Lung mesenchymal cells and method for producing lung mesenchymal cells

Abstract

Provided is a method for producing lung mesenchymal cells that can be used as supporting cells when inducing alveolar epithelial cells.　This method for producing lung mesenchymal cells involves a step for culturing mesoderm cells in the presence of a mesenchymal cell inducer, KGF, and FGF10, and inducing differentiation of same into lung mesenchymal cells.

Description

Method for producing pulmonary mesenchymal cells and pulmonary mesenchymal cells

[0001] The present invention relates to a method for producing pulmonary mesenchymal cells and pulmonary mesenchymal cells.

[0002] When using pluripotent stem cells to create alveolar organoids, the alveolar organoids can be created by co-culturing lung progenitor cells derived from the pluripotent stem cells with human fetal fibroblasts (HFLF) (Non-Patent Documents 1-2). However, although HFLF are allogeneic cells, they are not autologous cells, and transplantation of the resulting alveolar organoids poses the risk of graft rejection. In addition, HFLF are difficult to obtain and are fetal-derived cells, which raises ethical concerns.

[0003] Gotoh, Shimpei et al. “Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells.” Stem cell reports vol. 3,3 (2014): 394-403. doi:10.1016 / j.stemcr.2014.07.005Yamamoto, Yuki et al. “Long-term expansion of alveolar stem cells derived from human iPS cells in organoids.” Nature methods vol. 14,11 (2017): 1097-1106. doi:10.1038 / nmeth.4448

[0004] In order to solve these problems, the present inventors have developed a method for producing alveolar organoids without using HFLF. However, in the method for producing alveolar organoids, type II alveolar epithelial cells are mainly induced, and the induction of type I alveolar epithelial cells is difficult. Therefore, it has been suggested that HFLF is important as a supporting cell in the production of alveolar organoids. Therefore, a method for inducing mesenchymal cells that function as supporting cells, similar to HFLF, is required.

[0005] Therefore, an object of the present disclosure is to provide a method for producing pulmonary mesenchymal cells that can be used as support cells in the induction of alveolar epithelial cells.

[0006] To achieve the above object, the method for producing pulmonary mesenchymal cells of the present disclosure includes a step of culturing mesodermal cells in the presence of a mesenchymal cell inducer, KGF, and FGF10, to induce differentiation into pulmonary mesenchymal cells.

[0007] The cell population comprising mesenchymal cells of the present disclosure (hereinafter also referred to as "cell population") comprises pulmonary mesenchymal cells that express RSPO2 and / or RSPO3.

[0008] The cell population comprising mesenchymal cells of the present disclosure includes pulmonary mesenchymal cells that express at least one transcription factor selected from the group consisting of Forkhead box protein F1 (FOXF1), Transcription factor 21 (TCF21), T-Box Transcription Factor 4 (TBX4), and Odd-Skipped Related Transcription Factor (OSR1).

[0009] The method for producing pulmonary epithelial cells and / or airway epithelial cells of the present disclosure (hereinafter also referred to as the "production method") comprises a step of culturing pulmonary progenitor cells in the presence of pulmonary mesenchymal cells to induce differentiation into pulmonary epithelial cells and / or airway epithelial cells, wherein the pulmonary mesenchymal cells are pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure and / or a cell population containing the mesenchymal cells of the present disclosure.

[0010] The pharmaceutical composition of the present disclosure comprises pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure, and / or a cell population comprising mesenchymal cells of the present disclosure.

[0011] According to the present disclosure, a method for producing pulmonary mesenchymal cells that can be used as support cells in the induction of alveolar epithelial cells can be provided.

[0012] FIG. 1 is a schematic diagram outlining the method for inducing pulmonary mesenchymal cells and pulmonary progenitor cells and forming alveolar organoids in Example 1. FIG. 2 is a photograph showing a phase-contrast image and an Oil Red O stained image showing the differentiation state of cells after culture in Example 1. FIG. 3 is a graph showing flow cytometry analysis in Example 1. FIG. 4 is a graph showing gene expression of cells at each culture stage in Example 1. FIG. 5 is a photograph showing a fluorescent image of cells on day 7 of culture in Example 1. FIG. 6 is a graph showing the results of an investigation of alveolar organoids in Example 1. FIG. 7 is a photograph and graph showing the expression of various cell markers in alveolar organoids in Example 1. FIG. 8 is a schematic diagram outlining the assay system in Example 2. FIG. 9 is a diagram showing iMES marker expression in Example 2. FIG. 10 is a diagram showing the organoid formation ability in Example 2. FIG. 11 is a diagram showing the results of RNA-Seq analysis in Example 2. FIG. 12 is a diagram showing the relative expression levels of Wnt ligands in Example 2. Figure 13 shows the culture method and the analysis results of SFTPC-GFP-positive cells in Example 2. Figure 14 shows the results of cluster analysis of each mesenchymal cell in Example 2. Figure 15 shows the results of passaging type II alveolar epithelial cells in Example 3. Figure 16 shows the results of cluster analysis of scRNA-seq analysis in Example 4. Figure 17 shows the results showing ligand-receptor interactions in Example 4. Figure 18 is a graph showing the results of SFTPC-GFP-positive cells / EPCAM-positive cells in Example 5.

[0013] <Definitions> As used herein, "marker" refers to a nucleic acid, gene, polypeptide, or protein that is expressed at a different level in a cell of interest. When the marker is a positive marker, the different level refers to increased expression compared to undifferentiated cells. When the marker is a negative marker, the different level refers to decreased expression compared to undifferentiated cells.

[0014] As used herein, "positive (+)," "positive," or "expressing" means that a cell expresses a detectable marker. The "positive (+)" typically means that a higher signal is detected by an analytical method such as flow cytometry, which utilizes an antigen-antibody reaction, compared to negative control cells that do not express the antigen or a negative control reaction using an antibody that does not react with the antigen. The "expressing" means that an increase in the expression level of the marker gene in the subject sample is observed when the expression levels of the marker gene in a reference sample and the marker gene in the subject sample are compared by RT-PCR or the like. The expression level is corrected using an internal standard gene (e.g., the β-actin gene). When the subject sample is a cell or cell population induced from a pluripotent cell, induced pluripotent stem cells (iPS cells) can be used as the reference sample.

[0015] As used herein, "negative (-)," "negative," or "does not express" means that a cell does not express a detectable marker. The "negative (-)" typically means that an equivalent or lower signal is detected in an analytical method, such as flow cytometry, that utilizes an antigen-antibody reaction, compared to a negative control cell that does not express the antigen or a negative control reaction using an antibody that does not react with the antigen. The "does not express" means that a decrease in the expression level of the marker gene in the subject sample is observed when the expression levels of the marker gene in a reference sample and the marker gene in the subject sample are compared by RT-PCR or the like. The expression level is corrected using an internal standard gene (e.g., the β-actin gene). When the subject sample is a cell or cell population induced from a pluripotent cell, induced pluripotent stem cells (iPS cells) can be used as the reference sample.

[0016] As used herein, the term "pluripotent cells" refers to cells that have the ability to differentiate into ectodermal, mesodermal, and endodermal cells. When the pluripotent cells have the ability to self-renew, they can also be called pluripotent stem cells.

[0017] As used herein, the term "mesodermal cells" refers to cells that are destined to have the ability to differentiate into connective tissues such as bone, cartilage, blood vessels, lymphatic vessels, etc.; muscle tissue; etc., if they are given an appropriate developmental stimulus, and are cells that express mesodermal cell markers such as NCAM (neural cell adhesion molecule), PDGFRα (Platelet Derived Growth Factor Receptor α), KDR (Kinase Insert Domain Receptor), ISL1, NKX2-5, and / or OSR1, preferably cells that express NCAM, PDGFRα, and / or KDR, more preferably cells that express NCAM and / or PDGFRα.

[0018] As used herein, "definitive endoderm (DE)" refers to cells that, upon developmentally appropriate stimulation, are destined to have the ability to differentiate into the thymus; digestive organs such as the stomach, intestines, and liver; respiratory organs such as the trachea, bronchi, and lungs; and urinary organs such as the bladder and urethra; and are cells that express SRY (sex determining region Y)-box 17 (SOX17) and FOXA2 (Forkhead box protein A2).

[0019] As used herein, "anterior foregut endoderm cells" (AFE) (also referred to as anterior foregut cells) refer to cells that are destined to have the ability to differentiate into the thymus and respiratory organs such as the trachea, bronchi, and lungs if given appropriate developmental stimuli, and are cells that express SOX2, SOX17, and FOXA2.

[0020] As used herein, "ventral anterior foregut endoderm cells" (VAFE) (also referred to as ventral anterior foregut cells) refer to cells that are destined to differentiate into thyroid and lung cells when given appropriate developmental stimuli, and express NKX2.1, GATA-binding factor 6 (GATA6), and Homeodomain-only protein (HOPX).

[0021] As used herein, "mesenchymal cells" refer to cells derived from mesodermal cells that, upon developmentally appropriate stimulation, are destined to differentiate into connective tissues such as bone, cartilage, blood vessels, and lymphatic vessels, and are cells that express mesenchymal cell markers such as VIM (Vimentin), THY1 (Thy-1 Cell Surface Antigen, CD90), PDGFRα, COL1A1 (Collagen Type I Alpha 1 Chain), NCAM, and / or KDR, and are preferably cells that express VIM, THY1, and / or COL1A1. The mesenchyme is also called mesenchyme. For this reason, the mesenchymal cells are also called mesenchymal cells.

[0022] As used herein, "pulmonary mesenchymal cells" refer to cells derived from mesodermal cells that are destined to differentiate into pulmonary connective tissues upon developmentally appropriate stimulation, and express forkhead box protein F1 (FOXF1), transcription factor 21 (TCF21), and / or T-box transcription factor 4 (TBX4) in addition to the mesenchymal cell markers. When the pulmonary mesenchymal cells express fibroblast markers (e.g., neural cell adhesion molecule (NCAM), adipose differentiation-related protein (ADRP), and / or collagen type I alpha 1 (COL1A1), actin alpha 2 (ACTA2), etc.), the pulmonary mesenchymal cells can also be referred to as pulmonary fibroblasts.

[0023] As used herein, the term "pulmonary progenitor cells" refers to cells that are destined to differentiate into alveolar epithelial cells and / or airway epithelial cells upon developmentally appropriate stimulation. The pulmonary progenitor cells express carboxypeptidase M (CPM), NK2 homeobox 1 (NKX2.1 or NKX2-1), SRY-box 9 (SRY (sex determining region Y)-box 9, SOX9), SRY-box 2 (SRY (sex determining region Y)-box 2, SOX2), and / or forkhead box protein 2A (FOXA2). The pulmonary progenitor cells are preferably CPM- and / or NKX2.1-positive cells.

[0024] As used herein, "alveolar epithelial cells" refers to epithelial cells present in the alveoli of the lung. Examples of the alveolar epithelial cells include type I alveolar epithelial cells and / or type II alveolar epithelial progenitor cells.

[0025] As used herein, "type I alveolar epithelial cells" refers to epithelial cells that have a histologically flat shape and express PDPN (Podoplanin), AGER (Advanced Glycosylation End-Product Specific Receptor), CAV1 (Caveolin 1), HOPX (HOP Homeobox), and / or AQP5 (Aquaporin 5).

[0026] As used herein, "type II alveolar epithelial cells" refers to epithelial cells that produce pulmonary surfactant proteins such as SFTPC (Surfactant protein C) and SFTPB (Surfactant protein B), and are cells that express SFTPC (Surfactant protein C), SFTPB (Surfactant protein B), ABCA3 (ATP-binding cassette sub-family A member 3), DCLAMP (Lysosome-associated membrane glycoprotein 3), and / or SLC34A2 (Sodium-dependent phosphate transport protein 2B).

[0027] As used herein, the term "cell population" refers to a collection of cells that includes a desired cell and is composed of one or more cells. In the cell population, the proportion of the desired cells among all cells (also referred to as "purity") can be quantified, for example, as the proportion of cells that express one or more markers expressed by the desired cells. The purity is, for example, the proportion among live cells. The purity can be measured by methods such as flow cytometry, immunohistochemistry, in situ hybridization, RT-PCR, and single-cell analysis. The purity of the desired cells in the cell population is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0028] As used herein, "isolated" means identified and separated, or in an identified and separated state, and / or identified and recovered from components in their natural state, or in a recovered state from components in their natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0029] As used herein, "enrichment" refers to increasing the content rate of target cells compared to the state before treatment, or to a state in which the content rate is increased. The enrichment can also be referred to as concentration. The enrichment does not include, for example, culturing.

[0030] As used herein, "protein" or "polypeptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0031] As used herein, "nucleic acid molecule" or "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The nucleic acid molecule may be a single-stranded or double-stranded nucleic acid molecule.

[0032] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal. It is used to particularly include humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0033] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a patient who develops a target disease, or treatment of an animal model of the target disease.

[0034] Sequence information for the proteins described herein or the nucleic acids (eg, DNA or RNA) encoding them is available from sources such as Protein Data Bank, UniPort, or Genbank.

[0035] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure is not limited by the following explanation. Furthermore, each explanation in the present disclosure can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0036] <Method for producing pulmonary mesenchymal cells> In one aspect, the present disclosure provides a method for producing pulmonary mesenchymal cells or a method for producing pulmonary mesenchymal cells that can be used to form alveolar organoids. The method for producing pulmonary mesenchymal cells of the present disclosure includes culturing mesodermal cells in the presence of a mesenchymal cell inducer and KGF and / or FGF10 to induce differentiation into pulmonary mesenchymal cells. The method for producing pulmonary mesenchymal cells of the present disclosure can provide pulmonary mesenchymal cells that can be used to form alveolar organoids. Furthermore, when pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure are used to form alveolar organoids, the alveolar organoids contain, for example, airway epithelial cells such as type I alveolar epithelial cells, type II alveolar epithelial cells, and airway ciliated epithelial cells. Therefore, the method for producing pulmonary mesenchymal cells of the present disclosure can provide, for example, pulmonary mesenchymal cells as supporting cells that can replace the HFLF.

[0037] In the method for producing pulmonary mesenchymal cells of the present disclosure, the mesodermal cells used to induce the pulmonary mesenchymal cells can be induced, for example, from pluripotent cells. Thus, the method for producing pulmonary mesenchymal cells of the present disclosure may induce differentiation of the pluripotent cells into mesodermal cells prior to inducing the pulmonary mesenchymal cells from the mesodermal cells. In this case, the method for producing pulmonary mesenchymal cells of the present disclosure includes, for example, a step of culturing the pluripotent cells in the presence of a mesodermal induction factor to induce differentiation into the mesodermal cells (first induction step).

