] The HIPPO signaling pathway inhibitor may be, for example, an inhibitor of MST1 kinase or MST2 kinase (MST1 / 2 kinase inhibitor), or an inhibitor of LATS1 kinase or LATS2 kinase (LATS1 / 2 kinase inhibitor).

[0017] The NCBI accession numbers for the amino acid sequence of human MST1 kinase are NP_001380510.1, NP_001380511.1, NP_001380512.1, NP_001380513.1, NP_001380514.1, NP_066278.3, etc. Similarly, the NCBI accession numbers for the amino acid sequence of human MST2 kinase are NP_001243241.1, NP_001243242.1, NP_006272.2, etc.

[0018] The NCBI accession numbers for the amino acid sequence of human LATS1 kinase are NP_001257448.1, NP_001337268.1, NP_001337269.1, NP_001337321.1, NP_004681.1, etc. The NCBI accession numbers for the amino acid sequence of human LATS2 kinase are NP_055387.2, etc.

[0019] More specific examples of HIPPO signaling pathway inhibitors include, for example, LATS1 kinase and LATS2 kinase inhibitors such as TRULI (CAS number: 1424635-83-5), GA-017 (CAS number: 2351906-74-4), and TDI-011536 (CAS number: 2687970-96-1). These may be used individually or in combination of two or more. Among these, TRULI and TDI-011536 can be used particularly well.

[0020] The concentration of the HIPPO signaling pathway inhibitor in the culture medium may be, for example, 1 μM to 10 mM, for example, 1 μM to 1 mM, or for example, 1 to 100 μM.

[0021] It is preferable to add the HHO culture medium between 2 to 10 days before electroporation, for example, 4 to 8 days before electroporation, or for example, 5 days before electroporation, and between 2 to 20 days after electroporation, for example, 4 to 18 days after electroporation, or for example, 14 days after electroporation.

[0022] The construct for gene modification may be the CRISPR Cas protein or its expression vector, and gRNA or its expression vector. Alternatively, the construct for gene modification may be the expression vector of the target gene. That is, the CRISPR Cas protein or its expression vector, and gRNA or its expression vector may be introduced into HHO by electroporation. Foreign DNA may also be introduced at this time. This allows for genome editing to be induced in HHO, disrupting the target gene by InDel mutation, or introducing a foreign gene into the target locus to modify the target gene. The CRISPR Cas protein is not particularly limited, but examples include Cas9 and Cas3.

[0023] Alternatively, the expression vector of the target gene may be introduced into HHO by electroporation. This allows the target gene to be introduced into HHO. The target gene is not particularly limited, but examples include the GFP gene.

[0024] Human platelet lysate (hPL) is rich in various growth factors and cytokines necessary for cell culture, and is therefore used as an additive to culture media in various human cell cultures.

[0025] It is preferable to add platelet lysates to the culture medium at the stage in which HHO is dissociated into single cells and cloned. Commercially available platelet lysates may also be used. The concentration of platelet lysates in the culture medium is 1 volume (v / v)% to 10 volume (v / v)%, preferably 5 volume (v / v)%.

[0026] In this specification, the method for dissociating cells into single cells is not particularly limited and may be an enzymatic method, a physical method, or a combination of both. When dissociating enzymatically, enzymes such as collagenase, dispase I, liberase, trypsin, and TrypLE Express (Thermo Fisher Scientific) can be used. Physical dissociation can be performed by mechanical shearing, for example, by pipetting.

[0027] Human liver organoids may be proliferative liver organoids or metabolically activated liver organoids.

[0028] (Proliferative Liver Organoids) Proliferative liver organoids are HHOs that have the ability to proliferate, and refer to HHOs that are cultured in the growth medium (EM medium) described later. Proliferative liver organoids can also be said to be HHOs that are cultured in EM medium. Hereafter, proliferative liver organoids may be referred to as eHHOs.

