Method for producing iPS cell-derived chondrocyte construct

Abstract

A method for producing a three-dimensional structure of cartilage cells, the method being characterized by comprising layering cell aggregates that have been produced by culturing a mesenchymal stem cell using a culture medium for inducing the differentiation into a cartilage cell on the 9th or 10th day after the starting of the culture.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a chondrocyte construct. In particular, the present invention relates to a method for manufacturing a chondrocyte construct using a three-dimensional bioprinter.

Background Art

[0002] Research on producing cartilage from human induced pluripotent stem cells (iPS cells) has been conducted conventionally. There are established culture methods for producing cartilage tissue without using a scaffold from human iPS cell-derived chondrocytes (Yamashita A. et al., Stem Cell Reports, 2015 (Non-Patent Document 1)), methods for inducing somites and sclerotomes from iPS cells (Matsuda S. et al., Nature, 2020 (Non-Patent Document 2)), methods for inducing hypertrophic chondrocytes from proliferating chondrocytes (Pretemer Y. et al., Stem Cell Reports, 2021 (Non-Patent Document 3)), and the like. The chondrocytes produced in this way can be a clinically applicable tool because they produce a high dose of extracellular matrix.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in order to further find the clinical utility of chondrocytes induced from iPS cells, the three-dimensional culture of chondrocytes toward cartilage tissue is an issue.

Means for Solving the Problems

[0005] As a result of intensive studies to solve the above problems, the present inventors cultured mesenchymal stem cells induced from iPS cells, particularly mesenchymal stem cells induced from iPS cells, using a differentiation induction medium for chondrocytes to form cell aggregates (spheroids), and laminated these cell aggregates at a predetermined time from the start of the culture using the differentiation induction medium to three-dimensionally culture them, thereby succeeding in obtaining a three-dimensional structure of chondrocytes having high functionality and completing the present invention.

[0006] That is, the present invention is as follows. (1) A method for producing a three-dimensional structure of chondrocytes, comprising laminating cell aggregates obtained by culturing mesenchymal stem cells using a differentiation induction medium for chondrocytes on the 9th or 10th day after the start of the culture. (2) The method according to (1), wherein the mesenchymal stem cells are derived from pluripotent stem cells. (3) The method according to (2), wherein the pluripotent stem cells are induced pluripotent stem cells. (4) The method according to any one of (1) to (3), wherein the differentiation induction medium contains platelet-derived growth factor. (5) The method according to any one of (1) to (4), wherein the cells are cultured in the presence of a transforming growth factor from the 6th day after the start of the culture. (6) A method for producing a cartilage tissue, comprising further culturing the three-dimensional structure produced by the method according to any one of (1) to (5) in the presence of an osteogenic protein. (7) A three-dimensional structure of chondrocytes, comprising laminating cell aggregates obtained by culturing mesenchymal stem cells using a differentiation induction medium for chondrocytes on the 9th or 10th day after the start of the culture. (8) A cartilage tissue, comprising further culturing the three-dimensional structure according to (7) in the presence of an osteogenic protein.

Advantages of the Invention

[0007] According to the present invention, it has become possible to produce a three-dimensional structure of chondrocytes in which differentiation into cartilage is sufficiently carried out and an extracellular matrix is secreted. By further culturing this three-dimensional structure in the presence of bone morphogenetic protein using a bioreactor, it has become possible to obtain a cartilage tissue.

Brief Description of the Drawings

[0008]

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Mode for Carrying Out the Invention

[0009] Various studies have been made on methods for inducing differentiation of iPS cells into chondrocytes. For example, as a protocol of Kyoto University, a method (Non-Patent Document 1) of introducing 10 ng / ml TGFB1, 10 ng / ml BMP-2, 10 ng / ml GDF5, and 10 ng / ml bFGF into iPS cells to directly form a mass of chondrocytes is known. According to this method, since it exhibits characteristics of cartilage tissue such as producing an extracellular matrix, it is useful for treating partial defects and the like of cartilage tissue. However, in order to use a mass of chondrocytes for treatment to regenerate a wide articular surface having a curved surface, it is necessary to form a three-dimensional structure of chondrocytes. Therefore, in the present invention, during the multi-step induction of iPS into chondrocytes, the optimal conditions for three-dimensional formation with a bio-3D printer were found. The present invention relates to a three-dimensional structure of chondrocytes formed by laminating cell aggregates obtained by culturing mesenchymal stem cells using a differentiation-inducing medium for chondrocytes on the 9th or 10th day after the start of the culture, and a method for producing the same. Furthermore, the present invention relates to a cartilage tissue formed by culturing the above three-dimensional structure in the presence of a bone morphogenetic protein, and a method for producing the same.