[0038] In the first induction step, for example, the pluripotent cells are cultured in a medium containing the mesoderm induction factor to differentiate into cells expressing the mesoderm cell marker, i.e., mesoderm cells. In other words, the pluripotent cells are contacted with the mesoderm induction factor and cultured to differentiate into mesoderm cells. For the induction of pluripotent cells into mesoderm cells, see, for example, References 1 to 4 listed below. Specifically, in the first induction step, the mesoderm cells can be induced from the pluripotent cells by culturing using, for example, a GSK3β inhibitor, activin A, and / or BMP4 as the mesoderm induction factor. For example, one type of mesoderm induction factor may be used, or multiple types may be used. When one type of mesoderm induction factor is used, the GSK3β inhibitor is preferably used as the mesoderm induction factor. Furthermore, when a plurality of mesoderm induction factors are used in combination, examples of the mesoderm induction factors include a combination of the GSK3β inhibitor with activin A and / or BMP4; a combination of the GSK3β inhibitor with activin A and BMP4; etc. When the mesoderm induction factor is a peptide or protein, the mesoderm induction factor is, for example, a peptide or protein derived from an animal species different from or the same as that from which the pluripotent cells are derived. In the first induction step, Lefty may be used instead of activin A. In addition, in the first induction step, BMP2, BMP6, and / or BMP7 may be used instead of BMP4.Reference 1: Han, L., Chaturvedi et al. “Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis.” Nat Commun, 2020, 11, 4158. Reference 2: Kishimoto, K. et al, “Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells.” Nat Commun, 2020, 11, 4159. Reference 3: Loh, KM et al., “Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.” Cell, 2016, 166, 451-467. Reference 4: Xi, H. et al. “In Vivo Human Somitogenesis Guides Somite Development from hPSCs.”Cell Rep, 2017, 18, 1573-1585.

[0039] Examples of the pluripotent cells include totipotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); pluripotent stem cells such as tissue stem cells or somatic stem cells, including hematopoietic stem cells, neural stem cells, and mesenchymal stem cells; and the like.

[0040] As the ES cells, for example, human embryonic stem cell lines such as H1, H7, and H9 (available from WiCell Research Institute) can be used. The ES cells may be prepared, for example, by culturing cell masses isolated from animal blastocysts. As a specific example, the following Reference 5 can be referred to for the method of deriving the ES cells. Reference 5: Thomson JA et al., "Embryonic stem cell lines derived from human blastocysts," Science, 1998, vol. 282, pages 1145-1147

[0041] Examples of iPS cells that can be used include 201B7 (available from RIKEN BRC) and 604A1 (available from the Institute for iPS Cell Research, Kyoto University). The iPS cells can be prepared, for example, by introducing reprogramming factors into target cells. Examples of the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, and a specific example is a combination of Oct3 / 4, Sox2, Klf4, L-Myc, and Lin28.

[0042] The GSK3β inhibitor may be any substance that inhibits the kinase activity of GSK3β protein (for example, the ability to phosphorylate β-catenin), and specific examples thereof include indirubin derivatives such as BIO (GSK-3β inhibitor IX: 6-bromoindirubin 3'-oxime); maleimide derivatives such as SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione) and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione); and SK-3β inhibitor VII Examples of such GSK-3β inhibitors include phenyl-α-bromomethyl ketone compounds such as (4-dibromoacetophenone); cell membrane-permeable phosphorylated peptides such as L803-mts (GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2); CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), and nucleic acid molecules that inhibit the expression of GSK-3β protein (e.g., siRNA, shRNA, antisense). CHIR99021 is preferred as the GSK-3β inhibitor due to its high selectivity for GSK-3β. GSK-3β inhibitors are commercially available from, for example, Calbiochem, Biomol, and the like.

[0043] The activin A is a protein (SEQ ID NO: 49) encoded by a polynucleotide registered with NCBI under accession number NM_002192.

[0044] Activin A (SEQ ID NO: 49) MPLLWLRGFLLASCWIIVRSSTPPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDD IGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA RKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVN ICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

[0045] The activin A may be a functional equivalent of the activin A. The functional equivalent is a substance that can activate SMAD2 / 3 signaling via activin receptors (ACVR1 / 2) in the same way as activin A. Examples of the functional equivalent of activin A include Nodal and Lefty.

[0046] The BMP4 is a protein encoded by a polynucleotide registered with NCBI under accession numbers NM_001202, NM_001347914, NM_001347916, NM_130850, or NM_130851. An example of the BMP4 is a protein consisting of the amino acid sequence of SEQ ID NO: 50 below.

[0047] BMP4 (SEQ ID NO: 50) MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSFHHEEHLENIPGTSENSAFRFLFNLSSIPENEVISSAELRLFREQVDQGPDWERGFHRINIYEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTHLHQTRTHQGQHVRISRSLPQGSGNWAQLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR

[0048] The BMP4 may be a functional equivalent of the BMP4. The functional equivalent is a substance that can activate SMAD1 / 5 / 8 signaling via BMP receptors (BMPR1 / 2) in the same way as BMP4. Examples of the functional equivalent of BMP4 include BMP2, BMP6, and BMP7.

[0049] The concentration of the mesoderm induction factor in the first induction step is not particularly limited, as long as each factor is at an effective concentration at which it exhibits mesoderm cell induction activity. Specifically, when the GSK3β inhibitor CHIR99021 is used as the mesoderm induction factor, the concentration of CHIR99021 in the medium is, for example, 0.1 to 20 μmol / L. When activin A is used as the mesoderm induction factor, the concentration of activin A in the medium is, for example, 1 to 100 ng / ml. When BMP4 is used as the mesoderm induction factor, the concentration of BMP4 in the medium is, for example, 1 to 100 ng / ml.

[0050] The medium can be prepared using a medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (manufactured by Thermo Fisher Scientific), stem cell culture media (e.g., mTeSR-1 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), CDM-PVA, StemPRO hESC SFM (manufactured by Life Technologies), E8 (manufactured by Life Technologies)), and mixtures thereof. The medium may contain or not contain serum. The medium may contain serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The medium may also contain additives such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. When proliferation culture is performed using the construct, the medium is preferably a stem cell culture medium supplemented with glutamic acid and antibiotics.

[0051] The culture period in the first induction step may be any period that allows the mesodermal cells to differentiate, and may be, for example, 1 to 7 days, 1 to 5 days, or 2 to 4 days.

[0052] The culture conditions for the first induction step can be, for example, ordinary conditions for cell culture. Specific examples of the culture temperature include 25 to 40°C, 30 to 40°C, or about 37°C. The carbon dioxide concentration during culture is 1 to 10%, 3 to 7%, or about 5%. The culture is performed, for example, in a humid environment.

[0053] In the first induction step, the differentiation of the mesodermal cells can be detected, for example, by the expression of a marker for the mesodermal cells and / or the loss of expression of a marker for the pluripotent cells.

[0054] Examples of the mesodermal cell marker include NCAM, PDGFRα, KDR, ISL1, NKX2-5, and / or OSR1, etc. The mesodermal cell marker is preferably NCAM, PDGFRα, and / or KDR, more preferably NCAM and / or PDGFRα, or NCAM and PDGFRα.

[0055] Examples of the pluripotent stem cell marker include ABCG2, Cripto, FOXD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0056] After the first induction step, the content (lower limit) of the mesodermal cells in all cells (cell population) after the induction is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less, based on the number of cells. The numerical range of the content ratio can be, for example, any combination of the lower limit and the upper limit. As a specific example, when the cells are cultured for three days in the first induction step, the content ratio is, for example, 30 to 60%. The content ratio decreases, for example, by shortening the number of culture days in the first induction step. On the other hand, the content ratio increases, for example, by extending the number of culture days in the first induction step.

[0057] Next, in the method for producing pulmonary mesenchymal cells of the present disclosure, the mesodermal cells are cultured in the presence of a mesenchymal cell inducer, KGF, and FGF10 to induce differentiation into pulmonary mesenchymal cells (second induction step).

[0058] In the second induction step, for example, the mesodermal cells are cultured in a medium containing a mesenchymal cell inducer to differentiate into cells expressing the mesenchymal cell marker, i.e., mesenchymal cells such as pulmonary mesenchymal cells. In the second induction step, for example, the mesodermal cells are cultured in contact with the mesenchymal cell inducer to differentiate into mesenchymal cells. Specifically, in the second induction step, the pulmonary mesenchymal cells can be induced from the mesodermal cells by culturing using, for example, activin A, FGF2, BMP4, retinoic acid (RA), PDGFbb (platelet-derived growth factor bb), a Wnt inducer, and / or a GSK3β inhibitor, in combination with KGF (FGF7) and / or FGF10, as the mesenchymal cell inducer. In the second induction step, the pulmonary mesenchymal cells can be induced by allowing KGF and FGF10 to coexist in the mesenchymal cell induction method. For example, the following References 6 and 7 can be referred to for the method of inducing mesenchymal cells. For example, one type of mesenchymal cell induction factor may be used, or multiple types may be used. When one type of mesenchymal cell induction factor is used, the mesenchymal cell induction factor is preferably BMP4 or FGF2. When multiple mesenchymal cell induction factors are used in combination, examples of the mesenchymal cell induction factors include a combination of activin A, FGF2, and BMP4; a combination of retinoic acid, BMP4, and a Wnt inhibitor and / or a GSK3β inhibitor (Reference 6); and a combination of FGF2 and PDGFbb (Reference 7). When the mesenchymal cell induction factor is a peptide or protein, the mesoderm induction factor is, for example, a peptide or protein derived from the same or different animal species as the animal from which the mesodermal cells are derived. The KGF and FGF10 are, for example, peptides or proteins derived from the same or different animal species as the animal from which the mesodermal cells are derived. In the second induction step, FGF1 may be used instead of FGF2. In the second induction step, FGF3 and / or FGF22 may be used instead of KGF and FGF10 (References 8 to 9). Examples of the Wnt inhibitor and GSK3β inhibitor described below can be used.Reference 6: Han, Lu et al. “Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis.” Nature communications vol. 11,1 4158. 27 Aug. 2020, doi:10.1038 / s41467-020-17968-x Reference 7: Takebe, Takanori et al. “Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.” Cell reports vol. 21,10 (2017): 2661-2670. doi:10.1016 / j.celrep.2017.11.005 Reference 8: Morishu Konishi et al. “Regulation of biological functions by extracellular secreted factor FGF21”, Internet<https: / / seikagaku.jbsoc.or.jp / 10.14952 / SEIKAGAKU.2016.880086 / index.html> Reference 9: Hui, Qi et al. “FGF Family: From Drug Development to Clinical Application.” International journal of molecular sciences vol. 19,7 1875. 26 Jun. 2018, doi:10.3390 / ijms19071875.

[0059] The FGF2 is a protein (SEQ ID NO: 51) encoded by a polynucleotide registered with NCBI under accession number NM_002006. The FGF2 may be in an activated form after cleavage by a protease.

[0060] FGF2 (SEQ ID NO: 51) MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

[0061] The FGF2 may be a functional equivalent of the FGF2. The functional equivalent is a substance that can activate Ras-Raf via an FGF receptor (FGFR1 or FGFR4) like FGF2. An example of the functional equivalent of FGF2 is FGF1, which belongs to the FGF1 subfamily like FGF2.

[0062] The KGF is a protein encoded by a polynucleotide registered with NCBI under accession number NM_002009. The KGF may be in an activated form after being cleaved by a protease.

[0063] The KGF (FGF7) may be a functional equivalent of the KGF. The functional equivalent is a substance that can activate Ras-Raf via an FGF receptor (FGFR2b, FGFR1b, etc.) like KGF. Examples of the functional equivalent of KGF include FGF3, FGF10, FGF22, etc., which belong to the FGF7 subfamily like KGF. The KGF may be in an activated form after cleavage by a protease.

[0064] The FGF10 is a protein encoded by a polynucleotide registered with NCBI under accession number NM_004465. The FGF10 may be in an activated form after being cleaved by a protease.

[0065] The FGF10 may be a functional equivalent of the FGF10. The functional equivalent is a substance that can activate Ras-Raf via an FGF receptor (FGFR2b, FGFR1b) like FGF10. Examples of the functional equivalent of FGF10 include KGF, FGF3, FGF22, and the like, which belong to the FGF7 subfamily like FGF10. The FGF10 may be in an activated form after being cleaved by a protease.

[0066] The concentration of the mesenchymal cell induction factor in the second induction step is not particularly limited, as long as each factor exhibits an effective concentration at which it exhibits mesenchymal cell induction activity. Specifically, when activin A is used as the mesenchymal cell induction factor, the concentration of activin A in the medium is, for example, 0.01 to 1000 ng / ml, 0.1 to 100 ng / ml, or 0.2 to 10 ng / ml. When FGF2 is used as the mesenchymal cell induction factor, the concentration of FGF2 in the medium is, for example, 0.1 to 1000 ng / ml, 1 to 100 ng / ml, or 2 to 50 ng / ml. When BMP4 is used as the mesenchymal cell induction factor, the concentration of BMP4 in the medium is, for example, 0.1 to 1000 ng / ml, 1 to 100 ng / ml, or 2 to 50 ng / ml.

[0067] The concentrations of KGF and FGF10 in the second induction step are not particularly limited, as long as each factor is at an effective concentration at which it exhibits the activity of inducing pulmonary mesenchymal cells. Specific examples of the KGF concentration in the medium include 0.1 to 1000 ng / ml, 1 to 100 ng / ml, or 2 to 50 ng / ml. Specific examples of the FGF10 concentration in the medium include 0.1 to 1000 ng / ml, 1 to 100 ng / ml, or 2 to 50 ng / ml.

[0068] The medium used in the second induction step may be the same as or different from the medium used in the first induction step.

[0069] The culture period in the second induction step may be any period that allows the pulmonary mesenchymal cells to differentiate, and is, for example, 1 to 9 days, 3 to 7 days, or 4 to 6 days.

[0070] The culture conditions for the second induction step can be the same as those for the first induction step, and may be the same as or different from those for the first induction step.

[0071] In the second induction step, the differentiation of the pulmonary mesenchymal cells can be detected, for example, by the expression of a pulmonary mesenchymal cell marker and / or the loss of expression of a mesodermal cell marker.

[0072] Examples of the mesenchymal cell marker include PDGFRα, KDR, ISL1, NKX2-5, VIM, COL1A1, FOXF1, and / or TCF21, and preferably FOXF1 and TCF21.

[0073] Examples of the mesodermal cell marker include TBXT (T-box transcription factor T).

[0074] After the second induction step, the content (lower limit) of the pulmonary mesenchymal cells in all cells (cell population) after the induction is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less, based on the number of cells. The numerical range of the content ratio can be, for example, any combination of the lower limit and the upper limit. When the second culture step is performed for 3 days, the content ratio is, for example, 20 to 40%. When the second culture step is performed for 7 days, the content ratio is, for example, 60 to 90%. The content ratio decreases, for example, by shortening the number of culture days in the second induction step. On the other hand, the content ratio increases, for example, by extending the number of culture days in the first induction step.