[0029] Metabolic-activated liver organoids are HHOs with metabolic activity equivalent to that of hepatocytes, which constitute the liver tissue of living organisms, and possess gluconeogenic and urea-producing abilities equivalent to or greater than those of primary hepatocytes. Metabolic-activated liver organoids can be obtained by culturing and differentiating proliferative liver organoids in a differentiation medium (DM medium) described later. Metabolic-activated liver organoids can also be described as HHOs cultured in DM medium. Hereinafter, metabolic-activated liver organoids may be referred to as dHHOs.

[0030] Proliferative liver organoids can be obtained by culturing primary hepatocytes in EM medium in contact with the extracellular matrix (ECM). Primary hepatocytes may be cryopreserved cells.

[0031] One method for culturing primary hepatocytes in contact with an extracellular matrix is ​​to mix primary hepatocytes with an extracellular matrix precursor, polymerize the extracellular matrix precursor to form an extracellular matrix, and then immerse the extracellular matrix in EM medium for cultivation.

[0032] The extracellular matrix is ​​a substance that serves as a scaffold for cells in cell culture. Components of the extracellular matrix include, for example, components found in the basement membrane and glycoproteins present in the intercellular spaces. Examples of basement membrane components include type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. Examples of glycoproteins present in the intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, and heparin sulfate. One of these components may be used alone, or two or more may be used in combination. The extracellular matrix preferably contains laminin, and more preferably contains laminin-111.

[0033] Examples of commercially available extracellular matrix products include Matrigel®, Cultrex (Biotechne), Geltrex (Thermo Fisher Scientific), EHS Gel Basement Membrane Matrix (Fujifilm Wako Pure Chemical Industries, Sigma), and Collagen I (Nitta Gelatin).

[0034] Hydrogels made from artificial polymers can also be used as scaffolds for cells. Examples of commercially available hydrogels made from artificial polymers include Mebiol Gel (Mebiol), VitroGel (Zawell Biosciences), and GrowDex (UPM Biomedicals).

[0035] (Growth medium (EM medium)) The growth medium is a medium containing a Wnt agonist, a bone morphogenetic protein inhibitor, a growth factor, a TGF-β inhibitor, forskolin, and a STAT3 activating molecule. The inventors have found that by using the growth medium, it is possible to obtain proliferative liver organoids that can be cultured for a long period of time while maintaining high proliferative capacity.

[0036] The growth medium can be prepared by adding each component to a basal medium. Examples of the basal medium include Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Eagle's Basal Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12 (Advanced DMEM / F12), Iscove's Modified Dulbecco's Medium, Ham's F-10, Ham's F-12, Medium 199, RPMI 1640 medium, and the like.

[0037] Among these, DMEM / F12 and RPMI 1640 to which HEPES, glutamine, and penicillin / streptomycin are added are preferred. Also, Advanced DMEM / F12 or Advanced RPMI, which is optimized for serum-free culture and contains GlutaMAX (manufactured by Gibco, L-alanyl-L-glutamine) instead of glutamine, is also preferably used. It is preferable to add glutamine and penicillin / streptomycin to the Advanced DMEM / F12 or Advanced RPMI medium.

[0038] The growth medium preferably contains a Wnt agonist from the viewpoints of maintaining hepatic stem cells and improving cell proliferation. A Wnt agonist is an agonist that activates the Wnt signaling pathway.

[0039] Examples of the Wnt agonist include the Wnt family, the R-spondin family, Norrin, glycogen synthase (GSK) inhibitors, and the like.

[0040] Examples of the Wnt family include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Among them, Wnt3a is preferred.

[0041] Since it is known that afamin contributes to the stabilization and solubilization of the Wnt family, it is more preferable to use a complex of a Wnt family member and afamin as a Wnt agonist. The complex of a Wnt family member and afamin can be used as a conditioned medium containing the complex at a concentration of the Wnt family member of 18 ng / mL to 900 ng / mL.

[0042] Afamin means a glycoprotein belonging to the albumin family. In GenBank, the amino acid sequence of human afamin is registered as AAA21612, and the amino acid sequence of bovine afamin is registered as DAA28569.