[0010] 1. Mesenchymal stem cells In the present invention, the cells to be cultured using a differentiation-inducing medium for chondrocytes are mesenchymal stem cells (MSC). MSC is a pluripotent cell having self-renewal ability and differentiation ability, and since the risk of tumor formation is low, it can be expected as a tool for cell therapy and regenerative medicine. The MSC used in the present invention is not limited in its origin, and examples include adipose tissue, bone marrow, umbilical cord, pluripotent stem cells, deciduous dental pulp, etc. Also, MSC is commercially available and can be obtained from ATCC, Evercyte, CellSource, GeneTech Science, etc.

[0011] Among the above origins of MSC, pluripotent stem cells are stem cells having pluripotency to differentiate into all cells present in the living body and having proliferation ability, and examples include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, etc. A preferred pluripotent stem cell is an iPS cell. ES cells are stem cells established from cell masses of early embryos of mammals such as humans and mice. ES cells can be established by taking out the inner cell mass from the blastocyst of a fertilized egg of the target mammal and culturing the inner cell mass on a fibroblast feeder. Also, the maintenance of cells by subculture can be performed using a culture solution supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Also, human ES cell lines can be obtained from the Institute of Virus Research and Regenerative Medicine, Kyoto University (Kyoto, Japan).

[0012] Induced pluripotent stem (iPS) cells are somatic cell-derived artificial stem cells that have almost the same pluripotency of differentiation and proliferation ability by self-renewal as ES cells, and can be produced by introducing specific reprogramming factors into somatic cells (Yamanaka S. et al., Cell, 126:663-676, 2006; Okita K. et al., Nature 448, 2007; WO2007 / 069666, etc.). Examples of the genes included in the reprogramming factors are, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. These reprogramming factors can be used alone or in appropriate combinations, but Oct3 / 4, Sox2, Klf4 and c-Myc are preferred.

[0013] Methods for inducing iPS cells from somatic cells are well known (Yamanaka S. et al., Cell, 126:663-676, 2006; Okita K. et al., Nature 448, 2007; WO2007 / 069666, etc.). The tissues from which the iPS cells used in the present invention are derived include, for example, articular cartilage, bone, adipose tissue, ligament, tendon, tooth, auricle, nose, liver, pancreas, blood vessel, nerve, heart, etc. In addition, the spheroid does not necessarily have to be formed as an aggregate of a single type of cell. As long as the spheroid is formed, a plurality of cell types other than iPS cells, for example, undifferentiated cells such as cells derived from umbilical cord blood or their differentiated cells, can be mentioned.

[0014] In the present invention, methods for inducing mesenchymal stem cells (MSC) from pluripotent stem cells include, for example, a method of culturing iPS cells in the presence of factors such as TGFB1. The differentiation induction period from pluripotent stem cells to MSC is, for example, 10 days. At that time, it can pass through neural crest stem cells (NCC) once. In addition, MSC derived from iPS cells can also be obtained from Kyoto University.

[0015] 2. Differentiation into chondrocytes and formation of cell aggregates When the cell suspension of MSC obtained as described above is seeded on a plate for preparing cell aggregates (spheroid plate) and cultured using a differentiation-inducing medium, MSC is gradually induced to differentiate into chondrocytes. In parallel, cells aggregate to form cell aggregates (spheroids). In the present invention, a growth factor can be used as a factor to be contained in the differentiation-inducing medium. Examples of the growth factor include platelet-derived growth factor (PDGF), Transforming Growth Factor-β (TGFβ), Bone Morphogenetic Protein (BMP) factor, etc. These factors are contained in the medium at a predetermined time to induce differentiation of MSC into chondrocytes.