[0075] The method for producing pulmonary mesenchymal cells of the present disclosure can also enrich the pulmonary mesenchymal cells after the second induction step.Therefore, the method for producing pulmonary mesenchymal cells of the present disclosure, for example, when using the cell population containing the obtained pulmonary mesenchymal cells and the pulmonary progenitor cells described below to form alveolar organoids, can improve the induction efficiency of type II alveolar epithelial cells.In this case, the method for producing pulmonary mesenchymal cells of the present disclosure comprises, after the second induction step, enriching the pulmonary mesenchymal cells from the cell population induced from the mesodermal cells (enrichment step).

[0076] The enrichment of pulmonary mesenchymal cells can be carried out, for example, using as an indicator a marker (positive marker) that is expressed in the cell population by the pulmonary mesenchymal cells but not expressed in other cells or that is expressed at a low level by other cells, or a marker (negative marker) that is not expressed in the pulmonary mesenchymal cells but is expressed in other cells or that is expressed at a high level by other cells. The positive marker and the negative marker are preferably markers expressed on the cell surface. Examples of the positive marker include PDGFRα, KDR, VIM, THY1, and NCAM. Examples of the negative marker include EpCAM and E-Cadherin. The enrichment may be carried out using a combination of multiple markers. For example, the enrichment is carried out using the negative marker to suppress the occurrence of signal transduction mediated by the marker in the pulmonary mesenchymal cells.

[0077] The enrichment can be performed, for example, after recovering the cell population after the second induction step, using an antibody against the positive marker and / or an antibody against the negative marker with an automated magnetic cell separator (e.g., autoMACS), a magnetic cell separator (e.g., MACS), a closed magnetic cell separator (e.g., Prodigy), or a cell sorter (e.g., FACS).

[0078] After the enrichment step, the content (lower limit) of positive marker-positive pulmonary mesenchymal cells in the enriched cell population is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of the positive marker-positive pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less, based on the number of cells. The numerical range of the content ratio of the positive marker-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. Furthermore, after the enrichment step, the content (lower limit) of negative marker-negative pulmonary mesenchymal cells in the enriched cell population is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of the negative marker-negative pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less, based on the number of cells. The numerical range of the content ratio of the negative marker-negative pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit.

[0079] As a specific example, when the marker used for the enrichment is EpCAM, the content (lower limit) of EpCAM-negative pulmonary mesenchymal cells in the cell population after the enrichment step is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of EpCAM-negative pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The numerical range of the content ratio of EpCAM-negative pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit.

[0080] The EpCAM-negative pulmonary mesenchymal cells may be, for example, PDGFRα-, KDR-, VIM-, and / or THY1-positive. The content (lower limit) of the PDGFRα-positive and KDR-positive pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of the PDGFRα-positive and KDR-positive pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The numerical range of the content ratio of the PDGFRα-positive and KDR-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. The content (lower limit) of the VIM-positive and THY1-positive pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of the VIM-positive and THY1-positive pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The numerical range of the content ratio of the VIM-positive and THY1-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit.

[0081] Pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure may be identified, for example, by the expression of various nucleic acids and proteins.

[0082] The pulmonary mesenchymal cells express, for example, RSPO2 (R-Spondin 2) and / or RSPO3 (R-Spondin 3). Furthermore, the pulmonary mesenchymal cells do not express, for example, WNT2. The pulmonary mesenchymal cells express, for example, RSPO2 and / or RSPO3, but do not express WNT2. For example, when the pulmonary mesenchymal cells express RSPO2 and / or RSPO3, and a cell population containing the obtained pulmonary mesenchymal cells is formed using the pulmonary progenitor cells described below, type II alveolar epithelial cells can be induced.

[0083] The content (lower limit) of RSPO2-positive pulmonary mesenchymal cells among the EpCAM-negative pulmonary mesenchymal cells is, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, based on the number of cells. The content (upper limit) of RSPO2-positive pulmonary mesenchymal cells among the EpCAM-negative pulmonary mesenchymal cells is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less, based on the number of cells. The numerical range of the content of RSPO2-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. As a specific example, when the second culture step is performed for 7 days, the content is, for example, 20 to 40%.

[0084] Among the EpCAM-negative pulmonary mesenchymal cells, RSPO2 is particularly expressed in, for example, STC1 (Stanniocalcin-1)-positive pulmonary mesenchymal cells. Therefore, the RSPO2-positive cells can be enriched, for example, by using STC1. The content (lower limit) of RSPO2-positive pulmonary mesenchymal cells among the EpCAM-negative STC1-positive pulmonary mesenchymal cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content (upper limit) of RSPO2-positive pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The numerical range of the content of RSPO2-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. As a specific example, when cultured for 7 days in the second culture step, the content is, for example, 80% or more.

[0085] The content ratio (lower limit) of RSPO3-positive pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The content ratio (upper limit) of RSPO3-positive pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The range of the content of RSPO3-positive pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. Specifically, when the second culture step is performed for 7 days, the content is, for example, 70 to 90%.

[0086] The percentage (lower limit) of Wnt2-negative pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, based on the number of cells. The percentage (upper limit) of Wnt2-negative pulmonary mesenchymal cells in the EpCAM-negative pulmonary mesenchymal cells is, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less, based on the number of cells. The range of the content of Wnt2-negative pulmonary mesenchymal cells can be, for example, any combination of the lower limit and the upper limit. Specifically, when cultured for 7 days in the second culture step, the content is, for example, 95% or more.

[0087] The pulmonary mesenchymal cells express, for example, a transcription factor selected from the group consisting of FOXF1 (Forkhead box protein F1), TCF21 (Transcription factor 21), TBX4 (T-Box Transcription Factor 4), and OSR1 (Odd-Skipped Related Transcription Factor). The pulmonary mesenchymal cells may express, for example, one or more types of transcription factors, or may express all types of transcription factors. The pulmonary mesenchymal cells do not express, for example, TBXT as a transcription factor. The pulmonary mesenchymal cells express, for example, a transcription factor selected from the group consisting of FOXF1, TCF21, TBX4, and OSR1 as a transcription factor, but do not express TBXT. The pulmonary mesenchymal cells may further express, for example, a fibroblast marker described below and / or be positive for a mesenchymal cell marker.

[0088] The pulmonary mesenchymal cells express, for example, a fibroblast marker selected from the group consisting of NCAM, ADRP, COL1A1, and ACTA2. The pulmonary mesenchymal cells are preferably cells that express NCAM, ADRP, and / or COL1A1; NCAM, ADRP, and COL1A1. The pulmonary mesenchymal cells may express, for example, one or more types of fibroblast markers, or may express all types of fibroblast markers.

[0089] The pulmonary mesenchymal cells are positive for a mesenchymal cell marker selected from the group consisting of, for example, VIM (Vimentin), THY1 (Thy-1 Cell Surface Antigen, CD90), PDGFRα (Platelet Derived Growth Factor Receptor α), and KDR (Kinase Insert Domain Receptor). The pulmonary mesenchymal cells are preferably cells that express VIM, THY1, and / or COL1A1; or VIM, THY1, and COL1A1. The pulmonary mesenchymal cells may be positive for, for example, one or more types of mesenchymal cell markers, or may be positive for all types of mesenchymal cell markers.

[0090] The pulmonary mesenchymal cells can induce epithelial cells that constitute alveoli from, for example, the pulmonary progenitor cells described below. Therefore, for example, in an alveolar organoid formation assay by co-culture with the progenitor cells, the pulmonary mesenchymal cells can induce type I alveolar epithelial cells and / or type II alveolar epithelial cells from the pulmonary progenitor cells. The alveolar organoid formation assay can be performed in the same manner as in Example 1 (3) described below. Examples of the alveolar epithelial cells include type I alveolar epithelial cells and type II alveolar epithelial cells. The pulmonary mesenchymal cells can be, for example, capable of inducing type I alveolar epithelial cells or type II alveolar epithelial cells from the pulmonary progenitor cells, or can be capable of inducing type I alveolar epithelial cells and type II alveolar epithelial cells from the pulmonary progenitor cells.

[0091] For example, in the alveolar organoid formation assay, the pulmonary mesenchymal cells can induce a cell population from the pulmonary progenitor cells, in which the percentage (lower limit) of SFTPC-positive cells in an EpCAM-positive cell population is 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The upper limit of the percentage can be, for example, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, or 55% or less. The numerical range of the percentage can be, for example, any combination of the lower limit and the upper limit. As a specific example, when the pulmonary mesenchymal cells and the pulmonary progenitor cells are cultured for 14 days, the percentage of SFTPC-positive cells is 20 to 70%.

[0092] According to the method for producing pulmonary mesenchymal cells of the present disclosure, for example, when co-cultured with the pulmonary progenitor cells, it is possible to induce alveolar epithelial cells. The pulmonary mesenchymal cells are expected to be suitable for use, for example, in lung tissue regeneration.

[0093] <Cell Population> In another aspect, the present disclosure provides a cell population comprising pulmonary mesenchymal cells, which can also be used to generate alveolar organoids. The cell population comprising mesenchymal cells of the present disclosure comprises pulmonary mesenchymal cells that express RSPO2 and / or RSPO3.

[0094] The pulmonary mesenchymal cells of the present disclosure may be identified, for example, by the expression of various nucleic acids and proteins in the description of the pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure, i.e., the pulmonary mesenchymal cells after the second induction step or the enrichment step.

[0095] <Method for producing alveolar epithelial cells> In another aspect, the present disclosure provides a method for producing alveolar epithelial cells using the pulmonary mesenchymal cells. The method for producing alveolar epithelial cells of the present disclosure includes a step of culturing pulmonary progenitor cells in the presence of pulmonary mesenchymal cells to induce differentiation into alveolar epithelial cells, wherein the pulmonary mesenchymal cells are pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure and / or a cell population containing the mesenchymal cells of the present disclosure.

[0096] In the method for producing alveolar epithelial cells of the present disclosure, the pulmonary progenitor cells used to induce the alveolar epithelial cells can be induced from precursor cells of pulmonary progenitor cells, such as the pluripotent cells. Therefore, the method for producing alveolar epithelial cells of the present disclosure may induce the pulmonary progenitor cells from precursor cells of pulmonary progenitor cells prior to inducing the alveolar epithelial cells. In this case, the method for producing alveolar epithelial cells of the present disclosure includes, for example, a step of culturing the cells in the presence of an inducer for pulmonary progenitor cells and inducing differentiation into the pulmonary progenitor cells (a third induction step).

[0097] In the third induction step, for example, the precursor cells of the lung progenitor cells are cultured in a medium containing an inducer for the lung progenitor cells and differentiated into lung progenitor cells expressing the lung progenitor cell marker, i.e., the precursor cells of the lung progenitor cells are contacted with the inducer for the lung progenitor cells and cultured to differentiate into the lung progenitor cells. The induction of the precursor cells of the lung progenitor cells into the lung progenitor cells can be carried out by reference to, for example, the method for inducing alveolar epithelial progenitor cells described in International Publication No. 2014 / 168264, the method for inducing pulmonary airway progenitor cells described in International Publication No. 2019 / 217429, the method for isolating lung progenitor cells described in U.S. Patent No. 10,386,368, or the method for inducing NKX2-1 lung progenitor cells described in Reference 10 below. Reference 10: Hawkins et al., J Clin Invest. 2017 Jun 1;127(6):2277-2294. doi: 10.1172 / JCI89950.

[0098] Examples of the precursor cells of the lung progenitor cells include ventral anterior foregut endoderm cells, anterior foregut endoderm cells, and / or definitive endoderm cells. The precursor cells of the lung progenitor cells and the lung progenitor cells can be induced from the pluripotent cells or the pluripotent stem cells. Therefore, the precursor cells of the lung progenitor cells and the lung progenitor cells are preferably precursor cells induced from the pluripotent cells or the pluripotent stem cells. Examples of the pluripotent stem cells include totipotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); pluripotent stem cells such as tissue stem cells or somatic stem cells, including hematopoietic stem cells, neural stem cells, and mesenchymal stem cells; and the like.

[0099] The cell population obtained in the third induction step is a cell population containing the lung progenitor cells and / or precursor cells of the lung progenitor cells. In the method for producing alveolar epithelial cells of the present disclosure, the obtained cell population may be used as is, or the lung progenitor cells and / or precursor cells of the lung progenitor cells may be isolated from the obtained cell population and used. When isolating the lung progenitor cells, the lung progenitor cells can be isolated based on, for example, the expression of CPM, NKX2.1, SOX9, SOX2, and / or FOXA2. The lung progenitor cells are preferably isolated as CPM-positive cells using the cell surface marker CPM.

[0100] Next, in the method for producing alveolar epithelial cells of the present disclosure, the pulmonary progenitor cells are cultured in the presence of the pulmonary mesenchymal cells to induce differentiation into alveolar epithelial cells (fourth induction step).

[0101] In the fourth induction step, for example, the pulmonary progenitor cells are cultured in a medium containing an inducer for the pulmonary mesenchymal cells to differentiate into alveolar epithelial cells that express the alveolar epithelial cell marker, i.e., the pulmonary progenitor cells are contacted with the inducer for the pulmonary progenitor cells to differentiate the pulmonary progenitor cells. In the fourth induction step, the pulmonary progenitor cells may be cultured in the presence of the pulmonary mesenchymal cells and the inducer for the alveolar epithelial cells to induce differentiation into alveolar epithelial cells.

[0102] The alveolar epithelial cell induction factor can be set depending on the type of alveolar epithelial cells to be induced. When the alveolar epithelial cells are type I alveolar epithelial cells, the alveolar epithelial cell induction factor is an induction factor for type I alveolar epithelial cells, specifically, the Wnt inducer. The induction factor may be one type or multiple types. The induction factor is preferably multiple types, and more preferably all types.

[0103] The Wnt inducer is a substance that induces Wnt signaling. Examples of the Wnt inducer include IWP2 (N-(6-Methyl-2-benzothiazolyl)-2-(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno3,2-dpyrimidin-2-yl)thio), Dickkopf-related protein 1 (DKK1), XAV939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), and nucleic acid molecules that induce expression of Wnt proteins (such as siRNA, shRNA, and antisense), and preferably XAV939.

[0104] The concentration of the Wnt inducer in the medium is, for example, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 50 nmol / L to 30 μmol / L, 100 nmol / L to 25 μmol / L, or 500 nmol / L to 20 μmol / L.

[0105] The description of the medium in the first induction step can be applied to the medium.

[0106] The number of days for culture in the step of inducing type I alveolar epithelial cells can be set depending on the period for which the type I alveolar epithelial cells are induced. The lower limit of the number of days for culture can be, for example, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, or more. The upper limit of the number of days for culture can be, for example, 35 days or less, 30 days or less, 28 days or less, or 21 days or less.

[0107] The culture conditions in the step of inducing type I alveolar epithelial cells can be the same as those in the first induction step.

