Bone marrow stroma organoid production method, bone marrow stroma organoid, and use thereof

Abstract

The present invention provides a novel method for producing a bone marrow stroma organoid. A method for producing a bone marrow stroma organoid in accordance with an embodiment of the present invention is for producing a bone marrow stroma organoid from pluripotent stem cells, said method comprising: (1) a step A for performing mesoderm induction from pluripotent stem cells; and (2) a step B for culturing a cell mass obtained in step A in a culture medium that is obtained by adding blood serum or a blood serum substitute to a culture medium for proliferation / differentiation of hematopoietic stem cells.

Description

Method for producing bone marrow stromal organoids, bone marrow stromal organoids, and uses thereof

[0001] The present invention relates to a method for producing bone marrow stromal organoids, bone marrow stromal organoids, and uses thereof.

[0002] Organoids are three-dimensional organ-like structures created in vitro, also known as mini-organs. Because organoids can reproduce certain specific functions of the corresponding organs, they are being considered for application in, for example, developmental biology, medical and pharmaceutical research, and regenerative medicine.

[0003] Numerous attempts to induce various organoids from pluripotent stem cells have been reported, including, for example, brain organoids, cerebellar organoids, inner ear organoids, thyroid organoids, thymus organoids, testicular organoids, liver organoids, pancreatic organoids, intestinal organoids, epithelial organoids, lung organoids, cardiac organoids, kidney organoids, etc. These organoids are generally produced by three-dimensional culture in a three-dimensional medium such as hydrogel / Matrigel, or on hydrogel / Matrigel, laminin gel, etc.

[0004] On the other hand, there have been only a few reports on the production of bone marrow-related organoids at present. Non-Patent Document 1 describes the production of bone marrow-like organoids from human induced pluripotent stem cells (iPS cells).

[0005] Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov 13, 364-85, 2023

[0006] However, the method for producing bone marrow-like organoids described in Non-Patent Document 1 is a complicated method that involves a step of embedding the cultured material in an ECM (extracellular matrix) containing collagen and Matrigel. Furthermore, partly due to the embedding step, scalable culture is not easy.

[0007] In view of the above-mentioned problems, one aspect of the present invention aims to realize a method for efficiently producing bone marrow organoids using a simple technique.

[0008] In order to solve the above problems, the present inventors have conducted extensive research and have found that, unlike the approach of producing bone marrow organoids from the viewpoint of realizing a structure and function as close as possible to that of the entire corresponding organ, by adopting an approach of producing bone marrow organoids that are specialized for a portion of the corresponding organ, bone marrow organoids with the required properties can be reproducibly obtained, leading to the invention.

[0009] 1) Bone marrow stromal organoids derived from pluripotent stem cells. 2) A method for producing bone marrow stromal organoids from pluripotent stem cells, comprising: (1) step A of inducing mesoderm from pluripotent stem cells; and (2) step B of culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells supplemented with serum or a serum substitute. 3) A method for producing mesenchymal stromal cells, comprising a step of isolating bone marrow mesenchymal stromal cells from bone marrow stromal organoids produced by the method described in 2) above. 4) Bone marrow mesenchymal stromal cells produced by the method described in 3) above. 5) A method for producing blood cells, comprising a step of co-culturing hematopoietic stem cells / hematopoietic progenitor cells with the bone marrow mesenchymal stromal cells described in 4) above as feeder cells.

[0010] According to one aspect of the present invention, myeloid organoids can be efficiently produced from pluripotent stem cells using a simple method.

[0011] FIG. 1 is a schematic diagram showing an outline of the process for producing coral-like induced bone marrow stromal organoids (CLiBMSO) in one example of the present invention. FIG. 2 is a diagram showing microscopic images of CLiBMSO obtained in one example of the present invention. The left image shows a microscopic image (appearance) of CLiBMSO, and the right image shows a microscopic image (hematoxylin-eosin (HE)-stained image) of a section of CLiBMSO. FIG. 3 is a diagram showing microscopic images of CLiBMSO obtained in one example of the present invention, immunofluorescently stained with VE cad antibody and CD271 antibody. FIG. 4 is a diagram showing microscopic images of CLiBMSO obtained in one example of the present invention, immunofluorescently stained with UEA1 antibody and CD71 antibody. FIG. 5 is a diagram showing microscopic images of CLiBMSO obtained in one example of the present invention, immunostained with CD31 antibody and immunostained with CD34 antibody, respectively. FIG. 6 relates to one example of the present invention, and is a diagram showing a comparison of the expression of representative genes expressed in human bone marrow among bone marrow stromal organoids, human iPS cells, and human bone marrow as a result of RNA-seq analysis. FIG. 7 shows the results of flow cytometry analysis of CLiBMSO obtained in one example of the present invention dispersed into single cells. FIG. 8 shows another result of flow cytometry analysis of CLiBMSO obtained in one example of the present invention dispersed into single cells. FIG. 9 shows yet another result of flow cytometry analysis of CLiBMSO obtained in one example of the present invention dispersed into single cells. FIG. 10 shows the results of establishment of bone marrow mesenchymal stromal cells and the results of immunofluorescent staining of bone marrow mesenchymal stromal cells obtained by subculture. FIG. 11 shows the results of an osteogenic differentiation induction assay in one example of the present invention. FIG. 12 shows the results of an adipogenic differentiation induction assay in one example of the present invention. FIG. 13 shows the results of a chondrogenic differentiation induction assay in one example of the present invention. FIG. 14 shows the results (day 6 and day 10) of differentiation of T cells and dendritic cells (DCs) from hematopoietic stem cells / hematopoietic progenitor cells derived from human iPSCs by co-culture on CLiBM-MSC feeder cells.Figure 15 shows the results (day 6) of differentiation of CD8A single-positive T cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 16 shows the results (heat map) of array PCR of gene expression in blood cells differentiated from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 17 shows the results (day 6 and day 13) of differentiation of CD86-positive dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 18 shows the results (day 6 and day 13) of differentiation of HLA-DR-positive dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 19 shows the results (day 6 and day 13) of differentiation of CD4-positive T cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 20 shows the results (Giemsa staining results on day 6 and day 13) of differentiation of various blood lineage cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. FIG. 21 shows micrographs of human ES cell-derived coral-like bone marrow stromal organoids (CLeBMSO) obtained in one example of the present invention. The left image shows a micrograph (appearance) of CLeBMSO, and the right image shows a micrograph (hematoxylin-eosin (HE)-stained image) of a section of CLeBMSO. FIG. 22 shows micrographs of fluorescent immunostaining of CLeBMSO obtained in one example of the present invention using VEcad antibody and CD271 antibody. FIG. 23 shows micrographs of fluorescent immunostaining of CLeBMSO obtained in one example of the present invention using UEA1 antibody and CD235a antibody. FIG. 24 shows the results of flow cytometry analysis of CLeBMSO obtained in one example of the present invention, which were dispersed into single cells.Fig. 25 shows the results of establishing bone marrow mesenchymal stromal cells derived from CLeBMSO obtained in one example of the present invention, and Fig. 26 shows the results of flow cytometry analysis of cells differentiated by co-culture of iPS-derived hematopoietic stem cells with ES-derived bone marrow stromal feeder cells.

[0012] Hereinafter, one embodiment of the present invention will be described in detail.

[0013] 1. Method for Producing Bone Marrow Stromal Organoids (Summary of the Invention) A method for producing bone marrow stromal organoids according to one embodiment of the present invention is a method for producing bone marrow stromal organoids from pluripotent stem cells, and comprises the following steps A and B: (1) Step A: Inducing mesoderm from pluripotent stem cells. (2) Step B: Culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells supplemented with serum or a serum substitute.

[0014] (Example of Effect of the Invention) According to a method for producing bone marrow stromal organoids according to one embodiment of the present invention, 1) bone marrow stromal organoids can be produced from pluripotent stem cells using a very simple method, and 2) the proportion of cellular components in the produced bone marrow stromal organoids is high (compared to, for example, the method of Non-Patent Document 1), thereby increasing the efficiency of cell utilization. For example, bone marrow stromal organoids containing a relatively large amount of bone marrow mesenchymal stromal cells can be obtained. Furthermore, as described in detail below, the method for producing bone marrow stromal organoids according to one embodiment of the present invention may also achieve the following effects compared to the method described in Non-Patent Document 1: 1) Fewer types of cytokines are required. 2) The step of embedding in ECM is unnecessary, and ECM is not even required in the first place. In other words, one embodiment of the present invention is a method for producing bone marrow organoids using a more convenient method than the method described in Non-Patent Document 1, and is also compatible with scalable culture.

[0015] (Bone marrow stromal organoid) In one embodiment of the present invention, bone marrow stromal organoids are organ-like structures resembling bone marrow that are created in vitro in a three-dimensional (3D) fashion. Bone marrow stromal organoids are aggregates formed by a large number of cells specific to the bone marrow stroma, vascular cells, and some hematopoietic stem cells, and exhibit at least one of the specific functions of bone marrow stromal cells. Specific functions of bone marrow stromal cells include, for example, 1) supporting blood cells, 2) being usable as a site for inducing differentiation of hematopoietic stem cells (HSC) / hematopoietic progenitor cells (HPC) into blood cells, and 3) containing a cell portion capable of differentiating into at least one cell selected from the group consisting of osteocytes, chondrocytes, adipocytes, and skeletal muscle cells. Other characteristics that bone marrow stromal organoids in one embodiment of the present invention may possess are described in the section [2. Bone marrow stromal organoids] below.

[0016] (Step A) In the method for producing bone marrow stromal organoids according to one embodiment of the present invention, step A refers to a “step of inducing mesoderm from pluripotent stem cells.” Step A is carried out in vitro.