[0016] PDGF is a humoral factor stored in platelet α-granules. PDGF promotes the differentiation and proliferation of pluripotent stem cells into chondrocytes. Four types of genes encoding PDGF (A chain, B chain, C chain, D chain) are known, and there are four homodimers and an AB heterodimer of PDGF having biological activity. AA, AB, and BB are modified by protease in the cytoplasm and secreted as a mature form with a molecular weight of about 30,000, while CC and DD are secreted while retaining a CUB region that inhibits receptor binding.

[0017] There are three isoforms of TGFβ (TGFβ1, β2, β3) in mammals, and the structurally similar TGFβ superfamily includes activin, BMP (bone morphogenetic protein), etc. In recent studies, TGFβ has been shown to contribute to growth inhibition, cell differentiation, induction of apoptosis, etc. in many cell types. For example, while it promotes the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, it has been reported to act inhibitory on the proliferation of epithelial cells and osteoclasts. In the present invention, preferably TGFβ3 can be used.

[0018] Here, when the first day of seeding the cell suspension on the spheroid plate, that is, the first day of culture with the differentiation induction medium, is defined as the start day (day 0), after day 0, for example, a PDGF-containing medium is used, and TGFβ is added and cultured after day 6. When culturing MSCs in a PDGF-containing differentiation induction medium, gradually from around the third day (day 3), the MSCs differentiate into chondrocytes and form spheroids. These formed spheroids are stacked to produce a three-dimensional structure. In the present invention, the timing of stacking the spheroids is the ninth or tenth day (day 9 or day 10) from the start of the spheroid plate formation culture using a PDGF-containing differentiation induction medium.

[0019] In the present invention, chondrocytes mean cells that produce an extracellular matrix constituting cartilage, such as collagen, or their progenitor cells. Such chondrocytes may be cells that express chondrocyte markers, and examples of chondrocyte markers include type II collagen (COL2A1) or SOX9. In the present invention, COL2A1 includes a gene having a nucleotide sequence described as an accession number of NCBI, NM_001844 or NM_033150 in the case of humans, NM_001113515 or NM_031163 in the case of mice, a protein encoded by the gene, and naturally occurring variants having these functions. In the present invention, SOX9 includes a gene having a nucleotide sequence described as an accession number of NCBI, NM_000346 in the case of humans, NM_011448 in the case of mice, a protein encoded by the gene, and naturally occurring variants having these functions.

[0020] 3. Lamination of spheroids A method for producing a three-dimensional structure of cells by arranging cells in an arbitrary three-dimensional space is known (WO2008 / 123614). This method involves arranging acicular bodies on a substrate in a hedgehog shape and arranging cell aggregates (spheroids) on the acicular bodies by piercing them. In the present invention, a three-dimensional structure (three-dimensional structure) is produced by laminating spheroids using the above method. Since an automatic lamination robot for realizing the above method is already known (Bio 3D printer "Regenova" (registered trademark), Cyfuse Biomedical Co., Ltd.), the three-dimensional structure can also be produced using this robot.

[0021] The number and arrangement shape of the spheroids are not particularly limited and are arbitrary. After laminating the spheroids, they are cultured in the presence of bone morphogenetic protein (BMP) to form cartilage tissue. A reaction vessel for growing from a three-dimensional structure to cartilage tissue is called a bioreactor. BMP is a group of proteins identified as molecules that induce and promote the differentiation of bone tissue and cartilage. BMP belongs to the TGFβ superfamily, binds to type I and type II receptor dimers, and signals are transmitted into the nucleus through the phosphorylation of the transcription factor SMAD.. BMP belonging to the TGFβ superfamily is divided into the BMP2 / 4 group (BMP2, BMP4), the OP-1 group (BMP5, BMP6, BMP7, BMP8a, BMP8b), the BMP9 group (BMP9, BMP10), and the GDF5 group (GDF5, GDF6, GDF7).