[0108] When the alveolar epithelial cells are type II alveolar epithelial cells, the alveolar epithelial cell inducer is an inducer of type II alveolar epithelial cells, and specific examples include steroids, cAMP derivatives, phosphodiesterase inhibitors, KGF, GSK3β inhibitors, TGFβ inhibitors, ROCK inhibitors, and / or FGF10. The inducer may be one type or multiple types. The inducer is preferably multiple types, more preferably a combination of the steroids, the cAMP derivatives, the phosphodiesterase inhibitors, and the KGF. The KGF, GSK3β inhibitors, and FGF10 can be described in the same manner as above.

[0109] The steroid drug is a steroidal anti-inflammatory drug, and examples of the steroid drug include glucocorticoids and synthetic derivatives thereof, such as hydrocortisone, hydrocortisone succinate, prednisolone, methylprednisolone, methylprednisolone succinate, triamcinolone, triamcinolone acetonide, dexamethasone, and betamethasone, with dexamethasone and hydrocortisone being preferred.

[0110] The concentration of the steroid agent in the medium is, for example, 1 nmol / L to 100 μmol / L, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 10 nmol / L to 30 μmol / L, 10 nmol / L to 25 μmol / L, or 10 nmol / L to 20 μmol / L.

[0111] The cAMP derivative is a compound in which a substituent has been modified (added) to cyclic AMP. Examples of the cAMP derivative include cyclic adenosine monophosphate (cAMP), 8-bromo cyclic adenosine monophosphate (8-Br-cAMP), 8-chloro cyclic adenosine monophosphate (8-Cl-cAMP), 8-(4-chlorophenylthio)cyclic adenosine monophosphate (8-CPT-cAMP), and dibutyryl cyclic adenosine monophosphate (DB-cAMP), and 8-Br-cAMP is preferred.

[0112] The concentration of the cAMP derivative in the medium is, for example, 1 nmol / L to 100 μmol / L, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 50 nmol / L to 30 μmol / L, 100 nmol / L to 25 μmol / L, or 500 nmol / L to 20 μmol / L.

[0113] The phosphodiesterase inhibitor is a compound that inhibits phosphodiesterase (PDE) and thereby increases the intracellular concentration of cAMP or cGMP. Examples of the phosphodiesterase inhibitor include 1,3-dimethylxanthine, 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)isoquinoline, 4-{[3',4'-(Methylenedioxy)benzyl]amino}-6-methoxyquinazoline, 8-methoxymethyl-3-isobutyl-1-methylxanthine, and 3-isobutyl-1-methylxanthine (IBMX), and 1,3-dimethylxanthine is preferred.

[0114] The concentration of the phosphodiesterase inhibitor in the medium is, for example, 1 nmol / L to 100 μmol / L, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 50 nmol / L to 30 μmol / L, 50 nmol / L to 25 μmol / L, or 50 nmol / L to 20 μmol / L.

[0115] The TGFβ inhibitor is a substance that inhibits signal transduction mediated by SMADs, which occurs when TGFβ binds to a receptor. Examples of the TGFβ inhibitor include substances that inhibit binding to the ALK family, which is a TGFβ receptor, or substances that inhibit phosphorylation of SMADs by the ALK family. Specific examples of the TGFβ inhibitor include Lefty-1 (NCBI accession numbers: NM_010094 (mouse), NM_020997 (human)), SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridine-2-yl)-1H-imidazol-2-yl)benzamide), SB202190 (4-(4-Fluorophenyl)-2-(4 -hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB505124 (2-(5-Benzo1,3dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO2009 / 146408), and the like, preferably SB431542.

[0116] The concentration of the TGFβ inhibitor in the medium is, for example, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 50 nmol / L to 30 μmol / L, 100 nmol / L to 25 μmol / L, or 500 nmol / L to 20 μmol / L, preferably 1 nmol / L to 40 μmol / L.

[0117] The ROCK inhibitor is a substance capable of inhibiting the function of Rho kinase (ROCK). Examples of the ROCK inhibitor include Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), Fasudil / HA1077 (5-(1,4-Diazepane-1-sulfonyl)isoquinoline), H-1152 ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine), Wf-536 ((+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl)benzamide), and nucleic acid molecules that inhibit the expression of ROCK proteins (e.g., siRNA, shRNA, and antisense). Y-27632 is preferred.

[0118] The concentration of the ROCK inhibitor in the medium is, for example, 1 nmol / L to 50 μmol / L, 10 nmol / L to 40 μmol / L, 50 nmol / L to 30 μmol / L, 100 nmol / L to 25 μmol / L, 500 nmol / L to 20 μmol / L, or 750 nmol / L to 15 μmol / L, preferably 1 nmol / L to 40 μmol / L.

[0119] The description of the medium in the first induction step can be applied to the medium.

[0120] The number of days for culture in the step of inducing type II alveolar epithelial cells can be set depending on the period for which the type II alveolar epithelial cells are induced. The lower limit of the number of days for culture can be, for example, 2 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or more. The upper limit of the number of days for culture can be, for example, 35 days or less, 30 days or less, 28 days or less, or 21 days or less.

[0121] The culture conditions in the step of inducing type I alveolar epithelial cells can be the same as those in the first induction step.

[0122] In the fourth induction step, the differentiation of the alveolar epithelial cells can be detected, for example, by the expression of a marker for the alveolar epithelial cells and / or the loss of expression of a marker for the lung progenitor cells. The alveolar epithelial cells may be, for example, a cell population containing the type I alveolar epithelial cells or the type II alveolar epithelial cells, or a cell population containing the type I alveolar epithelial cells and the type II alveolar epithelial cells.

[0123] When the alveolar epithelial cells are type I alveolar epithelial cells, examples of the alveolar epithelial cell marker include PDPN, AGER, CAV1, HOPX, AQP5, etc. When the alveolar epithelial cells are type II alveolar epithelial cells, examples of the alveolar epithelial cell marker include SFTPC, SFTPB, ABCA3, DCLAMP, SLC34A2, etc.

[0124] According to the method for producing alveolar epithelial cells of the present disclosure, it is possible to induce the alveolar epithelial cells as, for example, organoids containing the alveolar epithelial cells.

[0125] <Method for maintaining and / or expanding alveolar epithelial cells> In another aspect, the present disclosure provides a method for maintaining and / or expanding type II alveolar epithelial cells. The method for maintaining and / or expanding type II alveolar epithelial cells of the present disclosure comprises a step (culturing step) of culturing type II alveolar epithelial cells in the presence of pulmonary mesenchymal cells to maintain or expand them, wherein the pulmonary mesenchymal cells are pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells of the present disclosure and / or a cell population containing mesenchymal cells of the present disclosure.

[0126] The type II alveolar epithelial cells are known to function as lung tissue stem cells. Therefore, in the culturing step, the type II alveolar epithelial cells can be maintained and / or expanded by, for example, inducing self-renewal (proliferation) of the type II alveolar epithelial cells, or inducing self-renewal (proliferation) and differentiation of the type II alveolar epithelial cells.

[0127] In the culturing step, for example, the pulmonary progenitor cells are cultured in a medium containing the pulmonary mesenchymal cells to maintain or proliferate the pulmonary progenitor cells expressing the pulmonary progenitor cell marker, or the pulmonary progenitor cells expressing the pulmonary progenitor cell marker are maintained or proliferated and differentiated into alveolar epithelial cells expressing the alveolar epithelial cell marker. In the culturing step, the pulmonary progenitor cells may be cultured in the presence of the pulmonary mesenchymal cells and an inducer for alveolar epithelial cells to maintain or expand the pulmonary progenitor cells.

[0128] The induction factor for alveolar epithelial cells can be determined depending on the type of alveolar epithelial cells to be induced, and the explanation of the method for producing alveolar epithelial cells disclosed herein can be used.

[0129] <Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition comprising pulmonary mesenchymal cells. The pharmaceutical composition of the present disclosure comprises the pulmonary mesenchymal cells of the present disclosure and a pharmaceutically acceptable carrier.

[0130] The pharmaceutical composition of the present disclosure may be administered, for example, intravenously. The pharmaceutical composition of the present disclosure may be administered, for example, in the form of an injection. In the case of the injection, the number of pulmonary mesenchymal cells contained in the injection may be, for example, 1×10 ６ The pharmaceutical composition may contain a pharmaceutically acceptable carrier. Examples of the carrier include physiological saline, phosphate-buffered saline (PBS), cell preservation solution, cell culture solution, hydrogel, extracellular matrix, and cryopreservation solution.

[0131] The pharmaceutical compositions of the present disclosure can be suitably used, for example, in the treatment of pulmonary diseases.

[0132] In another aspect, the present disclosure provides a culture medium for use in inducing pulmonary mesenchymal cells from mesodermal cells, comprising a culture medium, a mesenchymal cell inducer, KGF, and FGF10.

[0133] The types, combinations, and concentrations of the mesenchymal cell induction factors, KGF and FGF10, contained in the medium can be determined, for example, from the description of the method for producing pulmonary mesenchymal cells of the present disclosure.

[0134] <Kit> In another aspect, the present disclosure provides a kit for use in inducing pulmonary mesenchymal cells from mesodermal cells. The kit for use in inducing pulmonary mesenchymal cells from mesodermal cells of the present disclosure comprises a mesenchymal cell inducer, KGF, and FGF10.

[0135] The types and combinations of the mesenchymal cell induction factors, KGF, and FGF10 contained in the kit can be determined, for example, from the description of the method for producing pulmonary mesenchymal cells of the present disclosure. The contents of the mesenchymal cell induction factors, KGF, and FGF10 in the kit can be set, for example, so that when added to a predetermined amount of medium, the concentrations of each factor will be those described in the description of the method for producing pulmonary mesenchymal cells of the present disclosure.

[0136] Next, examples of the present disclosure will be described, but the present disclosure is not limited by the following examples. Commercially available reagents were used according to their protocols unless otherwise specified.

[0137] [Example 1] It was confirmed that pulmonary mesenchymal cells can be induced by the production method of the present disclosure, and that alveolar organoids can be formed by co-culturing the pulmonary mesenchymal cells and the pulmonary progenitor cells.

[0138] An outline of the method for inducing pulmonary mesenchymal cells and pulmonary progenitor cells and forming alveolar organoids is shown in FIG. 1 .

[0139] (1) Induction of Pulmonary Mesenchymal Cells As shown in Figure 1(B), the pulmonary mesenchymal cells were induced from human-derived iPS cells (iPSCs). Specifically, subcultured undifferentiated human iPSCs were washed with D-PBS (Nacalai Tesque, Cat. No.: 14249-24) and then incubated at 37°C for 20 minutes in the presence of protease (Accutase, Innivative Cell Technologies, Cat. No.: AT-104) to dissociate the iPSCs into single cells. An equal volume of mTeSR Plus (STEMCELL technologies, Cat. No.: ST-05825 or ST-100-0276) was added to neutralize the protease, and the cell suspension containing iPSCs was centrifuged to remove the supernatant. The collected cell suspension was then resuspended in iMatrix-511 (0.25 μg / cm２ ) and 10 μmol / L Y-27632 (LC Laboratories, Cat. No.: Y-5301). ４ Cells were seeded into 6-well plates at a cell density of 1000 kJ / well (culture day 1). On culture day 0, the medium was replaced with StemPro™-34 (Thermo Fischer Scientific, Cat. No. 10639011) supplemented with 150 ng / ml activin A (API, Cat. No. GF-001), 50 ng / ml BMP4 (R&D Systems, Cat. No. 314-BP), 1.5 μmol / L CHIR99021 (Axon Medchem, Cat. No. Axon1386), Glutamax™ (Thermo Fischer Scientific, Cat. No. 35050061), and 50 U / ml penicillin / streptomycin. On culture day 2, the medium was replaced with the same medium. This resulted in the induction of mesodermal cells from iPSCs. The differentiation state of the cells was then confirmed using a phase-contrast microscope.

[0140] On day 3 of culture, the medium was switched to StemPro™-34 supplemented with 30 ng / ml activin A, 10 ng / ml KGF (Prospec, Cat. No.: CYT-219), 25 ng / ml BMP4, 10 ng / ml bFGF (DS Pharma Biomedical, Cat. No.: KHFGF001), 10 ng / ml FGF10, Glutamax, and 50 U / ml penicillin / streptomycin. The medium was replaced with the same medium on day 5 of culture. On day 7 of culture, cells were detached from the plate by treatment with TrypLE Select Enzyme (Thermo Fischer Scientific, Cat. No.: 12563029) at 37°C for 10 minutes. The resulting cell suspension was washed with DMEM containing 2% FBS and then suspended in PBS containing 1% BSA. 1x10 ６1 μl of antibody solution was added per 100 μl of cells, and the cells were incubated in the resulting mixture at room temperature (approximately 25°C, hereinafter the same) for 20 minutes. The antibody used in the antibody solution was an anti-EPCAM antibody (Santa Cruz Biotechnology, Cat. No.: sc-66020 / EBA-1). Next, the incubated mixture was reacted with anti-mouse IgG microbeads (Miltenyi Biotec, Cat. No.: 130-048-401) as a secondary antibody. EPCAM-negative cells were then negatively sorted from the resulting reaction mixture using an LD column (Miltenyi Biotec, Cat. No.: 130-042-901) according to the attached protocol. The recovered mesenchymal cells were then used as pulmonary mesenchymal cells (iMES).

[0141] In addition, granules in the cells on day 7 of culture were stained with Oil Red O. Furthermore, cells on days 0, 1, 3, and 7 of culture were stained with anti-EPCAM-FITC antibody (Miltenyi Biotec, Cat. No.: 130-080-301), anti-NCAM-Alexa Fluor 647 antibody (BioLegend, Cat. No.: 362513), anti-T-Alexa Fluor 488 antibody (RD systems, Cat. No.: IC2085G), anti-KDR-BV421 antibody (BioLegend, Cat. No.: 393009), anti-THY1 (CD90)-BV421 antibody (BioLegend, Cat. No.: 328121), and anti-Vimentin-Alexa Fluor 647 antibody (Novus Biologicals, Cat. No.: Staining was performed using anti-PDGFRA antibody (NBP1-97670AF647) and anti-PDGFRA-Alexa Fluor 647 antibody (BD Biosciences, Cat. No. 562798). Specifically, single-cell suspensions were washed with 1% BSA-containing PBS and stained with primary antibodies at 4°C for 15 minutes. After two washes with 1% BSA-containing PBS, cells were stained with secondary antibodies at 4°C for 15 minutes, if necessary. After two washes with 1% BSA-containing PBS, cells were stained with propidium iodide (PI). For intracellular staining, the cell suspension was fixed with BD Cytofix / Cytoperm (BD Biosciences, Cat. No. 51-2090KZ) for 20 minutes and then permeabilized with BD Perm / Wash (BD Biosciences, Cat. No. 51-2091KZ) for 20 minutes. After the treatment, the cells were washed twice with BD Perm / Wash and then stained with a primary antibody for 15 minutes at 4°C. The stained cell suspension was then washed twice with BD Perm / Wash and stained with a secondary antibody for 15 minutes at 4°C. After washing twice with PBS containing 1% BSA, the cells were prepared in 1% BSA / PBS without PI.The stained samples were subjected to flow cytometry analysis using Melody (BD Biosciences). The results are shown in Figures 2 and 3.

