Cell aggregate and method for producing same

Abstract

[Problem] To obtain a cell aggregate in which the formation process of a bone, particularly cartilage tissue, is reproduced. [Solution] By using a method comprising co-culturing sclerotome cells and vascular endothelial cells expressing a vascular endothelial growth factor, it is possible to obtain a cell aggregate having a cartilage-like tissue. The cell aggregate includes chondrocytes and osteoblast progenitor cells.

Description

Cell aggregates and methods for producing same

[0001] The present invention relates to a cell aggregate containing chondrocytes and osteoblast precursor cells, and a method for producing the cell aggregate.

[0002] In recent years, attention has been focused on the development of highly biocompatible functional medical materials. Biocompatible functional medical materials include artificially cultured cell aggregates. To develop such cell aggregates, it is useful to use a culture model that reflects the types of biomolecules and cells observed during the development and differentiation of biological tissues into which the cell aggregates can be introduced in order to determine their functionality and biocompatibility.

[0003] Most bones, excluding the craniofacial region, such as the axial bones and limb bones, are formed by a process called endochondral ossification. In endochondral ossification, mesenchymal cells aggregate and differentiate into chondrocytes, forming cartilage. Subsequently, chondrocytes located in the center of the cartilage differentiate and hypertrophy. Hypertrophic chondrocytes secrete growth factors such as hedgehog, bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF). Osteoblast precursor cells reside in the perichondrium, and differentiate into osteoblasts upon receiving growth factors from hypertrophic chondrocytes. Osteoblasts present in the perichondrium then form bone called bone collars, which eventually become cortical bone. Growth factors from hypertrophic chondrocytes induce nearby vascular cells and promote vascular invasion. At this time, osteoblast precursor cells present in the perichondrium migrate into the bone tissue along with the blood vessels, where they form bone marrow (Non-Patent Document 1).

[0004] Thus, the process of endochondral ossification is made possible by the functioning of various cells at specific times and locations, but the underlying mechanisms remain unclear. Therefore, in vitro bone development models that can mimic endochondral ossification are being studied. For example, methods for inducing hypertrophic chondrocytes (Non-Patent Document 2) and transdifferentiation of hypertrophic chondrocytes into osteoblasts (Non-Patent Document 3) have been reported.

[0005] Kanczler et al., European Cells & Materials, 15, 100-114, 2008 Pretemer et al., STEM CELL REPORTS, Volume 16, 3, 610-625, 2021 Lamande et al., Proceedings of the National Academy of Sciences, 120, 19, e2211510120, 2023

[0006] However, in all of these cases, the tissues formed lacked not only the perichondrium that is naturally present in cartilage, but also osteoblast precursor cells, which are essential components of perichondrium, and therefore the degree of reproducibility of bone development was limited. In response to this problem, the present inventors conducted extensive research with the aim of obtaining a cell aggregate that would reproduce the process of bone, particularly cartilage tissue, formation.

[0007] By co-culturing vertebral disc cells with vascular endothelial cells expressing vascular endothelial growth factor, it became possible to obtain cell aggregates with cartilage-like tissue, which contain chondrocytes and osteoblast precursor cells.

[0008] That is, in representative aspects, the present invention includes the following inventions. [1] A cell aggregate comprising chondrocytes and osteoblast precursor cells. [2] The cell aggregate described above, wherein the chondrocytes comprise hypertrophic chondrocytes. [3] The cell aggregate described above, which is a cell aggregate comprising cartilage-like tissue. [4] The cell aggregate described above, further comprising vascular endothelial cells. [5] The cell aggregate described above in [4], wherein the vascular endothelial cells express vascular endothelial growth factor A (VEGF-A). [6] The cell aggregate described above in [4] or [5], wherein the vascular endothelial cells are umbilical vein endothelial cells. [7] The cell aggregate described above in [6], wherein the umbilical vein endothelial cells are human umbilical vein endothelial cells. [8] The cell aggregate described in any of [1] to [7], wherein the ratio of the total number of chondrocytes and osteoblast precursor cells to the total number of cells constituting the cell aggregate is 15% or more. [9] The cell aggregate according to any one of [1] to [8] above, which contains calcified tissue, and the calcified tissue is a site that is positive by von Kossa staining.

[10] The cell aggregate according to [9] above, wherein the positive site is present in a proportion of 10% or more of the area of ​​a section of the cell aggregate.

[11] A method for producing a cell aggregate containing cartilage-like tissue, the method comprising co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor.

[12] The method according to

[11] above, wherein the vertebral disc cells are cells obtained by induction from pluripotent stem cells.

[13] The method according to

[11] or

[12] above, wherein the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A).

[14] A method for promoting chondrocyte hypertrophy, the method comprising co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor.

[15] The method according to

[14] above, wherein the vertebral disc cells are cells obtained by induction from pluripotent stem cells.

[16] The method according to

[14] or

[15] above, wherein the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A).

[0009] The cell aggregate of this embodiment contains chondrocytes and osteoblast precursor cells, and the cell aggregate has cartilage-like tissue. Furthermore, the chondrocytes in the cell aggregate of this embodiment contain hypertrophic chondrocytes. Furthermore, the inventors have newly discovered that the cell aggregate of this embodiment can be obtained by a method comprising co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor. According to the method for producing the cell aggregate of this embodiment, a cell aggregate containing chondrocytes and osteoblast precursor cells, which are necessary components of perichondrium, can be obtained, and therefore the method can be effectively used as a culture model for bone development in the development of biocompatible medical materials, etc.

