Methods for producing osteoblast constructs using iPS cells

Abstract

Provided is a method for producing an osteoblast construct from an iPS cell, the method comprising the steps of: (1) inducing formation of embryoid bodies by subjecting undifferentiated iPS cells to non-adherent culture; (2) inducing differentiation of the iPS cells into mesoderm cells by subjecting the embryoid bodies of the iPS cells obtained in step (1) to non-adherent culture; and (3) inducing differentiation into osteoblasts by subjecting the mesoderm cells of the iPS cells obtained in step (2) to non-adherent culture, wherein both steps (1) and (2) are performed using a culture vessel comprising a bottom surface and a ring-shaped side wall arranged vertically on the bottom surface, the bottom surface having a plurality of recesses arranged independently of one another.

Description

TECHNICAL FIELD

[0001] [CROSS-REFERENCE TO RELATED PATENT APPLICATIONS]

[0002] This application claims the benefit of priority of Japanese Patent Application No. 2019-033277 filed on February 26, 2019, the entire contents of which are incorporated herein by reference. The present invention relates to a method for producing an osteoblast construct using iPS cells. BACKGROUND

[0003] There is an extremely high demand for artificial bone / bone replacement materials for replacing, for example, a defect site of bone loss caused by bone tumor resection, comminuted fracture, bone defects associated with rheumatoid arthritis fixation, alveolar ridge absorption, and the like.

[0004] Non-absorbable materials (such as hydroxyapatite) and absorbable materials (such as β-tricalcium phosphate) currently used as artificial bone / bone replacement materials in the clinic have problems such as lower bone inductive activity than autologous bone and not always providing a good prognosis in surgical intervention. In addition, hybrid artificial bone / bone replacement materials (next-generation type requiring further development) obtained by combining artificial bone with a growth factor protein such as bone morphogenetic protein (BMP) lack an "extracellular matrix" important for bone tissue regeneration and thus cannot provide sufficient bone regeneration effects.

[0005] Under such circumstances, the inventors of the present invention have successfully produced a bone regenerative agent including an inactivated cell construct using stem cells as a raw material, the inactivated cell construct containing at least a mineral and an extracellular matrix (Patent Documents 1 and 2).

[0006] LIST OF CITATIONS

[0007] PATENT LITERATURE

[0008] PTL 1: WO 2015 / 064705 Al

[0009] PTL 2: WO 2018 / 181960 Al

[0010] NON-PATENT LITERATURE

[0011] NPL 1: Stem Cell Reports Vol. 2, 751-760, June 3, 2014 SUMMARY

[0012] TECHNICAL PROBLEM

[0013] An object of the present invention is to provide a new method for producing an osteoblast construct capable of providing an osteoblast construct having high bone regenerative ability by using iPS cells as a raw material.

[0014] Solution to the problem

[0015] In the above circumstances, the inventors of the present application have made intensive studies and as a result, have surprisingly found that by using micro-space culture in each of (1) a step of inducing formation of a spheroid and (2) a step of inducing differentiation into mesoderm cells in a method of producing an osteogenic cell construct using an undifferentiated iPS cell as a raw material and including a step of inducing formation of a spheroid, a step of inducing differentiation into mesoderm, and a step of inducing differentiation into an osteogenic cell, an osteogenic cell construct having an extremely high bone regenerative ability is obtained. The inventors of the present application have further made intensive studies on culture conditions and the like based on this new finding, and thus have completed the present application.

[0016] Therefore, the present application provides a method, an osteogenic cell construct, and the like as described in the following items.

[0017] Item 1. A method of producing an osteogenic cell construct from an iPS cell, comprising the steps of:

[0018] (1) inducing formation of a spheroid by subjecting an undifferentiated iPS cell to non-adherent culture;

[0019] (2) inducing differentiation of the iPS cell into a mesoderm cell by subjecting the spheroid of the iPS cell obtained in step (1) to non-adherent culture; and

[0020] (3) inducing differentiation into an osteogenic cell by subjecting the mesoderm cell of the iPS cell obtained in step (2) to non-adherent culture,

[0021] wherein each of steps (1) and (2) is performed using a culture vessel including a bottom surface and a ring-shaped side wall arranged vertically on the bottom surface, the bottom surface having a plurality of recesses arranged independently of each other.

