Method for producing mesodermal lineage cells from embryoid body

Abstract

The present invention provides a method for producing mesodermal lineage cells from an embryoid body, the method comprising: (1) a step for inducing an embryoid body into a differentiated embryoid body in which differentiation into a mesodermal cell lineage is promoted; and (2) a step for determining the solidity of the induced differentiated embryoid body.

Description

Method for producing mesodermal lineage cells from embryoid bodies

[0001] The present invention relates to a method for producing mesodermal lineage cells from embryoid bodies using area envelope (solidity).

[0002] Methods for producing high-quality and stable target cells from pluripotent stem cells are attracting attention as a source of various tissue cells used in regenerative medicine and transplantation. In particular, hematopoietic stem cells that can be differentiated from embryoid bodies via mesoderm are known to have diverse applications. Not only are they used for transplantation in cases of hematopoietic failure, but immune cells such as T cells that can be further differentiated from hematopoietic stem cells are also used in immunotherapy for cancer and infectious diseases. Therefore, various methods are being researched to efficiently induce differentiation of mesodermal cells from embryoid bodies and produce a sufficient quantity of hematopoietic stem cells for therapeutic use.

[0003] A known method for efficiently differentiating induced pluripotent stem cells into target cells involves exposing cells to a specific low-molecular-weight compound (Patent Document 1) to produce hematopoietic stem cells. In known methods, the timing of changing the composition of the culture medium is generally controlled by the number of days or hours of culture, but it is unclear whether individual cells or cell populations are in a state suitable for differentiation at that time. Therefore, problems such as reduced yield and variability in quality may occur.

[0004] Furthermore, methods that control differentiation solely by the number of culture days cannot evaluate problems in the differentiation process until the cells have been differentiated into the target cells. If the differentiation induction efficiency can be controlled and predicted from the state of cells during differentiation, the target cells can be produced efficiently.

[0005] Therefore, as a less invasive and more efficient method of inducing differentiation, attempts are being made to control differentiation by extracting morphological characteristics of cells from image data of those cells.

[0006] Patent Document 2 discloses a method for differentiating cardiomyocytes from pluripotent stem cells by evaluating the content of target cells using the size change rate of embryoid bodies. However, there is no known method for appropriately controlling the timing of culture medium exchange during the differentiation process from pluripotent stem cells to target cells based on morphological characteristics other than the size change rate.

[0007] Patent No. 5928681 Patent No. 6786077

[0008] The present invention aims to provide a method for efficiently producing mesodermal lineage cells from embryoid bodies, a method for efficiently producing hematopoietic stem cells from embryoid bodies, and a method for efficiently inducing differentiation of target cells from embryoid bodies.

[0009] The inventors have discovered that when differentiating embryoid bodies created from induced pluripotent stem cells or the like into hematopoietic stem cells, which are mesodermal cells, the timing of exposing the differentiated embryoid bodies to components necessary for differentiation into mesodermal cell lineage cells can be optimized by using Solidity, which indicates the degree of unevenness of the contour of the differentiated embryoid body in a state where differentiation into the mesodermal cell lineage has been promoted, as an indicator. Based on this finding, the inventors have completed the present invention by further conducting research.

[0010] In other words, the present invention is as follows: [1] A method for producing mesodermal lineage cells from an embryoid body, comprising the following steps: (1) inducing the embryoid body into a differentiated embryoid body in which differentiation into mesodermal cell lineage is promoted; and (2) determining the area envelope (solidity) of the induced differentiated embryoid body. [2] The method according to [1], further comprising the following steps: (3) culturing the differentiated embryoid body having a solidity of 0.6 to 0.8 in a differentiation induction medium containing a differentiation induction factor. [3] The method according to [1] or [2], wherein step (1) is performed using a medium containing BMP4 and / or CHIR99021. [4] The method according to [2], wherein the differentiation induction factor comprises at least one selected from the group consisting of cytokines, growth factors, and intercellular signaling inhibitors. [5] The method according to [4], wherein the cytokine comprises at least one selected from the group consisting of SCF, Flt3-L, IL-3, and IL-6. [6] The method according to either [4] or [5], wherein the growth factor is VEGF and / or bFGF. [7] The method according to any one of [4] to [6], wherein the intercellular signaling inhibitor is SB431542. [8] A method for producing hematopoietic stem cells from embryoid bodies, comprising the following steps: (1) inducing embryoid bodies into differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted; (2) determining the area envelope (solidity) of the induced differentiated embryoid bodies; and (3) culturing the differentiated embryoid bodies having a solidity of 0.6 to 0.8 in a differentiation induction medium containing hematopoietic differentiation factors to differentiate them into hematopoietic stem cells. [9] The method according to any one of [1] to [8], wherein the differentiated embryoid bodies are derived from induced pluripotent stem cells or embryonic stem cells.

[10] A method for differentiating an embryoid body into target cells, comprising the following steps: (1) inducing the embryoid body into a differentiated embryoid body in which differentiation into a mesodermal cell lineage, an ectoderm cell lineage, or an endodermal cell lineage is promoted; and (2) determining the area envelope (solidity) of the induced differentiated embryoid body.

[0011] According to the present invention, the optimal timing for embryoid body differentiation induction can be optimized non-invasively and objectively using solidity as an indicator. Therefore, according to the present invention, target cells (e.g., hematopoietic stem cells) prepared by differentiation induction from embryoid bodies can be prepared efficiently and with high reproducibility.

[0012] This figure compares the number of hematopoietic stem cells obtained when the median solidity of the differentiated embryoid bodies analyzed in [Example 1], [Example 2], and [Example 3] was changed under conditions where the median solidity of the differentiated embryoid bodies analyzed was 0.8 or less, and under conditions where it was greater than 0.8. This figure compares the positive rates of hematopoietic stem cell markers CD34, CD90, or CD201, obtained by flow cytometry analysis of hematopoietic stem cells recovered after differentiation induction of differentiated embryoid bodies in [Example 1], [Example 2], and [Example 3], under conditions where the median solidity of the differentiated embryoid bodies analyzed was 0.8 or less, and under conditions where it was greater than 0.8. This figure compares the median solidity of the differentiated embryoid bodies analyzed in [Example 1], [Example 2], and [Example 3]. This is a line graph showing the change in median solidity of the differentiated embryoid bodies analyzed over time. The scale bar in the figure represents 5 hours. This figure compares the number of hematopoietic stem cells obtained when the median solidity of the differentiated embryoid bodies analyzed in the differentiation induction of differentiated embryoid bodies in [Example 1-2], [Example 2-2], and [Example 3-2] was changed under conditions where the median solidity of the differentiated embryoid bodies analyzed was 0.8 or less, and under conditions where the median solidity was greater than 0.8. This figure compares the positive rates of the hematopoietic stem cell markers CD34, CD90, or CD201, after flow cytometry analysis of hematopoietic stem cells recovered after differentiation induction of differentiated embryoid bodies in [Example 1-2], [Example 2-2], and [Example 3-2]. This figure compares the median solidity of the differentiated embryoid bodies analyzed in the differentiation induction of differentiated embryoid bodies in [Example 1-2], [Example 2-2], and [Example 3-2]. This figure compares the recovery rate of mesenchymal stem cells obtained when the median solidity of the differentiated embryoid bodies analyzed in the differentiation induction of differentiated embryoid bodies was changed under conditions where the median solidity of the differentiated embryoid bodies analyzed was 0.8 or less, and under conditions where the median solidity was greater than 0.8. This figure shows the positivity rates for the mesenchymal stem cell positive markers CD90, CD105, and CD73, as well as the negative markers CD11b, CD19, CD45, HLA-DR, CD14, and CD34, obtained by flow cytometry analysis of the mesenchymal stem cells obtained in [Example 4]. This figure compares the median solidity of the differentiated embryoids analyzed in the differentiation induction of differentiated embryoids in [Example 4] to [Example 6].This figure shows the positivity rates for mesenchymal stem cells obtained in [Example 4-2] using flow cytometry analysis, specifically for the positive markers CD90, CD105, and CD73, as well as the negative markers CD11b, CD19, CD45, HLA-DR, CD14, and CD34. This figure shows microscopic images of bone, cartilage, or adipocytes obtained by differentiation of mesenchymal stem cells obtained in [Example 4-2], stained with Alizarin Red, Alcian Blue, and Oil Red O, respectively. Negative Control is a sample of iPS-MSCs cultured in the same differentiation medium (3) for the same period and stained in the same manner. This figure shows the positivity rates for NK cell markers CD45 and CD56, as well as NK mature cell markers NKG2D, NKp44, NKp46, KIR, and CD16, obtained by flow cytometry analysis of NK cells obtained in [Example 11].