[0043] When using a conditioned medium with the concentration of the Wnt family within the above range as the Wnt family, the content of the conditioned medium contained in the growth medium is 1 volume (v / v)% to 50 volume (v / v)%, preferably 10 volume (v / v)% to 30 volume (v / v)%, 15 volume (v / v)% to 25 volume (v / v)% with respect to the total volume of the growth medium.

[0044] Examples of the R-spondin family include R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Among them, R-spondin 1 is preferred. When the R-spondin family binds to Lgr5 in the cell membrane, it is removed from the cell membrane by autoubiquitination. As a result, Frizzled, which induces the activation of the Wnt signaling pathway, is stabilized in the cell membrane and activates the β-catenin pathway. The R-spondin family can be used as a conditioned medium containing the complex at a concentration of 0.13 μg / mL to 6.μg / mL.

[0045] When using a conditioned medium in which the concentration of the R-spongin family member is within the above range as the R-spongin family member, the amount of conditioned medium contained in the growth medium is 1 volume (v / v)% to 50 volume (v / v)%, preferably 5 volume (v / v)% to 25 volume (v / v)%, or 8 volume (v / v)% to 20 volume (v / v)%, relative to the total volume of the growth medium.

[0046] GSK inhibitors are inhibitors of glycogen synthase 3β (GSK3β). Since GSK3β phosphorylates β-catenin and promotes its degradation, GSK inhibitors act as Wnt agonists.

[0047] Examples of GSK inhibitors include CHIR99021 (CAS number: 252917-06-9), SB216763 (CAS number: 280744-09-4), SB415286 (CAS number: 264218-23-7), CHIR98014 (CAS number: 252935-94-7), AZD1080 (CAS number: 612487-72-6), and LY2090314 (CAS number: 603288-22-8).

[0048] As a Wnt agonist, it is preferable to use a combination of the Wnt family and the R-spongin family, more preferably a combination of Wnt3a and R-spongin 1, and even more preferably a combination of a complex of Wnt3a and afamin and R-spongin 1.

[0049] Examples of bone morphogenetic protein (BMP) inhibitors include noggin, Differential screening-selected gene Aberrative in Neuroblastoma (DAN), and DAN-like proteins. Examples of DAN-like proteins include cerberus and gremlin. Among these, noggin is preferred.

[0050] The concentration of the BMP inhibitor in the growth medium is 10 ng / mL to 100 ng / mL, preferably 15 ng / mL to 50 ng / mL, and more preferably 20 ng / mL to 30 ng / mL.

[0051] Growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

[0052] EGF is a member of the EGF family and is a growth factor that activates the epidermal growth factor receptor (EGFR or ErbB1). Activated EGFR primarily activates the MAPK signaling pathway, as well as the PI3K signaling pathway and the Jak / stat signaling pathway.

[0053] The concentration of EGF contained in the growth medium is 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0054] HGF is a growth factor that activates Met receptors, and activated Met receptors activate the HGF-Met signaling pathway. Activation of the HGF-Met signaling pathway promotes activation of the β-catenin pathway, which in turn promotes angiogenesis and metalloproteinase production.

[0055] The concentration of HGF contained in the growth medium is 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0056] The FGF is preferably one that can bind to FGF receptor 2 (FGFR2) or FGF receptor 4 (FGFR4), and is preferably FGF-2, FGF-4, FGF-7, or FGF-10, with FGF-10 being particularly preferred.

[0057] The concentration of FGF contained in the growth medium is 20 ng / mL to 500 ng / mL, preferably 50 ng / mL to 300 ng / mL, and more preferably 80 ng / mL to 150 ng / mL or less.

[0058] Examples of TGF-β inhibitors include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD-208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2), with A83-01 being the preferred choice.

[0059] The concentration of the TGF-β inhibitor in the growth medium is 0.05 μM to 50 μM, preferably 0.5 μM to 30 μM, and more preferably 1 μM to 15 μM or less.

[0060] From the viewpoint of improving cell proliferation, the growth medium preferably further contains forskolin.

[0061] The concentration of forskolin in the growth medium is 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 15 μM.