[0142] Figure 2 shows phase-contrast and Oil Red O stained images showing the differentiation state of cells after culture. In Figure 2, (A) shows a phase-contrast image, and (B) shows an Oil Red O stained image. Each scale bar in Figure 2 represents 100 μm. In Figure 2(A), the photographs are from left to right, showing the cells on days 0, 1, 3, and 7 of culture. As shown in Figure 2(A), the cells retained a PSC morphology on day 0 of culture, but by day 1 of culture, the boundaries of the cell clusters became unclear, suggesting the start of differentiation. These are presumed to be cells expressing TBXT in the EpCAM-positive cell population described below. Furthermore, by day 3 of culture, the cells became flattened, confirming differentiation into mesodermal cells. Furthermore, by day 7 of culture, the cells had numerous granules in the cytoplasm, and as shown in Figure 2(B), these granules were stained with Oil Red O.

[0143] Figure 3 is a graph showing flow cytometry analysis. In Figure 3, the upper graph shows, from left to right, the results on days 0, 1, 3, and 7 of culture. The middle and lower graphs show, from left to right, the results for the EpCAM-negative cell population on days 0, 1, and 3 of culture, and the EpCAM-negative and EpCAM-positive cell populations on day 7 of culture. As shown in Figure 3, on day 1 of culture, EpCAM-positive and TBXT-positive cells were observed, suggesting that the cells had differentiated into cells similar to the primitive streak embryologically. Furthermore, on day 3 of culture, the EpCAM-negative cell population showed expression of mesodermal cell markers NCAM, PDGFRα, and KDR, confirming differentiation into mesodermal cells. Furthermore, on day 7 of culture, the induction of mesenchymal cells positive for mesenchymal cell markers VIM, THY1 (CD90), PDGFRα, and KDR was confirmed. These results confirmed that mesenchymal cells were induced even when KGF and FGF10 were added under the above-mentioned mesenchymal cell induction conditions.

[0144] Next, gene expression at each culture stage was examined using RT-qPCR. Specifically, gene expression was examined in cells on day 0 of culture, cells on day 3 of culture, iMES, HFLF, and HDF. Total RNA was extracted from each cell line using an RNA extraction kit (PureLink RNA mini kit, Thermo Fisher Scientific, Cat. No. 12183020). Next, cDNA was prepared from 80 ng of total RNA per sample using reverse transcriptase (SuperScript® III reverse transiptase, Thermo Fisher Scientific). The resulting cDNA was amplified using an RT-PCR kit (Power SYBR Green PCR Master Mix, Applied Biosystems) and quantified using QuantStudio 3 (Applied Biosystems). The expression level of each gene was normalized using the β-actin gene as an internal control gene. Furthermore, the gene expression levels were quantified as relative to the gene expression levels in the cells on day 0 of culture. The primer sets used for RT-qPCR are shown in Table 1 below. These results are shown in Figure 4.

[0145]

[0146] FIG. 4 is a graph showing gene expression in cells at each culture stage. In FIG. 4, the horizontal axis indicates the cell culture stage, and the vertical axis indicates relative expression levels. As shown in FIG. 4, after the start of culture, TBXT and EPCAM were expressed in cells at the early stage of culture, but not in iMES. Furthermore, expression of fibroblast markers VIM and COL1A1 was induced in iMES. Furthermore, expression of pulmonary mesenchymal cell markers FOXF1 and TBX4 was observed in iMES. Furthermore, increased expression of NCAM, PDGFRα, KDR, ISL1, NKx2-5, OSR1, and ADRP was observed in iMES. These results demonstrate that iMES induced by the method for producing pulmonary mesenchymal cells disclosed herein are pulmonary mesenchymal cells. Furthermore, it was found that the iMES can be distinguished from other cells using these markers.

[0147] Next, the cells on day 7 of culture were fixed for 15 minutes with 4% paraformaldehyde in PBS and then permeabilized for 15 minutes with 0.2% Triton™ X-100 in PBS. After this fixation, the cells were stained with primary and secondary antibodies as previously described (Reference 11 below). The primary antibodies used were anti-E-Cadherin antibody (eBiosience, Cat. No. 14-3249), anti-Vimentin antibody (CST, Cat. No. 49636), and anti-FOXF1 antibody (RD Systems, Cat. No. AF4798). The secondary antibodies used were anti-rat IgG Alexa Fluor 488 (Thermo Fisher Scientific, Cat. No.: A-21208), anti-mouse IgG Alexa Fluor 546 (Thermo Fisher Scientific, Cat. No.: A-10036), and anti-goat IgG Alexa Fluor 647 (Thermo Fisher Scientific, Cat. No.: A-21447). The stained samples were observed under a fluorescence microscope (BZ-X710, Keyence). The results are shown in Figure 5. Reference 11: Gotoh, S., Ito, I., Nagasaki, T., Yamamoto, Y., Konishi, S., Korogi, Y., Matsumoto, H., Muro, S., Hirai, T., Funato, M., et al. (2014). Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Reports 3, 394-403.

[0148] Figure 5 is a photograph showing a fluorescent image of cells on day 7 of culture. In Figure 5, the scale bar indicates 100 μm. As shown in Figure 5, E-cadherin-positive cells were FOXF1-negative, while E-cadherin-negative cells were VIM- and FOXF1-positive. These results demonstrated that iMES expresses VIM and FOXF1 at the protein level.

[0149] (2) Induction of Lung Progenitor Cells. Differentiation of human iPSCs into lung progenitor cells was performed according to Reference 11 (mentioned above) and References 12-13 (mentioned below). Specifically, undifferentiated human iPSCs were cultured on Geltrex-coated plates in the presence of definitive endoderm transformation medium for 6 days to differentiate into definitive endoderm cells (Step 1). For differentiation into definitive endoderm cells, RPMI1640 medium (Nacalai Tesque, Cat. No. 30264-56) containing 100 ng / ml activin A, 1 μmol / l CHIR99021, 2% B27 supplement (ThermoFisher, Cat. No. 17504-001), and 50 U / ml penicillin / streptomycin was used. Each medium was replaced every 2 days. Furthermore, as shown in Table 2 below, during differentiation into the definitive endoderm cells, Y-27632 was added on day 0 of culture, and sodium butyrate (Wako, Cat. 193-015122) was added on days 1, 2, and 4 of culture.Environment 12:Konishi, S., Gotoh, S., Tateishi, K., Yamamoto, Y., Korogi, Y., Nagasaki, T., Matsumoto, H., Muro, S., Hirai, T., Ito, I., et al. (2016). Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells. Stem Cell Reports 6, 18-25. 13:Yamamoto, Y., Gotoh, S., Korogi, Y., Seki, M., Konishi, S., Ikeo, S., Sone, N., Nagasaki, T., Matsumoto, H., Muro, S., et al. (2017). Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat Methods 14, 1097–1106.

[0150]

[0151] Next, from days 6 to 10 of culture, the definitive endoderm cells were cultured in anteriorization medium (Step 2). Then, on day 10 of culture, the medium was changed to posteriorization medium containing BMP4 (20 ng / ml) and the specified concentrations of ATRA (Sigma-Aldrich, Cat. No. R2625) and CHIR99021 (Step 3). When using B2-3 PSCs, the optimal concentrations of ATRA and CHIR99021 were 0.05 to 0.5 μmol / L. From days 14 to 21 of culture, the posteriorized cells were cultured in CFKD preconditioning medium (Step 4). On day 21 of culture, NKX2-1-positive lung progenitor cells were isolated using mouse anti-human CPM (Wako, Cat. No. 014-27501) and anti-mouse IgG-Alexa647 (Thermo Fischer Scientific, Cat. No. A-31571) to gate CPM-positive cells, as described in Reference 13. Note that some lung progenitor cells were derived from PSCs (B2-3 line) expressing SFTPC-GFP, and GFP expression was induced upon differentiation into alveolar epithelial cells.

[0152] (3) Alveolar organoid formation assay Alveolar organoids were prepared according to the method described in Reference 13 and Reference 14 below. ４ CPM-positive cells (derived from 201B7 PSC strain) and 5.0 × 10 ５Fetal fibroblasts (HFLF, DV Biologics, Cat. No. PP002-F-1349), human pediatric dermal fibroblasts (HDF, TIG120, available from the National Institutes of Biomedical Innovation, Japan), or iMES were mixed in 100 μl of alveolarization medium (Table 3) supplemented with Y-27632 (10 μmol / L) and 100 μl of Matrigel (Corning, Cat. No. 354230). The resulting mixture was introduced into a 12-well cell culture insert (Corning, Cat. No. 3513) and cultured for 14 days. The medium in the lower chamber was replaced every two days. The HFLF were cultured in 10% FBS-containing DMEM (Nacalai Tesque, Cat. No.: 08459-64) and used at passage 10. The TIG120 were cultured in 10% FBS-containing MEM medium (Nacalai Tesque, Cat. No.: 21442-25) and used cells with a PDL of 30 or less. The resulting alveolar organoids were dissociated using 0.1% Tripsin-EDTA at 37°C for 15 minutes and then washed twice with 1% BSA-containing PBS. After washing, the cells were immunostained with anti-EpCAM-APC antibody (Miltenyi Biotec, Cat. No.: 130-113-263). After staining, SFTPC-GFP-positive cells / EPCAM-positive cells were analyzed using a flow cytometer (FACS). The alveolar organoids were also observed using the fluorescence microscope. Furthermore, alveolar organoids were prepared in the same manner as above, except that the 604A1 cell line was used as the iPSC cell line. The results are shown in Figure 6.Reference 14: Korogi, Y., Gotoh, S., Ikeo, S., Yamamoto, Y., Sone, N., Tamai, K., Konishi, S., Nagasaki, T., Matsumoto, H., Ito, I., et al. (2019). In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids. Stem Cell Reports 12, 431-440.

[0153]

[0154] Figure 6 is a graph showing the results of an investigation into alveolar organoids. In Figure 6, (A) shows a fluorescent image of alveolar organoids, (B) shows the results of flow cytometry analysis, and (C) shows the proportion of SFTPC-GFP positive cells among EpCAM-positive cells in alveolar organoids. As shown in Figure 6(A), when iMES and lung progenitor cells were co-cultured, spheroids containing SFTPC-GFP positive cells were formed, confirming the differentiation of lung progenitor cells into alveolar epithelial cells. Although not shown, spheroids containing SFTPC-GFP positive cells were also formed when the 604A1 cell line was used. Furthermore, as shown in Figures 6(B) and (C), the proportion of SFTPC-GFP positive cells induced by iMES was equivalent to that of HFLF, which was used as a feeder cell. On the other hand, SFTPC-GFP positive cells were not induced with HDF.

[0155] Next, the constituent cells of the alveolar organoids were examined. Specifically, the alveolar organoids were fixed for 20 minutes using 4% paraformaldehyde in PBS, followed by overnight incubation (approximately 8 hours) in 30% sucrose in PBS. After incubation, the alveolar organoids were embedded in OCT compound (Sakura Finetek, Cat. No.: 4583) and frozen using liquid nitrogen. The frozen alveolar organoids were sliced ​​into 10 μm-thick sections and attached to slides. The resulting sections were permeabilized for 15 minutes using 0.2% Triton™ X-100 in PBS. After permeabilization, they were blocked for 30 minutes using 5% normal donkey serum (EMD-Millipore) and 1% BSA in PBS. After the blocking treatment, the sections were stained with primary and secondary antibodies against EPCAM, VIM, Pro-SFTPC, ABCA3, GFP, Mature-GFP, SFTPB, PDPN, and HT1-56. Hoechst-33342 (Dojindo, Cat. No.: H342) was added to the secondary antibody solution to label cell nuclei. The stained sections were observed using the fluorescence microscope. Furthermore, alveolar organoids were prepared in the same manner, except that the 604A1 cell line was used as the PSC cell line.

[0156] In addition, alveolar organoids formed using the 201B7 and 604A1 cell lines were used to measure the genes SFTPB, SFTPC, SFTPD, SFTPA2, ABCA3, SLC34A2, HOPX, AGER, and AQP5. The exogenous control was human fetal lung RNA (Agilent Technologies; #540177, lot 0006055802) from 17, 18, or 22 weeks of gestation, and the relative expression levels were quantified. Otherwise, the expression of these genes in the alveolar organoids was examined as described in Example 1(1). The results are shown in Figure 7.

[0157] Figure 7 shows photographs and graphs illustrating the expression of various cell markers in the alveolar organoids. In Figure 7, (A) shows a fluorescent image of the alveolar organoids, and (B) shows the relative expression level of each gene. In Figure 7(B), the horizontal axis indicates the iPSC lineage, and the vertical axis indicates the relative expression level of each gene. As shown in Figure 7(A), in alveolar organoids containing SFTPC-GFP-positive cells, VIM-positive iMES was spread throughout the alveolar organoids. Furthermore, as shown in Figures 7(A) and (B), type II alveolar epithelial cell markers Pro-SFTPC, ABCA3, SFTPC-GFP, and mature-SFTPC were detected in cuboidal cells in the alveolar organoids. In addition, flat cells positive for type I alveolar epithelial cell markers PDPN and HT1-56 were also observed in the alveolar organoids. As shown in Figure 7(B), type I and type II alveolar epithelial cell markers were detected in the alveolar organoids formed by co-culture with iMES (iMES-AO). These results demonstrate that alveolar organoids can be formed by 3D co-culture of iPSC-derived lung progenitor cells with iMES.

[0158] From the above, it is found that the manufacturing method of the present disclosure can induce pulmonary mesenchymal cells, and can form alveolar organoid by co-culturing said pulmonary mesenchymal cells and said pulmonary progenitor cells.In addition, since the pulmonary mesenchymal cells obtained by the manufacturing method of the present disclosure can form alveolar organoid, it is found that said pulmonary mesenchymal cells (iMES) can be used as feeder cells instead of HFLF.

[0159] [Example 2] We confirmed that alveolar organoids can be formed using iMES induced from iPSCs derived from other cells. Furthermore, we analyzed the expression profile of iMES before and after culture.

[0160] As shown in Figure 8, we induced iPSCs from different cells and confirmed whether alveolar organoids could be formed using iMES induced from these different cells. That is, we confirmed that the induced pulmonary mesenchymal cells can be used as feeder cells, regardless of the origin of the pluripotent cells.