[0010] Figure 1 is a schematic diagram of the method for preparing cell aggregates according to this embodiment. Figure 2 shows microscopic images of sections stained with Safranin O from cell aggregates obtained by monoculture of vertebral disc cells (left: S method) and cell aggregates obtained by coculture of vertebral disc cells with vascular endothelial cells (right: SH method). Figure 3 shows the perimeter and area of ​​cell aggregates obtained by monoculture of vertebral disc cells (D28 S) and coculture of vertebral disc cells with vascular endothelial cells (D28 SH) quantified from images of both sections on day 28 of culture (3A: perimeter, 3B: area). As a control, human chondrocytes and hypertrophic chondrocytes in human fetal tibia were also quantified in the same manner. The analysis results of the human fetus used as a control were calculated from image data published in the following paper. McAlinden A, Varghese N, Wirthlin L, Chang LW. Differentially Expressed MicroRNAs in Chondrocytes from Distinct Regions of Developing Human Cartilage. PLoS One. 2013;8(9). Figure 4 shows microscopic images of alkaline phosphatase (ALP) stained sections of cell aggregates from monoculture of vertebral disc cells (top: S method) and cell aggregates from coculture of vertebral disc cells with vascular endothelial cells (bottom: SH method) on day 28 of culture. The scale bars in the lower right corner of the images represent 100 μm. Figure 5 shows the results of a comparative analysis using real-time PCR of the mRNA expression of osteoblast differentiation markers RUNX2 and SP7 in cell aggregates from monoculture of vertebral disc cells (D28 S) and cell aggregates from coculture of vertebral disc cells with vascular endothelial cells (D28 SH) on day 28 of culture. The t-test showed that the expression of RUNX2 and SP7 in D28 SH was significantly higher than that in D28 S (significance level: 0.05).Figure 6 shows section images of cell aggregates obtained by SH analysis on day 28 of co-culture of HUVECs constitutively expressing VEGFA with vertebral disc cells (top row), and section images of cell aggregates on day 28 of co-culture of normal HUVECs with vertebral disc cells (second and subsequent rows). From left to right, bright-field, CD31 staining, Safranin O staining, and alkaline phosphatase (ALP) staining are shown. The bright-field image of HUVECs constitutively expressing VEGFA is overlaid with an image visualized by green fluorescent protein (GFP). rhVEGFA was added to the co-culture with normal HUVECs at 0, 5, 37.5, and 75 ng / mL (top to bottom: 0 ng / mL, 5 ng / mL, 37.5 ng / mL, and 75 ng / mL, respectively). The scale bars in the lower right corner of the images represent 100 μm. Figure 7 shows stained images of sections obtained by immunohistochemical evaluation of cell aggregates obtained by the S and SH methods on day 28 of culture. COL2, SOX9, and COL10 were stained red, and nuclei were stained blue with DAPI. The scale bars in the lower right corner of the images represent 100 μm. Figure 8 shows stained images of sections obtained by immunohistochemical evaluation of cell aggregates obtained by the S and SH methods on day 28 of culture. COL1, RUNX2, and SP7 were stained red, and nuclei were stained blue with DAPI. The scale bars in the lower right corner of the images represent 100 μm. Figure 9 shows stained images of sections obtained by immunohistochemical evaluation of cell aggregates obtained by the SH method on day 28 of culture. GFP and CD31 were stained red, and nuclei were stained blue with DAPI. The scale bars in the lower right corner of the images represent 100 μm. Figure 10 shows section images showing osteogenic induction by combinatorial treatment with small molecules using cell aggregates obtained by SH method on day 28 of culture. The schematic diagram on the left shows the timeline of osteogenic induction by different small molecule treatment groups. The images on the right are, from left to right, bright field, hematoxylin and eosin stained (H&E), alkaline phosphatase stained (ALP), and Safranin O stained micrographs of cell aggregates on day 42 of culture (D42).The top image is a micrograph taken on day 28 (D28) of culture. For the bright-field image, an image visualizing green fluorescent protein (GFP) is superimposed. The scale bar in the lower right corner of the image represents 500 μm for the bright-field image and 250 μm for the other images. Figure 11 is a schematic diagram showing the type and number of cell aggregates transplanted into mice and their transplantation locations. Figure 12 compares the calcification status of cell aggregates obtained by the S and SH methods after subcutaneous transplantation on day 28 of culture. The top row shows representative microCT images of mice taken at each time point, from day 0 (POD0) to day 70 (POD70). The white arrow on POD70 indicates the location of the calcified cell aggregate. The bottom row shows 3D reconstructed images of mice taken by microCT. The location of the cell aggregate obtained by the SH method is on the right side of the mouse when viewed from the dorsal side. The circled areas in the images indicate the calcified cell aggregates. Figure 13 shows a comparison of section images of cell aggregates obtained by the SH method and the S method on day 28 of culture, 70 days after subcutaneous implantation (POD70). The left side shows the results of Safranin O staining, the center shows ALP staining, and the right side shows von Kossa staining (SH method only). The scale bar in the lower right of the image represents 100 μm.

[0011] The following describes embodiments of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, appropriate modifications can be made and implemented without departing from the spirit of the present embodiments.

[0012] (1) Cell aggregate containing chondrocytes and osteoblast precursor cells The cell aggregate of this embodiment contains chondrocytes and osteoblast precursor cells. Furthermore, the chondrocytes in the cell aggregate of this embodiment include hypertrophic chondrocytes. Because chondrocytes, hypertrophic chondrocytes, and osteoblast precursor cells are all essential cells that appear in the process of cartilage formation, they can be effectively used as an in vitro culture model for bone development.

[0013] 1-1. Cell Aggregates In this specification, the term "cell aggregate" refers to a mass formed by the aggregation of cells, in which the cells are adhered to one another. Examples of cell aggregates include cell clusters, spheres, spheroids, organoids, and embryoid bodies. Preferably, in a cell aggregate, cells are adhered to one another via a surface. Furthermore, in some or all of the cell aggregate, cell-cell junctions and / or cell adhesions are included as modes of adhesion. Furthermore, culture includes two-dimensional culture and / or three-dimensional culture. From the viewpoint of promoting cell-cell junctions and creating an environment more similar to that in vivo, cell aggregates obtained by three-dimensional cell culture are preferred. Here, spheroids refer to spontaneous aggregation of cells during cell growth in three-dimensional culture. An organoid is a self-aggregation of stem cell-derived cells in the presence of an extracellular matrix in a three-dimensional culture, and is preferably a collection of cells that can exhibit the functions of an organ.

[0014] The cell aggregate of this embodiment preferably includes a tissue formed by the assembly of cells, or an aggregate of cells that can be considered histologically similar. Examples of tissue include epithelial tissue, connective tissue, muscle tissue, and nerve tissue. When the cell aggregate of this embodiment includes connective tissue, the cell aggregate may include, for example, fibrous connective tissue, adipose tissue, cartilage tissue, and bone tissue. Furthermore, examples of aggregates of cells that can be considered histologically similar include cartilage-like tissue, which will be described later.

[0015] The cell aggregate of this embodiment contains stem cells and cells induced to differentiate during the culture process (including precursor cells and hypertrophied cells), and these may coexist. Preferably, a plurality of different cell types are contained in the same cell aggregate. For example, hypertrophic chondrocytes and osteoblast precursor cells are contained in the same cell aggregate. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, and include embryonic stem (ES) cells, adult stem cells, and induced pluripotent stem (iPS) cells. Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Pluripotent stem cells also include Muse cells (Multi-lineage Differentiating Stress Enduring cells) obtained from mesenchymal stem cells and GS cells (Germline stem cells) created from germ cells. Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues or cells, although not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues or cells. Differentiation-induced cells refer to cells that have been induced to differentiate into different cells through contact with other cells or stimuli such as differentiation-inducing factors, and progenitor cells refer to cells that are in the middle stage of differentiating from stem cells into specific somatic cells or germ cells. Enlarged cells refer to mature cells whose individual cell volume has increased toward terminal differentiation. For example, in an ossification pattern called endochondral ossification, mesenchymal cells are induced to differentiate into chondrocytes. Examples of precursor cells include osteoblast precursor cells, which will be described later, and examples of hypertrophied cells include hypertrophic chondrocytes, which will be described later. The cell aggregate of this embodiment includes chondrocytes and osteoblast precursor cells, i.e., the above-described differentiation-induced cells, but is not limited to these cells and may also include other types of cells.

[0016] The cells contained in the cell aggregate of this embodiment are not particularly limited in terms of the origin of the cell type, but are preferably derived from animals, such as humans, mice, rats, birds, amphibians, fish, and insects.

[0017] The shape of the cell aggregate of this embodiment is not particularly limited, and may be approximately spherical. However, when the cell aggregate is adhered to a cell culture substrate, the cell aggregate may have a flat portion on the adhesive surface.

[0018] The size of the cell aggregate of this embodiment is not particularly limited, but if the cell aggregate is considered to be approximately spherical, the diameter at the maximum diameter of the spherical cross section (if the cell aggregate is considered to be approximately ellipsoidal, the major axis diameter at the maximum major axis of the ellipsoidal cross section) should be 500 μm or more. Considering cell necrosis in the center, the size is preferably 500 μm to 2000 μm, more preferably 600 μm to 1500 μm, and even more preferably 800 μm to 1000 μm. The diameter and major axis diameter can be measured using, for example, image analysis software or a particle size distribution analyzer.

