Method for inducing mesendodermal cells, method for producing mesendodermal cells, and agent for inducing mesendodermal cells

Abstract

This method for inducing mesendodermal cells comprises a step for increasing the amount of intercellular zinc finger protein 384 (ZNF384) existing in pluripotent stem cells.

Description

Method for inducing mesendodermal cells, method for producing mesendodermal cells, and agent for inducing mesendodermal cells The present invention relates to a method for inducing mesendodermal cells, a method for producing mesendodermal cells, and an inducer for mesendodermal cells. The present invention also relates to a method for inducing mesodermal cells, a method for inducing endodermal cells, a method for producing mesodermal cells, and a method for producing endodermal cells. This invention claims priority based on Japanese Patent Application No. 2024-044260 filed on March 19, 2024, and Japanese Patent Application No. 2024-186739 filed on October 23, 2024, the contents of which are incorporated herein by reference. The development of regenerative medicine and cell therapy using pluripotent stem cells has the potential to address unmet medical needs. In regenerative medicine, the development of retinal cells and cardiomyocytes derived from induced pluripotent stem cells (iPS cells) is progressing, and the possibility of clinical application has been demonstrated. Meanwhile, in cell therapy, the development of cancer treatments using iPS cell-derived T cells and NK cells is progressing. To date, research has been conducted to generate various types of cells from pluripotent stem cells, and a wide variety of cell differentiation methods have been established. Methods for inducing differentiation of pluripotent stem cells into mesendodermal cells, mesodermal cells, endodermal cells, and the like have also been developed. However, these differentiation induction methods often involve the use of a combination of feeder cells, recombinant proteins, compounds, and the like, which results in complicated culture conditions and medium compositions. Forced expression systems for transcription factors and the like have also been developed as differentiation induction methods. However, these differentiation induction methods require the combination of multiple genes, and the complexity of the conditions makes it difficult to efficiently obtain the target cells. An object of the present invention is to provide a method for inducing mesendodermal cells, which allows for the production of mesendodermal cells by a simple method, and the like. One aspect of the present invention is a method for inducing mesendodermal cells, which comprises the step of increasing the abundance of zinc finger protein 384 (ZNF384) in pluripotent stem cells. The present invention can provide a method for inducing mesendodermal cells, which allows for the production of mesendodermal cells by a simple method, and the like. 1 shows a schematic diagram of proteins expressed from the transgenes used in the examples. mCherry was used as a negative control. 1 shows the results of Western blotting in Experimental Example 1. 1 shows the protocols for gene transfer and subsequent culture performed in Experimental Examples 1 to 6. 1 shows the results of observing ZNF384 transfected cells and mCherry transfected cells (Control) using a phase-contrast microscope in Experimental Example 2. 1 shows the results of principal component analysis of NGS analysis for each sample in Experimental Example 3. 1 is a diagram explaining marker genes for mesendoderm, mesoderm, and endoderm, and terminally differentiated tissues. 1 shows the results of expression analysis of representative mesendoderm markers GSC, PDGFRA, and EOMES in Experimental Example 4. 1 shows the results of expression analysis of representative mesoderm / endoderm common markers GATA4 and GATA6 in Experimental Example 4. 1 shows the results of expression analysis of representative mesoderm markers HAND1, TBXT, and GATA2 in Experimental Example 4. 1 shows the results of expression analysis of representative endoderm markers FOXA2, CER1, SOX17, CXCR4, and KDR in Experimental Example 4. 1 shows the results of expression analysis of representative ectoderm marker PAX6 in Experimental Example 4. 1 shows the results of expression analysis of representative undifferentiation markers POU5F1 (OCT4), SOX2, and NANOG in Experimental Example 4. 1 shows the results of expression analysis of representative differentiation-inducing factors used to induce mesoderm or endoderm differentiation in Experimental Example 5. 1 shows the results of expression analysis of transgenes (left panel) and CD34 (right panel) in Experimental Example 6. "1st" and "2nd" indicate the results of two independent experiments. "NT" indicates cells that were not transfected with a gene. 10 The expression of transgenes (ZNF384, mCherry (Control)) was performed using primers specific to the coding sequence of the DYKDDDDK tag. 1 shows images captured with a phase-contrast microscope of iPS cell-derived dendritic cells (iDCs) induced from CD34-positive cells in Experimental Example 7. 1 shows the results of flow cytometry analysis of the expression of dendritic cell marker genes in iPS cells, primary dendritic cells (primary DCs), and iDCs in Experimental Example 7.In Experimental Example 7, the expression of dendritic cell marker genes HLA-DR and CD11c, and CD141, a marker gene for cDC1, a dendritic cell subtype, was analyzed by qRT-PCR for primary DCs and iDCs. In Experimental Example 7, iDCs were treated with lipopolysaccharide (LPS) or a combination of lipopolysaccharide and INF-γ (LPS + INF-γ), and the expression of cytokines IL-6, IL-12 p70, and TNF-α was analyzed by ELISA. In Experimental Example 7, E. coli that fluoresces under acidic conditions was added to iDCs, and the phagocytic activity was observed under a fluorescent microscope. The protocol for gene transfer and subsequent culture performed in Experimental Example 8 is shown. In Experimental Example 8, the results of expression analysis of representative hepatocyte markers ALB, HNF4A, and KRT17 are shown. [Method for inducing mesendodermal cells] A first aspect of the present invention is a method for inducing mesendodermal cells. The method for inducing mesendodermal cells comprises a step of increasing the amount of zinc finger protein 384 (ZNF384) present in pluripotent stem cells. In the present disclosure, "mesendodermal cells" refers to mesendodermal cells that have the property of differentiating into both mesoderm and endoderm during the early differentiation of pluripotent stem cells, and cells induced to differentiate from mesendodermal cells, such as mesodermal cells and endodermal cells. <Step of increasing the amount of ZNF384 present> An example of a method for increasing the amount of ZNF384 present in pluripotent stem cells is a method of introducing a nucleic acid containing a nucleotide sequence encoding ZNF384 (hereinafter referred to as a "ZNF384-encoding nucleic acid") into pluripotent stem cells. The ZNF384-encoding nucleic acid is a nucleic acid containing a coding sequence for ZNF384. (pluripotent stem cells) "Pluripotent stem cells" refer to cells that have pluripotency and the ability to self-replicate. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and iPS cells. The organism from which pluripotent stem cells are derived is not particularly limited. The organism from which pluripotent stem cells are derived is preferably a eukaryote, more preferably a mammal. The organism from which pluripotent stem cells are derived is preferably an organism having the ZNF384 gene or a homolog thereof. The pluripotent stem cells are preferably human pluripotent stem cells, more preferably human iPS cells. Pluripotent stem cells (hereinafter also referred to as "target cells") to be introduced with a nucleic acid encoding ZNF384 may be any of primary cultured cells, subcultured cells, and frozen cells. The target cells may be cultured in a maintenance medium for pluripotent stem cells. Any known maintenance medium for pluripotent stem cells can be used. Examples of maintenance media include media prepared by appropriately adding various components to a basal medium. The basal medium contains components necessary for cell survival (inorganic salts, organic substances, amino acids, vitamins, etc.). Examples of basal media include, but are not limited to, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's Modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The maintenance medium may be a basal medium supplemented with serum (e.g., fetal bovine serum (FBS)) or a serum substitute. Examples of serum substitutes include one or more selected from albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The maintenance medium may be a basal medium supplemented with one or more selected from lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, inhibitors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like. The maintenance medium may be a commercially available maintenance medium for pluripotent stem cells. Examples of commercially available maintenance media include, but are not limited to, StemFit (registered trademark) (e.g., StemFit (registered trademark) AK02N, etc.) (Ajinomoto Co., Inc.), Essential 8 Medium (Life Technologies Japan, Inc.) (Thermo Fisher Scientific), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan, Inc.), mTeSR1 (Veritas), and TeSR2 (Veritas). The maintenance medium may contain a kinase inhibitor such as a ROCK (Rho-associated protein kinase) inhibitor. The culture temperature for pluripotent stem cells is not particularly limited as long as it is a temperature at which the pluripotent stem cells can survive. Examples of the culture temperature include 30 to 40°C, preferably 35 to 39°C, more preferably 36 to 38°C, and even more preferably 36.5 to 37.5°C. In some embodiments, the culture temperature is 37°C. Pluripotent stem cells are cultured under CO ２ Using an incubator or the like, 1% to 10% CO ２ In an atmosphere preferably 5% CO ２ It is preferable to carry out the treatment in a high-concentration atmosphere. Pluripotent stem cells may be cultured by adhesion culture, which can be performed using a cell culture dish coated with a cell adhesion protein (laminin, vitronectin, fibronectin, matrigel, etc.). (ZNF384) Unless otherwise specified, the term "ZNF384" refers to zinc finger protein 384 (ZNF384) protein. The term "ZNF384 gene" refers to a nucleic acid (e.g., DNA) comprising an open reading frame that can be translated into functional ZNF384. The ZNF384 gene may comprise only exons, or may comprise exons and introns. The ZNF384 gene may comprise one or more selected from introns, 5'UTR, and 3'UTR in addition to exons. The term "ZNF384 mRNA" refers to an mRNA that can be translated into functional ZNF384. ZNF384 is a C2H2-type zinc finger protein and functions as a transcription factor. Examples of the nucleotide sequence of the cDNA of human ZNF384 (Gene ID: 171017) include, but are not limited to, NCBI accession numbers NM_001039920.3, NM_001135734.3, NM_001385739.1 to NM_001385801.1, and NM_133476.5. Human ZNF384 has multiple isoforms, and any of these may be used. In some embodiments, the nucleotide sequence of the cDNA of human ZNF384 is NCBI accession number NM_001385788.1 (SEQ ID NO: 1). Examples of the amino acid sequence of human ZNF384 include, but are not limited to, NCBI accession numbers NP_001035009.1, NP_001129206.1, NP_001372668.1 to NP_001372730.1, and NP_597733.2. In some embodiments, the amino acid sequence of human ZNF384 is NCBI accession number NP_001372717.1 (SEQ ID NO: 2). Sequence information for ZNF384 in other organisms can also be obtained from publicly known databases such as GenBank. <ZNF384-encoding nucleic acid> The ZNF384-encoding nucleic acid may be DNA or RNA. Examples of the ZNF384-encoding nucleic acid that is DNA include DNA containing the ZNF384 gene. Examples of the ZNF384-encoding nucleic acid that is RNA include ZNF384 mRNA. The ZNF384-encoding nucleic acid may be a wild-type or a modified wild-type, as long as it encodes a functional ZNF384. Examples of ZNF384-encoding nucleic acids include the following. (a) Wild-type ZNF384 gene or wild-type ZNF384 mRNA. (b) a nucleic acid comprising a nucleotide sequence encoding wild-type ZNF384. (c) A nucleic acid comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of wild-type ZNF384, and having a function equivalent to that of wild-type ZNF384. (d) A nucleic acid comprising a nucleotide sequence encoding a protein comprising an amino acid sequence having 70% or more sequence identity with the amino acid sequence of wild-type ZNF384 and having a function equivalent to that of wild-type ZNF384. (e) A nucleic acid comprising a nucleotide sequence in which one or more nucleotides are mutated in the nucleotide sequence of the wild-type ZNF384 gene or wild-type ZNF384 mRNA, and which encodes a protein having a function equivalent to that of wild-type ZNF384. (f) A nucleic acid comprising a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of a wild-type ZNF384 gene or wild-type ZNF384 mRNA and encoding a protein having a function equivalent to that of wild-type ZNF384. (g) A nucleic acid that hybridizes with the wild-type ZNF384 gene or wild-type ZNF384 mRNA under stringent conditions and that contains a nucleotide sequence encoding a protein having a function equivalent to that of the wild-type ZNF384. In the above (c) and (e), the "mutation" may be any of deletion, substitution, addition, and insertion, or a combination thereof. In the above (c), the "plurality" is not particularly limited as long as the resulting protein has a function equivalent to that of ZNF384. The "plurality" in (c) may be, for example, 2 to 50, and may be 2 to 30, 2 to 20, 2 to 10, 2 to 5, 2 to 3, or 2. In the above (e), the term "plurality" is not particularly limited as long as the resulting nucleic acid encodes a protein having a function equivalent to that of ZNF384. The term "plurality" in (e) may be, for example, 2 to 100, and may be 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 2 to 3, or 2. In the above (d) and (f), the sequence identity is preferably 85% or more, and may be 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The sequence identity between amino acid sequences or nucleotide sequences is determined by aligning two amino acid sequences or nucleotide sequences, with gaps inserted and deleted to maximize the number of corresponding amino acids or nucleotides, and calculating the percentage of matching amino acids or nucleotides relative to the entire amino acid sequence or the entire nucleotide sequence excluding gaps in the resulting alignment. The sequence identity between amino acid sequences or nucleotide sequences can be determined using various homology search software known in the art. For example, the sequence identity value of amino acid sequences can be calculated based on the alignment obtained using the known homology search software BLASTP. For example, the sequence identity value of nucleotide sequences can be calculated based on the alignment obtained using the known homology search software BLASTN. In (g) above, "stringent conditions" include, for example, those described in Molecular Cloning—A Laboratory Manual Third Edition (Sambrook et al., Cold Spring Harbor Laboratory Press). Stringent conditions include, for example, hybridization conditions in a hybridization buffer consisting of 6×SSC (20×SSC composition: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 5×Denhardt's solution (100×Denhardt's solution composition: 2% by mass bovine serum albumin, 2% by mass Ficoll, 2% by mass polyvinylpyrrolidone), 0.5% by mass SDS, 0.1 mg / mL salmon sperm DNA, and 50% by volume formamide, at 42 to 70° C. for several hours to overnight. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by mass of SDS, more preferably a 0.1×SSC solution containing 0.1% by mass of SDS. In the above (b) to (d), the degenerate codons may be those that are frequently used in the biological species from which the pluripotent stem cells into which the ZNF384-encoding nucleic acid is to be introduced are derived. For example, in the case of human pluripotent stem cells, codons frequently used in human cells may be used, for example, the ZNF384-encoding nucleic acid may be codon-optimized for human codons. In the above (b) to (g), a "protein having a function equivalent to that of wild-type ZNF384" (hereinafter also referred to as "mutant ZNF384") refers to a protein having one or more of the functions expressed in cells by wild-type ZNF384. Examples of the functions of wild-type ZNF384 maintained in mutant ZNF384 include the function of inducing differentiation of pluripotent stem cells into mesendodermal cells. It is preferable that mutant ZNF384 has the transcriptional control function possessed by wild-type ZNF384. Wild-type ZNF384, the wild-type ZNF384 gene, and wild-type ZNF384 mRNA refer to naturally occurring ZNF384, the ZNF384 gene, and ZNF384 mRNA, respectively. Examples of the amino acid sequence and nucleotide sequence of human wild-type ZNF384 and the ZNF384 gene include those shown by the above-mentioned NCBI accession numbers. Unless otherwise specified, the terms "ZNF384," "ZNF384 gene," or "ZNF384 mRNA" encompass both wild-type and mutant forms. The ZNF384-encoding nucleic acid is preferably a nucleic acid that encodes endogenous ZNF384 in the target cells, which can reduce the risk of an unexpected reaction in the target cells. <Method for introducing ZNF384 gene> Methods for introducing the ZNF384 gene into pluripotent stem cells include methods for introducing an expression vector for the ZNF384 gene into pluripotent stem cells. An expression vector for the ZNF384 gene refers to a vector equipped with a system that enables the ZNF384 gene to be expressed in cells into which the vector has been introduced. An expression vector for the ZNF384 gene includes, for example, a promoter that can function in target cells, and a ZNF384 gene (ZNF384 coding sequence) operably linked to the promoter. "The promoter is operable" means that the gene operably linked to the promoter can be expressed in target cells. "Operatively linked to the promoter" means that the gene is linked to the promoter so that the gene is expressed under the control of the promoter in the target cells. The promoter is not particularly limited as long as it has the function of expressing the ZNF384 gene in the target cell. Examples of promoters include Pol II promoters. Examples of Pol II promoters include, but are not limited to, the EF1α promoter, CMV promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, and CBh promoter. The promoter may be a constitutive promoter or an inducible promoter. An inducible promoter is a promoter that induces gene expression under specific conditions. The expression vector for the ZNF384 gene may contain other components in addition to the ZNF384 gene and promoter. These components include a terminator, an enhancer, a poly(A) addition signal, a marker gene, a replication origin, and a gene encoding a protein that binds to the replication origin and controls replication. The terminator is linked to the 3' end of the ZNF384 gene. Terminators commonly used in the biological species from which the target cells are derived can be used. The term "marker gene" refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Examples of drug resistance genes include puromycin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Examples of fluorescent protein genes include the green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, and red fluorescent protein (RFP) gene. Examples of luminescent enzyme genes include the luciferase gene. Examples of chromogenic enzyme genes include the β-galactosidase gene, β-glucuronidase gene, and alkaline phosphatase gene. The type of expression vector is not particularly limited, and any known expression vector can be used. Examples of expression vectors include plasmid vectors and viral vectors. The plasmid vector is not particularly limited as long as it can be expressed in the target cells. For example, when the target cells are animal cells, a plasmid vector commonly used for expression in animal cells can be used. Examples of plasmid vectors for expression in animal cells include, but are not limited to, pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Examples of viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, herpes viral vectors, vaccinia viral vectors, pox viral vectors, polio viral vectors, Silvis viral vectors, rhabdoviral vectors, paramyxoviral vectors, and orthomyxoviral vectors. The expression vector for the ZNF384 gene can be introduced into pluripotent stem cells by known methods. Examples of methods for introducing an expression vector into pluripotent stem cells include lipofection, microinjection, DEAE-dextran, gene gun, electroporation, and calcium phosphate. When the expression vector is a viral vector, methods of infecting cells with the viral vector (e.g., polybrene method) can be used. <Method for introducing mRNA> In addition to the coding sequence of ZNF384, the mRNA may contain other components, such as a 5' Cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and a poly(A) tail. The ZNF384 mRNA may contain a 5' Cap at the 5' end. Examples of 5' Caps include Cap0, Cap1, and Cap2 structures. The cap structure is typically a 7-methylguanine ribonucleotide, attached via a 5'-triphosphate to the 5' position of the first nucleotide in the 5'-3' direction of the mRNA, i.e., the first cap-proximal nucleotide. In the Cap0 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain 2'-hydroxyl. In the Cap1 structure, the riboses of the first cap-proximal nucleotide of the mRNA contain 2'-methoxy, and the riboses of the second nucleotide contain 2'-hydroxyl. In the Cap2 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain 2'-methoxy. The cap structure can be incorporated into the 5' end of mRNA during transcription by known methods. For example, a cap structure can be incorporated into mRNA by co-transcription using a commercially available capping kit, etc. The cap structure may also be added to mRNA after transcription or to chemically synthesized RNA using a capping enzyme. The mRNA of ZNF384 may include either or both of a 5'UTR and a 3'UTR. The 5'UTR and 3'UTR may be those of the wild-type ZNF384 mRNA or those of a different mRNA. The mRNA of ZNF384 may contain a Kozak sequence. The Kozak sequence may affect translation initiation and the total amount of protein translated from the mRNA. The Kozak sequence includes a methionine codon that can function as an initiation codon. The minimum Kozak sequence is NNNRUGN (N is any nucleotide residue, and R is a purine residue (A or G)). In the formula, the first N is preferably A or G, and the second N is preferably G. In one embodiment, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. The ZNF384 mRNA may include a poly(A) tail at the 3' end. The poly(A) tail may include a sequence of at least 8 consecutive adenine nucleotides, but may also include one or more non-adenine nucleotide residues (e.g., G, C, U). The length of the poly(A) tail may be, for example, 10 to 500 nucleotides, 30 to 300 nucleotides, or 60 to 250 nucleotides. ZNF384 mRNA can be introduced into pluripotent stem cells by known methods, such as a method using a commercially available RNA transfection reagent such as Lipofectamine (registered trademark) MessengerMAX (Life Technologies). <<Culture after introduction of ZNF384-encoding nucleic acid>> After introducing a nucleic acid encoding ZNF384 into pluripotent stem cells, the introduced cells may be cultured. By culturing, ZNF384 is expressed from the ZNF384-encoding nucleic acid introduced into the pluripotent stem cells, thereby increasing the amount of ZNF384 present in the pluripotent stem cells. After introduction of the ZNF384-encoding nucleic acid, the cells can be cultured using a maintenance medium for pluripotent stem cells. Examples of maintenance media for pluripotent stem cells include those similar to those described above. The medium may contain a kinase inhibitor such as a ROCK inhibitor. When the ZNF384-encoding nucleic acid contains an antibiotic resistance gene as a marker gene, the medium may contain the antibiotic. Culture temperature