[0062] Examples of STAT3 activating molecules include interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), leukemia suppressor factor (LIF), cardiotropin-1 (CT-1), and ciliary neurotrophic factor (CNTF). Among these, IL-6 or oncostatin M are preferred as the STAT3 activating molecule.

[0063] The concentration of STAT3 activating molecules in the growth medium is 10 ng / mL to 1 μg / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0064] The growth medium may contain other components. These other components may include Rho kinase (ROCK) inhibitors, gastrin, neurobiological supplements, and N-acetylcysteine.

[0065] Examples of ROCK inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), Azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Rho kinase α inhibitors (ROKα Examples include inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, Ki-23095, VAS-012, etc.

[0066] The concentration of the ROCK inhibitor in the growth medium is 1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM.

[0067] The gastrin content in the growth medium is between 5 nM and 15 nM.

[0068] Examples of neurobiological supplements include insulin-containing supplements such as B27 supplement (Thermo Fisher Scientific) and N2 supplement (Thermo Fisher Scientific).

[0069] B27 supplement is a composition containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin, and is commercially available as a 50x liquid concentrate.

[0070] N2 supplement is a composition containing 500 μg / mL human transferrin, 500 μg / mL bovine insulin, 0.63 μg / mL progesterone, 161 μg / mL putrescine, and 0.52 μg / mL sodium selenite, and is commercially available as a 100x liquid concentrate.

[0071] The concentration of N-acetylcysteine ​​in the growth medium is between 150 ng / mL and 250 ng / mL.

[0072] (Metabolic Activated Liver Organoids) By culturing the above-mentioned proliferative liver organoids in a differentiation medium, the proliferative liver organoids can be differentiated to obtain metabolically activated liver organoids with superior metabolic activity. Metabolic activated liver organoids exhibit improved expression of various metabolic enzymes.

[0073] It is preferable to differentiate proliferative liver organoids cultured in growth medium for two weeks or more into metabolically activated liver organoids in differentiation medium. The culture period in differentiation medium is 5 to 15 days, preferably 7 to 12 days.

[0074] It is preferable to culture the proliferative liver organoids in contact with the extracellular matrix (ECM). The extracellular matrix is ​​the same as described above. For example, in proliferative liver organoids cultured by layering growth medium on polymerized extracellular matrix, if necessary, the organoids may be physically dissociated to contain an appropriate number of proliferative liver organoids, and then cultured by layering differentiation medium instead of growth medium.

[0075] The differentiation of proliferative liver organoids into metabolically activated liver organoids can be determined or evaluated using indicators such as the expression of hepatocyte markers and drug metabolism activity. Examples of hepatocyte markers include albumin (ALB), alpha-fetoprotein (AFP), tyrosine aminotransferase (TAT), and pregnane X receptor (PXR). The expression levels of hepatocyte markers may be measured at the gene level or at the protein level.

[0076] Metabolic-activated liver organoids exhibit expression levels that are, for example, 50% or higher for albumin, 300% or higher for CYP2E1, 300% or higher for UGT1A1, and 500% or higher for NRP2 compared to human primary cryopreserved hepatocytes.

[0077] (Differentiation medium (DM medium)) The differentiation medium may be a medium that is substantially free of Wnt agonists and STAT3 activating molecules and contains growth factors, TGF-β inhibitors, forskolin, growth hormone, prolactin, glucocorticoids, and γ-secretase inhibitors.

[0078] The differentiation medium is substantially free of STAT3 activating molecules. This allows for the differentiation of proliferative liver organoids into metabolically activated liver organoids. "Substantially free" means that it is permissible to include a small amount of STAT3 activating molecules, such that their effect is not apparent. For example, it is permissible for trace amounts of STAT3 activating molecules to be inevitably introduced from previously used media. For instance, it is permissible for the differentiation medium to contain less than 1 ng / mL of STAT3 activating molecules.

[0079] Differentiation media can be prepared by adding various components to a basic medium. Examples of basic media include those used for growth media.