[0013] 1. Method for producing mesodermal lineage cells from embryoid bodies using solidity as an indicator The present invention provides a method for producing mesodermal lineage cells from embryoid bodies (hereinafter sometimes referred to as "production method 1 of the present invention") comprising the following steps: (1) a step of inducing embryoid bodies into differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted, and (2) a step of determining the area envelope degree (solidity) of the induced differentiated embryoid bodies.

[0014] In the first step of the manufacturing method 1 of the present invention, the embryoid body is induced into a differentiated embryoid body in which differentiation into the mesodermal cell lineage is promoted.

[0015] In the manufacturing method 1 of the present invention, "embryoid body" refers to a cell mass formed by the adhesion of undifferentiated pluripotent stem cells. The embryoid body has the ability (pluripotency) to differentiate into all cells that make up a living organism except the placenta (tissues derived from the three germ layers). Embryoid bodies can be produced and cultured by known methods and are derived from pluripotent stem cells. Examples of production and culture methods include those described in International Publication No. 2021 / 230304.

[0016] The size of the embryoid body in this invention is not particularly limited, but the embryoid body on day 1 after sowing is 8,000 to 20,000 μm. ２This is preferable. Here, the size of the embryoid body refers to the area of ​​the cross-section that maximizes the size of the embryoid body when a two-dimensional image is captured with the three-dimensional embryoid body as the focus. A larger size makes it easier to form differentiated embryoid bodies and improves the efficiency of differentiation induction, but if it is too large, the oxygen supply to the inside of the embryoid body may be insufficient, potentially leading to cell necrosis.

[0017] In the manufacturing method 1 of the present invention, "differentiated embryoid body" refers to a cell population formed by multiple cells of each of the three germ layers (ectoderm, mesoderm, and endoderm) densely packed together, which are in the process of differentiating from an embryoid body into the three germ layers (ectoderm, mesoderm, and endoderm). Differentiated embryoid bodies may be cultured attached to a culture vessel (hereinafter referred to as attached culture) or cultured in a suspended state (hereinafter referred to as suspension culture). In the case of attached culture, the cell population does not need to be circular or oval in shape, and may have a complex morphology in which the cells of the outline protrude outward in multiple directions. Differentiated embryoid bodies may contain embryoid bodies, and may contain cells in the process of differentiating from an embryoid body into the three germ layers, as well as cells of each of the three germ layers.

[0018] The culture method for forming differentiated embryoid bodies from embryoid bodies is not particularly limited, and either adherent culture or suspension culture may be used. However, from the viewpoint of measuring the area envelope (solidity) in the production method 1 of the present invention, adherent culture is preferred. Furthermore, in the adherent culture of differentiated embryoid bodies, a cell support substrate may be coated onto the surface of the culture vessel from the viewpoint of proliferation, etc.

[0019] Examples of cell-supporting substrates include, but are not limited to, collagen, gelatin, Matrigel, poly-L-lysine, poly-D-lysine, laminin (e.g., laminin-511), fibronectin, and retronectin. One cell-supporting substrate may be used, or two or more may be used.

[0020] The culture medium in which differentiated embryoid bodies are cultured is not particularly limited as long as it is a medium that can be used for culturing animal-derived stem cells. In one embodiment, StemFit iPS / ES cell proliferation medium (Ajinomoto) can be used. In another embodiment, the culture medium can be appropriately changed according to the cells to which the differentiated embryoid bodies are to be differentiated, or multiple culture media can be combined to differentiate the differentiated embryoid bodies into desired cells. For example, when differentiating mesodermal lineage cells from differentiated embryoid bodies, the culture medium can be changed from StemFit iPS / ES cell proliferation medium to X-VIVO. When differentiating ectoderm lineage cells, the culture medium can be changed from StemFit to PRIME-XV® MSC Expansion XSFM.

[0021] When culturing differentiated embryoid bodies, components that promote the maintenance, proliferation, and differentiation of the differentiated embryoid bodies can be added to the culture medium. Examples of components that promote the maintenance, proliferation, and differentiation of differentiated embryoid bodies include serum, low molecular weight compounds, and differentiation-inducing factors described later. Low molecular weight compounds include intercellular signaling inhibitors, antioxidants, and reducing agents. For example, it is preferable to add monothioglycerol (MTG) and / or 2-mercaptoethanol as a reducing agent and ascorbic acid as an antioxidant to the culture medium.

[0022] In the manufacturing method 1 of the present invention, "mesoderm cells" refers to cells derived from the primitive endoderm that have the ability to differentiate into various cells of the circulatory system, blood cells, skeletal cells, various cells of the urinary system, various cells of the reproductive system, various cells of connective tissue, and other cells such as neural microglia, dental dentin cells, peritoneal epithelial cells, and adrenal cortical cells, as well as precursor cells and mature cells. Cells that can differentiate from mesoderm cells in this way are called mesoderm lineage cells in the present invention. Various cells of the circulatory system include cardiac cells, vascular endothelial cells, spleen cells, bone marrow cells, etc. Cardiac cells include cardiomyocytes, cardiovascular progenitor cells, vascular smooth muscle cells, cardiomyocyte endothelial cells, etc. Blood cells include immune cells, hematopoietic stem cells, lymphocytes, myeloid cells, dendritic cells, macrophages, monocytes, megakaryocytes, T cells, B cells, neutrophils, eosinophils, granulocytes, NK cells, NKT cells, etc. To differentiate from mesoderm cells into blood cells, they must first differentiate through hematopoietic stem cells and vascular progenitor cells (hematopoietic precursor cells) into various blood cell types. Skeletal cells include osteocytes and chondrocytes. Cells of the urinary tract include kidney cells and ureteral cells. Cells of the reproductive tract include ovarian cells, uterine cells, and testicular cells. Cells of connective tissue include mesenchymal stem cells and stromal cells.

[0023] In the first step of the manufacturing method 1 of the present invention, differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted are induced. Induction of embryoid bodies into differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted can be carried out using methods known to the present day. Specifically, this can be achieved by culturing embryoid bodies in a medium containing one or a combination thereof of mesodermal differentiation factors. Mesodermal differentiation factors are not particularly limited as long as they are substances that can promote and / or maintain the induction of differentiation from embryoid bodies to mesodermal cells (including differentiated embryoid bodies). Examples of such substances include cytokines, growth factors, and intercellular signaling inhibitors. Preferably, BMP4 and CHIR99021 are used.

[0024] In the second step of the manufacturing method 1 of the present invention, the area envelope degree of the differentiated embryoid body induced in the first step is determined.

[0025] In the manufacturing method 1 of the present invention, "area envelope degree (Solidity)" is a numerical value representing the degree of unevenness of the contour of the differentiated embryoid in a two-dimensional image taken from above or below the incubator of the differentiated embryoid in a state where the differentiated embryoid is attached to the incubator. Solidity can be calculated by dividing the actual area of ​​the differentiated embryoid in the two-dimensional image by the area enclosed by the convex hulling process so that the indentation is eliminated (the area around which the envelope circumference is wrapped). The closer Solidity is to 1.0, the less unevenness the contour of the differentiated embryoid is and the smoother the morphology of the cell population. The lower the Solidity is, the more unevenness the contour of the differentiated embryoid is and the more distorted the morphology of the cell population. In one embodiment, the two-dimensional image of the differentiated embryoid is taken perpendicular to the surface to which the differentiated embryoid is attached.