[0019] 1-2 Chondrocytes In this embodiment, chondrocytes refer to cells that produce extracellular matrix components constituting cartilage, such as collagen and proteoglycans, or precursor cells that become such cells. Hypertrophic chondrocytes, as described below, are also included in chondrocytes. As a method for identifying chondrocytes, dye staining can be used, for example, from the viewpoint of visibility. Examples of dye staining include safranin O staining, which stains proteoglycans, which are the extracellular matrix of cartilage. Alternatively, chondrocytes may be detected using toluidine blue staining, which stains the matrix of chondrocytes, or may be used in combination with hematoxylin-eosin staining, which stains the nuclei of chondrocytes. Furthermore, the expression of chondrocyte marker proteins may be confirmed using immunohistochemical staining. Examples of chondrocyte marker proteins include type II collagen (COL2, COL2A1) and SOX9.

[0020] 1-3. Hypertrophic chondrocytes Hypertrophic chondrocytes refer to chondrocytes in a hypertrophied (matured) state. Hypertrophic chondrocytes secrete growth factors such as hedgehog, bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF), and it is known that when osteoprogenitor cells receive growth factors from hypertrophic chondrocytes, they differentiate into osteoblasts.

[0021] A method for confirming hypertrophic chondrocytes is, for example, to measure the cell area (μm ２ The cell area of ​​hypertrophic chondrocytes can be confirmed by calculating the cell area (μm) or the cell perimeter (μm). As an example of a calculation method, a slice of the cell aggregate is prepared, a plurality of cells (e.g., 20 cells) are randomly extracted from the slice, and the average cell area or the average cell perimeter is calculated using image analysis software or the like. The cell area of ​​hypertrophic chondrocytes is 250 μm ２ It is sufficient if it is equal to or greater than 350 μm, and preferably 350 μm ２ More preferably, 450 μm or more ２ That is all. The perimeter is sufficient as long as it is 60 μm or more, and preferably 70 μm or more. To determine whether cell aggregates produced by the same method have hypertrophied, the cell area (or perimeter) at a specified culture time can be calculated and compared before and after the elapsed time. If the average cell area (or perimeter) increases with the elapsed time of culture, it can be determined that hypertrophy has occurred. For cell aggregates produced by different methods, the culture time can be made uniform and the cell area (or perimeter) obtained by each method can be compared. Furthermore, the presence of hypertrophic chondrocytes can be confirmed by the expression of type X collagen (COL10), which is specific to hypertrophic chondrocytes. COL10 can be visualized by immunohistochemical evaluation.

[0022] 1-4. Osteoblast precursor cells Osteoblast precursor cells refer to cells before they differentiate into osteoblasts. In vivo, they exist in the perichondrium covering cartilage tissue. Osteoblasts exist on the surface of bone tissue in vivo and are characterized by containing large amounts of basic alkaline phosphatase. In the cell aggregate of this embodiment, the osteoblast precursor cells may be located outside the cartilage-like tissue in which hypertrophic chondrocytes exist. More specifically, taking the cell aggregate shown in the SH method of Figure 4 as an example, the osteoblast precursor cells exist inside the perichondrium (a layered structure observed outside the cartilage-like tissue in which hypertrophic chondrocytes exist). (Figure 4 SH method)

[0023] Methods used for detecting osteoblasts can be used to identify osteoblast precursor cells. For example, alkaline phosphatase staining can be used after preparing sections of cell aggregates. It is known that the same activity is also observed in hypertrophic chondrocytes. Alternatively, real-time PCR can be used to confirm the expression of one or more of RUNX2 and SP7 mRNAs. Alternatively, Alizarin Red S or von Kossa staining can be used to detect the production of mineralized bone matrix, i.e., calcium deposition.

[0024] The cell aggregate of this embodiment may contain osteoblasts in addition to chondrocytes and osteoblast precursor cells. Since bone is rich in type I collagen, differentiation into osteoblasts in the cell aggregate can be confirmed by confirming the expression of type I collagen (COL1).

[0025] 1-5. Cartilage Tissue and Cartilage-Like Tissue "Cartilage tissue" refers to connective tissue composed of cartilage extracellular matrix and chondrocytes, and includes tissues that behave as articular cartilage tissue and / or growth plate cartilage-like tissue. Here, "tissue" refers to a structure in which multiple types of cells with different morphologies and properties are arranged three-dimensionally in a specific pattern. Depending on the properties of the cartilage matrix, cartilage can be classified as hyaline cartilage (articular cartilage, epiphyseal plate, costal cartilage, tracheal cartilage, laryngeal cartilage, etc.), fibrocartilage (sacroiliac joint, temporomandibular joint, sternoclavicular joint, intervertebral disc, pubic symphysis, meniscus, articular disc, etc.), or elastic cartilage (external auditory canal, Eustachian tube, auricular cartilage, epiglottis cartilage, etc.). However, the cartilage tissue and cartilage-like tissue in the cell aggregate of this embodiment are not particularly limited to the type of cartilage. Herein, "cartilage-like tissue" means being histologically similar to cartilage tissue. The cartilage-like tissue contained in the cell aggregate of this embodiment can be expressed as, for example, a region containing cells in which proteoglycan, a cartilage extracellular matrix, is detected. Furthermore, the cartilage-like tissue contained in the cell aggregate of this embodiment may contain hypertrophic chondrocytes and / or chondrocytes, may contain osteoblasts and / or osteoblast precursor cells, or may contain hypertrophic chondrocytes and / or chondrocytes and osteoblasts and / or osteoblast precursor cells. Conventional methods such as those described above can be used to detect each cell type. Furthermore, the cartilage-like tissue contained in the cell aggregate of this embodiment may contain perichondrium (including a layered structure that can be considered as perichondrium).

[0026] 1-6. Vascular Endothelial Cells The cell aggregate of this embodiment may further contain vascular endothelial cells in addition to chondrocytes and osteoblast precursor cells. In the process of producing the cell aggregate of this embodiment, vertebral disc cells (which may be induced from stem cells) described below are co-cultured with vascular endothelial cells, thereby forming a cell aggregate in which cartilage-like tissue is spontaneously recognized. Furthermore, the vascular endothelial cells may be distributed on the outside or the center of the cell aggregate of this embodiment, and there are no particular limitations on the distribution of the vascular endothelial cells. Note that the vascular endothelial cells may be removed by washing or the like after co-culture.

[0027] When the cell aggregate of this embodiment contains vascular endothelial cells, it is preferable that the cell aggregate further contains vascular endothelial growth factor. The vascular endothelial growth factor may be added externally and incorporated into the cell aggregate as a result, resulting in the inclusion of the vascular endothelial growth factor in the cell aggregate. Alternatively, the vascular endothelial cells of this embodiment may express the vascular endothelial growth factor and be included in the cell aggregate. From the viewpoint of effectively obtaining a cell aggregate having cartilage-like tissue, it is preferable that the vascular endothelial cells of this embodiment express the vascular endothelial growth factor. The expression of the vascular endothelial growth factor may be forced. For example, a method may be used in which a gene encoding the vascular endothelial growth factor is introduced using a viral vector or the like. Note that, while it is desirable to use vascular endothelial cells expressing the vascular endothelial growth factor in the process of producing the cell aggregate of this embodiment, the resulting cell aggregate of this embodiment may or may not express the vascular endothelial growth factor.