[0161] (1) Induction of iPSCs iPSCs were induced from the HFLFs and HDFs used in Example 1. First, HFLF-iPSCs (HFA) were established from HFLFs (17.5 weeks pregnant, DV Biologics, Cat. No.: PP002-F-1349, Lot 121109VA). ６ An episomal vector mix for human iPSC generation (Takara, Cat. No.: 3673) containing cDNAs for OCT3 / 4, SOX2, KLF4, L-MYC, LIN28, mp53-DD, and EBNA1 was introduced into the HFLF cells by electroporation. 5 × 10 cells were cultured. ４ The cells were seeded into each well of a 6-well plate containing 10% FBS-containing DMEM (culture day 0). The medium was replaced with 10% FBS-containing DMEM on culture days 1, 3, and 5. On culture day 6, the medium in each well was replaced with StemFit AK02N (Ajinomoto Co., Inc., Cat. No.: AJ100). The resulting iPSC colonies were then picked and incubated with StemFit AK02N and iMatrix-511 (Takara Bio Inc., Cat. No.: 892021) (0.25 μg / cm ２ HFLF-iPSCs were seeded into each well of a 12-well plate transfected with HFLF-iPSCs. The resulting iPSCs were maintained and passaged in StemFit AK02N medium. After several passages, the medium in each well was changed to mTeSR Plus (STEMCELL Technologies, Cat. No. ST-05825 or ST-100-0276), and the iPSCs (HFLF-iPSCs) were then used to induce iMES.

[0162] HDF-iPSCs (GC23) were established from HDFs (TIG120) in a feeder cell-dependent manner using the episomal vectors for human iPSC generation (OCT3 / 4, SOX2, KLF4, L-MYC, LIN28, and short hairpin RNA for p53 (mp53-DD)) according to the method described in Reference 14. The resulting HDF-iPSCs were then expanded and frozen. After thawing, the HDF-iPSCs were maintained and passaged in a feeder cell-free environment using mTeSR Plus medium before induction into iMES.

[0163] To confirm the genomic identity of HFLF, HFLF-iPSCs (HFA), HDF (TIG120), and HDF-iPSCs (GC23) with their parental lines and to rule out cell misidentification or cross-contamination during processing, we analyzed 16 short tandem repeat loci (Table 4) in HFLF, HFLF-iPSCs (HFA), HDF, and HDF-iPSCs (GC23) using the PowerPlex® 16 HS System (Promega). The tandem repeats at each locus were completely identical between the iPSCs and their corresponding parental fibroblasts. Furthermore, although not shown, HFLF-iPSCs and HDF-iPSCs expressed undifferentiated markers (Nanog, OCT3 / 4), showed no abnormal karyotypes, and were capable of differentiation into three germ layers (ectoderm, mesoderm, and endoderm). Therefore, we prepared iMES using HFLF-iPSCs (HFA) and HDF-iPSCs (GC23).

[0164]

[0165] (2) Induction of pulmonary mesenchymal cells (iMES) Induction of iMES from iPSCs was performed in the same manner as in Example 1(1) above, except that HFLF-iPSCs (HFA) and HDF-iPSCs (GC23) were used as iPSCs. The resulting iMES, HFLF, and HDF were stained for E-cadherin, Vimentin (VIM), and FOXF1 in the same manner as in Example 1(1) above, and then observed under a fluorescent microscope. Furthermore, the relative expression levels of PFGFRA, VIM, COL1A1, FOXF1, and TBX4 mRNA were quantified in the same manner as in Example 1(1) above, using HFLF as an exogenous control, except that the resulting iMES and HDF were used. Furthermore, the expression of VIM, THY1, PDGFRA, and KDR in the obtained iMES was examined by flow cytometry in the same manner as in Example 1(1). The results are shown in Figure 9.

[0166] Figure 9 shows the marker expression of iMES. In Figure 9, (A) is a photograph showing the fluorescent image of each cell, (B) shows the gene expression of each cell, and (C) shows the results of flow cytometry analysis. As shown in Figure 9(A), VIM and FOXF1 were expressed at the protein level in iMES induced from HFLF-iPSCs (HFA) and HDF-iPSCs (GC23). Furthermore, as shown in Figure 9(B), similar to Example 1(1), both HFLF-iPSCs (HFA) and HDF-iPSCs (GC23) differentiated into iMES expressing VIM, THY1, PDGFRA, and KDR. Furthermore, similar to Example 1(1), high levels of FOXF1 were expressed in HFLF and iMES, but almost no expression was observed in HDF.

[0167] (3) Formation of Alveolar Organoids Furthermore, the obtained iMES, HFLF, and HDF were used to form alveolar organoids in the same manner as in Example 1 (3). The alveolar organoids were observed using the fluorescence microscope. Furthermore, in the same manner as in Example 1 (3), the cells constituting the alveolar organoids were dissociated, and the obtained cells were analyzed for SFTPC-GFP positive cells / EPCAM positive cells. These results are shown in Figure 10.

[0168] Next, Figure 10 shows the organoid formation ability. In Figure 10, (A) shows the results of flow cytometry analysis, and (B) shows the percentage of SFTPC-GFP positive cells among EpCAM-positive cells in alveolar organoids. As shown in Figures 10(A) and (B), SFTPC-GFP positive cells were induced in both iMES induced from HFLF-iPSCs (HFA) and HDF-iPSCs (GC23), demonstrating that these cells can be used to induce alveolar epithelial cells.

[0169] (4) iMES Transcriptome Analysis To analyze the factors that contribute to iMES function as feeder cells in alveolar organoid formation, RNA-Seq analysis was performed on HFLF, HFLF-iPSC-derived iMES, HDF-iPSC-derived iMES, and HDF before and after the alveolar organoid formation assay. Specifically, total RNA was extracted using an RNA extraction kit (RNeasy Micro Kit, Qiagen) according to the attached protocol. Libraries were prepared for each sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina). The resulting libraries were sequenced using a NovaSeq 6000 (Illumina) with 100-bp paired-end reads. The raw FASTQ data were trimmed using software (fastp 0.20.1, https: / / github.com / OpenGene / fastp#install-with-bioconda, 15) and then filtered to remove rRNA, tRNA, snRNA, snoRNA, Mt_rRNA, and Mt_tRNA using software (SortMeRna 2.1b, https: / / github.com / biocore / sortmerna, 16). The preprocessed data were aligned to GRCh38 using software (STAR ​​2.7.6a, https: / / github.com / alexdobin / STAR, 17). The resulting alignment data were used with software (RSEM 1.3.3, https: / / github.com / deweylab / RSEM, 18) to calculate transcripts per million (TPM) and read counts. These data were then analyzed using the software (tximport 1.20.0, https: / / github.com / mikelove / tximport; Ref. 19) in R 4.1.1 (http: / / www.R-project.The dataset was imported into a database called org. Next, lowly expressed genes with an average read count of 1 or less were excluded from the analysis, as they were considered below the detection limit. After this exclusion, DEGs (Reference 21) were identified using software (DESeq2 1.32.0, https: / / github.com / mikelove / DESeq2, Reference 20). Pre-ranked GSEA was then performed using genes sorted by p-value calculated by DESeq2. GO enrichment analysis was performed using software (clusterProfiler 4.0.5, https: / / github.com / YuLab-SMU / clusterProfiler, Reference 22) and org.Hs.eg.db 3.13.0 (https: / / anaconda.org / bioconda / bioconductor-org.hs.eg.db). The results are shown in Figure 11. Reference 15: Chen, S., Zhou, Y., Chen, Y., and Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884-i890. Reference 16: Kopylova, E., Noe, L., and Touzet, H. (2012). SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211-3217. Reference 17: Dobin, A., Davis, CA, Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, TR (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. Reference 18: Li, B., and Dewey, CN (2011).RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323. Reference 19: Soneson, C., Love, M.I., and Robinson, M.D. (2015). Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 4, 1521. Reference 20: Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545-15550. Reference 21: Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550. Reference 22: Wu, T., Hu, E., Xu, S., Chen, M., Guo, P., Dai, Z., Feng, T., Zhou, L., Tang, W., Zhan, L., et al. (2021). clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation 2.

[0170] Figure 11 shows the results of RNA-Seq analysis. As shown in Figure 11(A), principal component analysis (PCA) of the RNA-seq transcriptomes clearly separated the clusters for each condition, with the transcriptomes of iMES after alveolar organoid formation (hereinafter referred to as "3D culture") and HFLF after 3D culture plotted closely. Furthermore, HDF after 3D culture was separated from iMES and HFLF after 3D culture. Gene expression often depends on the medium composition and culture conditions (e.g., 2D or 3D). Therefore, to clarify the factors contributing to alveolar organoid formation in iMES, gene expression was analyzed by pairing the analysis results of samples after 3D culture with the parent strain (HDF-iMES vs. HDF, HFLF-iMES vs. HFLF). As shown in Figure 11(C), GO enrichment analysis revealed enrichment of "Embryonic development" and "Lung development" between HDF-iMES and HDF after 3D culture. DEGs annotated with "Lung development" were extracted and heat maps were created for four groups: HDF-iMES, HDF, HFLF-iMES, and HFLF. WNT5A, FGF7, and PDGFRA are known to be important factors in type II alveolar epithelial cells. As shown in Figure 11(C), unexpectedly, WNT5A, FGF7, and PDGFRA were elevated in HDF. Meanwhile, iMES showed increased expression of secreted proteins, including RSPO2, WNT11, CCN2, SPARC, BMP4, HHIP, LAMA5, and LOX. The expression levels of transcription factors (TFs), such as FOXF1 and TCF21, were higher in HFLF-iMES, HDF-iMES, and HFLF compared with HDF. This suggests that iMES possesses characteristics of lung fibroblasts. Furthermore, EPAS1 was a gene shared between HFLF-iMES and HFLF, but its expression level was higher in HDF than in HFLF, suggesting that it is not a marker specific to lung mesenchyme. Next, we selected the top 5,000 genes in HFLF-iMES and HFLF after 3D culture and created a Venn diagram.As shown in Figure 11(D), 4,220 genes were common, and even among these genes, "Lung development" was enriched (FDR q-value = 0.001). Furthermore, genes annotated to "Lung development" again included HHIP, CCN2, SPARC, BMP4, LAMA5, and LOX, suggesting that these genes are important factors in alveolar organoid formation. Furthermore, the transcription factors FOXF1, TCF21, and EPAS1 also annotated to "Lung development," suggesting that these may be markers of lung fibroblasts.

[0171] (5) Functional Analysis of RSPO2 and RSPO3 in iMES: The present inventors have found that Wnt ligands and antagonists of TGFβ family ligands contribute to the differentiation of type II alveolar epithelial cells. Therefore, we compared the expression of Wnt ligands after 3D culture using the RNA-seq data. The results are shown in Figure 12 and Table 5 below.

[0172]

[0173] Figure 12 and Table 5 show the expression levels (TPM) of Wnt ligands and TGFβ antagonists. As shown in Figure 12 and Table 5, after 3D culture, the expression levels of RSPO2 and RSPO3 were higher in HFLF-iMES, HDF-iMES, and HFLF compared with HDF. Therefore, the role of RSPO2 and RSPO3 in alveolar organoid formation was investigated. We also investigated the role of TGFβ antagonists (FST, FSTL1, FSTL3, and DCN), which were expressed at lower relative levels than in HFLF and HDF but still showed sufficient expression.

[0174] Next, as shown in FIG. ５The isolated CPM-positive lung progenitor cells and each factor (Y-27632 10 μmol / L, CHIR99021 3 μmol / L, RSPO2 200 ng / ml, RSPO3 200 ng / ml, SB431542 10 μmol / L, FST 200 ng / ml, FSTL1 200 ng / ml, FSTL3 200 ng / ml, DCN 200 ng / ml) were seeded onto a 96-well plate (Corning, Cat. No.: 4446) coated with poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich, Cat. No.: 192066) and incubated at 37°C, 5% CO ２ Cell aggregates (spheroids) were formed by culturing the resulting spheroids under these conditions for 24 hours. After centrifugation, the pellet containing the spheroids was gently resuspended in 20 μl of pre-chilled Matrigel and transferred to each well of a 24-well plate (Greiner Bio-One, Cat. No.: 662160). After transfer, the cells were incubated at 37°C for 20 minutes, and then 500 μl of the alveolarization medium supplemented with the aforementioned factors was added to the Matrigel-embedded spheroids. The medium was changed every two days. On day 5 after the initiation of spheroid formation, the cells were dissociated using 0.1% Tripsin-EDTA at 37°C for 15 minutes and then washed twice with PBS containing 1% BSA. The washed cells were immunostained with anti-EPCAM-APC antibody. Thereafter, the ratio of SFTPC-GFP positive cells to EpCAM positive cells under each condition was evaluated by flow cytometry.

[0175] Figure 13 shows the culture method and the analysis results of SFTPC-GFP-positive cells. In Figure 13, (A) shows the culture method, (B) shows the results of flow cytometry analysis, (C) shows the ratio of SFTPC-GFP-positive cells among EpCAM-positive cells, (D) shows the GFP-positive cells in each well, and (E) shows the ratio of SFTPC-GFP-positive cells among EpCAM-positive cells. As shown in Figures 13(B), (C), and (D), GFP-positive cells were confirmed under all conditions after 4 days of culture. As shown in Figure 13(B) and (C), the RSPO2 / SB431542, RSPO3 / SB431542, and RSPO2 / RSPO3 / SB431542 groups increased the ratio of SFTPC-GFP cells among EpCAM-positive cells. In particular, the RSPO2 / RSPO3 / SB431542 group increased the ratio of SFTPC-GFP cells among EpCAM-positive cells to a similar extent as 2i (CHIR99021 / SB431542). As shown in Figure 13(E), none of the TGFβ family ligand antagonists increased the ratio of SFTPC-GFP cells among EpCAM-positive cells. These results suggest that RSPO2 and RSPO3 in iMES contribute to alveolar organoid formation, and that SB431542 contributes to alveolar organoid formation through an endogenous mechanism other than its role in inhibiting the TGFβ pathway. The following endogenous mechanism is presumed to be another mechanism. Because SB431542 is an artificial low-molecular-weight compound, it is presumed that, in a living body environment, it induces type II alveolar epithelial cells through a mechanism other than TGFβ pathway inhibition, and that iMES also induces type II alveolar epithelial cells through a mechanism other than TGFβ pathway inhibition. It is also presumed that iMES secretes substances other than FST that more potently inhibit the TGFβ pathway. The present invention is not limited to this presumption.