[0017] -Pluripotent Stem Cells- As used herein, "pluripotent stem cells" refer to undifferentiated cells that have pluripotency and the ability to proliferate undifferentiatedly depending on the culture conditions. As used herein, "pluripotency" refers to the ability of cells to differentiate into all germ layers (i.e., ectoderm, mesoderm, and endoderm) that constitute an individual. Examples of "pluripotent stem cells" include embryonic stem cells (ES cells) and iPS cells from mammals such as humans. Pluripotent stem cells may be subcultured. The medium used for subculture may be, for example, a medium for the undifferentiated proliferation of ES cells or iPS cells. In one example, the medium is feeder-free and / or serum-free, preferably feeder-free and serum-free. The medium may contain additives such as a Rock inhibitor.

[0018] - iPS cells - As used herein, "iPS cells" refer to cells that have properties similar to embryonic stem cells (ES cells) and are obtained by artificially reprogramming somatic cells, and are also referred to as "artificial pluripotent stem cells" or "induced pluripotent stem cells." iPS cells may be obtained without any particular limitation; for example, they may be established by the patient themselves or purchased commercially.

[0019] Somatic cells used for induction into iPS cells may be any cells other than germ cells, and include, but are not limited to, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), adipose-derived mesenchymal stem cells, tissue-derived fibroblasts, hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc. Somatic cells may be derived from any organism, for example, from vertebrates, preferably from warm-blooded animals, more preferably from mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, and humans), even more preferably from primates, and particularly preferably from humans. Somatic cells may be, for example, somatic cells isolated from healthy individuals or from patients.

[0020] Specifically, iPS cells are established by reprogramming (reprogramming) any somatic cell and culturing it. The somatic cells to be reprogrammed are preferably dispersed into a single cell state. The reprogramming method is not particularly limited, but can be performed by introducing a factor (referred to as a reprogramming factor) that triggers the reprogramming of any somatic cell into the somatic cell. The reprogramming factor may be a "gene," a gene product of the "gene" (a protein or RNA encoded by the gene), or other factors (e.g., a drug, etc.). The reprogramming factor is preferably a "gene" or a "protein," more preferably a "gene." For example, a method of transforming somatic cells by introducing a reprogramming factor (gene) into the somatic cell using a gene expression vector can be employed. The reprogramming factor as a gene can be derived from any organism, for example, a vertebrate, preferably a warm-blooded animal, such as a mammal (e.g., mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, goat, monkey, or human), more preferably a primate, and particularly preferably a human.

[0021] The reprogramming factor as a gene is, for example, at least one gene selected from the group consisting of Klf family genes, Oct family genes, Sox family genes, and Myc family genes. Among these, it is preferable to use at least one gene from the Klf family genes and Oct family genes. Although not particularly limited, in a typical example, one gene from each of the Klf family genes, Oct family genes, and Sox family genes is used (a total of three genes are used), and in another example, one gene from each of the Klf family genes, Oct family genes, Sox family genes, and Myc family genes is used (a total of four genes are used).

[0022] Examples of Klf family genes include Klf1, Klf2, Klf4, and Klf5, with Klf4 being preferred. Examples of Oct family genes include Oct3 / 4, Oct1A, and Oct6, with Oct3 / 4 being preferred. Examples of Sox family genes include Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, and Sox18, with Sox2 being preferred. Examples of Myc family genes include c-Myc, N-Myc, and L-Myc. The expression products of Myc family genes can sometimes be replaced with cytokines. Examples of cytokines include, but are not limited to, SCF or bFGF, with bFGF being more preferred. Specific examples of the above-mentioned Oct family genes, Klf family genes, Sox family genes, and Myc family genes are given in, for example, International Publication WO2007 / 69666.

[0023] Other examples of reprogramming factors as genes include factors used in establishing iPS cells, such as Lin family genes (such as Lin28 and Lin28B), Nanog genes, Tbx family genes (such as Tbx3), UTF1 genes, SALL family genes (such as SALL4), Nr5A2 genes, Nr5a1 genes, Nr1i2 genes, Rem2 GTPase genes, TCL-1A genes, Esrr family genes (such as Esrrb and Esrrg), Prmt5 genes, Glis family genes (such as Glis1, Glis2, and Glis3), and the like, or genes similar thereto.

[0024] The type of gene expression vector is not particularly limited as long as it is capable of expressing a reprogramming factor in somatic cells, and examples include viral vectors (e.g., Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, herpes virus vectors, etc.), plasmid vectors, chromosomal vectors, episomal vectors, etc. From the viewpoint of efficiency of introduction into somatic cells, etc., the gene expression vector is preferably a viral vector.

[0025] Somatic cells into which reprogramming factors have been introduced are cultured to establish iPS cells. In this culture, iPS cells with stable traits (pluripotency and undifferentiated proliferation ability) are established from the somatic cells through reprogramming. The established iPS cells undergo undifferentiated proliferation under specified culture conditions to form cell masses (spheroids, etc.) of iPS cells. In a particularly preferred example, the somatic cells after introduction of the reprogramming factors are cultured in suspension in a cell culture vessel containing a medium.

[0026] -Mesoderm induction- Mesoderm induction from pluripotent stem cells can be performed using known methods, or the methods described in the Examples or methods based thereon. Although not particularly limited, preferred conditions include the following.

[0027] Culture Method: The preferred method for culturing pluripotent stem cells is suspension culture. As used herein, "suspension culture" refers to culturing cells or cell clusters while maintaining them suspended in a medium. In other words, suspension culture is performed under conditions that do not allow cells or cell clusters to adhere to a cell culture vessel or the like. Culture performed under conditions that allow cells or cell clusters to adhere to a cell culture vessel or the like (adhesion culture) is excluded from the category of suspension culture. "Suspension culture" also includes suspension culture using microcarriers, but preferably suspension culture does not use any carrier for cell adhesion, such as microcarriers. Culture is preferably feeder-free. While the type of cell culture vessel is not particularly limited, a relatively limited volume for cell culture may be preferred from the perspective of easily obtaining relatively homogeneous cell clusters through culture. A relatively limited volume for cell culture corresponds to the volume of medium, and may be, for example, 2 mL or less, 1.5 mL or less, 1 mL or less, 0.5 mL or less, 0.3 mL or less, 0.25 mL or less, or 0.2 mL or less. The lower limit of the volume for cell culture is not particularly limited, but is, for example, 0.05 mL or more, 0.09 mL or more, or 0.1 mL or more. From this perspective, a 48-well plate (the amount of medium per well is approximately 0.3 mL) or a 96-well plate (the amount of medium per well is approximately 0.2 mL) may be preferable, and the wells may preferably have a U-shaped bottom.

[0028] Culture medium: Various media for the proliferation and / or differentiation of stem cells can be used, but it is preferable to use a medium for the proliferation and / or differentiation of pluripotent stem cells or a medium for the proliferation and / or differentiation of hematopoietic stem cells. In one example, the medium is a chemically defined medium (CDM), which is a nutrient solution for culturing cells that contains only specific components, preferably components with known chemical structures. In a preferred example, the medium is a serum-free medium. In a preferred example, the medium is xeno-free (does not contain components of non-human origin).

[0029] Examples of commercially available serum-free media include, but are not limited to, 1) mTeSR (trademark) 1, TeSR (trademark) 2, TeSR (trademark)-E8, or Stem Span (trademark) (all manufactured by Stem Cell Technologies, Inc.), 2) primate ES / iPS cell culture medium (manufactured by ReproCell, Inc.: product number RCHEMD001), 3) Stem Pro (registered trademark)-34, Stem Pro (registered trademark) hESC SFM (both manufactured by Invitrogen), and 4) X-VIVO (trademark) (Lonza).

[0030] The medium may be supplemented with serum-free culture medium supplements and / or additional components, including, but not limited to, L-glutamine or L-glutamine substitutes (such as GlutaMAX-1™), ascorbic acid, monothioglycerol (MTG) or 2-mercaptoethanol, human serum albumin or recombinant human serum albumin (such as Cellastim™, Recombumin™, etc.), insulin, transferrin, sodium selenite, and sodium pyruvate.

[0031] Serum or serum substitute: Mesoderm induction is performed by adding serum (e.g., bovine serum, human serum, etc.) or a serum substitute to the medium as needed, and more preferably, by adding a serum substitute without adding serum. The serum substitute may be any substance that effectively replaces serum during mesoderm induction. For example, one or more of the serum-free culture medium supplements listed above (e.g., a combination of two or more, three or more, four or more, five or more, or six or more) and / or serum substitutes such as KnockOut Serum Replacement (KSR; Gibco) may be added. Serum substitutes can also be prepared, for example, according to known methods (e.g., WO1998 / 030679 (corresponding to Japanese Patent Publication No. 2001-508302)).

[0032] - Differentiation-inducing factors (cytokines, etc.) Step A (mesoderm induction) is carried out in the presence of a differentiation-inducing factor that directs the differentiation of pluripotent stem cells into mesoderm. An example of a preferred differentiation-inducing factor is a combination of BMP and FGF. An example of a preferred differentiation-inducing factor is a combination of BMP, FGF, and VEGF. When a combination of multiple differentiation-inducing factors is used, these multiple differentiation-inducing factors may be added to the medium all at the same time, or may be added stepwise (for example, BMP and FGF may be added first, and VEGF may be added later).