[0026] In the manufacturing method 1 of the present invention, the solidity of the differentiated embryoid body can be determined using a method known to the present invention. For example, the solidity of the differentiated embryoid body can be determined by the following steps: (1) Capture an image of the differentiated embryoid body. (2) Determine the area of ​​the differentiated embryoid body. (3) Determine the smallest convex hull surrounding the differentiated embryoid body and determine its area (hull processing). (4) Determine the area envelope degree from the values ​​determined in (2) and (3).

[0027] In (1) above, imaging of differentiated embryoid bodies is not particularly limited, and any means may be used, as long as the imaging device can acquire still images and / or videos with sufficient magnification and resolution to recognize the contour of the differentiated embryoid body and be suitable for image analysis. In one embodiment, an optical microscope can be used. Examples of optical microscopes include, but are not limited to, the BZX-810 (Keyence).

[0028] Furthermore, any means of processing and analyzing the captured images may be used, as long as it can determine the area of ​​the differentiated embryoid body and perform convex hull processing on the differentiated embryoid body. For example, the means used in the embodiment of this application (acquisition of tiling images of the differentiated embryoid body, binarization, and contour determination by a contour extraction algorithm) can be used, but is not limited to this. For example, the contour of the differentiated embryoid body can be determined using edge detection or the like instead of binarization.

[0029] In (2) above, the determination of the area of ​​the differentiated embryoid body can also be done using a method that is already known. If the image data of the differentiated embryoid body is represented as mesh data or pixel data (area pixels), the area can be calculated using the number of pixels inside or the vertex coordinates of the mesh.

[0030] In (3) above, the processing of the convex hull of the differentiated embryoid body can also be done using a method that is already known. Algorithms for calculating the two-dimensional convex hull are already known (e.g., Quickhull).

[0031] In (4) above, the area enveloping degree can be determined by dividing the area of ​​the differentiated embryoid obtained in (2) by the area of ​​the convex hull obtained in (3). The image processing software used to calculate Solidity is not particularly limited. For example, commercially available software such as OSS's OpenCV (Apache-License-2.0), ImageJ, Pillow, and scikit-image can be used.

[0032] In the manufacturing method 1 of the present invention, when the solidity of multiple differentiated embryoid bodies is determined, the solidity may be a statistical value such as the median, mean, or variance of the values ​​determined in the multiple differentiated embryoid bodies.

[0033] The manufacturing method 1 of the present invention is characterized by optimizing the timing of inducing further differentiation of the differentiated embryoid body based on the Solidity value. Although we do not wish to be bound by theory, it is conceivable that during the process of inducing differentiation of the embryoid body into a differentiated embryoid body, the migratory ability of cells present in the contour region of the embryoid body increases, and as the cells begin to migrate, Solidity may decrease. Therefore, it is presumed that Solidity may reflect the timing of embryoid body differentiation induction.

[0034] In one embodiment, the manufacturing method 1 of the present invention further comprises the following steps (3): (3) A step of culturing a differentiated embryoid having a solidity of 0.6 to 0.8 in a differentiation induction medium containing a differentiation induction factor.

[0035] In step (3) of the manufacturing method 1 of the present invention, the timing of differentiating the differentiated embryoids into mesodermal lineage cells can be optimized using the solidity of the differentiated embryoids as an indicator. In one embodiment, it is preferable to differentiate the differentiated embryoids into mesodermal lineage cells using the point in time when the solidity of the differentiated embryoids is 0.4 to 0.8 as an indicator. In a preferred embodiment, a population of differentiated embryoids in which the median solidity of differentiated embryoids contained per well is 0.5 to 0.8, preferably 0.6 to 0.8, can be efficiently differentiated into mesodermal lineage cells by culturing in a differentiation induction medium containing a differentiation induction factor. In another embodiment, a population of differentiated embryoids in which the median solidity of differentiated embryoids contained per well is 0.4 to 0.69, preferably 0.55 to 0.69, can be efficiently differentiated into mesodermal lineage cells by culturing in a differentiation induction medium containing a differentiation induction factor.

[0036] Methods for inducing differentiation into mesodermal lineage cells can be employed using known methods. Specifically, mesodermal lineage cells can be obtained by culturing differentiated embryoid bodies (containing mesodermal cells) in a culture medium containing differentiation-inducing factors.

[0037] The mesodermal lineage cells in the manufacturing method 1 of the present invention are not particularly limited as long as they are cells derived from mesodermal cells.

[0038] The differentiation-inducing factor in the production method 1 of the present invention is not particularly limited as long as it can promote and / or maintain the induction of differentiation from differentiated embryoid bodies into cells derived from each cell of the three germ layers (for example, mesodermal lineage cells). Examples of such substances include cytokines, growth factors, and intercellular signal inhibitors.

[0039] For example, differentiation-inducing factors (hematopoietic differentiation factors) that differentiate blood cells such as hematopoietic stem cells from differentiated embryoid bodies include BMP4, activin A, bFGF, VEGF, TPO, EPO, SCF, FMS-like tyrosine kinase 3 ligand (Flt-3L), IL-6, soluble interleukin-6 receptor complex (sIL-6R), Notch ligand, GSK3 inhibitor, TGFβ inhibitor, Wnt signaling protein and its inhibitor Dickkopf1 (DKK1), Inhibitor of the Wnt Pathway (IWP)-4, FICZ (6-formylindolyl(3,2-b)carbazole), thiazovivin, IL-3, IL-6, IL-11, IGF-1, IGF-2, IL-1α, TNF-α, TPO, NR101, eltrombopag, butyzamide, retinol, retinyl acetate, retinoic acid, StemRegenin-1, UM171, UM729, valproic acid, TEP A, 740Y-P, etc., but are not limited thereto.

[0040] Examples of the GSK3 inhibitor include CHIR99021, CHIR98014, SB216763, L803-mts, BIO, MeBIO, LY2090314, lithium chloride or indirubin, preferably CHIR99021, but are not limited thereto.

[0041] Examples of the intercellular signal inhibitor (e.g., TGFβ inhibitor) include, but are not limited to, SB431542 (activin receptor-like kinase (ALK) 5 inhibitor), A83-01, galunisertib, SB525334, LY2109761, E-616452, LY3200882, SB-505124, PF-06952229, SD-208, ML347, R-268712, fresolimumab, ITD-1, AZ12601011 or BIO-013077-01, preferably SB431542.

[0042] In the first step of Production Method 1 of the present invention, the induction of a differentiated embryoid body in which the differentiation into the mesodermal cell lineage of the embryoid body is promoted is as described in Production Method 1 of the present invention. For example, by culturing an embryoid body in a medium containing any one or a combination of mesoderm differentiation factors that activate the Wnt signaling pathway and the BMP signaling pathway, it is possible to achieve the induction of a differentiated embryoid body in which the differentiation into the mesodermal cell lineage is promoted.

[0043] The substance capable of activating the Wnt signaling pathway is not particularly limited as long as it exhibits the desired effect, and those known per se can be used. As an example, but not limited thereto, CHIR99021, Wnt3a, SB216763, 6-bromoidirubin-3-oxime (BIO), and CHIR98014 etc. can be mentioned.

[0044] The substance capable of activating the BMP signaling pathway is not particularly limited as long as it exhibits the desired effect, and those known per se can be used. As an example, but not limited thereto, BMP4, BMP2, and activin A etc. can be mentioned.

[0045] In this invention, cytokines are small proteins with a molecular weight of approximately 5 to 20 kDa that are involved in intracellular signal transduction such as cell growth, differentiation, proliferation, movement, survival, immune responses, and inflammatory responses. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor (TNF) α, and transforming growth factor (TGF). In this invention, growth factors are proteins or peptides that regulate cell growth, differentiation, proliferation, movement, survival, and repair. In this invention, intercellular signaling inhibitors are compounds or drugs used to inhibit intracellular and / or intercellular signaling pathways at specific points.

[0046] In one embodiment, differentiated embryoids can be differentiated into mesodermal lineage cells using solidity calculated from images of differentiated embryoids cultured in the presence of BMP4 and / or CHIR99021 as an indicator. In another embodiment, when the median solidity of the differentiated embryoids to be analyzed is 0.6 to 0.8, the culture medium containing BMP4 and / or CHIR99021 is partially or completely removed, and the differentiated embryoids are cultured in a medium containing bFGF, VEGF, SCF and / or SB431542, thereby efficiently inducing differentiation into mesodermal lineage cells.