[0080] The growth factors, TGF-β inhibitors, forskolin, and their respective content are the same as those in the growth medium described above.

[0081] Growth hormone is a hormone secreted from growth hormone-secreting cells in the anterior pituitary gland. Growth hormone mimes may be used as the growth hormone. Examples of growth hormone mimes include agonist antibodies, antibody fragments, and peptides targeting growth hormone receptors. Examples of NCBI accession numbers for human growth hormone cDNA include NM_000515.5, NM_022559.4, NM_022560.4, NM_022561.2, and NM_022562.2.

[0082] The concentration of growth hormone in the differentiation medium is preferably 0.01 ng / mL to 1,000 ng / mL, and may be, for example, 0.1 ng / mL to 500 ng / mL, or 1 ng / mL to 100 ng / mL.

[0083] Prolactin is a hormone primarily secreted by prolactin-secreting cells in the anterior pituitary gland. Prolactin mimes may also be used as prolactin. Examples of prolactin mimes include agonist antibodies, antibody fragments, and peptides targeting prolactin receptors. Examples of NCBI accession numbers for human prolactin cDNA include NM_000948.6 and NM_001163558.3.

[0084] The concentration of prolactin in the differentiation medium is preferably 0.01 ng / mL to 1,000 ng / mL, and may be, for example, 0.1 ng / mL to 500 ng / mL, or 1 ng / mL to 100 ng / mL.

[0085] Glucocorticoids, also known as adrenal cortical hormones, are a type of adrenocortical hormone produced in the zona fasciculata of the adrenal cortex. Glucocorticoid mimes may be used as substitutes for glucocorticoids. Examples of glucocorticoid mimes include synthetic steroids such as prednisolone (CAS number: 50-24-8), dexamethasone (CAS number: 50-02-2), and betamethasone (CAS number: 378-44-9); and agonist antibodies, antibody fragments, and peptides targeting glucocorticoid receptors. Examples of glucocorticoids include cortisol (CAS number: 50-23-7), corticosterone (CAS number: 50-22-6), and cortisone (CAS number: 53-06-5).

[0086] The concentration of glucocorticoids in the differentiation medium is preferably 10 ng / mL to 1,000 ng / mL, and may be, for example, 10 ng / mL to 500 ng / mL, or 10 ng / mL to 200 ng / mL.

[0087] Examples of γ-secretase inhibitors include DAPT (CAS number: 208255-80-5) and dibenzazepine (CAS number: 256-96-2).

[0088] The concentration of the γ-secretase inhibitor in the differentiation medium is preferably 0.05 μM to 50 μM, and may be, for example, 0.5 μM to 30 μM, or 1 μM to 15 μM.

[0089] The differentiation medium may contain other components. These other components include Wnt agonists, osteomorphogenetic protein inhibitors, and ROCK inhibitors. The Wnt agonists, osteomorphogenetic protein inhibitors, ROCK inhibitors, and their respective content are the same as those in the growth medium described above.

[0090] In the manufacturing method of this embodiment, when proliferative liver organoids are genetically modified, HHO having metabolic activity equivalent to that of hepatocytes constituting the liver tissue of a living organism can be obtained by culturing the proliferative liver organoids in DM medium and differentiating them into metabolically activated liver organoids.

[0091] In the manufacturing method of this embodiment, when a metabolically activated liver organoid is genetically modified, HHO having metabolic activity equivalent to that of hepatocytes constituting the liver tissue of a living organism can be obtained.

[0092] [Human Liver Organoids] In one embodiment, the present invention provides human liver organoids (HHO) produced by the manufacturing method described above.

[0093] The HHO in this embodiment may be genetically modified. HHO in which disease-related genes have been modified can be used as a disease model. In other words, in one embodiment, the present invention can be said to provide a human liver gene disease model produced by the production method described above.

[0094] For example, as described later in the examples, HHO may have the G6PC gene or OTC gene disrupted.

[0095] Dysfunction of the G6PC gene causes glycogen storage disease. Therefore, HHOs with disrupted G6PC genes can be used as a model for glycogen storage disease.