[0022] Item 2. The method according to item 1, wherein at least one of the plurality of recesses has a circular equivalent diameter of 200 μm to 900 μm and a depth of 200 μm to 1000 μm.

[0023] Item 3. The method according to item 1 or 2, wherein each of the plurality of recesses has an opening in a shape of a circle.

[0024] Item 4. The method according to any one of items 1 to 3, wherein the iPS cell is a human iPS cell or a mouse iPS cell.

[0025] Item 5. The method according to item 4, wherein the culture time of step (1) is 0.625 days to 3.5 days.

[0026] Item 6. The method according to any one of items 1 to 5, wherein the culturing in step (2) is performed in the presence of at least one selected from the group consisting of a Wnt signal activator and a Hedgehog signal inhibitor.

[0027] Item 7. The method according to item 6, wherein the Wnt signal activator is at least one selected from the group consisting of CHIR99021, 6-bromoindirubin-3'-oxime, kenpaullone, SB-216763, SKL2001, deoxycholic acid, WAY-316606, NSC-693868, ricinine, 7-oxo-β-sitosterol, IM-12, HLY78, and retinoic acid.

[0028] Item 8. The method according to item 6 or 7, wherein the Hedgehog signal inhibitor is at least one selected from the group consisting of cyclopamine, AY9944, GANT58, GANT61, jervine, SANT-1, SANT-2, U18666A, veratramine, vismodegib, Cur-61414, robotnikinin, JK184, and HPI-4.

[0029] Item 9. The method according to any one of items 1 to 8, wherein the culturing of step (3) is performed in the presence of at least one selected from the group consisting of a hypoxia mimetic compound, a statin compound, and retinoic acid.

[0030] Item 10. The method according to any one of items 1 to 9, further comprising, prior to step (1), a step of culturing the undifferentiated iPS cells without using feeder cells.

[0031] Item 11. The method according to any one of items 1 to 10, wherein step (1) is performed by placing a suspension of the undifferentiated iPS cells having a cell concentration of 1.5 x 10 5 cells / ml to 3.5 x 10 5 cells / ml in a culture vessel, and then culturing.

[0032] Item 12. The method according to any one of items 1 to 11,

[0033] wherein step (2) is performed using a culture vessel having at least one well, and

[0034] wherein step (3) is performed by placing a culture solution corresponding to 1 to 10 wells containing the mesoderm cells obtained in step (2) in a culture vessel, and then culturing.

[0035] Item 13. The method according to any one of items 1 to 12,

[0036] In step (1), the culture was carried out in the presence of a ROCK inhibitor; and

[0037] Each culture in steps (2) and (3) is carried out in the presence of retinoic acid.

[0038] Project 14: An osteoblast construct derived from human iPS cells and having a Feret's diameter of 1 mm to 4 mm.

[0039] Item 15. The osteoblast construct according to Item 14, wherein the osteoblast construct is obtained by any one of Items 1 to 13.

[0040] Beneficial effects of the invention

[0041] According to the present invention, a novel method for producing osteoblast constructs is provided, which can provide osteoblast constructs with high bone regeneration capacity by using iPS cells as raw materials. Attached Figure Description

[0042] Figure 1 A method for inducing mouse iPS cells into osteoblast constructs. Osteoblast constructs are generated by inducing germ-like structures from mouse iPS cells using Elplasia (trademark) (Kuraray, Japan), a micro-space-shaped low-attachment plate with multiple recesses on the bottom surface of each pore. Elplasia (trademark) plates with different recess pore diameters (400 μm: Elp400, 500 μm: Elp500, and 900 μm: Elp900) were used, and the effect of these differences on the induction of osteoblast constructs was investigated.

[0043] Figure 2 Top image: Phase contrast micrographs of mouse iPS cells seeded in Elp400, Elp500, and Elp900 cells and cultured in ES medium for 2 days. Bottom image: Enlarged images of a concave portion in the middle of each of the Elp400, Elp500, and Elp900 cells.

[0044] Figure 3 Left panel: Feret's diameter of iPS cell constructs during osteoblast differentiation induction (day 0 to day 35) using Elp400, Elp500, and Elp900 (n = 3, *P < 0.01: ANOVA and Tukey's multiple valence test). Right panel: Photographs of typical iPS cell constructs using Elp400, Elp500, and Elp900 during the same period.