[0047] In the present invention, embryoid bodies may be derived from pluripotent stem cells. "Pluripotent stem cells" refer to stem cells that can be cultured in vitro while maintaining an undifferentiated state and that possess pluripotency. Embryonic stem cells (ES cells) isolated from embryos before the formation of the three germ layers after fertilization, embryos or fetuses after the organogenesis stage including primordial germ cells, are also included in "pluripotent stem cells." Induced pluripotent stem cells (hereinafter referred to as iPSCs or iPS cells) are also included in "pluripotent stem cells." "Induced pluripotent stem cells" refer to cells in which pluripotency has been induced by directly reprogramming somatic cells that have lost their pluripotency by expressing several genes such as Oct3 / 4, Sox2, Klf4, and Myc.

[0048] Pluripotent stem cells can be produced and cultured using known methods. For example, for induced human pluripotent stem cells, methods described in Cell, 2007, 131(5) pp. 861-872 and Kitayama, S. et al. (Stem Cell Reports. 6:213-227, (2016)) can be used. "Pluripotent stem cells" can also be obtained from animals that have undergone embryonic manipulation techniques, including cloned embryos, transgenic animals, and genome-edited mutant animals. Pluripotent stem cells are known to be able to differentiate into various cell types, including hematopoietic stem cells (HSCs).

[0049] Pluripotent stem cells can be obtained from designated institutions, and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute of Medical and Biological Sciences, Kyoto University. Human induced pluripotent stem cells 253G1 and 201B7 are available from the Center for iPS Cell Research and Application (CiRA), Kyoto University. Furthermore, the HLA homogeneous stock strain Ff-I14s03, the HLA homogeneous stock strain with genome editing to knock out the HLA-A, HLA-B, and CIITA genes Ff-I14s04-ABII-KO-7, and their respective clinical-grade strains QHJI 14s03 and QHJI14s04-ABII-KO-03 are available from the Kyoto University iPS Cell Research Foundation.

[0050] 2. Method for producing hematopoietic stem cells from embryoid bodies using solidity as an indicator In one embodiment of the production method 1 of the present invention, differentiated embryoid bodies can be further induced to differentiate into hematopoietic stem cells. In this regard, the present invention provides a method for producing hematopoietic stem cells from embryoid bodies (hereinafter sometimes referred to as "production method 2 of the present invention").

[0051] The second method of production of the present invention specifically includes the following steps: (1) a step of inducing an embryoid body into a differentiated embryoid body in which differentiation into mesodermal cell lineage is promoted; (2) a step of determining the area envelope (solidity) of the induced differentiated embryoid body; and (3) a step of culturing the differentiated embryoid body having a solidity of 0.6 to 0.8 in a differentiation induction medium containing hematopoietic differentiation factors to differentiate it into hematopoietic stem cells.

[0052] Steps (1) and (2) in the manufacturing method 2 of the present invention are the same as those described in the manufacturing method 1 of the present invention.

[0053] In the manufacturing method 2 of the present invention, the method for inducing differentiation of mesoderm cells into hematopoietic stem cells can be any method known to the present day. Specifically, hematopoietic stem cells can be obtained by culturing differentiated embryoid bodies in a medium containing hematopoietic differentiation factors. To confirm whether the cells obtained by differentiation induction are hematopoietic stem cells, surface antigens known to be expressed on the cell surface of hematopoietic stem cells can be used as markers. Examples of surface antigens expressed on hematopoietic stem cells include, but are not limited to, CD34, CD45, CD90, CD38, HLA-DR, CD117, CD123, CD133, CD45, CD201, CD164, CD49f, CD150, Sca-1, and GPRC5C.

[0054] The hematopoietic differentiation factor used in the manufacturing method 2 of the present invention is not particularly limited as long as it is a substance that has the effect of inducing differentiation from differentiated embryoid bodies to hematopoietic stem cells, and is the same as that described above.

[0055] The appropriate concentration of hematopoietic differentiation factors can be set as appropriate depending on the type of target cells to be further differentiated from hematopoietic stem cells and the amount of differentiated embryoid bodies. In one embodiment, the bFGF used as the hematopoietic differentiation factor is preferably 1 ng / ml to 100 ng / ml, 5 ng / ml to 60 ng / ml, and more preferably 7 ng / ml, 14 to 15 ng / ml, 23 to 25 ng / ml, and 56 to 57 ng / ml. In another embodiment, the VEGF is preferably 10 ng / ml to 200 ng / ml, 25 ng / ml to 160 ng / ml, and more preferably 28 ng / ml to 29 ng / ml, 50 to 55 ng / ml, 80 to 83 ng / ml, and 157 to 158 ng / ml. In another embodiment, SCF is preferably 1 ng / ml to 300 ng / ml, 5 ng / ml to 250 ng / ml, and more preferably 9 ng / ml to 10 ng / ml, 28 ng / ml to 29 ng / ml, 50 ng / ml to 55 ng / ml, 150 ng / ml to 151 ng / ml, and 220 ng / ml to 225 ng / ml. In yet another embodiment, SB431542 is preferably 1 to 20 μM, 5 to 15 μM, and more preferably 5 to 7 μM, 8 to 9 μM, and 9 to 10 μM.

[0056] 3. Method for Producing Mesodermal Lineage Cells, Ectodermal Lineage Cells, or Endodermal Lineage Cells from Embryoid Bodies Using Solidity as an Indicator Up to this point, the explanation has focused on the differentiation of embryoid bodies into mesodermal lineage cells. However, the essence of the present invention lies in determining the optimal timing for differentiation induction using the solidity of differentiated embryoid bodies as an indicator. Considering this fact, the inventors' findings can be extended not only to differentiation into mesodermal lineage cells, but also to an invention for determining the optimal timing for differentiation induction of embryoid bodies into mesodermal lineage cells, ectodermal lineage cells, or endodermal lineage cells, using solidity as an indicator. From this viewpoint, the present invention also provides a method for differentiating an embryoid body into target cells (hereinafter sometimes referred to as "the differentiation method of the present invention"), comprising the following steps: (1) a step of inducing the embryoid body into a differentiated embryoid body in which differentiation into a mesodermal cell lineage, an ectoderm cell lineage, or an endoderm cell lineage is promoted; and (2) a step of determining the area envelope degree (solidity) of the induced differentiated embryoid body.

[0057] The methods for determining the embryoid body, differentiated embryoid body, and area envelope in the differentiation method of the present invention are the same as those described above.

[0058] In the first step of the differentiation method of the present invention, differentiated embryoid bodies are induced in which differentiation into mesodermal cell lineage, ectoderm cell lineage, or endodermal cell lineage is promoted. Induction of differentiated embryoid bodies in which differentiation into each lineage is promoted can be carried out using methods known to the present day. Induction of differentiated embryoid bodies in which differentiation into mesodermal cell lineage is promoted is as described above. Furthermore, induction of differentiated embryoid bodies in which differentiation into ectoderm cell lineage or endodermal cell lineage is promoted can be carried out using methods known to the present day. For example, in the case of ectoderm cell lineage, differentiated embryoid bodies in which differentiation into ectoderm cell lineage is promoted can be induced by culturing embryoid bodies in the presence of a substance that can promote the induction of differentiation of embryoid bodies into ectoderm cells (e.g., EGF, FGF2, GSK3 inhibitor, TGFβ inhibitor, etc.). Furthermore, in the case of endodermal cell lineages, differentiated embryoid bodies can be induced by culturing embryoid bodies in the presence of substances that can promote the differentiation of embryoid bodies into endodermal cells (e.g., BMP4, activin A, FGF4, HGF, oncostatin M, etc.), thereby promoting differentiation into endodermal cell lineages.