[0096] Dysfunction of the OTC gene causes X-linked urea cycle disorders. Therefore, HHO individuals with disrupted OTC genes can be used as a model for X-linked urea cycle disorders.

[0097] The genes associated with the disease are not limited to the G6PC gene or the OTC gene. Various HHO genes can be modified and used as disease models.

[0098] [Method for producing primary hepatocytes] In one embodiment, the present invention provides a method for producing primary hepatocytes with excellent organoid-forming ability, comprising the steps of subjecting human primary hepatocytes dissociated into single cells to flow cytometry and recovering cells whose lateral scattering values ​​are in the lower 30% or less of the population of human primary hepatocytes, wherein the recovered cells are primary hepatocytes with excellent organoid-forming ability.

[0099] As described later in the examples, the inventors revealed that human primary hepatocytes with lateral scattering values ​​in the lower 30% or less of the population of human primary hepatocytes have a higher organoid formation efficiency compared to human primary hepatocytes with larger lateral scattering values.

[0100] Therefore, the manufacturing method of this embodiment makes it possible to produce primary hepatocytes with excellent organoid-forming ability.

[0101] Lateral scattering (SSC), also known as back-lateral scattering (BSC) depending on the flow cytometer model, is a value that reflects information about the complexity of the intracellular environment and the properties of granules.

[0102] Primary hepatocytes may be cryopreserved cells. The method for dissociating human primary hepatocytes into single cells is the same as described above.

[0103] [Other Embodiments] In one embodiment, the present invention provides a method for treating liver disease, comprising the step of transplanting a genetically modified human liver organoid into a patient with liver disease, wherein the genetically modified human liver organoid is a human liver organoid obtained by a manufacturing method comprising the steps of: producing a human liver organoid from hepatocytes derived from the patient; performing electroporation on the human liver organoid in the presence of a HIPPO signaling pathway inhibitor to induce the repair of mutations in disease-causing genes by genome editing; and culturing the human liver organoid after electroporation in the presence of human platelet lysates.

[0104] The same applies to human liver organoids, HIPPO signaling pathway inhibitors, human platelet lysates, genome editing, etc., as described above.

[0105] The liver disease and the disease-causing gene are not particularly limited, but for example, the liver disease may be a glycogen storage disease and the disease-causing gene may be the G6PC gene. Alternatively, the liver disease may be an X-linked urea cycle disorder and the disease-causing gene may be the OTC gene.

[0106] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0107] [Materials and Methods] (Establishment and maintenance of human hepatocyte organoids) Primary human hepatocytes (PHH) that were cryopreserved were purchased from Biopredic International, Bio IVT, Cytes Biotechnologies, Discovery Life Sciences, Sekisui XenoTech, Veritas, or Lonza. The PHH were thawed using cryopreserved hepatocyte recovery medium (Thermo Fisher Scientific).

[0108] Live cells were sorted using flow cytometry (MA900, Sony) with a 130 μm tip in "Purity" mode. In some experiments, healthy liver cells were purified using backside scattering (BSC). A total of 1,000 PHH cells were embedded in Matrigel (Corning) in 48-well plates (Corning), and growth medium (EM medium) was added. Table 1 below shows the composition of the basic medium, and Table 2 below shows the composition of the EM medium.

[0109]

[0110]

[0111] Plate 5% CO ２ , 20% ２ The cells were incubated in a humidified incubator. Once human liver organoids (HHO) formed and expanded, they were isolated using TripLE Express (Thermo Fisher Scientific) and subcultured every two weeks.

[0112] The viability of HHO cells was assessed using the CellTiter-Glo 3D Cell Viability Assay (Promega), and cell counts were estimated using a standard curve based on ATP emission from a specified number of PHH cells.

[0113] To differentiate into metabolically activated liver organoids (differentiated HHO, dHHO), eHHO cells were dissociated into single cells, embedded in Matrigel, and pre-cultured in EM medium for 7–14 days, followed by further culture in differentiation medium (DM medium) for 10–14 days. The composition of the DM medium is shown in Table 3 below.