and CO ２ The concentration may be the same as that described above. The culture may be performed by adhesion culture using a cell culture dish coated with a cell adhesion protein (laminin, vitronectin, fibronectin, matrigel, etc.). The culture period may be, for example, 1 to 10 days, preferably 2 to 8 days, and more preferably 3 to 7 days. The induction of mesendodermal cells can be confirmed by the expression of mesendodermal markers. Examples of mesendodermal markers include eomesodermin (EOMES), goosecoid homeobox (GSC), and platelet-derived growth factor receptor alpha (PFGFRA). The expression of mesendodermal markers can be confirmed by known methods such as qRT-PCR, northern blotting, western blotting, and FACS analysis. According to the method for inducing mesendodermal cells of this embodiment, mesendodermal cells can be induced in pluripotent stem cells simply by increasing the abundance of a single factor, ZNF384. When introducing a ZNF384-encoding nucleic acid, only the introduction of the ZNF384-encoding nucleic acid is sufficient; there is no need to introduce any other genes. Furthermore, after the introduction of the ZNF384-encoding nucleic acid, mesendodermal cells can be induced by culturing the cells using a known maintenance medium for pluripotent stem cells. Co-culturing with feeder cells is not required in the culture after the introduction of the ZNF384-encoding nucleic acid. Furthermore, there is no need to add recombinant proteins such as growth factors in the culture after the introduction of the ZNF384-encoding nucleic acid. Furthermore, there is no need to use recombinant proteins such as growth factors in the culture after the introduction of the ZNF384-encoding nucleic acid. [Method for inducing mesodermal cells] A second aspect of the present invention is a method for inducing mesodermal cells. The method for inducing mesodermal cells comprises the steps of inducing mesendodermal cells by the method for inducing mesendodermal cells according to the first aspect, and inducing the mesendodermal cells into mesodermal cells. In the present disclosure, "mesodermal cells" refers to mesodermal cells and cells differentiated from mesodermal cells. <Step of inducing mesodermal cells> A known method for inducing mesodermal cells can be selected depending on the type of terminally differentiated cells to be ultimately obtained. Examples of mesodermal cells include organ and tissue cells described in the "Terminal Differentiation" section of "Mesoderm" in Figure 6. Specific examples of mesodermal cells include muscle cells (myoblasts, muscle satellite cells, etc.), skeletal cells (osteoblasts, osteocytes, chondrocytes, etc.), circulatory system cells (cardiomyocytes, hematopoietic stem cells, erythrocytes, platelets, dendritic cells, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, natural killer cells, etc.), urogenital system cells (renal tubular cells, mesangial cells, juxtaglomerular cells, testes, ovaries, etc.), and connective tissue. Mesodermal cells may be hematopoietic cells. In the present disclosure, hematopoietic cells refer to cells derived from hematopoietic stem cells that are differentiated from lymphoid progenitor cells or myeloid progenitor cells. Hematopoietic cells may be CD34-positive cells or cells differentiated from CD34-positive cells. Examples of hematopoietic cells include hematopoietic stem cells, erythrocytes, platelets, dendritic cells, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, and natural killer cells. The induction of mesodermal cells can be confirmed by the expression of mesodermal markers. Examples of mesodermal markers include HAND1 (heart and neural crest derivatives expressed 1), TBXT (T-box transcription factor T), GATA2 (GATA binding protein 2), GATA4 (GATA binding protein 4), and GATA6 (GATA binding protein 6). The expression of mesendodermal markers can be confirmed by known methods such as qRT-PCR, Northern blotting, Western blotting, and FACS analysis. The induction of hematopoietic cells can be confirmed by the expression of hematopoietic cell markers. An example of a hematopoietic stem cell marker is CD34. Examples of dendritic cell markers include HLA-DR and CD11c. Examples of helper T cell markers include CD4. Examples of killer T cell markers include CD8. Examples of B lymphocyte markers include CD19. Examples of natural killer cell markers include CD56. The expression of these markers can also be confirmed by known methods such as qRT-PCR, northern blotting, western blotting, and FACS analysis. [Method for inducing endodermal cells] A third aspect of the present invention is a method for inducing endodermal cells. The method for inducing endodermal cells comprises the steps of inducing mesendodermal cells by the method for inducing mesendodermal cells according to the first aspect, and inducing the mesendodermal cells into endodermal cells. In the present disclosure, "endodermal cells" refers to endoderm cells and cells differentiated from endodermal cells. <Step of inducing endodermal cells> A known method for inducing endodermal cells can be selected depending on the type of terminally differentiated cells to be ultimately obtained. Examples of such endodermal cells include cells of organs and tissues listed under "Terminal Differentiation" in "Endoderm" in Figure 6. Specific examples of endodermal cells include digestive system cells (hepatocytes, bile duct cells, pancreatic endocrine cells, acinar cells, duct cells, absorptive cells, goblet cells, Paneth cells, enteroendocrine cells, etc.), lung tissue cells, and thyroid tissue cells. The endodermal cells may be digestive system cells. In the present disclosure, "digestive system cells" refers to cells of organs that constitute the digestive tract and digestive glands. The digestive system cells may be hepatocytes or hepatic progenitor cells. The induction of endodermal cells can be confirmed by the expression of endodermal markers. Examples of mesodermal markers include SOX17 (SRY-box transcription factor 17), FOXA2 (forkhead box A2), CXCR4 (C-X-C motif chemokine receptor 4), CER1 (cerberus 1, DAN family BMP antagonist), GATA4 (GATA binding protein 4), and GATA6 (GATA binding protein 6). The expression of endodermal markers can be confirmed by known methods such as qRT-PCR, northern blotting, western blotting, and FACS analysis. [Methods for producing mesendodermal cells, methods for producing mesodermal cells, methods for producing endodermal cells] A fourth aspect of the present invention is a method for producing mesendodermal cells. The method for producing mesendodermal cells comprises the step of inducing mesendodermal cells by the method for inducing mesendodermal cells according to the first aspect. The obtained mesendodermal cells can be used to produce mesodermal cells or endodermal cells. A fifth aspect of the present invention is a method for producing mesodermal cells. The method for producing mesodermal cells comprises the step of inducing mesodermal cells by the method for inducing mesodermal cells according to the second aspect. Mesodermal cells can be produced by obtaining mesendodermal cells by the method for inducing mesendodermal cells according to the first aspect, and then inducing mesodermal cells from the mesendodermal cells by a known method. A sixth aspect of the present invention is a method for producing endodermal cells. The method for producing endodermal cells comprises the step of inducing endodermal cells by the method for inducing endodermal cells according to the third aspect. The endodermal cells can be produced by obtaining mesendodermal cells by the method for inducing mesendodermal cells according to the first aspect, and then inducing endodermal cells from the mesendodermal cells by a known method. [Mesendodermal cell inducer] A seventh aspect of the present invention is an agent for inducing mesendodermal cells, which comprises a nucleic acid encoding ZNF384. Examples of ZNF384-encoding nucleic acids include those described above. The ZNF384-encoding nucleic acid may be DNA containing the ZNF384 gene or ZNF384 mRNA. The DNA containing the ZNF384 