[0059] In the second step of the differentiation method of the present invention, the area envelope degree of the differentiated embryoid body induced in the first step is determined. The method for measuring the area envelope degree is as described above. Similar to the manufacturing method 1 of the present invention described above (i.e., the embodiment intended for differentiation into mesodermal cells), the timing of differentiation into target cells should be optimized using solidity as an indicator in the differentiated embryoid body in which differentiation into the ectoderm cell lineage or endodermal cell lineage has been promoted. Those skilled in the art can appropriately determine the solidity for each target cell.

[0060] In the differentiation method of the present invention, the target cells refer to progenitor cells or mature cells of a specific tissue, and mean cells derived from each of the three germ layers (ectoderm lineage cells, mesoderm lineage cells, and endoderm lineage cells). Differentiated embryoid bodies can be further induced to differentiate into target cells using known methods and conditions. Specifically, target cells can be obtained by culturing differentiated embryoid bodies in a culture medium containing the aforementioned differentiation-inducing factors.

[0061] Mesodermal cells are as described above.

[0062] Ectoderm cells are cells derived from the ectoderm of the embryo that have the ability to differentiate into precursor cells and mature cells such as mesenchymal stem cells, neural progenitor cells, various cells of the digestive system, various cells of the urinary system, various cells of the respiratory system, various cells of endocrine tissue, various cells of muscle tissue, various cells of the sensory organs, and various cells of the reproductive system. Cells that can differentiate from ectoderm cells in this way are called ectoderm lineage cells in this invention. Mesenchymal stem cells can further differentiate into osteoblasts, adipocytes, etc. Neural progenitor cells can further differentiate into central nervous system cells, peripheral nerve cells, neural crest cells, axon cells, myelin cells, etc. Various cells of the digestive system include oral epithelial cells, tongue cells, dental enamel cells, and lateral rectal epithelial cells. Various cells of the urinary system include lateral urethral epithelial cells. Various cells of the endocrine tissue include nasal cavity epithelial cells, pituitary cells, pineal gland cells, and adrenal medulla cells. Examples of muscle tissue cells include pupillary sphincter cells and pupillary dilator cells. Examples of sensory organ cells include skin cells, corneal cells, retinal cells, inner ear cells, and outer ear cells. Examples of reproductive organ cells include vaginal epithelial cells.

[0063] Endoderm cells are cells derived from the primitive endoderm that have the ability to differentiate into precursor cells and mature cells of various organs, such as cells of the digestive system, urinary system, respiratory system, endocrine tissue, and sensory organs. In this invention, cells that can differentiate from endoderm cells in this way are called endodermal lineage cells. Cells of the digestive system include liver cells, islet cells, esophageal epithelial cells, gastric epithelial cells, and gastrointestinal epithelial cells. Liver cells include hepatocytes, Kupffer cells, and stellate cells. Cells of the urinary system include bladder epithelial cells and posterior urethral epithelial cells. Cells of the respiratory system include tonsil cells, pharyngeal epithelial cells, laryngeal epithelial cells, tracheal epithelial cells, and alveolar epithelial cells. Cells of the endocrine tissue include thyroid cells, parathyroid cells, and thymocytes. Cells of the sensory organs include Eustachian tube cells and tympanic cell cells.

[0064] In the differentiation method of the present invention, further differentiation induction of each cell of the three germ layers into target cells, which is determined to be the optimal differentiation induction timing based on the Solidity value determined in step (2), can be achieved by methods known to those skilled in the art, depending on the type of target cell. Several examples are given below, but are not limited to these.

[0065] When producing mesenchymal stem cells from differentiated embryoid bodies whose differentiation into mesodermal cell lineages has been promoted, the following is an example: (1) Culture the embryoid bodies in the presence of BMP4 and / or CHIR99021. (2) Determine the solidity of the differentiated embryoid bodies. (3) Culture the differentiated embryoid bodies with appropriate solidity in a differentiation-inducing medium containing differentiation-inducing factors. (4) After culturing in (3) for a certain period, remove part or all of the medium, change to a medium for mesenchymal stem cell differentiation, and culture further.

[0066] When producing mesenchymal stem cells from differentiated embryoid bodies whose differentiation into ectoderm cell lineages has been promoted, the following is an example: (1) Culture the embryoid bodies in the presence of SB431542 and / or CHIR99021. (2) Determine the solidity of the differentiated embryoid bodies. (3) Culture the differentiated embryoid bodies with appropriate solidity in a medium containing SB431542, EGF and / or FGF2. (4) After culturing in (3) for a certain period, remove part or all of the medium, change to a medium for mesenchymal stem cell differentiation, and culture further.

[0067] Examples of nerve cell production include: (1) culturing embryoid bodies in the presence of N-2 supplement, B27 supplement, non-essential amino acids (NEAA), sodium pyruvate, L-glutamine, SB431542 and / or dolsomorphine; (2) determining the solidity of differentiated embryoid bodies; (3) culturing differentiated embryoid bodies with appropriate solidity in a medium containing retinoic acid and purmorphamine.

[0068] When producing cardiac cells, the following is an example: (1) Culturing embryoid bodies in the presence of BMP4 and / or Y-27632 (ROCK inhibitor); (2) Determining the solidity of differentiated embryoid bodies; (3) Culturing differentiated embryoid bodies with appropriate solidity in a medium containing BMP4, bFGF and / or activin A; (4) After culturing in (3) for a certain period, removing part or all of the medium and culturing in a medium containing IWP-4 and / or VEGF.

[0069] Examples of hepatocyte production include: (1) culturing embryoid bodies in the presence of activin A; (2) determining the solidity of differentiated embryoid bodies; (3) culturing differentiated embryoid bodies with appropriate solidity in a medium containing BMP4 and / or FGF4; (4) after culturing in (3) for a certain period, removing part or all of the medium and culturing in a medium containing OsM.

[0070] The present invention will be described in more detail in the following examples, but the present invention is not limited in any way by these examples.

[0071] In Examples 1 to 3 below, the efficiency of differentiation induction from differentiated embryoid bodies to hematopoietic stem cells was evaluated using solidity as an indicator. In Examples 1-2, 2-2, and 3-2, the efficiency of differentiation induction from differentiated embryoid bodies to hematopoietic stem cells was evaluated using a culture medium with a different composition than that used in Examples 1 to 3, with solidity as an indicator. In Examples 4 to 6, the efficiency of differentiation induction from embryoid bodies to mesenchymal stem cells was evaluated using solidity as an indicator. In Example 4-2, the differentiation potential of mesenchymal stem cells differentiated from embryoid bodies to bone, cartilage, or adipocytes was evaluated using a culture medium with a different composition than that used in Example 4, with solidity as an indicator. In Examples 7 to 10, the efficiency of differentiation induction of mesodermal cells was evaluated by changing the concentration of hematopoietic differentiation factors. Furthermore, the differentiation efficiency of mesoderm cells was evaluated by the yield and recovery rate of hematopoietic stem cells and mesenchymal stem cells further differentiated from mesoderm cells. In addition, in [Example 11], hematopoietic stem cells obtained by the same method as in [Example 1] were further differentiated into NK cells, and it was evaluated whether functional hematopoietic stem cells were obtained.

[0072] [Example 1] 1. Materials and Methods (Induction of Differentiation of Embryoid Bodies into Hematopoietic Stem Cells) Embryoid bodies (hereinafter referred to as EB) prepared from human iPS cells by the method described in International Publication No. 2021 / 230304 were seeded at a concentration of 10-20 EB / well in a polystyrene tissue culture 6-well plate (Thermo Scientific: 130184) with culture medium (1) added, and adherent culture was performed to form differentiated embryoid bodies. The culture medium (1) used StemFit AK02N (Ajinomoto: AJ100) containing 50 ng / mL BMP-4 (R&D SYSTEMS: 314-BP-010), 24 μg / well retronectin (TaKaRa: T100B), 9.6 μL / well iMatrix-511 (Nippi: 892012), and 5 μM CHIR99021 (GSK3 inhibitor) (Nacalai Tesque: 18764-44). Images of differentiated embryoid bodies were acquired using an all-in-one fluorescence microscope BZ-X810 (Keyence), and the solidity of the differentiated embryoid bodies was calculated using the image analysis method described later. When the median solidity of the differentiated embryoid bodies to be analyzed in one well reached 0.691–0.769 (I in Figure 4), the above medium (1) was removed and replaced with medium (2). The culture medium (2) used was X-VIVO15 (Lonza: BEBP02-054Q) containing 225 μM Monothiogenic Cerol Solution (MTG) (SIGMA: M6145-25ML), 25 μg / mL Ascorbic Acid (SIGMA: A92902-25G), 25 ng / mL bFGF (Wako: 060-05383), 50 ng / mL VEGF (Ajinomoto: 8019173), 50 ng / mL SCF (Wako: 193-15513), and 10 μM SB431542 (ALK5 inhibitor) (Selleckchem: S1067).