[0114]

[0115] (Genome Editing) Knockout of the G6PC and OTC genes was performed by electroporation. Organoids were treated with LATSi (TDI-011536, 3 μM) for 3–5 days before electroporation and 7–14 days after electroporation. sgRNA was introduced together with a GFP-puro piggyBAC transposon vector (PB513B-1, System Biosciences). To enhance genome editing efficiency, a 3-day low-temperature incubation was performed at 30°C. On day 7 after electroporation, organoids were selectively treated with puromycin (2 μg / mL, Thermo Fisher Scientific) for 3 days. Puromycin-resistant clones were manually selected and individually grown in EM medium containing 5% human platelet lysate (AventaCell BioMedical). G6PC and OTC knockouts were verified by extracting genomic DNA from cloned genetically modified organoids, performing PCR amplification of target loci, Sanger sequencing, and allele deconvolution of the PCR product using ICE version 2 online software (Synthego). In all knockout strains, the loss of G6PC or OTC protein expression was confirmed by Western blotting or capillary immunoassay.

[0116] [Experimental Example 1] (Introduction of GFP gene into HHO) An expression vector for the GFP gene was introduced into HHO by electroporation. As a result, efficient GFP overexpression was observed. However, the GFP-expressing HHO underwent growth arrest. Growth arrest was thought to be mediated probably by the activation of TP53.

[0117] [Experimental Example 2] (Knockout of TP53 in HHO) The TP53 gene was knocked out in HHO using genome editing by electroporation. Two knockout clones were generated from two different donors (PY9 and PY30).

[0118] Figure 1 is a graph showing the percentage of proliferating cells measured by 5-ethynyl-2'-deoxyuridine (EDU) staining in HHO derived from donor (PY9). The culture period from PHH is shown below. Cells were analyzed from n=47 (218 days) and n=73 (345 days).

[0119] Figure 2 is a graph showing the results of measuring albumin secretion, glucose production, and urea synthesis from TP53 knockout HHO. The culture period from PHH is shown at the bottom.

[0120] Figures 1 and 2 suggest that while TP53 knockout (KO) HHO cells achieved stable proliferation, they were prone to bile duct metaplasia, limiting their usefulness for further functional analysis.

[0121] [Experimental Example 3] (Knockout of G6PC and OTC in HHO) The results of the study revealed that gene-edited organoids can be efficiently proliferated without causing premature growth arrest or bile duct metaplasia by using a LATS inhibitor (LATSi) and human platelet lysate.

[0122] Using this protocol, we introduced sgRNAs targeting G6PC, which causes glycogen storage disease, and OTC, which causes X-linked urea cycle disorders, along with a Cas9 expression vector.

[0123] Sanger sequencing and immunoassay of the HHO clone confirmed that the gene was correctly knocked out. Figure 3 (left) shows the results of Western blotting to confirm the expression of the G6PC protein in the G6PC-KO HHO clone. Figure 3 (right) is a representative image of the G6PC-KO HHO clone.

[0124] Figure 4 (left) shows the results of confirming OTC protein expression in OTC-KO HHO using Western blotting. Figure 4 (right) shows a representative image of an OTC-KO HHO clone.

[0125] Figure 5 is a graph showing the results of measuring albumin production using G6PC-KO HHO and OTC-KO HHO.

[0126] G6PC-KO HHO and OTC-KO HHO maintained the morphology of liver organoids and also produced large amounts of albumin.

[0127] Figure 6 is a graph showing the results of measuring glucose production by G6PC-KO HHO. G6PC-KO HHO showed a decrease in gluconeogenesis, indicating functional inhibition of this metabolic pathway.

[0128] Figure 7 is a graph showing the results of measuring the amount of citrulline produced by OTC-KO HHO. OTC-KO HHO was unable to produce citrulline. This result is consistent with the role of OTC in catalyzing citrulline synthesis from carbamoyl phosphate and ornithine.