[0028] The vascular endothelial cells may be brain microvascular endothelial cells, vein-derived endothelial cells, or artery-derived endothelial cells, and are preferably vein-derived endothelial cells. The origin of the vascular endothelial cells is not particularly limited, and vascular endothelial cells derived from humans or animals other than humans can be used. Examples of human-derived vascular endothelial cells that can be used include human umbilical vein endothelial cells (HUVECs).

[0029] The vascular endothelial cell growth factor is not particularly limited, but is preferably a protein belonging to the VEGF family, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF (placental growth factor)-1, and PlGF-2.

[0030] 1-7. Proportion of Each Cell in the Cell Aggregate The cell aggregate of this embodiment contains chondrocytes and osteoblast precursor cells. In the cell aggregate of this embodiment, when cartilage-like tissue can be confirmed, the total number of cells contained in the cell aggregate is taken as 100(%). The proportion of chondrocytes and osteoblast precursor cells that constitute the cartilage-like tissue is not particularly limited, but is preferably 15% or more, more preferably 20% or more, and even more preferably 25% or more. Among the cartilage cells, the proportion of hypertrophic chondrocytes is not particularly limited, but is preferably 5% or more, more preferably 10% or more, and even more preferably 14% or more. The cell aggregate of this embodiment may further contain vascular endothelial cells. When vascular endothelial cells are contained, the proportion of vascular endothelial cells is not particularly limited, but is preferably 0.1% or more, more preferably 1% to 2%, and even more preferably 2% to 3% when the total number of cells contained in the cell aggregate is taken as 100(%).

[0031] When the cell aggregate of this embodiment is considered to be approximately spherical, when a section of the cell aggregate (preferably a section including the center) is taken as 100% of the cross-sectional area of ​​the cell aggregate at the time when cartilage-like tissue can be confirmed, the proportion of chondrocytes is not particularly limited, but is preferably 12% or more, more preferably 18% or more. The proportion of hypertrophic chondrocytes among the chondrocytes is not particularly limited, but is preferably 10% or more, more preferably 15% or more. The combined proportion of chondrocytes and osteoblast precursor cells is not particularly limited, but is preferably 50% or more, more preferably 55% or more. When vascular endothelial cells are further included, the proportion of vascular endothelial cells is not particularly limited, but is preferably 0.1% to 0.5%, more preferably 0.3% to 0.5% of the cross-sectional area of ​​the cell aggregate taken as 100%.

[0032] 1-8. Vascular-like network in cell aggregates When the cell aggregates of this embodiment contain vascular endothelial cells, it is more preferable that a vascular-like network be observed. A vascular-like network refers to a state in which intercellular adhesion proteins (e.g., CD31) used as an evaluation index for angiogenesis are expressed in multiple continuous cells within the cell aggregates of this embodiment. Intercellular adhesion proteins can be visualized by immunohistochemical evaluation.

[0033] 1-9. Osteogenic Induction Factors and Bone Tissue Maturation in Cell Aggregates The cell aggregate of this embodiment may contain an osteogenic induction factor. The osteogenic induction factor preferably contains at least one selected from the group consisting of triiodothyronine (T3), smoothened agonist (SAG), which is a hedgehog agonist, CHIR99021 (CHIR), which is a GSK-3β inhibitor, and Bone Morphogenetic Protein 2 (BMP2). Among these osteogenic induction factors, it is more preferable to contain at least SAG, more preferably a combination of SAG and CHIR, or a combination of SAG and BMP2, and particularly preferably a combination of SAG, CHIR, and BMP2. These osteogenic induction factors may be incorporated into the cell aggregates by being added externally during culture, or may be expressed or secreted within the cell aggregates. When such osteogenic induction factors are contained in the cell aggregates, differentiation into osteoblasts and formation of cartilage components are promoted.

[0034] 1-10. Calcified Tissue in Cell Aggregates The cell aggregate of this embodiment preferably includes calcified tissue. Calcified tissue is a region in the cell aggregate where calcium deposits are observed. A method for confirming such calcium deposits includes, for example, von Kossa staining. Since calcified bones and calcium deposits are stained brown to black, regions that exhibit a brown to black color in von Kossa staining can be considered to be positive for von Kossa staining, and calcified tissue in the cell aggregate exhibits this positivity. Furthermore, the proportion of calcified tissue in the cell aggregate can be determined by calculating the ratio (%) of the area of ​​regions that exhibit positive von Kossa staining to the area of ​​a section of the cell aggregate. More specifically, for example, sections of the cell aggregate are prepared, microscopic images are obtained, and the ratio (%) of the area of ​​the cell aggregate to the area positive for von Kossa staining (= von Kossa staining positive area / total area of ​​the cell aggregate × 100) is calculated using image analysis software (e.g., "ImageJ"). The lower limit of this ratio is not particularly limited, but is preferably 10% or more, and more preferably 15% or more. The upper limit is also not particularly limited, but can be, for example, 99% or less, 80% or less, or 50% or less. Calcified tissue in the cell aggregate can be confirmed by obtaining an X-ray opaque image by CT scan.

[0035] (2) Method for Producing Cell Aggregates The cell aggregates of this embodiment can be produced by co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor.

[0036] 2-1. Vertebral disc cells Vertebral disc cells (sclerotomes) are mesenchymal stem cells present in the vertebral disc. The vertebral disc is known to give rise to other tissues, such as the vertebrae and associated vomers, tendons, and vascular cells of the dorsal aorta, intervertebral blood vessels, and meninges.

[0037] The origin of the vertebral disc cells of this embodiment is not particularly limited, and they may be induced from pluripotent stem cells via paraxial mesoderm. For example, vertebral disc cells can be induced from human pluripotent stem cells (hPSCs) under known culture conditions. When vertebral disc cells are induced from pluripotent stem cells, it is sufficient that only a portion of the cultured cells are induced into vertebral disc cells, but it is preferable that 70% or more of the cells constituting the cultured cells are PDGFRA-positive cells.

[0038] 2-2. Vascular endothelial cells expressing vascular endothelial growth factor The vascular endothelial cells expressing vascular endothelial growth factor of this embodiment are the same as those described above. The method for culturing vascular endothelial cells and the method for expressing vascular endothelial growth factor can be any known method, such as the culture method described in the instruction manual for commercially available HUVECs or the gene transfer method described in the instruction manual for commercially available lentiviral expression systems, and are not particularly limited. Cryopreserved vascular endothelial cells may also be used.

[0039] When preparing vascular endothelial cells in which vascular endothelial growth factor is forcibly expressed, whether or not vascular endothelial growth factor is expressed can be confirmed by inserting a fusion gene of the vascular endothelial growth factor and green fluorescent protein (GFP) into a viral vector and then measuring the fluorescence intensity. The insertion of green fluorescent protein is merely a step used to confirm the expression of the target gene and is not an essential step for producing the cell aggregate of this embodiment.

[0040] 2-3. Co-culture The method for producing a cell aggregate of the present invention includes co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor, and as a result of the co-culture, chondrocytes and osteoblast precursor cells are present in the same cell aggregate, making it possible to produce a cell aggregate in which cartilage-like tissue is recognized.

[0041] Specifically, the above-mentioned vertebral disc cells and vascular endothelial cells expressing vascular endothelial growth factor are prepared in separate containers, and then these are placed in the same container, brought into contact, and co-cultured to obtain the cell aggregate of this embodiment.