[0073] After switching to medium (2) and culturing for another two days, medium (2) was removed and replaced with medium (3). Medium (3) (control conditions) used X-VIVO15 containing 450 μM MTG, 50 μg / mL ascorbic acid, 50 ng / mL bFGF, 50 ng / mL VEGF, 50 ng / mL SCF, 5 ng / mL Flt3-L (Wako: 067-05393), and 20 ng / mL IL-3 (Wako: 09605703). After culturing in medium (3) for three days, the obtained hematopoietic stem cells were harvested, and the cell count was measured and the expression of cell surface markers was confirmed by Flow Cytometry (FCM).

[0074] 2. Analysis (Image Analysis) Images were acquired using a Keyence BZX-810 with a 4x objective lens. Images were acquired starting approximately 24 hours after the start of culture in medium (1). For the output of images, 100 tiling images were acquired for any well of a 6-well plate, 10 horizontally and 10 vertically, at 1920 pixels × 1440 pixels and 256 grayscale levels. (Note that, considering that the colony size of differentiated embryoid bodies is sufficiently large, the pixel length is not limited to the specific pixel length value (3.77442 um / px) used in this embodiment, and may be changed as appropriate as long as the present invention is feasible.) Image processing was performed using OSS OpenCV (Apache License 2.0). For the tiling image, a binarized image was created by masking cell populations derived from differentiated embryoid bodies as Regions of Interest (ROIs). Cell populations derived from differentiated embryoid bodies were extracted using the following method: First, the regions recognized as ROIs were sorted by area. Next, the number of cells (denoted as 'a') representing 80% of the number of seeded embryoid bodies was calculated. The top 'a' cell populations from the area-sorted data were used for analysis. Solidity was calculated using OpenCV functions on the binarized image created by image processing.

[0075] The statistics were calculated as the median, mean, and variance of the ROI in a given incubator (or any single well if using a 6-well plate). In the box plot, the box range (IQR) represents the first to third quartiles, and the divider within the box represents the median (Figure 3). The upper limit of the whiskers is the third quartile plus 1.5 times the IQR, and the lower limit is the first quartile minus 1.5 times the IQR. Data points not included in the whisker range are indicated as outliers with white circles. In the statistical analysis, the null hypothesis was "there is no difference between the two conditions being compared for a given parameter," and Welch's t-test was performed. The null hypothesis was rejected when the p-value was less than 0.05.

[0076] (Analysis of surface antigens in hematopoietic stem cells) Hematopoietic stem cells were collected by pipetting. The collected cells were stained with anti-CD34 antibody (BioLegend: 343506), anti-CD90 antibody (BioLegend: 328108), and anti-CD201 antibody (BioLegend: 351906). Subsequently, the cells were washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer (product name: BD Accuri C6 Flow Cutometer, Beckton Dickinson).

[0077] 3. Results In [Example 1], approximately 1.2 x 10⁻¹⁰ units were obtained from 1 well. ５ A cell population of cells was obtained (Figure 1), and the average positive rate for CD34, a hematopoietic stem cell marker, within this cell population was 61.6% (Figure 2).

[0078] [Example 1-2] 1. Materials and Methods In the above-mentioned (induction of differentiation of embryoid bodies into hematopoietic stem cells), the following media were used instead of media (1), media (2), and media (3).

[0079] Culture medium (1): StemFit AK02N Solution A (Ajinomoto, 400 mL) was mixed with StemFit For Difference (Ajinomoto, 100 mL), and further mixed with 50 ng / mL BMP-4 (R&D SYSTEMS: 314-BP-010), 24 μg / well retronectin (TaKaRa: T100B), 9.6 μL / well iMatrix-511 (Nippi: 892012), and 5 μM CHIR99021 (GSK3 inhibitor) (Nacalai Tesque: 18764-44).

[0080] Culture medium (2): To StemFit AK02N Solution A (Ajinomoto, 400 mL), StemFit For Difference (Ajinomoto, 100 mL) was added, and further, 225 μM Monothioglycerol Solution (MTG) (SIGMA: M6145-25 mL), 25 μg / mL Ascorbic acid (SIGMA: A92902-25 G), 25 ng / mL bFGF (Wako: 060-05383), 50 ng / mL VEGF (Ajinomoto: 8019173), 50 ng / mL SCF (Wako: 193-15513), 10 μM A culture medium supplemented with SB431542 (ALK5 inhibitor) (Selleckchem: S1067).

[0081] Culture medium (3): StemFit AK02N Solution A (Ajinomoto, 400 mL) was mixed with StemFit For Difference (Ajinomoto, 100 mL), and then 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL Flt3-L, 20 ng / mL IL-3, 10 ng / mL TPO (R&D Systems: 288-TP), and 50 ng / mL IL-6 (R&D Systems: 206IL / CF) were added to the culture medium.

[0082] When the median solidity of the differentiated embryoids to be analyzed in one well reached 0.775, medium (1) was removed and replaced with medium (2). After culturing for another day after switching to medium (2), medium (2) was removed and replaced with medium (3). Other conditions were carried out in the same manner as in [Example 1].

[0083] 2. Analysis The analysis was performed using the same method as in [Example 1]. The statistical values ​​of the solidity of the differentiated embryoid bodies to be analyzed in 1 well are shown in Figure 7.

[0084] 3. Result approximately 1.5x10 ４ Cells were obtained (Figure 5), and the positive rate for CD34, a hematopoietic stem cell marker, was 55.7% within this cell population (Figure 6).

[0085] [Example 2] 1. Materials and Methods (Induction of Differentiation of Embryoid Bodies into Hematopoietic Stem Cells) When the median solidity of the differentiated embryoid bodies to be analyzed in 1 well was 0.896 to 0.922 (a point earlier than shown in Figure 4, I), medium (1) was removed and replaced with medium (2). Otherwise, the procedure was the same as in [Example 1].

[0086] 2. Analysis The analysis was performed using the same method as in [Example 1]. The statistical values ​​of the solidity of the differentiated embryoid bodies to be analyzed in 1 well are shown in Figure 3.

[0087] 3. Results: Although the cells were cultured in medium (3) for 3 days, no hematopoietic stem cells were obtained (Figure 1).

[0088] [Example 2-2] 1. Materials and Methods In [Example 1-2], when the median solidity of the differentiated embryoid bodies to be analyzed in one well reached 0.878, the above medium (1) was removed and replaced with medium (2). Otherwise, the procedure was the same as in [Example 1-2].

[0089] 2. Analysis The analysis was performed using the same method as in [Example 1]. The statistical values ​​of the solidity of the differentiated embryoid bodies to be analyzed in 1 well are shown in Figure 7.

[0090] 3. Results: Approximately 1.0 x 10 from 1 well ４ Cells were obtained (Figure 5), and among this cell population, the positive rate for CD34, a hematopoietic stem cell marker, was 21.9% (Figure 6).

[0091] [Example 3] 1. Materials and Methods (Induction of Differentiation of Embryoid Bodies into Hematopoietic Stem Cells) When the median Solidity of the differentiated embryoid bodies to be analyzed in the well phase, obtained from image analysis, reached 0.825 to 0.832 (II in Figure 4), medium (1) was removed and replaced with medium (2). The rest of the procedure was the same as in [Example 1].

[0092] 2. The analysis was performed using the same method as in Analysis Example 1. The statistical values ​​of the solidity of the differentiated embryoid bodies to be analyzed in 1 well are shown in Figure 3.