[0033] BMP is a bone morphogenetic protein. A suitable BMP is any member of the BMP family, for example, at least one selected from the group including BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7. Among these, BMP2 or BMP4 is preferred, and BMP4 may be more preferred. The concentration of BMP in the culture medium is not particularly limited, but is, for example, within the range of 1 to 500 ng / mL, preferably within the range of 5 to 100 ng / mL, and more preferably within the range of 10 to 100 ng / mL (e.g., 30 to 70 ng / mL, or 40 to 60 ng / mL).

[0034] FGF is a fibroblast growth factor. A suitable FGF is any member of the FGF family, for example, at least one selected from the group including FGF1 to FGF23, preferably at least one selected from the group including FGF1 to FGF14, more preferably at least one selected from the group including FGF2 (bFGF), FGF7, and FGF10, and particularly preferably FGF2. The concentration of FGF in the medium is not particularly limited, but is, for example, within the range of 5 to 100 ng / mL, more preferably within the range of 10 to 100 ng / mL (e.g., 10 to 30 ng / mL, 30 to 70 ng / mL, or 40 to 60 ng / mL).

[0035] VEGF is a vascular endothelial growth factor. A suitable VEGF is any member of the VEGF family, for example, at least one selected from the group including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF. Preferably, the VEGF is VEGF-A (also simply referred to as VEGF). The concentration of VEGF in the culture medium is not particularly limited, but is, for example, within the range of 5 to 100 ng / mL, more preferably within the range of 10 to 100 ng / mL (e.g., 10 to 30 ng / mL, 30 to 70 ng / mL, or 40 to 60 ng / mL). Note that a VEGF activator or agonist may be used instead of VEGF, or neither VEGF nor a VEGF activator or agonist may be used.

[0036] Preferred examples of differentiation-inducing factors used in step A satisfy at least one, two, three, or all of the following conditions 1) to 4). Preferred examples of differentiation-inducing factors used in step A satisfy all of the following conditions 1) to 4) and also satisfy the following condition 5) or 6). 1) Activin (Activin A, etc.) is not used as a differentiation-inducing factor. Nodal is not used as a differentiation-inducing factor. 2) Various interleukins (IL), such as IL-3, are not used as differentiation-inducing factors. 3) SCF is not used as a differentiation-inducing factor. 4) Thrombopoietin (TPO) is not used as a differentiation-inducing factor. 5) Only BMP and FGF are used as differentiation-inducing factors. 6) Only BMP, FGF, and VEGF are used as differentiation-inducing factors.

[0037] Use of Various Inhibitors in Step A In step A, an inhibitor such as a Rock inhibitor (Rho-associated kinase (ROCK) inhibitor) may be added. The type of Rock inhibitor is not particularly limited, and examples include thiazovivin, Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), Fasudil, AR12286, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, and 3-(4-pyridyl)-1H-indole. In step A, the Rock inhibitor may not be added, or may be added in the early stage of step A (for example, from the start of step A until day 2, until day 1.5, until day 1, or until day 0.5) but not added (removed) midway through step A.

[0038] In step A, inhibitors other than Rock inhibitors can be used as needed, but it may be preferable to satisfy at least one, two, or all of the following conditions 1) to 3): 1) No GSK-3 inhibitor is used. 2) No MEK inhibitor is used. 3) No ALK inhibitor is used.

[0039] Culture Period The period for carrying out step A (culture period) is not particularly limited, but in one example it is within the range of 1 to 7 days, preferably within the range of 1 to 5 days, and more preferably within the range of 2 to 4 days. As will be described later, steps A and B can also be carried out consecutively using the same basic medium. Therefore, as long as step A is directed toward induction into mesoderm, it is possible to move on to step B without providing a step for confirming that mesoderm has actually been induced (for example, confirming the appearance of mesodermal markers). The shape of the cell aggregates obtained in step A is not particularly limited, but may be, for example, approximately spherical.

[0040] (Step B) In the method for producing bone marrow stromal organoids according to one embodiment of the present invention, step B refers to a step of culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells to which serum or a serum substitute has been added. By performing step B, it is possible to produce organoids as described below in the section [2. Bone marrow stromal organoids]. Step B is performed in vitro.

[0041] - Induction of bone marrow stromal organoids - In the method for inducing bone marrow stromal organoids from the cell mass obtained in step A, preferred conditions include the following.

[0042] - Culturing Method The method for culturing the cell mass obtained in step A to obtain bone marrow stromal organoids is preferably suspension culture, and more preferably, both steps A and B are performed in suspension culture. Unless otherwise specified, the "suspension culture" conditions described in step A can be suitably employed in step B as well. Meanwhile, as for the type of cell culture vessel, in the early stage of step B, it may be preferable to use one with a relatively limited volume for cell culture, as described in step A. On the other hand, in step B, a cell culture vessel with a larger volume can also be used to improve the productivity of bone marrow stromal organoids. In one example, in the early stage of step B, a cell culture vessel with a relatively limited volume for cell culture (e.g., a 48-well or 96-well plate) can be used, and then the cells can be transferred to a larger volume cell culture vessel to continue culturing. Alternatively, a large-volume cell culture vessel can be used from start to finish, or a cell culture vessel with a relatively limited volume for cell culture can be used from start to finish. The initial stage of process B refers to, for example, the first half (including part of the first half) when process B is divided into the first half, middle, and second half. In terms of the number of days, it refers to, for example, up to the first day, the second day, the third day, the fourth day, or the fifth day after the start of process B.

[0043] The culturing in step B does not involve embedding the culture (e.g., the cell mass obtained in step A or the cell mass during step B) in or arranging it on an extracellular matrix (e.g., Matrigel, etc.). As described below, even without embedding in or arranging it on an extracellular matrix, an organ-like structure (bone marrow stromal organoid) resembling bone marrow is formed in vitro in a three-dimensional (3D) manner. The cell mass obtained in step A is, for example, approximately spherical, but by performing step B, the cell mass becomes non-spherical, e.g., coral-like. The fact that embedding in an extracellular matrix is ​​not required facilitates culturing and also makes it easier to accommodate scalable (expanded scale, etc.) culturing.

[0044] Culture Medium In step B, as in step A, various culture media for proliferation and / or differentiation of stem cells can be used. Preferably, a culture medium for proliferation and / or differentiation of pluripotent stem cells or a culture medium for proliferation and / or differentiation of hematopoietic stem cells is used, and more preferably a culture medium for proliferation and / or differentiation of hematopoietic stem cells is used (note that the same medium may serve as both a culture medium for proliferation and / or differentiation of pluripotent stem cells and a culture medium for proliferation and / or differentiation of hematopoietic stem cells). In one example, the culture medium is a chemically defined medium (CDM), which is a nutrient solution for culturing cells that contains only specific components, preferably components with known chemical structures. In a preferred example, the culture medium is serum-free. In a preferred example, the culture medium is xeno-free (does not contain non-human-derived components).

[0045] As an example of a commercially available serum-free medium, for example, the medium specifically exemplified in step A can also be used in step B. The medium may be supplemented with serum-free culture medium supplements and / or additional components. Suitable supplements and additional components, for example, those specifically exemplified in step A, can also be used in step B. In a preferred example, the medium used in step A and the medium used in step B are substantially the same medium except for the fact that they contain different differentiation-inducing factors. In this case, step A and step B can be easily carried out consecutively using a common basic medium.

[0046] Serum or serum substitute Step B is performed in a medium supplemented with serum (e.g., bovine serum, human serum, etc.) or a serum substitute, more preferably in a medium supplemented with a serum substitute without serum. Examples of serum substitutes that can be used include KnockOut Serum Replacement (KSR; Gibco).

[0047] Differentiation-inducing factors (cytokines, etc.) Step B can also be considered a step of culturing the cell mass obtained in step A (i.e., cell masses directed to differentiate into mesoderm) under conditions for vascular and / or hematopoietic stem cell induction. Therefore, step B is performed in the presence of a differentiation-inducing factor that directs this differentiation induction. An example of a preferred differentiation-inducing factor is a combination of VEGF, SCF, and FLT. An example of a preferred differentiation-inducing factor is a combination of BMP, FGF, VEGF, SCF, and FLT. When using a combination of multiple differentiation-inducing factors, these multiple differentiation-inducing factors may be added to the medium simultaneously or in stages. The same BMP, FGF, and VEGF as those described in step A can also be used in step B. It may be preferable to use BMP, FGF, and VEGF, especially FGF and VEGF, at higher concentrations than in step A (e.g., 1.3- to 3-fold, 1.5-fold, or 2- to 3-fold concentrations).

[0048] Stem cell factor (SCF) is a cytokine that binds to the c-KIT receptor and is involved in hematopoiesis. The concentration of SCF in the culture medium is not particularly limited, but may be, for example, within the range of 1 to 1000 ng / mL, preferably within the range of 10 to 200 ng / mL, or within the range of 10 to 100 ng / mL (e.g., 30 to 70 ng / mL, or 40 to 60 ng / mL).

[0049] FLT is a cytokine with hematopoietic activity that binds to the FLT receptor (FLT3 receptor) and stimulates the proliferation and differentiation of progenitor cells. It is also called FLT ligand (FLT3 ligand) or Flt ligand (Flt3 ligand). The concentration of FLT in the culture medium is not particularly limited, but may be, for example, in the range of 0.25 to 250 ng / mL, preferably in the range of 10 to 100 ng / mL, or in the range of 10 to 50 ng / mL (e.g., 15 to 35 ng / mL).