[0045] Figure 4Survival and death of cells in iPS cell constructs. Investigation using LIVE / DEAD (trademark) Viability / Cytotoxicity Kit after 14 days (day 14) from the start of osteoblast differentiation induction with Elp400, Elp500 and Elp900 (day 0). Green indicates live cells and red indicates dead cells.

[0046] Figure 5 Real-time RT-PCR analysis of the expression of osteoblast-specific marker genes (Runx2, Osterix, Collagen 1a1, Bone sialoprotein, Osteopontin, Osteocalcin) in cell constructs after 10 days of osteoblast differentiation induction with Elp400, Elp500 and Elp900 (n=3, *P<0.05: ANOVA with Tukey's multiple comparison test).

[0047] Figure 6 Images of hematoxylin-eosin (HE) staining and von Kossa and methylene blue double staining of cell construct sections after 35 days of osteoblast differentiation induction with Elp400, Elp500 and Elp900.

[0048] Figure 7 A method of inducing human iPS into osteoblast cell constructs. By using a microspatial low attachment plate Elplasia (trademark) (Elp500) having a plurality of recesses each with a diameter of 500 μιη on the bottom surface of each well, embryoid bodies and mesoderm were induced from human iPS cells and osteoblast cell constructs were generated. As a comparative control, a case where a general low-attachment culture dish was used instead of Elplasia (trademark) was set.

[0049] Figure 8 Human iPS cells were induced into embryoid bodies and mesoderm cells using a low-attachment culture dish or Elp500, and then subjected to osteoblast differentiation induction for 30 days, followed by counting the number of cell constructs in the culture flask. When Elp500 was used, the generation efficiency of osteoblast cell constructs from human iPS cells was increased by about 9 times (n=5).

[0050] Figure 9 Images of von Kossa and methylene blue double staining of cell construct sections of human iPS cell constructs subjected to osteoblast differentiation induction for 30 to 90 days after induction into embryoid bodies and mesoderm cells using Elp500.

[0051] Figure 10Real-time RT-PCR analysis of the expression of osteoblast-specific marker genes (Runx2, Osterix, Collagen la 1 and Osteocalcin) in human iPS cell constructs after induction of embryoid bodies and mesodermal cells with low attachment culture dishes (low attachment culture dishes) or Elp500 (Elplasia (trademark)) and subjected to osteoblast differentiation induction for 30 days, and in undifferentiated human iPS cells just before embryoid body culture (n=3, *P<0.05: ANOVA with Tukey's multiple comparison test).

[0052] Figure 11 Results of analysis of the expression of undifferentiated marker gene (Nanog), mesoderm (mesenchymal stem cell)-osteoblast progenitor cell marker genes (Brachyury, Runx2 and Osterix) and osteoblast-specific genes (Collagen la 1 and Osteocalcin) in human iPS cell constructs after induction of embryoid bodies and mesodermal cells with Elp500 and subjected to osteoblast differentiation induction for 60 days, and in undifferentiated human iPS cells just before embryoid body culture by real-time RT-PCR method (n=3) are shown.

[0053] Figure 12 Composition analysis of human iPS cell-derived osteoblast constructs using FTIR. Human iPS cell constructs after induction of embryoid bodies and mesodermal cells with Elp500 and subjected to osteoblast differentiation induction for 0 to 60 days were dried and subjected to FTIR analysis. As a comparative control, human freeze-dried allogeneic bone graft (FDBA) was used. Arrows indicate FTIR spectral peaks (Am I, Am II, PO4 3- and CO3 2- ) possessed by natural bone tissue.

[0054] Figure 13 (A): Feret's diameters of human iPS cell constructs after induction of embryoid bodies and mesodermal cells with Elp500 and subjected to osteoblast differentiation induction for 3 to 30 days (n=25). After osteoblast differentiation induction for 30 days, various sizes of cell constructs with Feret's diameters of about 0.5 μm to about 3 μm were found (photograph inserted). Figure 13 (B) shows HE staining images (left column) and von Kossa and methylene blue double staining images (right column) of typical cell construct sections of each cell construct group grouped according to Feret's diameters of 0.5 mm to less than 1 mm, 1 mm to less than 2 mm and 2 mm to 3 mm after osteoblast differentiation induction for 30 days. As the size of the cell constructs increased, more apparent mineralization was shown.