[0176] (6) Mesenchymal Cell Types. Different types of mesenchymal cells have been identified in mice (myofibroblasts, mesenchymal alveolar niche cells, Refs. 23-24). Therefore, we analyzed the publicly available scRNA-seq data published in Refs. 23-24 to determine which type of mesenchymal cell iMES resembles. Specifically, we reclustered three types of mesenchymal cells: secondary crest myofibroblasts (SCMFs), Wnt2-Pα cells, and mesenchymal alveolar niche cells (MANCs), and identified genes that were significantly elevated in each cell cluster. Next, we constructed gene sets characteristic of each mesenchymal cell cluster. Using software (biomaRt, https: / / github.com / grimbough / biomaRt, Ref. 25), we converted mouse genes to their corresponding human counterparts and then performed GSEA using the scRNA-seq data from iMES, HFLFs, and HDFs. Specifically, scRNA-seq data were downloaded from GSE149563. Gene expression data normalization, dimensionality reduction, and data visualization were performed using Seurat 4.0.5 and Plotly 4.9.4.1. Upregulated genes in each cluster were identified using the Seurat function FindAllMarkers using a Wilcoxon rank-sum test with a P<0.05 cutoff value, and genes expressed in 25% or more of the cells were extracted. After extraction, the extracted mouse genes were converted to human genes using biomaRt 2.48.3. The results are shown in Figure 14.Reference 23: Zepp, J.A., Morley, M.P., Loebel, C., Kremp, M.M., Chaudhry, F.N., Basil, M.C., Leach, J.P., Liberti, D.C., Niethamer, T.K., Ying, Y., et al. (2021). Genomic, epigenomic, and biophysical cues controlling the emergence of the lung alveolus. Science 371. Reference 24: Zepp, J.A., Zacharias, W.J., Frank, D.B., Cavanaugh, C.A., Zhou, S., Morley, M.P., and Morrisey, E.E. (2017). Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell 170, 1134-1148 e1110. Reference 25: Durinck, S., Spellman, P.T., Birney, E., and Huber, W. (2009). Mapping identifiers for the integration of genomic datasets with the R / Bioconductor package biomaRt. Nat Protoc 4, 1184-1191.。

[0177] Figure 14 shows the results of cluster analysis of each mesenchymal cell. As shown in Figures 14(A)-(C), the mesenchymal cell-specific markers were consistent with those described in References 23 and 24. Furthermore, as shown in Figure 14(D), Wnt2 was highly expressed in the Wnt2-Pα cluster, Stc1 in the SCMF cluster, and Mfap5 in the MANC cluster. Furthermore, as shown in Figure 14(E), the SCMF gene set was enriched in iMES compared to HFLF and HDF after 3D culture. Meanwhile, as shown in Figure 14(E), the MANC gene set was enriched in HDF, and the Wnt2-Pα gene set was enriched in HFLF. Furthermore, as shown in Figure 14(F), STC1 was highly expressed in iMES, WNT2 in HFLF, and MFAP5 in HDF after 3D culture. Furthermore, as shown in Figure 14(G), immunofluorescence staining detected STC1-positive and VIM-positive cells in iMES after alveolar organoid formation, but some epithelial cells were also stained. These results suggest that "muscle system processes" were enriched in iMES after 3D culture compared to before 3D culture, suggesting that they acquired characteristics of mesenchymal cells of the muscle system (SCMF) during 3D culture. Furthermore, as shown in Figure 14(H), "canonical Wnt signaling pathways" were enriched in HFLF after 3D culture compared to before 3D culture, suggesting that they acquired characteristics of mesenchymal cells, such as Wnt2-Pα.

[0178] From the above, we found that alveolar organoids can be formed using iMES, that iMES contributes to the differentiation of alveolar epithelial cells through at least one of RSPO2 and RSPO3, and that the gene expression profile of iMES is similar to that of SCMF.

[0179] [Example 3] It was confirmed that alveolar epithelial cells can be maintained and cultured using iMES.

[0180] Alveolar organoids were formed in the same manner as in Example 1(3). SFTPC-GFP-positive type II alveolar epithelial cells were isolated from the obtained alveolar organoids, and an investigation was conducted into whether they could be maintained and cultured in iMES. Specifically, this was carried out as shown in Figure 15(A). First, alveolar organoids obtained in the same manner as in Example 1(3) were converted into single cells using PBS containing 0.1% Trypsin-EDTA. Immunostaining was performed using an anti-EPCAM-APC antibody (Miltenyi Biotec, Cat No.: 130-113-263), and SFTPC-GFP+ / EPCAM+ cells were collected using FACS. After the collection, 1 x 10 of the collected type II alveolar epithelial cells were collected. ５ cells and iMES 5 × 10 5 The cells were mixed with 100 μl of alveolarization medium supplemented with Y-27632 (10 μmol / L) and 100 μl of Matrigel. After mixing, the cells were embedded in 3D in a 12-well cell culture insert and cultured for 14 days (P0). The alveolarization medium was used for the culture. After the culture, SFTPC-GFP-positive cells were collected, the number of type II alveolar epithelial cells was counted, and the cells were cultured with iMES under the same conditions (P1). The same passages were repeated (P2 to P3). The passages were performed every two weeks. The cells at P0 to P3 were analyzed for SFTPC-GFP-positive cells / EPCAM-positive cells using a flow cytometer (FACS) as described in Example 1(3). Furthermore, for cells at P0 to P3, the expression of SFTPB, SFTPC, SFTPD, ABCA3, SFTPA2, SLC34A2, HOPX, AGER, and AQP5 was quantified in the same manner as in Example 1(1). Furthermore, in the same manner as in Example 1(3), alveolar organoids were stained with primary and secondary antibodies against Mature-GFP, PDPN, and HT1-56.

[0181] In addition, electron microscopy was performed to confirm the presence of lamellar structures in type II alveolar epithelial cells. Specifically, small pieces of alveolar organoids obtained from each culture were incubated overnight at 4°C in a fixative consisting of 2.5% glutaraldehyde, 4% paraformaldehyde, 1% tannic acid, and 0.1 mol / L phosphate buffer (pH 7.4). The next day, the fixative was changed to one without tannic acid. Specifically, the sections were washed three times for 20 minutes with 0.1 mol / L phosphate buffer (pH 7.4). After washing, the sections were fixed with 1% osmium tetroxide for 2 hours, gradually dehydrated, and embedded in pure Epon as previously described (see Reference 12). After embedding, ultrathin sections were stained with uranyl acetate and lead citrate and analyzed using a transmission electron microscope (JEOL; JEM-1400). These results are shown in Figure 15.

[0182] Figure 15 shows the results of passaging type II alveolar epithelial cells. As shown in Figure 15(B), EpCAM-positive cells increased linearly from P0 to P3. Furthermore, as shown in Figures 15(C) and (D), SFTPC-GFP-positive cells increased at the first passage, and then significantly increased from P0 to P2, reaching a plateau. Furthermore, as shown in Figure 15(E), ABCA3, SLC34A2 (type II alveolar epithelial cell markers), and HOPX (type II alveolar epithelial cell marker) significantly increased from P0 to P3, indicating that not only type II alveolar epithelial cells but also type I alveolar epithelial cells had matured. Furthermore, the expression of other alveolar epithelial cell markers, SFTPB, SFTPD2, SFTPA2, AGER, and AQP5, was maintained during passage, indicating that the various epithelial cells that form alveolar organoids were maintained. Furthermore, as shown in Figure 15(F), both SFTPC-GFP-positive type II alveolar epithelial cells and PDPN-positive, HT1-56-positive type I alveolar epithelial cells were observed in the P2 alveolar organoids. Furthermore, as shown in Figure 15(G), lamellar bodies, which are structures specific to type II alveolar epithelial cells, were confirmed in the alveolar organoids. These findings demonstrate that iMES can maintain and culture type II alveolar epithelial cells, which are lung tissue stem cells, and can induce differentiation of the various epithelial cells that make up alveolar organoids.

[0183] Example 4 The ligand-target and ligand-receptor interactions between iMES and alveolar epithelial cells were analyzed.

[0184] Using the alveolar organoids obtained after P2 culture in Example 3, we analyzed ligand-target and ligand-receptor interactions, which are important for interactions with alveolar epithelial cells. Specifically, we performed scRNA-seq analysis on iMES after P2 culture. Single-cell RNA libraries were prepared from iMES, HFLF, and HDF using a 10xgenomics Chromium device according to the accompanying protocol (Single Cell 3' Reagent Kits v3.1). The resulting libraries were sequenced using NovaSeq 6000 (Illumina). The resulting reads were then mapped to GRCh38, and a count matrix was created using Cell Ranger. The resulting single-cell data were processed using software (Seurat 4.0.4, Reference 26). During this processing, dead and low-quality cells were excluded by deleting data from cells expressing mitochondrial genes at more than 20% and less than 1.5%. To exclude cell doublets and low-quality cells, we also removed cells with UMIs greater than 140,000 or less than 5,000 and fewer than 2,000 expressed genes. The UMI counts were then normalized using SCTransform. The resulting data were then subjected to principal component (PC) analysis using the Seurat function RunPCA, and embedded into a UMAP with 17 PCs and a resolution of 1 using the Seurat function RunUMAP. UMAP plots were visualized using Plotly 4.9.4.1, and violin plots were drawn using Seurat. Analysis of ligand activity and prediction of active ligands, their target genes, and receptors were performed using software (nichenetr 1.0.0, 27). Trajectory inference among epithelial cells, including type I alveolar epithelial cells, type II alveolar epithelial cells, ASCL1-positive cells, and ciliated cells, was performed using software (monocle3 1.0.0, 28). The results are shown in Figure 16.References 26: Hao , Y. , Hao , S. , Andersen-Nissen , E. , Mauck , WM , 3rd , Zheng , S. , Butler , A. , Lee , MJ , Wilk , AJ , Darby , C. , Zager , M. , et al. (2021). Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587–3529. References 27: Browaeys, R., Saelens, W., & Saeys, Y. (2020). NicheNet: modeling intercellular communication by linking ligands to target genes. Nat Methods 17, 159–162. References28:Cao , J. , Spielmann , M. , Qiu , X. , Huang , X. , Ibrahim , DM , Hill , AJ , Zhang , F. , Mundlos , S. , Christiansen , L. , Steemers , FJ , et al. (2019). The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496–502.

[0185] Figure 16 shows the results of cluster analysis of scRNA-seq analysis. As shown in Figure 16(A), alveolar epithelial cells and iMES were separated by high expression of NKX2-1 and COL1A1, respectively. Furthermore, as shown in Figure 16(B), the cluster analysis results indicated that the alveolar organoids after P2 culture were annotated into 15 clusters. Each cluster was determined to represent the following cell clusters based on the genes highly expressed in that cluster. Specifically, as shown in Figure 16(C), cluster 12 was considered to represent type I alveolar epithelial cells due to high expression of AGER and CAV1. Clusters 1, 8, and 14 were considered to represent type II alveolar epithelial cells due to high expression of SFTPC. Regarding other epithelial cell clusters, cluster 5 was considered to represent ASCL1-positive cells, clusters 2 and 7 were considered to represent SOX9-positive cells, cluster 6 was considered to represent SOX2-positive cells, and cluster 0 was considered to represent cells undergoing cell division due to expression of MKI67. Cluster 13 showed high expression levels of FOXJ1, SNTN, and SFTPC, indicating that SFTPC-positive distal tip cells were differentiating into ciliated cells. Meanwhile, as shown in Figure 16(D), iMES were divided into five clusters. Specifically, as shown in Figure 16(B), cluster 9 was STC1-positive iMES, cluster 4 was FSTL1-positive iMES, cluster 10 was THY1-positive iMES, cluster 3 was WT1-positive iMES, and cluster 11 was considered to be iMES undergoing cell division. Furthermore, among the genes identified in the transcriptome of iMES after 3D culture, FOXF1, RSPO2, and RSPO3 were examined. FOXF1-positive and RSPO3-positive iMES were widely distributed in the mesenchymal cluster. On the other hand, as shown in FIG. 16(E), RSPO2 was specifically expressed in STC1-positive iMES.

[0186] Next, we analyzed intercellular communication between iMES and alveolar epithelial cells using software (NicheNet 1.0.0, https: / / github.com / saeyslab / nichenetr, see Reference 27). For this analysis, gene sets for type I alveolar epithelial cells (AT1) and type II alveolar epithelial cells (AT2) were defined as shown in Table 6. Representative genes for type I alveolar epithelial cells (AT1) and type II alveolar epithelial cells (AT2) were obtained from the Lung Gene Expression Analysis Web Portal (Reference 29). Representative lung genes listed using the Seurat function FindAllMarkers (adjusted P value < 0.05) and common genes with elevated expression in the AT1 and AT2 clusters were extracted. Among these genes, NAMPT, HMGB1, and TGFB1, expressed in iMES, were highly ranked for ligand activity and broadly covered representative genes for AT1. Furthermore, in AT2 cells, TGFB2, HAS2, and CTF1 were speculated to broadly regulate representative AT2 genes. These iMES-derived ligands (iMES-ligands) were expressed on various clusters of iMES, suggesting that each type of iMES acts cooperatively during the development of alveolar epithelial cells. NicheNet was also used to infer ligand-receptor interactions, considering only those reported in the literature and public databases. Reference 29: Du, Y., Ouyang, W., Kitzmiller, JA, Guo, M., Zhao, S., Whitsett, JA, and Xu, Y. (2021). Lung Gene Expression Analysis Web Portal Version 3: Lung-at-a-Glance. Am J Respir Cell Mol Biol 64, 146-149.

[0187]

[0188] Figure 17 shows the results of ligand-receptor interactions. As shown in Figure 17(A), in type I alveolar epithelial cells, NAMPT and TGFB1 interacted with INSR and TGFBR1 / 2 / 3, respectively. Furthermore, as shown in Figure 17(B), in type II alveolar epithelial cells, TGFB2, HAS2, and CTF1 interacted with TGFBR1 / 2 / 3, CD44, and IL6ST / LIFR, respectively. These results support the idea that ligands expressed in iMES interact with alveolar epithelial cells and are involved in the expression of each target marker gene. Furthermore, as shown in Figure 17(C), when the differentiation trajectories of type I pneumocytes, type II pneumocytes, ASCL1-positive cells, and ciliated pneumocytes were estimated, type I pneumocytes, ASCL1-positive cells, and ciliated pneumocytes branched around the cluster of type II pneumocytes, suggesting that they are cells derived from type II pneumocytes, i.e., cells differentiated from type II pneumocytes.

[0189] Example 5 It was confirmed that pulmonary mesenchymal cells can be efficiently induced from mesodermal cells by combining a mesenchymal cell induction factor with KGF and FGF10.

[0190] (1) Induction of Pulmonary Mesenchymal Cells Pulmonary mesenchymal cells were induced from the mesodermal cells in the same manner as in Example 1(1) above, except that any one of activin A (AA), KGF, BMP4, FGF2, and FGF10 was omitted.

[0191] (2) Induction of Lung Progenitor Cells The induction of lung progenitor cells was carried out in the same manner as in Example 1(2) above, except that the B2-3 strain was used as iPSCs.

[0192] (3) Alveolar organoid formation assay: Alveolar organoids were formed in the same manner as in Example 1 (3), except that the pulmonary mesenchymal cells of Example 5 (1) and the pulmonary progenitor cells of Example 5 (2) were used. The cells constituting the obtained alveolar organoids were then isolated, and SFTPC-GFP-positive cells / EPCAM-positive cells were analyzed in the same manner as in Example 1 (3). The results are shown in Figure 18.