[0050] Preferred examples of differentiation-inducing factors used in step B satisfy at least one, two, three, four, five, six, or all of the following conditions 1) to 7). Preferred examples of differentiation-inducing factors used in step B satisfy all of the following conditions 1) to 7) and also satisfy the following condition 8) or 9). 1) Activin (Activin A, etc.) is not used as a differentiation-inducing factor. Nodal is not used as a differentiation-inducing factor. 2) Various interleukins (IL), such as IL-3, are not used as differentiation-inducing factors. 3) Thrombopoietin (TPO) is not used as a differentiation-inducing factor. 4) Erythropoietin (EPO) is not used as a differentiation-inducing factor. 5) IGF-1 is not used as a differentiation-inducing factor. 6) Sonic hedgehog (SHH) is not used as a differentiation-inducing factor. 7) Angiotensin is not used as a differentiation inducer. 8) Only VEGF, SCF, and FLT are used as differentiation inducers. 9) Only BMP, FGF, VEGF, SCF, and FLT are used as differentiation inducers.

[0051] Use of Various Inhibitors in Step B Preferably, various inhibitors are not used in Step B. Specifically, no Rock inhibitor is used. No GSK-3 inhibitor is used. No MEK inhibitor is used. No ALK inhibitor is used.

[0052] Culture Period The period for carrying out step B (culture period) is not particularly limited, but in one example, it is 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. From the viewpoint of sufficient induction of bone marrow stromal organoids, the period for carrying out step B is preferably 4 days or more, more preferably 5 days or more. There is no particular upper limit to the period for carrying out step B, and it may be determined depending on the desired production amount of bone marrow stromal organoids, for example, 50 days or less, 40 days or less, 30 days or less, 20 days or less, 15 days or less, 14 days or less, 13 days or less, or 12 days or less. The period for carrying out step B can also be determined by observing the morphological changes of the culture. For example, the culture may be continued until the cell mass of the culture exhibits a coral-like shape.

[0053] 2. Bone Marrow Stromal Organoids (Overview) In one embodiment of the present invention, bone marrow stromal organoids are organ-like structures resembling bone marrow that are created in vitro in a three-dimensional structure. Bone marrow stromal organoids are aggregates formed by a large number of cells specific to bone marrow stroma, as well as vascular cells and some hematopoietic stem cells, and exhibit at least one of the specific functions of bone marrow stromal cells.

[0054] (Functional Characteristics) Specific functions of bone marrow stromal cells include, for example, 1) supporting blood cells, 2) being usable as a site for inducing differentiation of hematopoietic stem cells (HSCs) / hematopoietic progenitor cells (HPCs) into blood cells, and 3) containing a cellular portion that can differentiate into at least one cell (preferably two or more, three or more types of cells) selected from the group consisting of bone cells, chondrocytes, adipocytes, and skeletal muscle cells. In one embodiment of the present invention, bone marrow stromal organoids exhibit at least one of these specific functions, preferably two or more, and more preferably all of functions 1) to 3).

[0055] (Morphological Characteristics) In one embodiment of the present invention, the bone marrow stromal organoids have a non-spherical shape. More specifically, when viewed from a certain direction, the bone marrow stromal organoids have a non-spherical, amorphous shape and one or more curved portions (coral-like shape). The curved portion is, for example, a curved portion in an arm-like structure (for example, a curved bend in the middle of an arm-like structure). In one example of the bone marrow stromal organoids, they have one or more arm-like structures, or two or more, three or more, or four or more arm-like structures. When the bone marrow stromal organoids have two or more arm-like structures, some or all of them may have curved portions. In one example of the bone marrow stromal organoids, the tip of the arm-like structure is wider, and there is a portion that is narrower toward the base than the tip. In one example of the bone marrow stromal organoid, there is one or more, or two or more, three or more, or four or more, arm-like structures with wider tip portions. In one example, the bone marrow stromal organoid in one embodiment of the present invention has a non-spherical, amorphous shape when viewed from a certain direction, and one or more arm-like structures (longitudinal structures with a length greater than a width). The arm-like structures may have a curved portion as described above.

[0056] (Cell Markers) In one embodiment of the present invention, the bone marrow stromal organoids express at least one cell surface marker selected from the group shown below, preferably three or more, more preferably five, six, seven, or eight or more, and even more preferably all of the cell surface markers. In one embodiment of the present invention, the bone marrow stromal organoids express at least one cell marker selected from the group shown below, preferably two or more, more preferably three or four or more, and even more preferably all of the cell markers. Examples of expressed cell surface markers: VEcad (CD144), CD271, CD31, CD34, CD56 (NCAM: neural cell adhesion molecule), CD90, CD140a, UEA1. VEcad is a vascular marker, CD271 is a stromal cell marker, UEA1 is a vascular marker expressed in bone marrow, CD31 is a vascular endothelial marker, CD34 is a hematopoietic stem cell marker, CD56 is a bone marrow stromal cell marker, CD90 is a bone marrow stromal cell marker, and CD140a is a marker for bone marrow progenitor cells and other cells (including bone marrow stromal cells). All of these are cell surface markers that are also expressed in isolated human bone marrow. Examples of expressed cell markers (including genes): PDGFRA, EOMES, ANGPT, Col3A1, LUM, APLNR, RP11-85G21.3, CTD-2540B15.13, MYL4, FMOD, ANKRD1, SNAI2, BMPER, SLPI, GYPB, LRRC32, NRP1, SOX17, TNFRSF19, IGFBP7, SPNS2, GMPPR, ADAMTS9, IFI16. All of these are cell markers that are also expressed in isolated human bone marrow.

[0057] In one embodiment of the present invention, bone marrow stromal organoids do not substantially express at least one cell marker selected from the group consisting of the following, preferably two or more, more preferably three or more, four or more, or five or more, and even more preferably none of the cell markers are expressed. Examples of cell markers (including genes) that are not substantially expressed include SLC38A4, GDA, SLITRK3, RORB, RP11-185E8.2, GRIA4, RP11-60L3.6, RP11-145M4.2, SOX14, MIR4479, NKX2-5, EEF1A1P22, TBX5, FOXI2, ST8SIA5, and GFY. These are all cell markers that are expressed in human iPS cells but not in isolated human bone marrow.

[0058] In one embodiment of the present invention, bone marrow stromal organoids do not substantially express at least one cell marker (including genes) selected from the group shown below, preferably two or more, more preferably three or more, four or more, or five or more, and even more preferably none of the cell markers are expressed. An example of a cell surface marker that is not substantially expressed is CD71. This is a cell surface marker that is expressed in isolated human bone marrow. An example of a cell marker (including genes) that is not substantially expressed is HBB, IGKC, DEFA1, IGLC2, and IGHJ4. All of these are cell markers that are expressed in isolated human bone marrow. (Types of Constituent Cells) In one embodiment of the present invention, bone marrow stromal organoids are composed of, for example, the following types of cells. Of these cells, the cells included in (1-3), particularly (1-3-1), are bone marrow mesenchymal stromal cells. The proportion of bone marrow mesenchymal stromal cells in the total cells constituting bone marrow stromal organoids is not particularly limited, but may be, for example, 1% or more, 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 50% or more, 60% or more, 70% or more, or 80% or more. The upper limit of the proportion of bone marrow mesenchymal stromal cells in the total cells constituting bone marrow stromal organoids is not particularly limited, but may be, for example, 90% or less, 80% or less, 70% or less, 50% or less, or 40% or less. In addition, as cells other than bone marrow mesenchymal stromal cells, the following UEA1-positive cells are vascular cells, and CD34-positive cells are considered to be hematopoietic stem cells.

[0059] (1) CD56 positive cells. - (1-1) CD56 positive, CD45 negative cells. ...(1-1-1) CD56 positive, CD45 negative, CD14 negative cells. - (1-2) CD56 positive, CD16 negative cells. ...(1-2-1) CD56 positive, CD16 negative, CD271 weakly positive (+ / -) cells. ...(1-2-1-1) CD56 positive, CD16 negative, CD271 weakly positive (+ / -), CD140a weakly positive (+ / -) cells. ...(1-2-1-2) CD56 positive, CD16 negative, CD271 weakly positive (+ / -), CD90 positive cells. ...(1-2-3) CD56 positive, CD16 negative, CD34 negative, CD45 negative cells. - (1-3) CD56 positive, CD90 positive cells. ...(1-3-1) CD56 positive, CD90 positive, CD271 weakly positive (+ / -), CD140a weakly positive (+ / -) cells. (2) CD56 negative cells. - (2-1) CD56 negative, CD45 negative cells. ...(2-1-2) CD56 negative, CD45 negative, CD14 negative cells. - (2-2) CD56 negative, CD16 negative cells. (3) CD31 positive cells. (4) CD34 positive cells. (5) UEA1 positive cells.

[0060] [3. Bone marrow mesenchymal stromal cells and production method thereof] (Summary) In one embodiment of the present invention, bone marrow mesenchymal stromal cells are obtained by isolating them from the bone marrow stromal organoids described in the above section [2. Bone marrow stromal organoids]. That is, these bone marrow mesenchymal stromal cells are produced from the bone marrow stromal organoids by an in vitro method, via pluripotent stem cells to bone marrow stromal organoids.

[0061] (Method for producing bone marrow mesenchymal stromal cells) In one embodiment of the present invention, the method for producing bone marrow mesenchymal stromal cells comprises the following steps A and B, and further step C. Steps A and B have been explained in detail in the above section [1. Method for producing bone marrow stromal organoids], and therefore, step C will be particularly explained below.

[0062] This method comprises: (1) step A of inducing mesoderm from pluripotent stem cells; (2) step B of culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells to which serum or a serum substitute has been added; and (3) step C of isolating bone marrow mesenchymal stromal cells from the bone marrow stromal organoids obtained in step B.

[0063] In step C, the method for isolating bone marrow mesenchymal stromal cells from bone marrow stromal organoids is not particularly limited, and examples thereof include a method of dispersing cells that constitute bone marrow stromal organoids and culturing the dispersed cells in a medium.