[0042] Although it depends on the culture conditions, mature chondrocytes and osteoblast precursor cells can be confirmed after 28 days from the start of co-culture, and hypertrophic chondrocytes can be confirmed after 42 days from the start of co-culture.

[0043] In the method for producing cell aggregates of the present invention, an osteogenic induction factor may be added to the medium in the co-culture step. The osteogenic induction factor may be any of the osteogenic induction factors described above, and preferably at least one selected from the group consisting of triiodothyronine (T3), smoothened agonist (SAG), a hedgehog agonist, CHIR99021 (CHIR), a GSK-3β inhibitor, and Bone Morphogenetic Protein 2 (BMP2). Among these osteogenic induction factors, it is more preferable to use at least SAG, and more preferably to use a combination of SAG and CHIR, or a combination of SAG and BMP2, and it is particularly preferable to use a combination of SAG, CHIR, and BMP2. When the osteogenic induction factor can be expressed or secreted within the cell aggregate, a gene corresponding to the induction factor may be introduced in advance into the vertebral disc cells or vascular endothelial cells used for co-culture so that the osteogenic induction factor is expressed during the culture process.

[0044] (3) Method for Promoting Chondrocyte Hypertrophy According to the method for producing a cell aggregate of the present invention, the method including the co-culturing promotes chondrocyte hypertrophy compared to a method for culturing vertebral disc cells (which may be derived from stem cells) alone. Furthermore, the method for producing a cell aggregate of the present invention enables the appearance of chondrocyte precursor cells in the cell aggregate, enabling the formation of cartilage-like tissue.

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0046] Example 1 In one embodiment, the cell aggregate of the present embodiment is produced through the following two steps. Specifically, the production method includes a step (first step) of inducing vertebral disc cells from human pluripotent stem cells via paraxial mesoderm, and a step (second step) of co-culturing the vertebral disc cells obtained in the first step with human umbilical vein endothelial cells that constitutively express vascular endothelial growth factor A (VEGF-A), but the present invention is not limited thereto.

[0047] 1. Preparation of Cell Aggregates Figure 1 shows a schematic diagram of the method for preparing cell aggregates. First, sclerotomes are induced from human pluripotent stem cells (hPSCs). A lentiviral vector (pLenti-VEGF-EGFP) carrying both vascular endothelial growth factor (VEGF) and enhanced green fluorescent protein (EGFP) genes is introduced into human umbilical vein endothelial cells (HUVECs). The HUVECs and the induced sclerotomes are then co-cultured to obtain cell aggregates.

[0048] 1-1 Culture and Maintenance of Human Embryonic Stem Cells (hESCs) In this series of experiments, hESCs were used as hPSCs. Experiments using hESCs were conducted in compliance with the Ministry of Health, Labor and Welfare guidelines and with approval from the University of Tokyo Ethics Committee. The SEES3 hESC line was used. Initial adaptation and maintenance of hESCs were performed in a commercially available xeno-free culture system using StemFit AK02N medium (product number AK02N, Ajinomoto) on dishes coated with 5 μg / mL recombinant human vitronectin (VTN) (product number A14700, Gibco). Cells were cultured under optimal conditions at 37°C, 5% CO2, and in a humid environment. For efficient cell dissociation, 0.5 M EDTA (product number 15575-020; Gibco) was diluted to 0.5 mM with PBS (product number 049-29793; Wako). To increase cell viability during seeding and passaging, 1 μM Y-27632 (product number 034-24024, Fujifilm Wako) was used to prevent apoptosis.

[0049] 1-2 Induction of vertebral disc cells from human pluripotent stem cells. hESCs, a type of human pluripotent stem cells (hPSCs), were cultured under the following culture conditions to induce vertebral disc cells (sclerotomes).

[0050] Subconfluent undifferentiated hESCs were carefully dissociated (approximately 10 pipetting cycles) until they were finely clumped. Depending on the colony size and viability, they were diluted and passaged at a ratio of 1:16 to 1:24. This process was performed on culture dishes coated with Vitronectin (VTN-N: A14700; Thermo Fisher Scientific) and cultured overnight in StemFit AK02N medium (AK02N, Ajinomoto) supplemented with 1 μM Y-27632 (ROCK inhibitor: Product No. 034-24024; Fujifilm Wako). Diluting and seeding hESCs prior to differentiation is essential to reduce the risk of cell overgrowth, especially over long differentiation periods. Differentiation into vertebral disc cells was systematically performed in a serum-free, xeno-free, feeder-free, monolayer environment using chemically defined B27 / ITS medium (BIM). Preparation of BIM and induction of vertebral disc cells were performed using established and published methods. Briefly, BIM was supplemented with 1% B27 Supplement Xeno-Free (Product No. A1486701; Thermo Fisher Scientific), 1% ITS Liquid Media Supplement (Product No. I3146; Sigma-Aldrich), 1% MEM Non-Essential Amino Acids Solution (Product No. 11140050; Thermo Fisher Scientific), and 55 μM 2-mercaptoethanol (prepared by adding 55 mM 2-mercaptoethanol in DPBS (Product No. 21985023; Thermo Fisher Scientific) to DMEM / F12 (Product No. 11330032; Thermo Fisher Scientific) to a concentration of 0.1%).Primitive striatal (PS) cells were derived from hESCs using 5 μM CHIR99021 (Product No. 4423; Tocris), axial mesoderm (PM) was induced using 5 μM CHIR99021, 1 μM A 83-01 (Product No. 2939; Tocris), and 0.25 μM LDN193189 (Product No. SML0559; Sigma), somitic mesoderm (SM) was induced using 1 μM C59 (Product No. C7641-2S; Cellagen Technology), 1 μM A 83-01, and 0.25 μM LDN193189, and vertebral disc cells (SCL) were induced using 1 μM SAG (Product No. AG-CR1-3585; AdipoGen), 1 μM C59, and 0.25 μM LDN193189. SCL induction was performed for 2 days, and all other inductions were performed for 24 hours.

[0051] 1-3 Preparation of human umbilical vein endothelial cells (HUVECs) constitutively expressing vascular endothelial growth factor A (VEGF-A). 1 Preparation of lentivirus for VEGFA-EGFP expression. HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. They were maintained at 37°C in a tissue culture incubator with 5% CO2. After the cells reached 60-70% confluence, HEK293T cells were transfected using FuGENE® HD (Product No. E2312, Promega) and Opti-MEM (Product No. 31985-070; ThermoFisher) according to the manufacturer's instructions. Lentivirus production plasmids were used, including pCCLc-MNDU3-VEGFA-PGK-EGFP-WPRE (Addgene Plasmid Product No. 89609), pMD2.G (Addgene Plasmid Product No. 12259), and psPAX2 (Addgene Plasmid Product No. 12260). FuGENE HD Reagent and Opti-MEM were incubated at room temperature for 30 minutes. For one 10 cm dish, 8.5 μg of pCCLc-MNDU3-VEGFA-PGK-EGFP-WPRE, 2.1 μg of pMD2.G, and 6.4 μg of psPAX2 were mixed. The volume was adjusted to 799 μL with Opti-MEM, and then 51 μL of FuGENE HD Transfection Reagent was added to bring the total volume to 850 μL. This mixture was then added to HEK293 cells in a 100 mm dish containing 10 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Forty-eight hours after transfection, the cell culture medium was harvested and filtered through a 0.45 μm filter. Lentivirus was stored and concentrated using a Lenti-X concentrator (Product No. 631231; Takara) according to the manufacturer's protocol. The concentrated lentivirus suspension was used to transduce HUVEC cells.2. Lentiviral transduction of HUVECs for VEGFA-EGFP overexpression. HUVECs were cultured in Endothelial Growth Medium 2 (EGM2) and maintained in a tissue culture incubator at 37°C with 5% CO2. The next day, when HUVECs reached the optimal confluence range of 40-50%, lentiviral transduction was performed. For lentiviral transduction, 6 μg / mL polybrene (product number 107689-10G; Sigma) was added. After 48-72 hours, GFP expression could be observed under a fluorescent microscope. VEGFA overexpression was confirmed by RTqPCR.