gene may be an expression vector for the ZNF384 gene. The mesendodermal cell inducer may contain other components in addition to the ZNF384-encoding nucleic acid, including, but not limited to, a buffer, a transfection agent, a preservative, a stabilizer, a pH adjuster, etc. The mesendodermal cell inducer may be liquid or solid. When mesendodermal cell induction is in liquid form, for example, a solution in which ZNF384-encoding nucleic acid (and optional components) is dissolved in a buffer solution or the like can be used. Examples of buffer solutions include phosphate buffer, phosphate-buffered saline (PBS), Tris buffer, citrate buffer, etc. When mesendodermal cell induction is in solid form, the solution may be obtained by drying the above-mentioned solution of ZNF384-encoding nucleic acid (and optional components). Examples of drying methods include freeze-drying. The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. [Experimental Example 1] (Introduction of ZNF384 gene) <<Transduced gene>> The ZNF384 gene introduced into iPS cells was human ZNF384 cDNA (NCBI accession number NM_001385788.1; nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2). The EF1α promoter was used. The ZNF384 gene used for the transfer contained a coding sequence for a tag (DYKDDDDK) at the C-terminus of ZNF384 (see Figure 1), which enabled the expression of the ZNF gene transferred into iPS cells to be confirmed at the protein level using an anti-DYKDDDDK tag antibody (Merck). mCherry was used as a negative control (see Figure 1). The mCherry gene was modified so that a tag (DYKDDDDK) was added to the C-terminus of mCherry, and was introduced into iPS cells in the same manner as the ZNF384 gene. <Introduction of ZNF384 gene> The introduction of the ZNF384 gene into iPS cells and the subsequent culture were carried out according to the protocol shown in Figure 3. The cells were cultured at 37°C, 5% CO ２ It was carried out under the following conditions. [Day 0] Subcultured human iPS cells (1383D2) were detached using a mixture of equal volumes of TrypLE Select Enzyme (Thermo Fisher Scientific Inc.) and 0.5 mM EDTA, and the number of cells was counted. The number of cells was determined based on the type of culture dish to be seeded (for example, 2 × 10 for a 6-well plate). ４Cells / well). A predetermined number of cells were placed in StemFit AK02N (Ajinomoto, AJ100, hereinafter also referred to as "StemFit"), which was a mixture of ROCK inhibitor (Fujifilm Wako Pure Chemical Industries, Ltd.) and laminin (Nippi Corporation), and mixed thoroughly with lentivirus carrying the ZNF384 gene (pPACK Lentivector Packaging Kit, System Biosciences, LLC), and then seeded onto a culture dish. Lentivirus preparation was performed according to the manufacturer's instructions. Human iPS cells were subcultured using StemFit. [Day 1] 24 hours after seeding, the culture medium was completely replaced. [Day 3] Half of the culture medium was discarded, and half of a medium for blood cells (X-VIVO15, Lonza) was added. [Day 4] The culture medium was completely discarded, and a medium for blood cells containing 1 μg / mL of puromycin (Fujifilm Wako Pure Chemical Industries, Ltd.) was added. [Day 7] Half of the culture medium was discarded, and half of a medium for blood cells containing 1 μg / mL of puromycin was added. [Day 9] Half of the culture medium was discarded, and half of a medium for blood cells containing 1 μg / mL of puromycin was added. Western blotting Ten days after the introduction of the ZNF384 gene, the cells were collected and subjected to Western blotting using an anti-DYKDDDDK tag antibody. The results are shown in Figure 2. In the ZNF384 gene-transfected cells (ZNF384-tag), a band corresponding to the protein size of ZNF384 was detected. On the other hand, in the mCherry gene-transfected cells (mCherry-tag), no band corresponding to the protein size of ZNF384 was detected. These results confirmed that ZNF384 was expressed from the introduced ZNF384 gene in the ZNF384 gene-transfected cells. [Experimental Example 2] (Cell morphology observation) After introduction of the ZNF384 gene, ZNF384 gene-transfected cells were cultured according to the protocol shown in Figure 3. Cells were observed under a phase-contrast microscope (CKX53, Olympus Corporation) on days 7 and 10 after ZNF384 gene introduction. As a negative control, mCherry gene-transfected cells were cultured in the same manner and observed. The results are shown in Figure 4. Conventional iPS cells form colonies in a monolayer, as shown in the phase-contrast microscope image of mCherry transfected cells (control). On the other hand, ZNF384 transfected cells showed a significant change in cell morphology compared to mCherry transfected cells. On Day 7, ZNF384 transfected cells were not monolayered, but rather cells were stacked on top of each other, resulting in an increase in floating cells. On Day 10, ZNF384 transfected cells showed an increase in floating cells, and their morphology changed to that of blood cells. This phenomenon is a morphological change often observed when pluripotent stem cells differentiate into blood cells. On Day 10, ZNF384 transfected cells showed cells attached to the bottom that resembled cobblestones. [Experimental Example 3] (Principal Component Analysis) After introduction of the ZNF384 gene, ZNF384 gene-transfected cells were cultured according to the protocol shown in Figure 3. Cells were harvested 10 days after introduction of the ZNF384 gene (Day 10). RNA was extracted from the cells and subjected to next-generation sequencing (NGS) analysis using a next-generation sequencer (NextSeq 500, Illumina). Principal component analysis was performed using the data obtained by NGS analysis. The results are shown in Figure 5. The ZNF384 gene-transfected cells (ZNF384) exhibited a gene expression profile different from that of non-treated cells (Non-Treatment) and mCherry gene-transfected cells (Control). From these results, it was inferred that the ZNF384 gene-transfected cells had escaped from an undifferentiated state and differentiated into some type of cell. [Experimental Example 4] (Marker gene expression analysis) During embryonic development, cells branch into three germ layers (ectoderm, endoderm, and mesoderm). Among these, there is a developmental stage called mesendoderm, which is a precursor to mesoderm and endoderm. In differentiation induction from pluripotent stem cells, there is also a mesendoderm stage, which is a precursor to mesoderm and endoderm (see Figure 6). Figure 6 shows a list of marker genes expressed at each stage and terminally differentiated tissues. In Experimental Example 4, expression analysis of marker genes at each stage was performed in ZNF384-introduced cells. After introduction of the ZNF384 gene, the ZNF384 gene-transduced cells were cultured according to the protocol shown in Figure 3. The cells were harvested 10 days (Day 10) after introduction of the ZNF384 gene. RNA was extracted from the cells, and expression analysis of mesendoderm marker genes, mesoderm / endoderm common marker genes, mesoderm marker genes, endoderm marker genes, ectoderm marker genes, and undifferentiation marker genes was performed by qRT-PCR (quantitative reverse transcription-polymerase chain reaction). <Expression analysis of mesendoderm marker genes> As representative marker genes of mesendoderm, expression analysis of the GSC, PDGFRA, and EOMES genes was performed. The results are shown in Figure 7. In ZNF384 transfected cells (ZNF384), expression of all mesendoderm marker genes (GSC, PDGFRA, EOMES) was increased compared to mCherry transfected cells (Control) and cells that had not been transfected with the gene (Non-Treatment). From these results, it was inferred that the ZNF384 transfected cells had differentiated to the mesendoderm stage. <Expression analysis of mesoderm / endoderm common marker genes> Expression analysis of the GATA4 and GATA6 genes was carried out as representative marker genes common to both mesoderm and endoderm. The results are shown in Figure 8. In ZNF384 transfected cells (ZNF384), expression of all mesoderm / endoderm common marker genes (GATA4, GATA6) was increased compared to mCherry transfected cells (Control) and cells that had not been transfected with a gene (Non-Treatment). From these results, it was inferred that the ZNF384 transfected cells had differentiated into mesoderm / endoderm. <Expression analysis of mesodermal marker genes> As representative marker genes of the mesoderm, expression analysis of the HAND1, TBXT, and GATA2 genes was carried out. The results are shown in Figure 9. In ZNF384 transfected cells (ZNF384), expression of all mesoderm marker genes (HAND1, TBXT, GATA2) was increased compared to mCherry transfected cells (Control) and cells not subjected to gene transfection (Non-Treatment). From these results, it was inferred that the ZNF384 transfected cells had differentiated into mesoderm. <Expression analysis of endoderm marker genes> As representative marker genes of endoderm, expression analysis was performed on the genes FOXA2, CER1, SOX17, CXCR4, and KDR. The results are shown in Figure 10. In ZNF384 transfected cells (ZNF384), expression of all endoderm marker genes (FOXA2, CER1, SOX17, CXCR4, KDR) was increased compared to mCherry transfected cells (Control) and cells not subjected to gene transfection (Non-Treatment). From these results, it was inferred that the ZNF384 transfected cells had differentiated into endoderm. <Expression analysis of ectodermal marker genes> As a representative marker gene of the ectoderm, expression analysis of the PAX6 gene was carried out. The results are shown in Figure 11. The expression level of the ectoderm marker gene (PAX6) in the ZNF384 transfected cells (ZNF384) was unchanged compared to the mCherry transfected cells (Control) and cells that had not been transfected with the gene (Non-Treatment). From these results, it was inferred that the ZNF384 transfected cells had not differentiated into ectoderm. <Expression analysis of undifferentiated marker genes> As representative marker genes for undifferentiation, expression analysis of the POU5F1 (OCT4), SOX2, and NANOG genes was performed. The results are shown in Figure 12. In ZNF384 transfected cells (ZNF384), expression of all undifferentiated marker genes (POU5F1, SOX2, NANOG) was reduced compared to mCherry transfected cells (Control) and cells not subjected to gene transfection (Non-Treatment). From this result, it was inferred that the ZNF384 transfected cells had escaped from the undifferentiated state. [Experimental Example 5] (Gene expression analysis of factors inducing differentiation into mesoderm and endoderm) Protein addition is a widely known method for inducing differentiation of pluripotent stem cells into mesoderm or endoderm. Proteins (hereinafter referred to as "differentiation-inducing factors") used in this process include BMP4, VEGFA, bFGF (FGF2), KITLG (SCF), and Activin A. Protein addition methods vary widely, including combinations of two or more of the above proteins, or combinations of the above proteins with inhibitors, etc. In Experimental Example 5, gene expression analysis of differentiation-inducing factors used to induce mesoderm or endoderm differentiation was performed. After introduction of the ZNF384 gene, the ZNF384 gene-transfected cells were cultured according to the protocol shown in Figure 3. The cells were harvested 10 days after introduction of the ZNF384 gene (Day 10). RNA was extracted from the cells, and gene expression analysis of differentiation-inducing factors (BMP4, VEGFA, FGF2, KITLG) was performed by qRT-PCR. The results are shown in Figure 13. In ZNF384 transfected cells (ZNF384), expression of the BMP4 gene and KITLG gene was increased compared to mCherry transfected cells (Control) and cells not transfected with the gene (Non-Treatment). In ZNF384 transfected cells (ZNF384), expression of the FGF2 gene was decreased compared to mCherry transfected cells (Control) and cells not transfected with the gene (Non-Treatment). It was suggested that FGF2 may not be essential for induction of differentiation into endodermal cells by ZNF384 transfection. [Experimental Example 6] (Induction of differentiation into CD34-positive cells) We investigated whether CD34-positive cells could be obtained as mesodermal cells. CD34 is a hematopoietic stem cell marker. <<Induction of differentiation into CD34-positive cells>> After the introduction of the ZNF384 gene, the ZNF384 gene-introduced cells were cultured according to the protocol shown in FIG. <Expression analysis of the introduced ZNF384 gene and CD34 gene> Cells were harvested 10 days after ZNF384 gene transfection (Day 10). RNA was extracted from the cells, and CD34 gene expression was analyzed by qRT-PCR. Furthermore, transgene expression was analyzed using primers specific to the coding sequence of the DYKDDDDK tag. The results are shown in Figure 14. Figure 14 shows the results of two experiments. The coding sequence of the DYKDDDDK tag was detected in both ZNF384 transfected cells (ZNF384) and mCherry transfected cells (Control) (left panel). On the other hand, the coding sequence of the DYKDDDDK tag was not detected in non-transfected cells (NT). These results confirmed the expression of the transgene in the transfected cells. In ZNF384 transfected cells (ZNF384), CD34 gene expression was significantly elevated compared to mCherry transfected cells (Control) and non-transfected cells (NT). This result suggests that ZNF384 transfected cells are differentiated into mesoderm. [Experimental Example 7] (Induction of differentiation from CD34-positive cells to dendritic cells) An investigation was conducted to determine whether dendritic cells could be obtained from CD34-positive cells. <Differentiation of CD34-positive cells into dendritic cells> A known method (Zhan X et al., Lancet 2004:364(9429); Sachamitr P et al., Front Immunol 2018:8:1935; Alsinet C. et al., Nat Commun. 2022: 13(1):2885) was used to induce dendritic cells from CD34-positive cells. The known method involves culturing CD34-positive cells in a medium containing recombinant GM-CSF and IL-4 proteins to differentiate them into dendritic cells. Flt3L may be added during this process. In this example, the cells were cultured in GM-CSF (50 μg / mL), IL-4 (20 μg / mL), and Flt3L (50 μg / mL). The concentrations shown are merely examples, and the range may be adjusted during culture; for example, GM-CSF (50-150 μg / mL), IL-4 (20-50 μg / mL), and Flt3L (50-150 μg / mL) may be used. The medium used for inducing dendritic cells was RPMI-1640 supplemented with 10% FBS. Cells differentiated into CD34-positive cells according to the method of Experimental Example 6 were cultured for four or more days in a medium containing the recombinant protein. The resulting cells were photographed using a phase-contrast microscope. The results are shown in Figure 15. The cells obtained by the above method resembled the morphology of human dendritic cells, and dendritic processes were observed extending from the cells. These results confirmed that iPS cell-derived dendritic cells (iDCs) were successfully produced. <Flow cytometry analysis of dendritic cell marker genes> The generated iDCs were collected and subjected to flow cytometry analysis to analyze the expression of dendritic cell marker genes. The results are shown in Figure 16. 