[0064] The cells constituting bone marrow stromal organoids can be dispersed by a method for dispersing cells from organoids or tissues, such as treating the collected bone marrow stromal organoids with a protease used for cell dispersion. The type of protease used for cell dispersion is not particularly limited, and examples include trypsin, dispase, hyaluronidase, collagenase, elastase, and combinations of these enzymes, and alternatives to these enzymes (e.g., TrypLESelect, etc.) can also be used as protease.

[0065] The concentration of the protease used to disperse the cells may be an appropriate concentration depending on the type of enzyme, for example, in the range of 0.1 U / mL to 10 U / mL, preferably 1 U / mL to 5 U / mL. The conditions for incubating the bone marrow stromal organoids are not particularly limited as long as they allow cell dispersion, but for example, a temperature range of 25°C to 40°C, preferably 30°C to 40°C, and a treatment time range of 5 to 30 minutes.

[0066] Bone marrow stromal organoids are dispersed into smaller units (at the level of a cell group consisting of a small number of cells, or preferably at the level of individual cells) through the dispersion process of step C described above. Then, in step C, these dispersed cells are cultured in a medium to finally isolate bone marrow mesenchymal stromal cells. The cell culture is, for example, adhesion culture. These dispersed cells can also be cultured as subcultures, and the proportion of bone marrow mesenchymal stromal cells increases with each subculture, allowing for purification. The number of subcultures is not particularly limited, but for example, 1 to 20 subcultures are sufficient, with 1 to 10 subcultures being more preferred. The medium in which the dispersed cells are cultured is not particularly limited as long as it is a medium in which mesenchymal stromal cells (e.g., isolated from bone marrow or fat) can be cultured in an undifferentiated state (e.g., B0 medium or a modified medium thereof), but is preferably a medium that allows undifferentiated proliferation. Furthermore, the medium in which the dispersed cells are cultured is not particularly limited as long as it allows mesenchymal stromal cells to be cultured in an undifferentiated state.

[0067] (Bone marrow mesenchymal stromal cells and their uses) As explained in the section [2. Bone marrow stromal organoids], bone marrow mesenchymal stromal cells in one embodiment of the present invention express, for example, the following cell surface markers: - CD56-positive, CD90-positive cells. - Furthermore, they are weakly CD271-positive (+ / -) and weakly CD140a-positive (+ / -) cells. - On the other hand, bone marrow mesenchymal stromal cells hardly express VE cad or CD34. - Bone marrow mesenchymal stromal cells hardly express CD133 and CD117. - Bone marrow mesenchymal stromal cells in one embodiment of the present invention exhibit, for example, at least one of the following properties 1) to 3), and preferably all of properties 1) to 3). 1) They are capable of undifferentiated proliferation in a medium capable of culturing mesenchymal stromal cells (e.g., those isolated from bone marrow or adipose tissue) in an undifferentiated state. 2) Under appropriate differentiation-inducing conditions, they are capable of differentiating into at least one cell (preferably two or more, three or more types of cells) selected from the group consisting of bone cells, chondrocytes, adipocytes, and skeletal muscle cells. 3) They have the ability to differentiate into blood cells when co-cultured with hematopoietic stem cells / hematopoietic progenitor cells.

[0068] In one embodiment of the present invention, bone marrow mesenchymal stromal cells are established as bone marrow stromal organoid-derived mesenchymal stromal cells (stromal cells) from pluripotent stem cells such as human pluripotent stem cells, via bone marrow stromal organoids, and can be used in the same fields as mesenchymal stromal cells derived from living organisms. While biologically derived mesenchymal stromal cells can be obtained, for example, by initial culture of bone marrow cells, the bone marrow mesenchymal stromal cells according to one embodiment of the present invention have the advantages of unlimited supply of materials (pluripotent stem cells such as iPS cells) and easy quality control.

[0069] In one embodiment of the invention, bone marrow mesenchymal stromal cells can be used in a variety of fields, including, but not limited to, 1) use in various mesenchymal stem cell therapies, 2) use in tissue engineering as part of regenerative medicine, 3) use as a vector, and 4) use in the differentiation of various blood cells.

[0070] An example of the use of mesenchymal stem cells in therapy is regenerative medicine, in which bone marrow mesenchymal stromal cells themselves or tissue precursor cells derived from such stromal cells (e.g., preadipocytes, prehepatocytes, prevascular endothelial cells, neuroblasts, myoblasts, chondroblasts, osteoblasts, etc., preferably preadipocytes, chondroblasts, osteoblasts, etc.) are transplanted (allotransplantation) into the affected area. Tissues that can be regenerated using this regenerative medicine include at least a portion of adipose tissue, liver tissue, vascular endothelium, nerve, muscle, cartilage, bone, etc. Mesenchymal stem cells have been approved as a therapeutic agent for spinal cord injury (Stemilac®) and can be used to treat spinal cord injury. Furthermore, because mesenchymal stem cells have a cytoprotective effect, they can also be used to treat ischemic diseases such as angina pectoris, myocardial infarction, and cerebral infarction.

[0071] Another example of the use of mesenchymal stem cells in therapy is their use in the treatment of inflammatory immune disorders. Mesenchymal stem cells have been approved as a treatment for graft-versus-host disease (GVHD) (Temcel®). They have also been reported to be effective in treating inflammatory immune disorders such as sepsis, enteritis, hepatitis, nephritis, and rheumatoid arthritis (Song N, et al., Trends in Pharmacological Sciences. 2020; 41: 653-664). Examples of inflammatory immune diseases include sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease, rheumatoid arthritis, systemic inflammatory disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, immune-mediated thrombocytopenia, immune-mediated hemolytic anemia, diabetes, systemic lupus erythematosus, atopic dermatitis, collagen disease, steroid-responsive meningoarteritis, multiple sclerosis, psoriasis, autoimmune bullous disease, polyarthritis, pneumonia, infectious osteomyelitis, and periodontitis.

[0072] One example of their use in tissue engineering is to use cells differentiated from bone marrow mesenchymal stromal cells to create chondrocyte cell sheets, which can then be used to treat cartilage damage. One example of their use as vectors is to take advantage of the property of mesenchymal stem cells to accumulate in specific tissues (e.g., tumors). That is, bone marrow mesenchymal stromal cells can be used as vectors to deliver substances (e.g., cancer gene therapy drugs, small molecule drugs, and other drugs) targeting tumors.

[0073] In one example of use in differentiating various blood cell types, bone marrow mesenchymal stromal cells according to one embodiment of the present invention are co-cultured with hematopoietic stem cells to differentiate various blood cell types. More details are provided below in section [4. Production of Hematopoietic Cells]. Bone marrow stromal cells are known to provide a niche for hematopoietic stem cells and maintain long-term hematopoiesis by expressing CD56. The bone marrow mesenchymal stromal cells according to one embodiment of the present invention also express CD56, presumably providing a suitable environment for the maintenance and differentiation of hematopoietic stem cells. Therefore, the use of bone marrow stromal cells according to one embodiment of the present invention has the advantage of providing a hematopoietic environment closer to that of the living body than methods for inducing differentiation of blood cell types without feeders, making it suitable for the mass production of blood cell types. Furthermore, compared to Non-Patent Document 1 (Cancer Discov 13, 364-85, 2023), the method according to one embodiment of the present invention does not require a step of embedding cells in an ECM (extracellular matrix) and can use hematopoietic stem cells induced from pluripotent stem cells, making it suitable for the mass production of blood cell types.

[0074] By using bone marrow mesenchymal stromal cells according to one embodiment of the present invention as a feeder, it is possible to promote the differentiation and induction of various blood cells, including lymphocytes (e.g., T cells, dendritic cells (DCs), etc.), and this is expected to be applied to clinical practice. Examples of applications include cell production in CAR-T cell immunotherapy using regenerative medicine technology, production of blood cells from hematopoietic stem cells, and production of immune cells, and the cells will also make a significant contribution to individualized immunotherapy, allogeneic transplantation, mixed lymphocyte regeneration (MLR) tests, etc.

[0075] [4. Production of Hematopoietic Cells] A method for producing hematopoietic cells according to one embodiment of the present invention comprises a step of co-culturing bone marrow mesenchymal stromal cells, as described in the section [3. Bone marrow mesenchymal stromal cells and production method thereof], as feeder cells with hematopoietic stem cells / hematopoietic progenitor cells.

[0076] The origin of hematopoietic stem cells / hematopoietic progenitor cells is not particularly limited; for example, cells isolated from a biological sample (e.g., human peripheral blood or umbilical cord blood) may be used, or cells induced in vitro from pluripotent stem cells may be used. From the perspective of ease of obtaining hematopoietic stem cells / hematopoietic progenitor cells and homogenizing the quality of the produced blood cells, cells induced in vitro from pluripotent stem cells are preferably used. Note that the pluripotent stem cells described in the section "1. Method for producing bone marrow stromal organoids" can be used. Hematopoietic stem cells / hematopoietic progenitor cells can be induced from pluripotent stem cells by known methods or modifications thereof, as appropriate (see also the description in the Examples).

[0077] Conditions for co-culturing bone marrow mesenchymal stromal cells with hematopoietic stem cells / hematopoietic progenitor cells can be appropriately selected from, for example, conventional culture methods using feeder cells to differentiate hematopoietic stem cells / hematopoietic progenitor cells into hematopoietic cells, or modified methods thereof. Conventional feeder cells include, for example, OP9 cells, MS5 cells, and mFL stromal cells. The medium for co-culturing can be based on, for example, a medium capable of culturing mesenchymal stromal cells in an undifferentiated state (e.g., B0 medium or a modified version thereof). Hematopoietic differentiation-inducing factors added to the medium can be appropriately selected depending on the type of hematopoietic cells to be induced.