[0052] 1-4 Co-culture of vertebral disc cells with human umbilical vein endothelial cells (HUVEC) constitutively expressing vascular endothelial growth factor A (VEGF-A) Human umbilical vein endothelial cells (HUVEC) transfected with both vascular endothelial growth factor A (VEGF-A) and enhanced green fluorescent protein (EGFP) genes were mixed and co-cultured with the vertebral disc cells obtained in 1-2 above. The specific co-culture method is described below.

[0053] Human ESC-derived disc cells and human umbilical vein endothelial cells (VEGF-A-overexpressing HUVECs) that constitutively express vascular endothelial growth factor A (VEGF-A) were dissociated using Accutase (Product No. AT104; Innova Cell Technologies) and 2.5% Trypsin-EDTA solution (Product No. T4174-100mL; Wako), respectively. Disc cells and HUVECs were counted, centrifuged, and collected in SCL induction medium supplemented with 1 μM Y-27632 and EGM2, respectively. Disc cells and HUVECs were mixed in a 4:1 ratio in SCL induction medium to prepare SH cell aggregates. 3 x 10 cells were plated per well in a 96-well ultra-low attachment plate (Product No. 7007; Corning). 4 Cells were seeded onto the cells. The SH medium was prepared by mixing EGM2 and SCL induction medium containing 1 μM Y-27632 at a 9:1 ratio. After the initial 24-hour culture period, the medium was replaced with EGM2 medium. EGM medium was replaced every other day.

[0054] The cell aggregates obtained by co-culture are approximately spherical and have a diameter of 600 μm to 1500 μm.

[0055] Hereinafter, the method for producing the cell culture mass of this embodiment, which includes co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor, will be referred to as the "SH method" for convenience.

[0056] (Comparative Example) As a comparative example, cell aggregates were obtained by culturing the vertebral disc cells induced in 1-2 alone without co-culturing them with HUVECs. Hereinafter, for convenience, the method for obtaining cell aggregates in the comparative example will be referred to as the "S method." (Culture conditions for vertebral disc cells in the S method) Human ESC-derived vertebral disc cells were collected, centrifuged, and adjusted in SCL induction medium supplemented with 1 μM Y-27632 and EGM2. 3 x 10 cells were plated per well in a 96-well ultra-low attachment plate (product number 7007; Corning). 4 Cells were seeded onto the cells. Similar to the SH method, EGM2 and SCL induction medium containing 1 μM Y-27632 were mixed at a 9:1 ratio to prepare a medium. After the initial 24-hour culture period, the medium was replaced with EGM2 medium. EGM medium was replaced every other day.

[0057] Example 2: Cell aggregates obtained by co-culturing vertebral disc cells with normal HUVECs, which do not constitutively express VEGFA, were designated Example 2. When normal HUVECs were used for co-culturing vertebral disc cells, recombinant human VEGF-A (rhVEGFA, Product No. 100-20, Peprotech) was added to EGM2 medium at concentrations of 0-75 ng / mL. The following examples were designated in descending order of the amount of rhVEGFA added: Example 2-1 (0 ng / mL), Example 2-2 (5 ng / mL), Example 2-3 (37.5 ng / mL), and Example 2-4 (75 ng / mL).

[0058] 2. Confirmation of cartilage-like tissue The cell aggregates obtained by the above co-culture were evaluated using the method described below to determine whether cells or tissues reproducing the process of bone development were observed, for example, whether they had the characteristics of cartilage-like tissue.

[0059] Histological Analysis 1. General Preparation: 3D cell aggregates prepared using the S or SH method were transferred to a microtube, washed with DPBS, and then fixed in 4% PFA at 4°C for 1 hour. The fixed cell aggregates were washed twice with DPBS and stored at 4°C. To prepare frozen sections, the cell aggregates were incubated in OCT Compound (Sakura Finetech Japan) at room temperature for 5 minutes and then frozen at -100°C. 2. Section Preparation: Sections were prepared for each cell aggregate in the Examples and Comparative Examples to observe the state of cells and tissues through cell area measurement, Safranin O staining, and alkaline phosphatase staining. Sections were prepared to include the center of the cell aggregate. Frozen blocks were precisely sliced ​​to 10 μm using a LEICA CM3050IV cryotome and air-dried at room temperature before staining. 3. Safranin O Staining: The presence and size of chondrocytes were confirmed by staining with Safranin O, which specifically stains proteoglycans, a component of the cartilage extracellular matrix. Specifically, frozen sections were immersed in hematoxylin for 2 minutes, tap water for 10 minutes, 0.001% Fast Green (Product No. 1040220025; Sigma-Aldrich) for 5 minutes, 1% acetic acid (Product No. 012-00245; Wako) for 10 seconds, and Safranin O (Product No. 1B-463; Waldeck) for 5 minutes. Then, they were washed three times in 100% ethanol for 5 minutes each, and immersed three times in xylene for 5 minutes each before mounting. 4. ALP Staining. ALP staining was performed by rinsing frozen sections twice in PBS containing 0.1% Tween 20 (PBT) for 5 minutes at room temperature. Sections were then incubated twice in NTMT (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20) for 5 minutes each, followed by another 30 minutes at room temperature. The sections were incubated in NBT / BCIP (product number 11697471001, Roche) staining solution for 40 minutes at room temperature to initiate the color reaction. The sections were then washed in PBT for 5 minutes, Kernechtrot stain (product number 4087-2; Muto Pure Chemicals Co. Ltd.) for 10 seconds, and tap water for 5 minutes. Then, the sections were washed three times in 100% ethanol for 5 minutes each, and immersed three times in xylene for 5 minutes each before mounting.5. Immunohistochemical Staining. Antigen retrieval for matrix staining was performed using 20 μg / mL Proteinase K (Recombinant) in PBS (Product No. 1567906, Nacalai). Sections were blocked with Blocking One Histo (Product No. 06349-64, Nacalai). Primary antibodies against COL2 (Product No. MAB8887, Sigma-Aldrich), COL1 (Product No. ab6308, Abcam), COL10 (Product No. 14-9771-82, Invcam), SOX9 (Product No. ab5535, Millipore), RUNX2 (Product No. ab192256, Abcam), and CD31 (Product No. ab192256, Abcam) were then applied. Fluorescence visualization was performed using Alexa Fluor 564 anti-mouse IgG antibody (Product No. A-11030, Invitrogen) and Alexa Fluor 546 anti-rabbit IgG antibody (Product No. A-11035, Invitrogen). The antibody dilution ratio was maintained at 1:500. Bright-field and fluorescent images were captured using a BZ-X700 fluorescence microscope (Keyence) and a confocal microscope (LSM 880; Zeiss). 6. von Kossa staining. Frozen sections were washed twice with distilled water for 5 minutes each. Then, an outline was drawn around the section with a hydrophobic pen, and a small amount of 1% silver nitrate solution (Product No. 196-00831, Wako) was added and incubated under UV light for 10–20 minutes. The sections were then washed twice with distilled water for 5 minutes each. To remove unreacted silver nitrate, sections were stained with 5% sodium thiosulfate (product number 197-03605, Wako) for 5 minutes, followed by a 5-minute wash in distilled water. After counterstaining with Kern Echtrot stain (Fast Red) for 5-10 seconds, sections were washed in tap water for 5 minutes. After three 5-minute washes in ethanol and three 5-minute washes in xylene, sections were mounted with Marinol.