[0093] 3. Results: Although culture was performed in medium (3) for 3 days, no suspended hematopoietic stem cells were obtained (Figure 1).

[0094] [Example 3-2] 1. Materials and Methods In [Example 1-2], when the median solidity of the differentiated embryoid bodies to be analyzed in one well reached 0.801, the above medium (1) was removed and replaced with medium (2). Otherwise, the procedure was the same as in [Example 1-2].

[0095] 2. Analysis The analysis was performed using the same method as in [Example 1]. The statistical values ​​of the solidity of the differentiated embryoid bodies to be analyzed in 1 well are shown in Figure 7.

[0096] 3. Results: Although the cells were cultured in medium (3) for 3 days, no hematopoietic stem cells were obtained (Figure 5).

[0097] [Example 4] 1. Materials and Methods (Induction of Differentiation of Embryoid Bodies into Mesenchymal Stem Cells) Embryoid bodies (hereinafter referred to as EBs) prepared from human iPS cells by the method described in International Publication No. 2021 / 230304 were seeded at a concentration of 10-20 EBs / well in a polystyrene tissue culture 6-well plate (Thermo Scientific: 130184) with culture medium (1) added, and adherent culture was performed to form differentiated embryoid bodies. The culture medium (1) used StemFit AK02N (Ajinomoto: AJ100) containing 50 ng / mL BMP-4 (R&D SYSTEMS: 314-BP-010), 24 μg / well retronectin (TaKaRa: T100B), 9.6 μL / well iMatrix-511 (Nippi: 892012), and 5 μM CHIR99021 (GSK3 inhibitor) (Nacalai Tesque: 18764-44).

[0098] Images of differentiated embryoid bodies were acquired using an all-in-one fluorescence microscope BZ-X810 (Keyence), and the solidity of the differentiated embryoid bodies was calculated using the same image analysis method as in [Example 1] (Figure 10). When the median solidity of the differentiated embryoid bodies to be analyzed in one well reached 0.715 ("Best condition"), the above culture medium (1) was removed and replaced with culture medium (2). The culture medium (2) used StemFit AK02N, which contains 50 ng / mL bFGF (Wako: 060-05383), 50 ng / mL VEGF (Ajinomoto: 8019173), 50 ng / mL SCF (Wako: 193-15513), 10 μM SB431542 (ALK5 inhibitor) (Selleckchem: S1067), and 20% StemFit For Difference (Ajinomoto).

[0099] After switching to medium (2) and culturing for another two days, medium (2) was removed and replaced with medium (3). Medium (3) used StemFit AK02N containing 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL Flt3-L (Wako: 067-05393), 20 ng / mL IL-3 (Wako: 09605703), 10 ng / mL TPO (R&D Systems: 288-TP), 50 ng / mL IL-6 (R&D Systems: 206IL / CF), and 20% StemFit For Difference (Ajinomoto).

[0100] After culturing in medium (3) for 3 days, cells adhering to the bottom were detached using TripLE and re-seed onto another adherent culture plate containing aMEM with 10% fetal bovine serum for further culturing. After 1 day, the medium was changed and floating cells were removed. Cells cultured for 8 days were collected, and the cell count was measured and the expression of cell surface markers was confirmed by Flow Cytometry (FCM). The analysis method is as follows.

[0101] 2. Surface Antigen Analysis of Mesenchymal Stem Cells Mesenchymal stem cells were collected by pipetting. The collected cells were stained with anti-CD90 antibody (BioLegend: 328108), anti-CD105 antibody (BioLegend: 323205), anti-CD73 antibody (BioLegend: 344005), anti-CD11b antibody (BioLegend: 301317), anti-CD19 antibody (BioLegend: 302208), anti-CD45 antibody (BioLegend: 304017), anti-HLA-DR antibody (BioLegend: 307606), anti-CD14 antibody (BioLegend: 325604), and anti-CD34 antibody (BioLegend: 343506). Subsequently, the cells were washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer (product name: BD Accuri C6 Flow Cutometer, Beckton Dickinson). The results of the analysis are shown in Figure 9.

[0102] 3. Results: Approximately 6 x 10 from 1 well ４ Cells mesenchymal stem cells were obtained, and the recovery rate from seeded cells was 24% (Figure 8). In [Example 4], the presence of hematopoietic stem cells suspended in the culture medium was confirmed during culture in medium (3).

[0103] [Example 4-2] 1. Materials and Methods (Induction of Differentiation of Embryoid Bodies into Mesenchymal Stem Cells) Differentiated embryoid bodies were formed using the same method as in [Example 4], except that StemFit AK02N containing 50 ng / mL BMP-4 (R&D SYSTEMS: 314-BP-010), 24 μg / well retronectin (TaKaRa: T100B), 9.6 μL / well iMatrix-511 (Nippi: 892012), 5 μM CHIR99021 (GSK3 inhibitor) (Nacalai Tesque: 18764-44) and 20% StemFit For Difference (Ajinomoto) was used in medium (1).

[0104] At the same time as in [Example 4], culture medium (1) was removed and replaced with culture medium (2). Culture medium (2) was the same as culture medium (2) in [Example 4].

[0105] After switching to medium (2) and culturing for another day, medium (2) was removed and replaced with medium (3). Medium (3) was the same as medium (3) in [Example 4].

[0106] After culturing in medium (3) for 10 days, cells adhering to the bottom were detached using TrypLE and re-seed onto another adherent culture plate containing aMEM with 10% fetal bovine serum for further cultivation. After 2 days, the medium was changed and floating cells were removed. Cells were cultured for another 2 days, and the cell count was measured and the expression of cell surface markers was confirmed using Flow Cytometry (FCM).

[0107] 2. Surface antigen analysis of mesenchymal stem cells Surface antigen analysis was performed using the same method as in [Example 4]. The results of the analysis are shown in Figure 11.

[0108] 3. Differentiation Test into Bone, Cartilage, or Adipocytes Furthermore, cells cultured in the above-mentioned medium (3) for 4 days were harvested and a differentiation test into bone, cartilage, or adipocytes was performed to confirm whether they possessed the ability to differentiate into mesenchymal stem cells. The differentiation induction method was as follows.

[0109] Osteogenesis was performed using the StemPro Osteogenesis Difference Kit (gibco: A10072-01) following the attached protocol. 7.2 × 10⁴ cells were placed in a polystyrene tissue culture 6-well plate (Thermo scientific: 130184) containing aMEM with 10% fetal bovine serum. ４ Cells / well cells were seeded and cultured for 10 days. The culture medium was then changed to Complete Osteogenesis medium, and the cells were cultured for a further 21 days. After culturing, calcium deposition associated with osteocyte differentiation was detected using an Alizarin Red S staining kit (Funakoshi: BMK-R009).

[0110] Adipogenic differentiation was performed using the StemPro Adipogenesis Differentiation Kit (Gibco: A10070-01) according to the following procedure with reference to the attached protocol. 7.2×10 ４ cells / well were seeded into a polystyrene tissue culture 6-well plate (Thermo Scientific: 130184) containing αMEM with 10% fetal bovine serum and cultured for 10 days. Then, the medium was changed to Complete Adipogenesis Medium and cultured for an additional 21 days. After culturing, the Lipid Assay Kit (Cosmo Bio: AK02F) was used to detect the fat accumulated in adipocytes.

[0111] Chondrogenic differentiation was performed using the StemPro Chondrogenesis Differentiation Kit (Gibco: A10071-01) according to the following procedure in accordance with the attached protocol. A cell suspension of 1.6×10 ７ cells / well was prepared and seeded into a polystyrene tissue culture 48-well plate (Thermo Scientific: 130187) by making 5 μL / well droplets. PBS was added to the wells around the wells where the droplets were formed at 1 mL per well, and incubated at 37°C in a 5% CO ２ environment for 2 hours. After 2 hours, 1 mL of Complete Chondrogenesis Medium was gently added, and cultured for 14 days while changing the medium once every 2-3 days. After culturing, the Alcian Blue Staining Kit (Funakoshi: BMK-R011) was used to detect the production of acidic mucopolysaccharides (chondroitin sulfate, hyaluronic acid, etc.) associated with chondrocyte differentiation. The results of the differentiation test are shown in Figure 12.