[0078] The produced blood cells are broadly classified into myeloid blood cells such as erythrocytes, megakaryocytes / platelets, granulocytes (eosinophils, basophils, neutrophils), monocytes / macrophages, etc., and lymphoid blood cells such as B cells, T cells, NK cells, dendritic cells (DCs), etc. Examples of blood differentiation-inducing factors used to produce B cells include: 1) a combination of some or all of BMP4, VEGF, FGF1, bFGF, SCF, Flt3-L (Flt3 ligand), TPO, GM-CSF, IL-2, IL-4, IL-15, G-CSF, IL-3, IL-6, and IL-7, and 2) a combination of some or all of IL-7, IL-3, SCF, and Flt3-L. Examples of blood cell differentiation-inducing factors used in the production of T cells include: 1) a combination of some or all of Flt3-L, IL-7, and SCF; 2) a combination of some or all of BMP4, bFGF, Activin A, VEGF, IL-6, IGF-1, IL-11, SCF, EPO, TPO, Flt3-L, IL-7, and IL-15; 3) a combination of some or all of BMP4, bFGF, Activin A, VEGF, IGF-1, IL-6, IL-11, SCF, IL-3, EPO, TPO, IL-3, Flt3-L, and IL-7; and 4) a combination of some or all of BMP4, bFGF, VEGF, TPO, SCF, IL-6, IL-3, IL-7, and Flt3-L. Examples of hematopoietic differentiation-inducing factors used in the production of NK cells include 1) a combination of some or all of BMP4, bFGF, Activin A, VEGF, IGF-1, IL-6, IL-11, SCF, IL-3, EPO, TPO, IL-13, Flt3-L, and IL-15, and 2) a combination of some or all of BMP4, VEGF, SCF, IL-3, IL-6, TPO, EPO, IL-7, Flt3-L, and IL-15. Examples of hematopoietic differentiation-inducing factors used in the production of dendritic cells (DCs) include 1) a combination of some or all of GM-CSF, IL-4, and TNF-α, and 2) a combination of some or all of SCF, Flt3-L, GM-CSF, IL-3, TPO, IL-4, and TNF-α. The blood cell differentiation inducer used to produce eosinophils is, for example, a combination of IL-3 and IL-5.Examples of blood cell differentiation-inducing factors used in the production of red blood cells include: 1) a combination of some or all of bFGF, VEGF, EPO, SCF, Flt3-L, IL-3, IL-6, G-CSF, and TPO; 2) a combination of some or all of SCF, IL-3, IL-6, TPO, G-CSF, and EPO; and 3) a combination of some or all of EPO, TPO, IL-3, IL-6, Flt3-L, SCF, and Dex. Examples of blood cell differentiation inducers used to produce granulocytes include: 1) a combination of some or all of IGF-II, VEGF, SCF, Flt3-L, TPO, and G-CSF, 2) a combination of some or all of BMP4, VEGF, SCF, TPO, Flt3-L, IL-3, and G-CSF, and 3) a combination of IL-3 and G-CSF (or GM-CSF). Examples of blood cell differentiation inducers used to produce macrophages include a combination of M-CSF and IL-1β. Examples of blood cell differentiation inducers used in the production of megakaryocytes / platelets include 1) a combination of some or all of BMP4, VEGF, bFGF, SCF, TPO, and IL-3, 2) a combination of some or all of BMP4, VEGF, IL-3, Flt3-L, TPO, SCF, and EPO, and 3) a combination of some or all of BMP4, VEGF, bFGF, TPO, SCF, Flt3-L, IL-3, IL-6, and IL-9. Examples of blood cell differentiation inducers used in the production of neutrophils include 1) a combination of some or all of SCF, Flt3-L, IL-6, IL-6 receptor, TPO, IL-3, and G-CSF, and 2) G-CSF.

[0079] [5. Summary] The scope of the present invention also includes, for example, the following: 1) Bone marrow stromal organoid derived from pluripotent stem cells. 2) Bone marrow stromal organoid according to 1), expressing at least one cell marker selected from the group consisting of VEcad (CD144), CD271, CD31, CD34, CD56 (NCAM), CD90, CD140a, and UEA1. 3) Bone marrow stromal organoid according to 1) or 2), having a coral-like shape. 4) Bone marrow stromal organoid according to any one of 1) to 3), comprising bone marrow mesenchymal stromal cells. 5) Bone marrow stromal organoid according to 4), wherein the bone marrow mesenchymal stromal cells have i) the ability to differentiate into bone tissue, ii) the ability to differentiate into adipose tissue, iii) the ability to differentiate into cartilage tissue, and iv) the ability to differentiate into blood cells when co-cultured with hematopoietic stem cells / hematopoietic progenitor cells. 6) The bone marrow stromal organoid according to any one of 1) to 5), wherein the pluripotent stem cells are human cells. 7) A method for producing bone marrow stromal organoids from pluripotent stem cells, comprising: (1) step A of inducing mesoderm from pluripotent stem cells; and (2) step B of culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells supplemented with serum or a serum substitute. 8) The method according to 7), wherein at least one of steps A and B is suspension culture. 9) The method according to 7) or 8), wherein step B does not involve embedding in an extracellular matrix. 10) The method according to any one of 7) to 9), wherein step B continues culturing until the cell mass assumes a coral-like shape. 11) The method according to any one of 7) to 10), wherein step A is performed in a medium containing BMP and FGF as differentiation inducers. 12) The method according to 11) above, wherein step A is carried out in a medium further containing VEGF as the differentiation-inducing factor. 13) The method according to any one of 7) to 12), wherein step A is carried out in a medium supplemented with serum or a serum substitute. 14) The method according to any one of 7) to 13), wherein step B is carried out by culturing the cell mass obtained in step A under conditions for inducing vascular and / or hematopoietic stem cells. 15) The method according to 14), wherein step B is carried out in a medium containing VEGF, SCF, and FLT as differentiation-inducing factors.16) The method according to 14), wherein step B is performed in a medium containing BMP, FGF, VEGF, SCF, and FLT as differentiation-inducing factors. 17) The method according to any one of 7) to 16), wherein the medium used in step A and the medium used in step B are substantially the same medium except for the contained differentiation-inducing factors. 18) A method for producing mesenchymal stromal cells, comprising a step of isolating bone marrow mesenchymal stromal cells from bone marrow stromal organoids produced by the method according to any one of 7) to 17). 19) Bone marrow mesenchymal stromal cells produced by the method according to 18). 20) A method for producing blood cells, comprising a step of co-culturing hematopoietic stem cells / hematopoietic progenitor cells using the bone marrow mesenchymal stromal cells according to 19) as feeder cells. 21) A frozen storage product of bone marrow stromal organoids obtained according to one embodiment of the present invention, or a frozen storage product of bone marrow mesenchymal stromal cells obtained according to one embodiment of the present invention. In one example, these frozen storage products are produced using an appropriate tissue / cell cryopreservation solution. 22) A combination of differentiation-inducing factors used in step A and / or step B of the method for producing bone marrow stromal organoids according to one embodiment of the present invention. These combinations of differentiation-inducing factors constitute a kit for producing bone marrow stromal organoids.

[0080] Examples of the present invention will be described below. Note that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0081] =Materials and Methods= <a. Human iPSC-derived bone marrow stromal organoids > (1) Culture of human iPSCs At least three human iPSC cell lines were used and cultured in Stemflex complete medium (Stemflex basal medium (A33493-01, Gibco) supplemented with Stemflex supplement (A33492-01, Gibco). All human iPSC cell lines were established at the National Center for Child Health and Development and transfected with Klf4, Oct3 / 4, Sox2, and c-Myc as reprogramming factors. For human iPSC passage, dispersed human iPSCs were seeded onto 6-well plates coated with rhVTN-N (A14700, Gibco) and filled with Stemflex complete medium containing a Rock inhibitor (Y27632) at a concentration of 10 μM. The cells were incubated at 37°C in 5% CO. ２ The cells were incubated under 5% CO₂ at 4°C for 1 hour. One day later, the Rock inhibitor was removed by a complete medium change. Human iPSCs were cultured for 1 week with medium changes every 1-3 days until the next passage.

[0082] (2) Preparation of bone marrow stromal organoids To obtain bone marrow stromal organoid cultures, dispersed human iPSCs were seeded into each well of a U-bottom 96-well plate (174929, Thermo Fisher Scientific) and cultured in bone marrow basal medium (BMBM; Stempro34 complete medium (10639-011, Gibco), 20% KSR (10828-028, Gibco), penicillin / streptomycin, monothioglycerol (MTG), Glutamax, L-ascorbic acid, and insulin-transferrin-selenite solution with sodium pyruvate) supplemented with BMP4, bFGF, VEGF, and Y27632 at 37°C in 5% CO. ２The organoids were incubated under 5% CO₂ (suspension culture). One day later, Y27632 was removed by a complete medium change. On day 3, the medium was replaced with BMBM supplemented with BMP4, bFGF, VEGF, SCF, and FLT3. On day 7, the coral-shaped bone marrow stromal organoids (CLiBMSO) were transferred to 6-well plates or 10 cm dishes and further cultured with BMBM supplemented with BMP4, bFGF, VEGF, SCF, and FLT3 (suspension culture) with multiple medium changes. After culture, the resulting CLiBMSO were harvested for further analysis and / or to establish novel in vitro mesenchymal stromal cells derived from the CLiBMSO. Figure 1 shows a schematic diagram of the process up to this point. The three panels on the left side of Figure 2 show microscopic images (appearance) of the obtained CLiBMSO over time (day 3, day 9, and day 14). The two panels on the right side of Figure 2 show microscopic images (hematoxylin-eosin (HE)-stained images) of sections of the obtained CLiBMSO. As shown on the left side of Figure 2, CLiBMSO exhibited a coral-like shape, suggesting morphological superiority over existing bone marrow organoids, which have a nearly spherical shape.