[0060] Confirmation of chondrocytes by Safranin O staining As a result of Safranin O staining, microscopic observation revealed that sections obtained by the SH method were stained more intensely than those obtained by the S method on the 28th day of culture. Furthermore, while no cell hypertrophy was observed with the S method even on the 35th day of culture, enlarged cells were observed with the SH method on the 42nd day of culture (Figure 2).

[0061] To investigate cell size in more detail, sections of cell aggregates obtained by the S and SH methods were prepared using the method described above on day 28 of culture. Twenty cells were randomly selected from the red-stained areas on each section, and their perimeter and area were measured. The perimeter and area of ​​each of the 20 cells were plotted for each of the S and SH methods. (Figure 3A: Cell perimeter, 3B: Cell area) (Method for measuring cell perimeter and area) Both the cell perimeter and area were quantified for cells on tissue sections using Image J software.

[0062] As can be seen from Figures 2 and 3, it was revealed that the cells present in the cell aggregates obtained by the SH method had larger cell perimeters and cell areas than those obtained by the S method, suggesting that the method for producing cell aggregates of this embodiment accelerates the maturation (hypertrophy) of hypertrophic chondrocytes.

[0063] Identification of osteoblast precursor cells by alkaline phosphatase staining Alkaline phosphatase is activated when osteoblast precursor cells differentiate into osteoblasts and is used as a marker enzyme for osteoblasts. It is generally known that its activity is high in osteoblasts, but that it is also found in hypertrophic chondrocytes. Therefore, the presence of osteoblast precursor cells and hypertrophic chondrocytes was confirmed by checking alkaline phosphatase activity in sections of cell aggregates obtained by the S method and SH method. In addition, perichondrium (a layered structure observed outside the cartilage-like tissue where hypertrophic chondrocytes exist) was observed (Figure 4).

[0064] In Figure 4, areas stained dark purple indicate areas that were positive for alkaline phosphatase activity. While no cells positive for alkaline phosphatase activity were observed with the S method, positive cells were observed around the cartilage-like tissue where hypertrophic chondrocytes were present with the SH method.

[0065] When the area of ​​a section of a cell aggregate obtained by the SH method was taken as 100%, the proportion of chondrocytes was 15%, and the proportion of hypertrophic chondrocytes among the chondrocytes was 14%. Furthermore, when the total number of cells in the cell aggregate was taken as 100%, the proportion of chondrocytes was estimated to be 3% to 6%, and the proportion of hypertrophic chondrocytes among the chondrocytes was estimated to be 3% to 5%. Similarly, when the area of ​​a section of a cell aggregate obtained by the SH method was taken as 100%, the proportion of osteoblast precursor cells was 40%. Furthermore, when the total number of cells in the cell aggregate was taken as 100%, the proportion of osteoblast precursor cells was estimated to be 20% to 30%. When the area of ​​a section of a cell aggregate obtained by the SH method was taken as 100%, the combined proportion of chondrocytes and osteoblast precursor cells was 50%. Furthermore, when the total number of cells in the cell aggregate was taken as 100%, the combined proportion of chondrocytes and osteoblast precursor cells was estimated to be 23% to 36%. When the area of ​​the section of the cell aggregate obtained by the SH method was taken as 100%, the proportion of HUVEC cells was 0.2%. Furthermore, when the total number of cells in the cell aggregate was taken as 100, the proportion of HUVEC cells was 0.2%.

[0066] Confirmation of osteoblast precursor cells by real-time PCR: The mRNA expression of osteoblast differentiation markers RUNX2 and SP7 was analyzed comparatively using real-time PCR for cell aggregates obtained by the S and SH methods (Figure 5). It was confirmed that both genes were expressed at relatively high levels in the SH method.

[0067] Constitutive Expression of VEGFA in Cell Aggregates (Figure 6) Cell aggregates obtained by the SH method (Example 1) as a positive control were compared with cell aggregates obtained by co-culture of vertebral disc cells with normal HUVECs (not HUVECs that constitutively express VEGFA) to determine the formation of osteoblast precursor cells, cartilage-like tissue, and vascular-like networks. After 28 days of culture, cell aggregates with hypertrophic chondrocytes at the core were formed in Example 2, similar to Example 1. CD31-positive cells were observed in the positive control group (Example 1) and the groups containing rhVEGFA (5-75 ng / mL, Examples 2-2 to 2-4), suggesting the formation of vascular networks around the cell aggregates. However, when evaluating the induction of osteoblast precursor cells using alkaline phosphatase staining, ALP-positive cells were sparsely induced and did not exhibit the same ALP expression pattern as the cell aggregates obtained by the SH method in Example 1. These results indicate that, in order to effectively obtain cell aggregates having cartilage-like tissue, it is more preferable to constitutively express VEGFA in HUVECs than to add rhVEGFA to the culture medium.

[0068] Immunohistochemical Staining of Cell Aggregates (Figure 7) To further verify the modeling of endochondral ossification in cell aggregates obtained by the SH method on day 28 of culture, immunohistochemical staining for cartilage and bone markers was performed. Expression of type II collagen (COL2), the master regulator of chondrocyte differentiation (SOX9), and type X collagen (COL10), which is specific to hypertrophic chondrocytes, was examined. Cell aggregates obtained by the SH method expressed COL2, COL10, and SOX9, whereas cell aggregates obtained by the S method expressed COL2 and SOX9 but not COL10. COL2 expression was more pronounced in cell aggregates obtained by the SH method than in those obtained by the S method. These findings suggest that the SH method promotes endochondral ossification, particularly inducing hypertrophic chondrocytes.

[0069] Osteoblast Differentiation in Cell Aggregates (Figure 8) To further investigate osteoblast differentiation in cell aggregates obtained by the SH method, immunostaining was performed for the bone marker type I collagen (COL1) and the osteoblast precursor cell markers RUNX2 and SP7. COL1 expression was observed in both cell aggregates obtained by the S method and those obtained by the SH method. However, only cell aggregates obtained by the SH method showed both RUNX2 and SP7 expression. RUNX2 was detected in both the core and outer layer of cell aggregates obtained by the SH method, while SP7 was localized in the outer layer surrounding the core, particularly outside the cartilage-like tissue.

[0070] Furthermore, we examined the presence of a vascular network within the cell aggregates obtained by SH by staining with CD31 and GFP. Both markers were localized outside the cartilage-like tissue where osteoblast precursor cells were detected, confirming the presence of a vascular network.