16 shows, from the left, the expression levels of dendritic cell marker genes HLA-DR (Y axis) and CD11c (X axis) in iPS cells, primary dendritic cells (Primary DCs), and iDCs. As cell populations with high expression of both genes were observed in the prepared iDCs, similar to Primary DCs, it was possible to infer that the prepared iDCs were dendritic cells. <qRT-PCR analysis of dendritic cell marker genes> The expression of dendritic cell marker genes HLA-DR and CD11c, and CD141, a marker gene for cDC1, a subtype of dendritic cells, was analyzed by qRT-PCR in the primary DCs and the prepared iDCs. The results are shown in Figure 17. Figure 17 shows the expression levels of HLA-DR, CD11c, and CD141 in primary DCs and iDCs. HLA-DR expression in the prepared iDCs was lower than in primary DCs, but the expression of other marker genes was equal to or higher than that in primary DCs. The results of qRT-PCR analysis were similar to those of flow cytometry analysis. <ELISA analysis of dendritic cell marker genes> The generated iDCs were treated with lipopolysaccharide (LPS) or a combination of lipopolysaccharide and INF-γ (LPS+IFN-γ), and the expression of cytokines IL-6, IL-12 p70, and TNF-α was analyzed by ELISA. The results are shown in Figure 18. From left to right, each graph shows the expression levels of IL-6, IL-12 p70, and TNF-α in the prepared iDCs. In each graph, NT, LPS, and LPS + INF-γ represent no treatment (NT), lipopolysaccharide (LPS) treatment, and combined treatment with lipopolysaccharide and INF-γ (LPS + INF-γ), respectively. An increase in the expression of all types of cytokines was confirmed by LPS + INF-γ treatment. iDC phagocytosis assay E. coli, which exhibits fluorescence under acidic conditions, was added to the prepared iDCs, and the phagocytic ability of the iDCs was observed under a fluorescence microscope. The results are shown in Figure 19. It was found that the intracellular fluorescence intensity of the iDCs increased over time, confirming that the iDCs had phagocytic ability. [Experimental Example 8] (Induction of differentiation into hepatic progenitor cells and hepatocytes) We investigated whether hepatic progenitor cells and hepatocytes could be obtained as endodermal cells. <<Induction of differentiation into hepatic progenitor cells and stem cells>> The transgene used was the same as in Experimental Example 1. Introduction of the ZNF384 gene into iPS cells and subsequent culture were carried out according to the protocol shown in Figure 20. Cell culture was carried out at 37°C, 5% CO ２ It was carried out under the following conditions. [Day 0] iPS cells (1383D2) that had been subcultured were detached using a mixture of equal volumes of TrypLE Select Enzyme (Thermo Fisher Scientific Inc.) and 0.5 mM EDTA, and the number of cells was counted. The number of cells was determined based on the type of culture dish to be seeded (for example, 2 × 10 for a 6-well plate). ４ (cells / well). A predetermined number of cells were placed in StemFit containing a Rock inhibitor and laminin, mixed thoroughly with lentivirus carrying the ZNF384 gene, and then seeded onto a culture dish. Lentivirus was prepared according to the manufacturer's instructions. iPS cells were subcultured using StemFit. [Day 1] 24 hours after seeding, the culture medium was completely replaced. [Day 3] Half of the culture medium was discarded, and half of a cell maintenance medium (StemFit) was added. [Day 4] The culture medium was completely discarded, and a cell maintenance medium containing 1 μg / mL of puromycin was added. [Day 7] Half of the culture medium was discarded, and a hepatocyte culture medium (Hepatocyte Culture Media Bullet Kit, Lonza) containing 1 μg / mL puromycin was added. [Day 8] The culture medium was completely discarded, and half of the culture medium for hepatocytes containing 1 μg / mL of puromycin was added. <NGS analysis of hepatocyte marker genes> The cells were collected 10 days after ZNF384 gene transfer (Day 10). RNA was extracted from the cells and subjected to NGS analysis to analyze the expression of hepatocyte marker genes. The results are shown in Table 1. In Table 1, "Fold Change" indicates the value obtained by dividing the expression level in ZNF384 transfected cells by the expression level in mCherry transfected cells (control). If there was a significant difference (P<0.05) between the expression levels in both cells, a check mark was placed in the "P<0.05" column. In the ZNF384 gene-transfected cells, the expression of many hepatocyte marker genes was increased compared to the mCherry gene-transfected cells (control). <Expression analysis of hepatocyte marker genes by qRT-PCR> The cells were collected 10 days after ZNF384 gene transfer (Day 10). RNA was extracted from the cells, and expression of hepatocyte marker genes (ALB, HNF6A, KRT18) was analyzed by qRT-PCR. The results are shown in Figure 21. In ZNF384 transfected cells (ZNF384), expression of hepatocyte marker genes was elevated compared to mCherry transfected cells (Control) and non-transfected cells (NT, NT day 10). In Figure 21, "NT" indicates non-transfected cells on day 4 of culture. "NT day 10" indicates non-transfected cells on day 10 of culture. <Next Generation Sequencing analysis of liver-specific apolipoprotein genes> The cells were collected 10 days after ZNF384 gene transfer (Day 10). RNA was extracted from the cells and subjected to NGS analysis to analyze the expression of liver-specific apolipoprotein genes. The results are shown in Table 2. In Table 2, "Fold Change" indicates the value obtained by dividing the expression level in ZNF384 transfected cells by the expression level in mCherry transfected cells (control). If there was a significant difference (P<0.05) between the expression levels in both cells, a check mark was placed in the "P<0.05" column. In ZNF384 gene-transfected cells, the expression of many liver-specific apolipoprotein genes was increased compared to mCherry gene-transfected cells (control). These results confirmed that the ZNF384 gene-transfected cells were differentiated into hepatic progenitor cells and hepatocytes. [Experimental Example 9] (Induction of Differentiation into Hepatic Progenitor Cells and Hepatocytes (2)) We investigated whether hepatic progenitor cells and hepatocytes could be obtained by using a cell maintenance medium (StemFit) instead of the hepatocyte medium. The ZNF384 gene was introduced in the same manner as in Experimental Example 7, except that StemFit was used instead of the hepatocyte medium. Increased expression of hepatocyte marker genes was confirmed, as in Experimental Example 7. From these results, it was confirmed that hepatic progenitor cells and hepatocytes could be obtained even when culture was continued in a cell maintenance medium after introduction of the ZNF384 gene. [Experimental Example 10] (Transfection test of ZNF384 mRNA) An experiment was carried out in the same manner as in Experimental Example 1, except that ZNF384 mRNA was used instead of the ZNF384 gene. The mRNA was introduced by adding mRNA encapsulated in lipid nanoparticles (LNPs) to cells. Expression analysis of mesendodermal marker genes was performed using the same method as in Experimental Example 4. As a result, increased expression of mesendodermal marker genes was confirmed. These results confirmed that mesendodermal cells could be obtained even when ZNF384 mRNA was used. Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims. The present invention includes the following aspects. [1] A method for inducing mesendodermal cells, comprising the step of increasing the amount of zinc finger protein 384 (ZNF384) present in pluripotent stem cells. [2] The method for inducing mesendodermal cells according to [1], wherein the increasing step comprises introducing a nucleic acid encoding ZNF384 into the pluripotent stem cells. [3] A method for inducing mesodermal cells, comprising the steps of: inducing mesendodermal cells by the method for inducing mesendodermal cells according to [1] or [2]; and inducing the mesendodermal cells into mesodermal cells. [4] The method for inducing mesodermal cells described in [3], wherein the mesodermal cells are hematopoietic cells. [5] The method for inducing mesodermal cells described in [4], wherein the hematopoietic cells are CD34-positive cells. [6] A method for inducing endoderm cells, comprising the steps of inducing mesendodermal cells by the method for inducing mesendodermal cells according to [1] or [2], and inducing the mesendodermal cells into endoderm cells. [7] The method for inducing endodermal cells described in [6], wherein the endodermal cells are digestive system cells. [8] The method for inducing endodermal cells described in [7], wherein the digestive system cells are hepatic progenitor cells or hepatic cells. [9] A method for producing mesendodermal cells, comprising a step of inducing mesendodermal cells by the method for inducing mesendodermal cells according to [1] or [2].