[0193] Figure 18 is a graph showing the results for SFTPC-GFP-positive cells / EPCAM-positive cells. Figure 18(A) shows the results of flow cytometry analysis, and (B) shows the proportion of SFTPC-GFP-positive cells among EpCAM-positive cells in alveolar organoids. In Figure 18(B), the horizontal axis indicates the added factor (AKB210) or the removed factor (-X), and the vertical axis indicates the proportion of SFTPC-GFP-positive cells / EPCAM-positive cells. Note that AKB210 represents the case where activin A, KGF, BMP4, FGF2, and FGF10 were all added. As shown in Figures 18(A) and (B), the removal of any factor reduced the induction efficiency of alveolar epithelial cells, indicating that pulmonary mesenchymal cells can be efficiently induced by combining mesenchymal cell induction factors with KGF and FGF10.

[0194] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0195] This application claims priority based on Japanese Patent Application No. 2022-014212, filed February 1, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0196] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendixes. <Method for producing pulmonary mesenchymal cells> (Appendix 1) A method for producing pulmonary mesenchymal cells, comprising the step of culturing mesodermal cells in the presence of a mesenchymal cell inducer, KGF, and FGF10, to induce differentiation into pulmonary mesenchymal cells. (Appendix 2) A method for producing pulmonary mesenchymal cells according to Appendix 1, comprising the step of enriching EpCAM- and / or E-cadherin-negative pulmonary mesenchymal cells from a cell population induced from the mesodermal cells. (Appendix 3) A method for producing pulmonary mesenchymal cells according to Appendix 2, wherein the enrichment is to enrich for a cell population containing 50% or more EpCAM- and / or E-cadherin-negative pulmonary mesenchymal cells. (Appendix 4) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 3, wherein the pulmonary mesenchymal cells express RSPO2 (R-spondin 2) and / or RSPO3 (R-spondin 3). (Appendix 5) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 4, wherein the pulmonary mesenchymal cells express a transcription factor selected from the group consisting of Forkhead box protein F1 (FOXF1), Transcription factor 21 (TCF21), T-Box Transcription Factor 4 (TBX4), and Odd-Skipped Related Transcription Factor (OSR1). (Appendix 6) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 3, wherein the pulmonary mesenchymal cells express at least one factor selected from the group consisting of RSPO2 (R-spondin 2), RSPO3 (R-spondin 3), FOXF1 (Forkhead box protein F1), TCF21 (Transcription factor 21), TBX4 (T-Box Transcription Factor 4), and OSR1 (Odd-Skipped Related Transcription Factor). (Appendix 7) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 6, wherein the pulmonary mesenchymal cells do not express WNT2.(Appendix 8) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 7, wherein the pulmonary mesenchymal cells do not express TBXT (T-box transcription factor T). (Appendix 9) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 8, wherein the pulmonary mesenchymal cells express a fibroblast marker selected from the group consisting of NCAM (neural cell adhesion molecule), ADRP (Adipose differentiation-related protein), COL1A1 (Collagen, type I, alpha 1), and ACTA2 (actin alpha 2). (Appendix 10) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 9, wherein the pulmonary mesenchymal cells are positive for a mesenchymal cell marker selected from the group consisting of VIM (Vimentin), THY1 (Thy-1 Cell Surface Antigen, CD90), PDGFRα (Platelet Derived Growth Factor Receptor α), and KDR (Kinase Insert Domain Receptor). (Appendix 11) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 10, wherein the mesenchymal cell induction factor comprises a factor selected from the group consisting of activin A, FGF2, and BMP4. (Appendix 12) The method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 11, wherein the pulmonary mesenchymal cells are capable of inducing type I alveolar epithelial cells and / or type II alveolar epithelial cells from pulmonary progenitor cells in an alveolar organoid formation assay by co-culture with the pulmonary mesenchymal cells. (Appendix 13) The method for producing mesenchymal cells according to any one of Appendices 1 to 12, comprising, prior to inducing the pulmonary mesenchymal cells, a step of culturing pluripotent cells in the presence of a mesoderm induction factor to induce differentiation into the mesoderm cells. (Appendix 14) The method for producing mesenchymal cells according to Appendices 13, wherein the mesoderm induction factor comprises a factor selected from the group consisting of a GSK3β inhibitor, activin A, and BMP4. (Appendix 15) The method for producing mesenchymal cells according to Appendix 14, wherein the GSK3β inhibitor is CHIR99021.<Pulmonary Mesenchymal Cells> (Appendix 16) A cell population containing mesenchymal cells, comprising pulmonary mesenchymal cells expressing RSPO2 (R-Spondin 2) and / or RSPO3 (R-Spondin 3). (Appendix 17) The cell population according to Appendix 16, wherein the pulmonary mesenchymal cells express a transcription factor selected from the group consisting of Forkhead box protein F1 (FOXF1), Transcription factor 21 (TCF21), T-Box Transcription Factor 4 (TBX4), and Odd-Skipped Related Transcription Factor (OSR1). (Appendix 18) The cell population according to Appendix 16 or 17, wherein the pulmonary mesenchymal cells express RSPO2 and RSPO3. (Appendix 19) A cell population containing mesenchymal cells, comprising pulmonary mesenchymal cells expressing at least one transcription factor selected from the group consisting of Forkhead box protein F1 (FOXF1), Transcription factor 21 (TCF21), T-Box Transcription Factor 4 (TBX4), and Odd-Skipped Related Transcription Factor (OSR1). (Appendix 20) The cell population according to any of Appendices 16 to 19, wherein the pulmonary mesenchymal cells are EpCAM and / or E-cadherin negative. (Appendix 21) The cell population according to Appendices 20, wherein the proportion (cell number) of EpCAM and / or E-cadherin negative pulmonary mesenchymal cells to the total cells of the cell population is 50% or more. (Appendix 22) The cell population according to any of Appendices 16 to 21, wherein the pulmonary mesenchymal cells do not express Wnt2. (Appendix 23) The cell population described in any one of Appendices 16 to 22, wherein the pulmonary mesenchymal cells do not express TBXT (T-box transcription factor T).(Appendix 24) The cell population according to any one of Appendices 16 to 23, wherein the pulmonary mesenchymal cells express at least one fibroblast marker selected from the group consisting of NCAM (neural cell adhesion molecule), ADRP (Adipose differentiation-related protein), COL1A1 (Collagen, type I, alpha 1), and ACTA2 (actin alpha 2). (Appendix 25) The cell population according to any one of Appendices 16 to 24, wherein the pulmonary mesenchymal cells are positive for at least one mesenchymal cell marker selected from the group consisting of VIM (Vimentin), THY1 (Thy-1 Cell Surface Antigen, CD90), PDGFRα (Platelet-Derived Growth Factor Receptor α), and KDR (Kinase Insert Domain Receptor). (Appendix 26) The cell population according to any one of Appendices 16 to 25, wherein the pulmonary mesenchymal cells are capable of inducing type I alveolar epithelial cells and / or type II alveolar epithelial cells from the pulmonary progenitor cells in an alveolar organoid formation assay by co-culture with pulmonary progenitor cells. <Method for producing alveolar epithelial cells> (Appendix 27) A method for producing pulmonary epithelial cells, comprising a step of culturing pulmonary progenitor cells in the presence of pulmonary mesenchymal cells to induce differentiation into alveolar epithelial cells, wherein the pulmonary mesenchymal cells are a cell population containing pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 15 and / or the mesenchymal cells according to any one of Appendices 16 to 26. (Appendix 28) The production method according to Appendice 27, wherein the alveolar epithelial cells are type I alveolar epithelial cells and / or type II alveolar epithelial cells. (Appendix 29) The production method according to Appendices 27 or 28, wherein the alveolar epithelial cells are alveolar epithelial cells that constitute alveolar organoids. (Appendix 30) The method of production according to any one of Appendices 27 to 29, wherein the lung progenitor cells are NKX2-1 positive and / or CPM positive. (Appendix 31) The method of production according to any one of Appendices 27 to 30, wherein the lung progenitor cells are cultured in the presence of an inducer of the lung mesenchymal cells and alveolar epithelial cells to induce differentiation into the alveolar epithelial cells.(Appendix 32) The method for producing type II alveolar epithelial cells according to Appendix 31, wherein the inducer of alveolar epithelial cells is a Wnt promoter, a steroid, a cAMP derivative, a phosphodiesterase inhibitor, KGF, a GSK3β inhibitor, a TGFβ inhibitor, a ROCK inhibitor, and / or FGF10. <Pharmaceutical Composition> (Appendix 33) A pharmaceutical composition comprising pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any of Appendices 1 to 15 and / or a cell population containing the mesenchymal cells according to any of Appendices 16 to 26, and a pharmaceutically acceptable carrier. <Method for Maintaining and Culturing Type II Alveolar Epithelial Cells> (Appendix 34) A method for maintaining and culturing type II alveolar epithelial cells, comprising a step of culturing and maintaining type II alveolar epithelial cells in the presence of pulmonary mesenchymal cells, wherein the pulmonary mesenchymal cells are a cell population containing pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any of Appendices 1 to 15 and / or the mesenchymal cells according to any of Appendices 16 to 26. (Appendix 35) The maintenance culture method according to Appendix 34, wherein the type II alveolar epithelial cells are SFTPC-positive cells. (Appendix 36) The maintenance culture method according to Appendix 34 or 35, wherein the type II alveolar epithelial cells are cultured in the presence of an inducer of the pulmonary mesenchymal cells and alveolar epithelial cells for maintenance culture. (Appendix 37) The maintenance culture method according to Appendix 36, wherein the inducer of the alveolar epithelial cells is a Wnt promoter, a steroid, a cAMP derivative, a phosphodiesterase inhibitor, KGF, a GSK3β inhibitor, a TGFβ inhibitor, a ROCK inhibitor, FGF10, and / or EGF. <Method for expanding type II alveolar epithelial cells> (Appendix 38) A method for expanding type II alveolar epithelial cells, comprising the step of culturing type II alveolar epithelial cells in the presence of pulmonary mesenchymal cells to perform expansion, wherein the pulmonary mesenchymal cells are a cell population containing pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any one of Appendices 1 to 15 and / or the mesenchymal cells according to any one of Appendices 16 to 26. (Appendix 39) The expansion method according to Appendix 38, wherein the type II alveolar epithelial cells are SFTPC-positive cells. (Appendix 40) The expansion method according to Appendix 38 or 39, wherein the type II alveolar epithelial cells are cultured in the presence of an inducer for the pulmonary mesenchymal cells and alveolar epithelial cells to perform expansion.(Appendix 41) The expansion culture method according to Appendix 40, wherein the alveolar epithelial cell inducing factor is a Wnt promoter, a steroid, a cAMP derivative, a phosphodiesterase inhibitor, KGF, a GSK3β inhibitor, a TGFβ inhibitor, a ROCK inhibitor, FGF10, and / or EGF. <Culture Medium> (Appendix 42) A culture medium for inducing pulmonary mesenchymal cells from mesodermal cells, comprising a culture medium (basal medium), a mesenchymal cell inducing factor, KGF, and FGF10. (Appendix 43) The culture medium according to Appendix 42, wherein the mesenchymal cell inducing factor comprises a factor selected from the group consisting of activin A, FGF2, and BMP4. <Kit> (Appendix 44) A kit for inducing pulmonary mesenchymal cells from mesodermal cells, comprising a mesenchymal cell inducing factor, KGF, and FGF10. (Appendix 45) The kit according to Appendix 44, wherein the mesenchymal cell induction factor comprises a factor selected from the group consisting of activin A, FGF2, and BMP4.

[0197] As described above, the present disclosure enables the production of pulmonary mesenchymal cells that can also be used to create alveolar organoids. Therefore, the present disclosure is extremely useful in, for example, the fields of regenerative medicine and cell therapy.

Claims

1. A method for producing pulmonary mesenchymal cells, comprising the step of culturing mesodermal cells in the presence of a mesenchymal cell inducer, KGF, and FGF10, to induce differentiation into pulmonary mesenchymal cells.

2. The method for producing pulmonary mesenchymal cells according to claim 1, comprising a step of enriching EpCAM and / or E-cadherin negative pulmonary mesenchymal cells from a cell population derived from said mesodermal cells.

3. The method for producing pulmonary mesenchymal cells according to claim 2, wherein the enrichment is to enrich the cell population to contain EpCAM and / or E-cadherin negative pulmonary mesenchymal cells at 50% or more.

4. 4. The method for producing pulmonary mesenchymal cells according to claim 1, wherein the pulmonary mesenchymal cells express at least one factor selected from the group consisting of R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), Forkhead box protein F1 (FOXF1), Transcription factor 21 (TCF21), T-box transcription factor 4 (TBX4), and Odd-skipped related transcription factor (OSR1).

5. The method for producing pulmonary mesenchymal cells according to any one of claims 1 to 3, wherein the mesenchymal cell induction factor comprises a factor selected from the group consisting of activin A, FGF2, and BMP4.

6. The method for producing mesenchymal cells according to any one of claims 1 to 3, comprising, prior to inducing the pulmonary mesenchymal cells, a step of culturing pluripotent cells in the presence of a mesoderm induction factor to induce differentiation into the mesoderm cells.

7. A cell population containing mesenchymal cells, including lung mesenchymal cells that express RSPO2 (R-Spondin 2) and / or RSPO3 (R-Spondin 3).

8. A cell population containing mesenchymal cells, comprising pulmonary mesenchymal cells that express at least one transcription factor selected from the group consisting of FOXF1 (Forkhead box protein F1), TCF21 (Transcription factor 21), TBX4 (T-Box Transcription Factor 4), and OSR1 (Odd-Skipped Related Transcription Factor).

9. The cell population of claim 7 or 8, wherein the pulmonary mesenchymal cells are EpCAM-negative.

10. The cell population according to claim 7 or 8, wherein the pulmonary mesenchymal cells are capable of inducing type I alveolar epithelial cells and / or type II alveolar epithelial cells from pulmonary progenitor cells in an alveolar organoid formation assay by co-culture with the pulmonary progenitor cells.

11. The method comprises culturing lung progenitor cells in the presence of lung mesenchymal cells to induce differentiation into alveolar epithelial cells, A method for producing pulmonary epithelial cells, wherein the pulmonary mesenchymal cells are pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any one of claims 1 to 3 and / or a cell population comprising the mesenchymal cells according to claim 7 or 8.

12. Culturing type II alveolar epithelial cells in the presence of pulmonary mesenchymal cells and expanding the cells, 9. A method for expanding type II alveolar epithelial cells, wherein the pulmonary mesenchymal cells are a cell population comprising pulmonary mesenchymal cells obtained by the method for producing pulmonary mesenchymal cells according to any one of claims 1 to 3 and / or the mesenchymal cells according to claim 7 or 8.