[0071] Example 3: Bone Tissue Maturation in Cell Aggregates and In Vitro Differentiation Induction of Cell Aggregates (Figure 10). To confirm whether osteogenic induction factors could induce bone tissue maturation in cell aggregates obtained by SH, in vitro experiments were performed using various combinations of osteogenic induction factors selected from the group consisting of T3, SAG, CHIR, and BMP2. For this experiment, EGM-2 medium was used, containing a final concentration of 10 mM β-glycerol phosphate and 10 nM triiodothyronine (T3) (Product No. T-074, Sigma-Aldrich). The osteogenic induction agents used were 3 μM CHIR99021, 1 μM SAG, and 100 ng / mL BMP2 (Product No. 7510050, Medtronic). Osteogenic induction medium containing these ingredients was prepared and replaced every other day. Osteogenic induction was performed for 1 week, after which only osteogenic induction medium (EGM-2) was used for long-term culture. Cell aggregates obtained by SH were harvested on day 42 of culture and histologically analyzed. The following samples were further added to a medium containing EGM-2, b-glycerol phosphate, and T3 (Example 3-1) between days 28 and 35 of culture: Example 3-2 (CHIR), Example 3-3 (SAG), Example 3-4 (BMP2), Example 3-5 (SAG and CHIR), Example 3-6 (SAG and BMP2), Example 3-7 (CHIR and BMP2), and Example 3-8 (SAG, CHIR, and BMP2). Treatment with specific combinations (CHIR + BMP2 and SAG + CHIR + BMP2) demonstrated favorable induction of ALP activity (Examples 3-7 and 3-8). These results suggest that the combination of CHIR and BMP2 is important for osteoblast induction in cell aggregates obtained by SH. Regarding chondrogenesis, treatment with SAG, CHIR, and BMP2 induced more Safranin-O-positive cartilage areas than treatment with CHIR and BMP2, suggesting that SAG induced chondrogenesis. Consistent with this, a single administration of SAG induced chondrogenesis better than a single administration of CHIR or BMP2.This suggests that treatment with SAG, CHIR, and BMP2 may mimic bone formation.

[0072] Example 4: Maturation Potential of Cell Aggregates and In Vivo Differentiation Induction of Cell Aggregates (Figure 11) To examine the in vivo maturation potential of cell aggregates obtained by the SH method and cell aggregates obtained by the S method on day 28, both samples were subcutaneously implanted into immunodeficient mice. Figure 10 shows a schematic diagram of the subcutaneous implantation of one or more cell aggregates obtained by the SH method into mice. As a control, cell aggregates obtained by the S method were also implanted into the same mice. Specifically, cell aggregates obtained by the S method or SH method on day 28 of culture were first collected from the 96-well 3D culture plate and stored in 1.5 mL tubes with EGM-2 medium. These cell aggregates were then stored on ice until subcutaneous implantation and implanted into 8- to 12-week-old immunodeficient male mice (NOD.CB17-Prkdcscid / J; NOD SCID; The Jackson Laboratory Japan). For subcutaneous implantation, mice were anesthetized with isoflurane during surgery. The hair on the back was gently removed, and a 1 cm dorsal incision was made. A 200 μL pipette and 200 μL tip were used to harvest the cell aggregates. Cell aggregates obtained from the SH method and the S method on day 28 of culture were transplanted singly or multiple times to the left and right sides of the back. Transplants were performed at three sites on each side of the back. After transplantation, the skin was sutured with 6-0 nylon thread. A heating plate was used to maintain the mouse's body temperature during the procedure. These cell aggregates were harvested from the skin 70 days after transplantation and analyzed histologically.

[0073] Changes in Cell Aggregates Obtained by SH Method Using In Vivo Live MicroCT Scanning (Figure 12). In vivo live MicroCT scans (R_mCT2-FX; Rigaku) ​​were performed every two weeks after transplantation to track the time course of changes in the transplanted cell aggregates obtained by the SH method and the S method. Scanning conditions were 90 kV, 160 UA, 60 FOV, and 17 seconds. MicroCT analysis revealed radiopaque images suggestive of calcified tissue in the transplanted cell aggregates obtained by the SH method on postoperative day 70 (POD70) (Figure 12). In contrast, no X-ray signals suggestive of calcification were observed in the cell aggregates obtained by the S method. 3D reconstruction confirmed that calcification occurred in one of the six transplanted cell aggregates obtained by the SH method.

[0074] Histological analysis of cell aggregates obtained by the SH method on day 70 after transplantation (POD70) (Figure 13, left and center). Histological analysis of the samples obtained on day 70 after transplantation (POD70) showed that both the cell aggregates obtained by the SH method and the cell aggregates obtained by the S method on day 28 of culture exhibited a Safranin O-positive matrix. However, the size of chondrocytes in the cell aggregates obtained by the SH method was larger than that in the cell aggregates obtained by the S method, suggesting the presence of hypertrophic chondrocytes in the tissue derived from the cell aggregates obtained by the SH method. ALP staining revealed intense ALP staining around the cartilage-like tissue of the cell aggregates obtained by the SH method on POD70, suggesting the presence of osteoblasts in the cartilage-like tissue. Some hypertrophic chondrocyte-like cells within the cell aggregates obtained by the SH method also expressed ALP, consistent with physiological hypertrophic chondrocyte activity. In contrast, no ALP-positive areas were observed in the cell aggregates obtained by the S method.

[0075] Confirmation of mineralized tissue by von Kossa staining (Figure 13, bottom right). Furthermore, we confirmed the mineralized tissue using von Kossa staining, which shows mineralized tissue in brown to black. As a result, a positive area (calcified area) was observed in the central region of the cell aggregates obtained by the SH method on POD70, which contained hypertrophic chondrocytes and perichondrium tissue. This calcified area occupied an area equivalent to 19.9% ​​of the cross-sectional area of ​​the cell aggregate (Figure 13, bottom right). These findings suggest that when cell aggregates obtained by the SH method on day 28 of culture are transplanted subcutaneously, endochondral bone-like tissue is formed by POD70.

Claims

1. A cell aggregate comprising chondrocytes and osteoblast precursor cells.

2. The cell aggregate of claim 1, wherein the chondrocytes include hypertrophic chondrocytes.

3. The cell aggregate according to claim 1 or claim 2, which is a cell aggregate containing cartilage-like tissue.

4. The cell aggregate according to claim 1 or claim 2, further comprising vascular endothelial cells.

5. The cell aggregate according to claim 4, wherein the vascular endothelial cells express vascular endothelial growth factor A (VEGF-A).

6. The cell aggregate according to claim 4, wherein the vascular endothelial cells are umbilical vein endothelial cells.

7. The cell aggregate of claim 6, wherein the umbilical vein endothelial cells are human umbilical vein endothelial cells.

8. A cell aggregate according to claim 1 or 2, wherein the ratio of the total number of chondrocytes and osteoblast precursor cells to the total number of cells constituting the cell aggregate is 15% or more.

9. A cell aggregate according to claim 1 or 2, which comprises calcified tissue, and the calcified tissue is a site that shows positivity in von Kossa staining.

10. The cell aggregate described in claim 9, wherein the positive area is present at a rate of 10% or more relative to the area of ​​a section of the cell aggregate.

11. A method for producing a cell aggregate containing cartilage-like tissue, comprising co-culturing vertebral disc cells and vascular endothelial cells that express vascular endothelial growth factor.

12. The method according to claim 11, wherein the vertebral disc cells are cells obtained by induction from pluripotent stem cells.

13. The method of claim 11 or 12, wherein the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A).

14. A method for promoting chondrocyte hypertrophy, comprising co-culturing vertebral disc cells with vascular endothelial cells that express vascular endothelial growth factor.

15. The method according to claim 14, wherein the vertebral disc cells are cells obtained by induction from pluripotent stem cells.

16. The method of claim 14 or 15, wherein the vascular endothelial growth factor is vascular endothelial growth factor A (VEGF-A).

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