[0129] Figure 8 is a graph showing the results of measuring the percentage of proliferating cells in OTC-KO HHO by EDU staining. The culture period from PHH is shown at the bottom. The results showed that OTC-KO HHO maintained its proliferative capacity for at least 100 days.

[0130] These results demonstrate that, for the first time, genome editing in human adult liver cells has been successfully and stably performed using the method described in this experiment.

[0131] [Experimental Example 4] (Sorting of PHH and Production of HHO) Conventionally, clonal proliferation of human adult hepatocytes has been difficult. In contrast, the inventors have found that sorted single ASGPR1 cells can be used regardless of the donor's age or dissemination. ＋ / EpCAM － We demonstrated that HHO can be derived from PHH.

[0132] Figure 9 shows time-lapse snapshots of organoid growth using OSM derived from a single PHH. The same organoid was photographed at the same magnification. The scale bar represents 100 μm. Similar results were obtained with two PHHs from different donors.

[0133] The inventors noticed that about half of the sorted hepatocytes exhibited granular structures in their cytoplasm and invariably died within six hours of seeding. These hepatocytes were identified as high-side-scattering (BSC) populations by flow cytometry.

[0134] Figure 10 shows a graph and sorted images of PHH cells, illustrating the classification of lateral scattering values ​​of DAPI-surviving cells into three levels. The scale bar is 10 μm.

[0135] Figure 11 is a graph showing the results of measuring the organoid formation efficiency when each PHH with Low, Middle, and High lateral scattering values ​​was cultured in EM medium. The top approximately 30% of the PHH population were selected as BSC. ｈｉｇｈ The group is designated as PHH, and the bottom approximately 30% of the PHH group is designated as BSC. ｌｏｗ Set the remaining portion to PHH and BSC. ｍｉｄｄｌｅ It was set to PHH.

[0136] As a result, BSC ｌｏｗ PHH formed organoids with an efficiency of 10–30%. This was a high efficiency, comparable to that of other human tissue stem cells.

[0137] According to the present invention, a technology for genetically modifying human liver organoids can be provided.

Claims

1. A method for producing genetically modified human liver organoids, comprising the steps of: introducing a construct for genetic modification into the genome of a human liver organoid in the presence of a HIPPO signaling pathway inhibitor; and culturing the human liver organoid after the introduction of the construct in the presence of human platelet lysate.

2. The manufacturing method according to claim 1, wherein the step of introducing a construct for gene modification is performed by electroporation.

3. The manufacturing method according to claim 1, wherein the construct for gene modification is a CRISPR Cas protein or its expression vector, and a gRNA or its expression vector, or is an expression vector.

4. The method for producing a human liver organoid according to claim 1 or 2, wherein the human liver organoid is a proliferative liver organoid or a metabolically activated liver organoid, the proliferative liver organoid is obtained by culturing human primary hepatocytes in a growth medium, the metabolically activated liver organoid is obtained by culturing the proliferative liver organoid in a differentiation medium, the growth medium is a medium containing a Wnt agonist, a bone morphogenetic protein inhibitor, a growth factor, a TGF-β inhibitor, forskolin, and a STAT3 activating molecule, and the differentiation medium is a medium substantially free of a STAT3 activating molecule and containing a growth factor, a TGF-β inhibitor, forskolin, growth hormone, prolactin, a glucocorticoid, and a γ-secretase inhibitor.

5. The manufacturing method according to claim 4, wherein the STAT3 activating molecule is IL-6 or oncostatin M.

6. A human liver organoid produced by the manufacturing method described in claim 1 or 2.

7. A human liver gene disease model produced by the manufacturing method described in claim 3.

8. The human liver gene disease model according to claim 7, wherein the G6PC gene or the OTC gene is disrupted.

9. A method for producing primary hepatocytes with excellent organoid-forming ability, comprising the step of subjecting human primary hepatocytes dissociated into single cells to flow cytometry and recovering cells whose lateral scattering values ​​are in the lower 30% or less of the population of human primary hepatocytes, wherein the recovered cells are primary hepatocytes with excellent organoid-forming ability.

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