[0083] (3) Immunofluorescence Staining. Coral-like bone marrow stromal organoids (CLiBMSO) were harvested after 14 days of culture and fixed in 4% paraformaldehyde (PFA)-PBS to prepare paraffin blocks. 5 μm-thick sections were cut from the paraffin blocks using a microtome. The deparaffinized and rehydrated sections were subjected to microwave heat-induced epitope retrieval using Histofine, an antigen retrieval buffer. The sections were incubated in blocking buffer and then incubated overnight at 4°C with primary antibodies in dilution buffer. The sections were washed with PBS and incubated with secondary antibodies at room temperature for 1 to 2 hours. Slides were counterstained with DAPI (dilution 1:300-1:1000) and mounted with mounting medium. Primary antibodies used were: anti-VE cadherin (CD144) (Thermo Fisher Scientific, MA1-198), anti-CD271 (NGFR) (Thermo Fisher Scientific, MA5-31968), anti-CD34 (BioLegend, 343502), anti-CD71 (Thermo Fisher Scientific, 14-0719-82), anti-biotinylated Ulex Europaeus Agglutinin (UEA) I (Vector Laboratories, B-1065-2), and anti-CD90 (Thy1) (BioLegend, 328102). Secondary antibodies used were: Alexa 568 goat anti-mouse IgG1(H+L) (Invitrogen, A11004), Streptavidin Alexa 488 (Invitrogen, S11223), Alexa 568 goat anti-rabbit IgG (H+L) (Invitrogen, A11036), and Alexa 488 goat anti-mouse IgG (H+L) (Invitrogen, A28175).

[0084] FIG. 3 shows microscopic images of fluorescent immunostaining using VEcad antibody and CD271 antibody. As shown in FIG. 3, the obtained CLiBMSO was confirmed to express VEcad, a vascular marker, and CD271, a stromal cell marker. FIG. 4 shows microscopic images of fluorescent immunostaining using UEA1 antibody and CD71 antibody. As shown in FIG. 4, the obtained CLiBMSO was confirmed to express UEA1, a vascular marker expressed in bone marrow, and CD71, an erythrocyte marker. Furthermore, although only the results are shown in FIG. 5, immunostaining using CD31 antibody and immunostaining using CD34 antibody were performed. The results shown in FIG. 5 revealed that the obtained CLiBMSO contained CD31-positive cells, a vascular endothelial marker, and CD34-positive cells, a hematopoietic stem cell marker.

[0085] (4) RNA-seq Analysis. After 14 days of culture, coral-like bone marrow stromal organoids (CLiBMSO) were harvested (n = 8) and flash-frozen in liquid nitrogen. Human iPS cells (two cell lines) and human bone marrow RNA were used as controls. Total RNA was extracted using the miRNeasy micro Kit (QIAGEN) according to the manufacturer's protocol. Libraries for RNA-seq analysis were then prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina). The resulting libraries were sequenced using a NovaSeq 6000 and X (pair-end 100-bp reads). Sequencing data were mapped to the human reference genome (hg38), with the number of reads and transcripts per million (TPM) calculated for each gene. Figure 6 shows the results of RNA-seq analysis, comparing the expression of representative genes (PDGFRA, EOMES, ANGPT, and Col3A1) expressed in human bone marrow between bone marrow stromal organoids, human iPS cells, and human bone marrow. In addition to those shown in Figure 6, the results of the RNA-seq analysis are specifically described below. For example, SLC38A4, GDA, SLITRK3, RORB, RP11-185E8.2, GRIA4, RP11-60L3.6, RP11-145M4.2, SOX14, MIR4479, NKX2-5, EEF1A1P22, TBX5, FOXI2, ST8SIA5, and GFY are all expressed in human iPS cells, but are not substantially expressed in isolated human bone marrow or the resulting bone marrow stromal organoids. These cell markers are also expressed in isolated human bone marrow, but are not substantially expressed in the resulting bone marrow stromal organoids.

[0086] (5) Flow Cytometry Analysis For flow cytometry analysis, coral-like bone marrow stromal organoids (CLiBMSO) were harvested after 17 days of culture and treated with collagenase I and dispase II at 37°C for 20 minutes. Subsequently, they were treated with diluted TrypLESelect (Gibco) at 37°C for 5-10 minutes. The CLiBMSO were dispersed into single cells by pipetting. The cells were centrifuged, resuspended in 2% FBS-PBS, stained with diluted antibodies, and analyzed using a FACS Aria II (BD). Figures 7, 8, and 9 show the results of flow cytometry analysis of CLiBMSO dispersed into single cells. As shown in Figure 7, CLiBMSO contained abundant CD56-positive cells. As shown in Figures 8 and 9, the CD56-positive (+) cells contained in the CLiBMSO were CD56-positive (+) CD16-negative (-) cells, and were also CD90-positive (+), CD271+ / -, and CD140a+ / -. Furthermore, they were negative for the blood cell marker CD45 (-). These results demonstrate that the CD56-positive (+) cells contained in the CLiBMSO are bone marrow mesenchymal stromal cells, since they are positive for bone marrow stromal cell markers CD271 and CD90. Flow cytometry analysis revealed that the proportions of bone marrow mesenchymal stromal cells in the constituent cells of the multiple CLiBMSOs obtained were 30.5%, 6.67%, 18.25%, 8.54%, 82.6%, 7.91%, 63.1%, and 5.81%, respectively.

[0087] (6) Establishment of novel in vitro mesenchymal stromal cells derived from CLiBMSO: To establish novel in vitro mesenchymal stromal cells (CLiBM-MSCs), coral-like bone marrow stromal organoids (CLiBMSO) were harvested after 17 days of culture and treated with collagenase I and dispase II at 37°C for 20 minutes. Subsequently, they were treated with diluted TrypLESelect (Gibco) at 37°C for 5-10 minutes. The dispersed cells were cultured in 6-well plates and / or 10-cm dishes using B0 modified medium (Knockout DMEM plus Ham's F-12 medium (mixing ratio 2:1), 10% human serum, penicillin-streptomycin, monothioglycerol (MTG), ascorbic acid, sodium selenite, insulin, and transferrin) at 37°C in 5% CO ２ The cells were cultured in an incubator. The left side of Figure 10 shows the results of establishing bone marrow mesenchymal stromal cells, and the right side of the figure shows the results of immunofluorescent staining of the bone marrow mesenchymal stromal cells obtained by subculture, using the samples as samples, according to the method described in "(3) Immunofluorescent Staining" above. As shown in Figure 10, these cells showed almost no expression of vascular markers VEcad and CD34 (-), while they showed expression of stromal cell markers CD271 and CD90 (+). Subculture enabled the production (purification) of nearly pure bone marrow mesenchymal stromal cells.

[0088] (7) In Vitro Differentiation of CLIB-MSCs To examine the differentiation potential of CLIB-MSCs, adipogenic, chondrogenic, and osteogenic assays were performed. These assays were performed using the hMSC differentiation BulletKit-adipogenic (Lonza, PT-3004), hMSC differentiation BulletKit-chondrogenic (Lonza, PT-3003), and hMSC differentiation BulletKit-osteogenic (Lonza, PT-3002), respectively. Figure 11 shows the results of the osteogenic differentiation assay, Figure 12 shows the results of the adipogenic assay, and Figure 13 shows the results of the chondrogenic assay. As shown in Figure 11, osteogenic differentiation of CLIB-MSCs resulted in changes in the spindle shape of the cells and the presence of AP-positive cells. As shown in Figure 12, when CLiBM-MSCs were induced to differentiate into adipocytes, they differentiated into cells containing lipid vacuoles. As shown in Figure 13, when CLiBM-MSCs were induced to differentiate into chondrocytes, they differentiated into chondrocytes that stained positive with Alcian blue. (8) Differentiation of T cells and dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells (HSCs) / hematopoietic progenitor cells (HPCs) using CLiBM-MSC feeder cells. Hematopoietic stem cells / hematopoietic progenitor cells were differentiated from human iPSCs via embryoid body (EB) formation using a modified version of a previously published protocol (Iriguchi et al. Nat. Commun. 2021 Jan. 18; 12(1):430). EBs derived from hematopoietic stem / progenitor cells were harvested after 18 days (or more) of culture and transferred onto CLiBM-MSC feeder cells derived from CLiBMSO for differentiation of T cells and dendritic cells (DCs).The cells were then co-cultured for 5 days in B0 modified medium (Knockout DMEM plus Ham's F-12 medium (mixing ratio 2:1), 10% human serum, penicillin-streptomycin, monothioglycerol (MTG), ascorbic acid, sodium selenite, insulin, and transferrin) supplemented with BMP4, VEGF, SCF, and FLT3. On day 6, FLT3 and IL-7 were added to induce differentiation into T cells, and GM-CSF and IL-4 were added to induce differentiation into dendritic cells (DCs). Differentiated hematopoietic cells were collected on day 6, day 10, and / or day 13 for further analysis. The results are shown in Figures 14 to 20. Figure 14 shows the results (day 6 and day 10) of differentiation of T cells and dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 15 shows the results (day 6) of differentiation of CD8A single-positive T cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 16 shows the results (heat map) of array PCR of hematopoietic cell marker gene expression in blood cells differentiated from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 17 shows the results (day 6 and day 13) of differentiation of CD86-positive dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 18 shows the results (day 6 and day 13) of differentiation of HLA-DR-positive dendritic cells (DCs) from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells. Figure 19 shows the results (day 6 and day 13) of differentiation of CD4-positive T cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells.FIG. 20 shows the results of differentiation of various blood lineage cells from human iPSC-derived hematopoietic stem cells / hematopoietic progenitor cells by co-culture on CLiBM-MSC feeder cells (Giemsa staining results on day 6 and day 13).