[0112] 4. Results Approximately 1.44×10 ４ mesenchymal stem cells were obtained from 1 well, and the recovery rate with respect to the seeded cells was 57.6%. Similar to [Example 4], the presence of hematopoietic stem cells floating in the medium was confirmed during culturing in Medium (3).

[0113] [Example 5] 1. Materials and Methods Mesenchymal stem cells were induced using the same method as in [Example 4], except that when the median solidity of the differentiated embryoid bodies to be analyzed in 1 well was 0.901 ("Early condition"), medium (1) was removed and replaced with medium (2).

[0114] 2. Analysis The solidity was calculated using the same method as in [Example 4]. In this example, surface antigen analysis of mesenchymal stem cells and determination of the recovery rate were not performed (because mesenchymal stem cells could not be obtained).

[0115] 3. Results In Example 5, mesenchymal stem cells could not be obtained (Figure 8). The results of the Solidity calculation are shown in Figure 10. In addition, in Example 5, the presence of hematopoietic stem cells in the culture medium (3) could not be confirmed.

[0116] [Example 6] 1. Materials and Methods The procedure was the same as in [Example 4], except that when the median solidity of the differentiated embryoid bodies to be analyzed in one well reached 0.858 ("Late condition"), medium (1) was removed and replaced with medium (2).

[0117] 2. Analysis The calculation of solidity, analysis of surface antigens of mesenchymal stem cells, and determination of the recovery rate were performed using the same method as in [Example 4].

[0118] 3. Results In [Example 6], mesenchymal stem cells were obtained (confirmed by cell expression antigen analysis; data not shown). However, the recovery rate was 17%, which was lower than that of [Example 4] (Figure 8). Also, in [Example 6], the presence of hematopoietic stem cells in the culture medium (3) could not be confirmed.

[0119] Consideration of Examples 1 to 6: From Examples 1 to 6 described above, when differentiation induction of embryoid bodies was performed when the median solidity was 0.8 or less, differentiation induction was achieved efficiently, and as a result, mesoderm cells, and even hematopoietic stem cells and mesenchymal stem cells, could be efficiently prepared. On the other hand, when differentiation induction was performed when the median solidity was 0.8 or more (i.e., "Early conditions" or "Late conditions"), the efficiency of differentiation induction into the desired cells decreased, or differentiation could not be induced at all.

[0120] [Examples 7] to [Examples 10] 1. Materials and Methods (Induction of differentiation of embryoid bodies into hematopoietic stem cells) was carried out in the same manner as in [Example 1], except that the concentration of culture medium (2) was adjusted as shown in Table 1 below.

[0121] 2. Analysis The analysis was performed using the same method as in [Example 1].

[0122] 3. Results The CD34 positivity rate is shown in Table 1 below. When the culture medium was changed under conditions where the median solidity was 0.8 or less, it was shown that hematopoietic stem cells could be differentiated with high efficiency even when the concentration of culture medium (2) was adjusted.

[0123]

[0124] [Example 11] 1. Materials and Methods (Induction of differentiation of embryoid bodies into hematopoietic stem cells) The procedure was the same as in Example 1, except that serum-free medium containing 50 ng / mL VEGF, 50 ng / mL SCF, 5 ng / mL Flt3-L (Wako: 067-05393), and 20 ng / mL IL-3 (Wako: 09605703) was used in medium (3). (Induction of differentiation of hematopoietic stem cells into NK cells) After switching to medium (3) and culturing for a further 3 days, the obtained hematopoietic stem cells were harvested and seeded in medium (4). Medium (4) was a serum-free medium containing 50 ng / mL IL-2 (Miltenyie Biotech: 130-097-748), 50 ng / mL IL-7 (PEPROTECH: 200-07-50UG), 50 ng / mL IL-15 (PEPROTECH: 200-15-50UG), 20 ng / mL IL-3, 50 ng / mL SCF, and 50 ng / mL Flt3-L. (Expanded culture of NK cells) After switching to medium (4) and culturing for a further 5 days, the obtained NK cells were harvested, seeded in medium (5), and cultured for 14 days. The culture medium (5) used was a serum-free medium containing 50 ng / mL IL-2, 50 ng / mL IL-7, 50 ng / mL IL-15, 20 ng / mL IL-3, 50 ng / mL SCF, and 50 ng / mL Flt3-L.

[0125] 2. Analysis (Image Analysis) was performed in the same manner as in [Example 1]. (Surface antigen analysis of NK cells) NK cells were collected by pipetting. The collected cells were stained with anti-CD45 antibody (BioLegend: 304017), anti-CD56 antibody (BioLegend: 362508), anti-KIR antibody (BioLegend: 312604), anti-CD16 antibody (BioLegend: 302006), anti-NKG2D antibody (BioLegend: 320808), anti-NKp44 antibody (BioLegend: 325110), and anti-NKp46 antibody (BioLegend: 331918). The cells were then washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer (product name: BD Accuri C6 Flow Cytometer, manufactured by Beckton Dickinson).

[0126] 3. Results The positive rates for the NK cell markers CD45 and CD56, and the NK mature cell markers NKG2D, NKp44, NKp46, KIR, and CD16 are shown in Figure 13. It was shown that hematopoietic stem cells differentiated under the same conditions as in [Example 1] could be further differentiated into NK cells. Specifically, it was shown that functional hematopoietic stem cells could be differentiated when the culture medium was changed under conditions where the median solidity was 0.8 or less.

[0127] According to the present invention, the optimal timing for embryoid body differentiation induction can be optimized non-invasively and objectively using solidity as an indicator. Therefore, according to the present invention, target cells (e.g., hematopoietic stem cells) prepared by differentiation induction from embryoid bodies can be prepared efficiently and with high reproducibility. Accordingly, the present invention is extremely useful, for example, in the field of cell therapy.

[0128] This application is based on Japanese Patent Application No. 2024-193944 (filed November 5, 2024) and Japanese Patent Application No. 2025-146382 (filed September 3, 2025), both of which are fully incorporated herein by reference.

Claims

1. A method for producing mesodermal lineage cells from embryoid bodies, comprising the following steps: (1) inducing embryoid bodies into differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted; and (2) determining the area envelope degree (solidity) of the induced differentiated embryoid bodies.

2. The method according to claim 1, further comprising the following steps: (3) A step of culturing a differentiated embryoid having a solidity of 0.6 to 0.8 in a differentiation-inducing medium containing a differentiation-inducing factor.

3. The method according to claim 1, wherein step (1) is performed using a culture medium containing BMP4 and / or CHIR99021.

4. The method according to claim 2, wherein the differentiation-inducing factor comprises at least one selected from the group consisting of cytokines, growth factors, and intercellular signaling inhibitors.

5. The method according to claim 4, wherein the cytokine comprises at least one selected from the group consisting of SCF, Flt3-L, IL-3, and IL-6.

6. The method according to claim 4, wherein the growth factor is VEGF and / or bFGF.

7. The method according to claim 4, wherein the intercellular signaling inhibitor is SB431542.

8. A method for producing hematopoietic stem cells from embryoid bodies, comprising the following steps: (1) inducing embryoid bodies into differentiated embryoid bodies in which differentiation into mesodermal cell lineages is promoted; (2) determining the area envelope (solidity) of the induced differentiated embryoid bodies; and (3) culturing the differentiated embryoid bodies having a solidity of 0.6 to 0.8 in a differentiation induction medium containing hematopoietic differentiation factors to differentiate them into hematopoietic stem cells.

9. The method according to claim 1 or 8, wherein the differentiated embryoid body is derived from induced pluripotent stem cells or embryonic stem cells.

10. A method for differentiating an embryoid body into target cells, comprising the following steps: (1) inducing the embryoid body into a differentiated embryoid body in which differentiation into a mesodermal cell lineage, an ectoderm cell lineage, or an endoderm cell lineage is promoted; and (2) determining the area envelope (solidity) of the induced differentiated embryoid body.

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