[10] A method for producing mesodermal cells, comprising a step of inducing mesodermal cells by the method for inducing mesodermal cells according to any one of [3] to [5].

[11] A method for producing endodermal cells, comprising a step of inducing endodermal cells by the method for inducing endodermal cells according to any one of [6] to [8].

[12] An inducer of mesendodermal cells, comprising a nucleic acid encoding ZNF384. JP 2023-068362 A Japanese Patent No. 6076625 A Japanese Patent No. 6076624 A Patrick Guye et al., Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6. Nature Communications volume 7, Article number: 10243 (2016)

Claims

1. A method for inducing mesendodermal cells, comprising the step of increasing the abundance of zinc finger protein 384 (ZNF384) in pluripotent stem cells.

2. The method for inducing mesendodermal cells according to claim 1, wherein the step of increasing comprises introducing a nucleic acid encoding ZNF384 into the pluripotent stem cells.

3. A method for inducing mesodermal cells, comprising the steps of: inducing mesendodermal cells by the method for inducing mesendodermal cells according to claim 1 or 2; and inducing the mesendodermal cells into mesodermal cells.

4. The method for inducing mesodermal cells according to claim 3, wherein the mesodermal cells are hematopoietic cells.

5. The method for inducing mesodermal cells according to claim 4, wherein the hematopoietic cells are CD34-positive cells.

6. A method for inducing endodermal cells, comprising the steps of: inducing mesendodermal cells by the method for inducing mesendodermal cells according to claim 1 or 2; and inducing the mesendodermal cells into endodermal cells.

7. The method for inducing endodermal cells according to claim 6, wherein the endodermal cells are digestive system cells.

8. The method for inducing endodermal cells according to claim 7, wherein the digestive system cells are hepatic progenitor cells or hepatic cells.

9. A method for producing mesendodermal cells, comprising the step of inducing mesendodermal cells by the method for inducing mesendodermal cells described in claim 1 or 2.

10. A method for producing mesodermal cells, comprising the step of inducing mesodermal cells by the method for inducing mesodermal cells according to claim 3.

11. A method for producing endodermal cells, comprising the step of inducing endodermal cells by the method for inducing endodermal cells according to claim 6.

12. An inducer of mesendodermal cells, comprising a nucleic acid encoding ZNF384.

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