[0022] Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples. [Example 1]

[0023] Method The induction of differentiation from MSC to chondrocytes, spheroid formation, and the schedule of lamination are shown in Fig. 1. An example of the production of a three-dimensional chondrocyte structure is shown using mesenchymal stem cells (hereinafter iMSC) induced to differentiate from human iPS cells provided by Kyoto University via neural crest stem cells as raw materials. iMSC was seeded at 4.5x10 4 cells per well on Sumilon (registered trademark) PrimeSurface (registered trademark) 96 (manufactured by Sumitomo Bakelite Co., Ltd., spheroid plate), and spheroid formation was performed using an induction medium in which a differentiation induction supplement PT-4121 was added to a chondrogenic differentiation induction basal medium PT-3925 (manufactured by Lonza).

[0024] The day of seeding on the spheroid plate was set as Day0, the medium was changed approximately every 3 days, and from Day0 to Day6, PDGF was added to the differentiation induction medium, from Day6 to Day10, the differentiation induction medium + PDGF + TGFβ3, and from Day10 to the final day (24), the cells were cultured in a medium of differentiation induction medium + TGFβ3 + BMP-4. Platelet-Derived Growth Factor BB: PDGF-BB (R&D systems) 520-BB-050 Transforming Growth Factor-β3: TGF-β3 (Peprotech) 100-36E Bone Morphogenetic Protein 4: BMP-4 (R&D systems) 314-BP-050

[0025] The timing of setting each cell mass (spheroid) on the bio-3D printer and three-dimensionalizing it was divided into four conditions for layering (printing). Condition 1: Day 3 Condition 2: Day 6 Condition 3: Day 10 Condition 4: Day 16 After printing, all were cultured with the spheroids stuck on the pin cushion. On the 24th day, the three-dimensional cell structure was removed from the pin cushion and evaluated using pathological tissues, etc.

[0026] The results are shown in Figures 2 to 4. Figure 2 is a photograph of the three-dimensional structure on the 24th day under Conditions 1 and 2. Both had smoothly healed, but there was no strength under Condition 1, and it was softer than rubber under Condition 2. For pathological tissues, staining that dyes cartilage-specific proteoglycan red (safranin O-fast green staining) and type II collagen immunostaining were performed.

[0027] Under Condition 1, almost no staining of proteoglycan was observed. Also, the expression of type II collagen specific to articular cartilage was poor. Under Condition 2, some staining of proteoglycan was observed, but it was very faint. The expression of type II collagen was firm.

[0028] Figure 3 is a photograph of the three-dimensional structure on the 24th day under Condition 4. When the culture period of the spheroids was extended, the induction of differentiation into cartilage proceeded too far, and it was occasionally observed that the spheroids were no longer pierced by the needles of the pincushion. Under Condition 4, the fusion of the spheroids was poor, and a smooth curved surface was not formed, and a structure with severe unevenness was obtained. Even if this is transplanted into articular cartilage, the function as a joint cannot be expected. Pathologically, the expression of safranin O was prominent, and the expression of type II collagen was also strong. The strength of the structure is very strong.

[0029] Figure 4 is a photograph of the three-dimensional structure on the 24th day under Condition 3. When printing was performed on the 9th or 10th day after the start of spheroid formation, the fusion of the spheroids was also good, and a structure with sufficient strength was obtained. A firm expression of proteoglycan and an expression of type II collagen were also observed in the pathological tissue sections, and it was confirmed that these are effective conditions for the regeneration of articular cartilage (Figure 4).

Claims

1. A method for producing a three-dimensional structure of chondrocytes, comprising laminating cell aggregates obtained by culturing mesenchymal stem cells using a differentiation induction medium for chondrocytes on the 9th or 10th day after the start of the culture, wherein the differentiation induction medium contains platelet-derived growth factor and the culture is carried out in the presence of a transforming growth factor from the 6th day after the start of the culture.

2. The method according to claim 1, wherein the mesenchymal stem cells are derived from pluripotent stem cells.

3. The method according to claim 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

4. A method for producing a cartilage tissue, comprising culturing the three-dimensional structure produced by the method according to any one of claims 1 to 3 in the presence of an osteogenic protein.

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