[0089] <b. Human ESC-Derived Bone Marrow Stromal Organoids> (1) Culture of Human ES Cells and Preparation of Bone Marrow Stromal Organoids (CLeBMSO) Bone marrow stromal organoids based on human ES cells were prepared using the same methods as in (1) and (2) of the above section <a. Human iPSC-Derived Bone Marrow Stromal Organoids>. Specifically, two human ES cell lines established at the National Center for Child Health and Development were subcultured using the same method as in (1) of the section <a. Human iPSC-Derived Bone Marrow Stromal Organoids>. Then, using the same method as in (2) of the section <a. Human iPSC-Derived Bone Marrow Stromal Organoids>, suspension culture was performed using bone marrow basal medium supplemented with differentiation-inducing factors, and coral-like bone marrow stromal organoids (CLeBMSO) were collected. The four panels on the left side of Figure 21 show microscopic images (appearance) of CLeBMSO obtained from two cell lines over time (Day 3, Day 7, Day 11, and Day 14), and the panel on the right side of Figure 21 shows a microscopic image (hematoxylin-eosin (HE) stained image) of a section of the obtained CLeBMSO. As shown on the left side of Figure 21, CLeBMSO exhibited a coral-like shape, suggesting morphological superiority over existing bone marrow organoids, which exhibit a nearly spherical shape.

[0090] (2) Immunofluorescence Staining. Coral-like bone marrow stromal organoids (CLeBMSO) were harvested after 14 days of culture and fixed in 4% paraformaldehyde (PFA)-PBS to prepare paraffin blocks. Five-micrometer-thick sections were cut from the paraffin blocks using a microtome and subjected to immunofluorescence staining as described in (3) of the "Human iPSC-derived bone marrow stromal organoids" section. Primary antibodies used were anti-VE cadherin (CD144) (Thermo Fisher Scientific, MA1-198), anti-CD271 (NGFR) (Thermo Fisher Scientific, MA5-31968), anti-biotinylated Ulex Europaeus Agglutinin (UEA) I (Vector Laboratories, B-1065-2), and anti-CD235a (Glycophorin A) (Abcam, ab129024). Secondary antibodies used were: Alexa 568 goat anti-mouse IgG1(H+L) (Invitrogen, A11004), Streptavidin Alexa 488 (Invitrogen, S11223), Alexa 568 goat anti-rabbit IgG (H+L) (Invitrogen, A11036), and Alexa 488 goat anti-mouse IgG (H+L) (Invitrogen, A28175).

[0091] Figure 22 shows microscopic images of fluorescent immunostaining using VEcad antibody and CD271 antibody. As shown in Figure 22, the obtained CLeBMSO was confirmed to express VEcad, a vascular marker, and CD271, a stromal cell marker. Figure 23 shows microscopic images of fluorescent immunostaining using UEA1 antibody and CD235a antibody. As shown in Figure 4, the obtained CLeBMSO was confirmed to express UEA1, a vascular marker expressed in bone marrow, and CD235a (glycophorin-A), a marker for erythroid progenitor cells and erythrocytes.

[0092] (3) Flow Cytometry Analysis For flow cytometry analysis, coral-like bone marrow stromal organoids (CLeBMSO) were collected after 17 days of culture and treated with collagenase I and dispase II at 37°C for 20 minutes. Subsequently, they were treated with diluted TrypLESelect (Gibco) at 37°C for 5-10 minutes. The CLeBMSO were dispersed into single cells by pipetting. The cells were centrifuged, resuspended in 2% FBS-PBS, stained with diluted antibodies, and analyzed using a FACS Aria II (BD). Figure 24 shows the results of flow cytometry analysis of CLeBMSO dispersed into single cells. CLeBMSO contained abundant CD56-positive cells, which were CD90-positive (+), CD271+ / -, and negative (-) for the blood cell marker CD45. These results demonstrated that the CD56-positive (+) cells contained in CLeBMSO were bone marrow mesenchymal stromal cells, as they were positive for bone marrow stromal cell markers CD271 and CD90. Flow cytometry analysis revealed that the percentages of bone marrow mesenchymal stromal cells in the constituent cells of the multiple CLeBMSO samples obtained were 6.23%, 8.39%, 9.99%, and 27.8%, respectively.

[0093] (4) Establishment of novel in vitro mesenchymal stromal cells (CLeBM-MSCs) derived from CLeBMSO To establish novel in vitro mesenchymal stromal cells (CLeBM-MSCs) derived from CLeBMSO, coral-like bone marrow stromal organoids (CLeBMSOs) were harvested after 17 days of culture and treated with collagenase I and dispase II at 37°C for 20 minutes. Subsequently, they were treated with diluted TrypLESelect (Gibco) at 37°C for 5-10 minutes. The dispersed cells were cultured in 6-well plates and / or 10-cm dishes using B0 modified medium (Knockout DMEM plus Ham's F-12 medium (mixing ratio 2:1), 10% human serum, penicillin-streptomycin, monothioglycerol (MTG), ascorbic acid, sodium selenite, insulin, and transferrin) at 37°C in 5% CO ２ The left side of Figure 25 shows the results of establishing bone marrow mesenchymal stromal cells (CLeBM-MSCs), and the right side of the same figure shows the bone marrow mesenchymal stromal cells (CLeBM-MSCs) obtained by subculture.

[0094] (5) T Cell Differentiation by Co-Culture with Human iPSC-Derived Hematopoietic Stem Cells (HSCs) Using CLeBM-MSCs as Feeder Cells. CLeBM-MSCs obtained in (4) above were used as feeder cells to co-culture with human iPSC-derived hematopoietic stem cells (HSCs) in an attempt to induce T cells. The hematopoietic stem cells and co-culture techniques used were the same as those described in (8) under "a. Human iPSC-Derived Bone Marrow Stromal Organoids." The results are shown in Figure 26. Figure 26 shows the results of differentiation of human iPSC-derived hematopoietic stem cells into T cells by co-culture on CLeBM-MSC feeder cells, as determined by flow cytometry analysis on days 13 and 20 of co-culture. T cells induced from hematopoietic stem cells were either CD4 single positive or CD4 / CD8a double positive.

[0095] The present invention can be used, for example, in the medical field.

Claims

1. Bone marrow stromal organoids derived from pluripotent stem cells.

2. The bone marrow stromal organoid of claim 1, which expresses at least one cell marker selected from the group consisting of VEcad (CD144), CD271, CD31, CD34, CD56 (NCAM), CD90, CD140a, and UEA1.

3. The bone marrow stromal organoid of claim 1, which has a coral-like shape.

4. The bone marrow stromal organoid of claim 1, comprising bone marrow mesenchymal stromal cells.

5. The bone marrow stromal organoid of claim 4, wherein the bone marrow mesenchymal stromal cells have the ability to 1) differentiate into bone tissue, 2) differentiate into adipose tissue, 3) differentiate into cartilage tissue, and 4) differentiate into blood cells by co-culture with hematopoietic stem cells / hematopoietic progenitor cells.

6. The bone marrow stromal organoid of claim 1, wherein the pluripotent stem cells are human cells.

7. A method for producing bone marrow stromal organoids from pluripotent stem cells, comprising: (1) step A of inducing mesoderm from pluripotent stem cells; and (2) step B of culturing the cell mass obtained in step A in a medium for proliferation and differentiation of hematopoietic stem cells to which serum or a serum substitute has been added.

8. The method according to claim 7, wherein at least one of step A and step B is suspension culture.

9. The method according to claim 7, wherein step B does not involve embedding in an extracellular matrix.

10. The method according to claim 7, wherein step B continues culturing the cell mass until it assumes a coral-like shape.

11. The method according to claim 7, wherein step A is carried out in a medium containing BMP and FGF as differentiation-inducing factors.

12. The method according to claim 11, wherein step A is carried out in a medium further containing VEGF as the differentiation-inducing factor.

13. The method of claim 7, wherein step A is carried out in a medium supplemented with serum or a serum substitute.

14. The method according to claim 7, wherein step B comprises culturing the cell mass obtained in step A under conditions for inducing vascular and / or hematopoietic stem cells.

15. The method according to claim 14, wherein step B is carried out in a medium containing VEGF, SCF, and FLT as differentiation-inducing factors.

16. The method according to claim 14, wherein step B is carried out in a medium containing BMP, FGF, VEGF, SCF, and FLT as differentiation-inducing factors.

17. The method according to claim 7, wherein the medium used in step A and the medium used in step B are substantially the same medium except for the fact that they contain different differentiation-inducing factors.

18. A method for producing mesenchymal stromal cells, comprising a step of isolating bone marrow mesenchymal stromal cells from bone marrow stromal organoids produced by the method of claim 7.

19. Bone marrow mesenchymal stromal cells produced by the method of claim 18.

20. A method for producing blood cells, comprising the step of co-culturing hematopoietic stem cells / hematopoietic progenitor cells with the bone marrow mesenchymal stromal cells according to claim 19 as feeder cells.

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