[0055] Figure 14 : Photographs of human iPS cell constructs subjected to osteoblast differentiation induction for 120 days after induction of embryoid bodies and mesoderm cells using Elp500 (A) and photographs thereof after lyophilization (B).

[0056] Figure 15 : Photographs of human iPS cell-derived lyophilized osteoblast cell constructs (A) or (B) human lyophilized allogeneic bone graft (FDBA) implanted into rat skulls 4 weeks after the generation of a 5-mm-diameter bone defect site. Figure 14 : HE staining images and micro-CT imaging images taken 4 weeks after implantation of human iPS cell-derived lyophilized osteoblast cell constructs or (B) human lyophilized allogeneic bone graft (FDBA) into rat skulls.

[0057] Figure 16 : Schematic diagram of a micro-space culture vessel 1 in a typical embodiment of the present application.

[0058] Figure 17 : Cross-sectional view of the face of the micro-space culture vessel 1 including the dashed line A-B in Figure 16 : Enlarged view of the area 4 of the bottom face 2 in : Cross-sectional view of the area 4 shown in

[0059] : Enlarged view of the area 4 in an embodiment in which the shape of each recess 6 is a regular hexagon. Figure 18 : Cross-sectional view of the area 4 shown in Figure 16 : Enlarged view of the area 4 in an embodiment in which the shape of each recess 6 is a regular hexagon. : Cross-sectional view of the area 4 shown in

[0060] : Enlarged view of the area 4 in an embodiment in which the shape of each recess 6 is a regular hexagon. Figure 19 : Cross-sectional view of the area 4 shown in Figure 18 : Enlarged view of the area 4 in an embodiment in which the shape of each recess 6 is a regular hexagon. : Enlarged view of the area 4 in an embodiment in which the shape of each recess 6 is a regular hexagon.

[0061] : Schematic diagram for explaining an embodiment in which one cell construct is housed in each recess of the micro-space culture vessel 1. Figure 20 : Schematic diagram for explaining an embodiment in which one cell construct is housed in each recess of the micro-space culture vessel 1. : Profile diagram of a seesaw-type bioreactor used in the example.

[0062] : Profile diagram of a seesaw-type bioreactor used in the example. Figure 21 : Schematic diagram of a flask that can be used for shaking culture. : Schematic diagram of a flask that can be used for shaking culture.

[0063] : Schematic diagram of a flask that can be used for shaking culture. Figure 22 : Schematic diagram of a flask that can be used for shaking culture. : Schematic diagram of a flask that can be used for shaking culture.

[0064] : Schematic diagram of a flask that can be used for shaking culture. Figure 23 : Schematic diagram of a flask that can be used for shaking culture. : Schematic diagram of a flask that can be used for shaking culture.

[0065] : Schematic diagram of a flask that can be used for shaking culture. Figure 24 : Schematic diagram of a flask that can be used for shaking culture. : Schematic diagram of a flask that can be used for shaking culture.

[0066] : Schematic diagram of a flask that can be used for shaking culture. Figure 25 : Schematic diagram of a flask that can be used for shaking culture.

[0067] Figure 26 The Feret's diameter (n=10) of the cell constructs on day 30 of osteoblast differentiation induction is shown. Number of cells seeded: The number of cells seeded in one well of a 24-well Elplasia (trademark) culture plate during the embryoid formation step. Well: The number of wells from which mesodermal cell cultures were transferred to one flask at the start of the osteoblast differentiation induction step.

[0068] Figure 27 The results of real-time RT-PCR analysis of the expression of osteoblast-specific marker genes (Runx2 and osteocalcin) in cell constructs at days 0 and 30 of osteoblast differentiation induction are shown (n=3, *P<0.05: ANOVA and Tukey's multiple valence test). Significant differences exist between different letters. Number of cells seeded: The number of cells seeded in one well of a 24-well Elplasia (trademark) culture plate during the embryoid formation step. Well: The number of wells in which mesodermal cells were transferred to a flask for culture at the start of the osteoblast differentiation induction step.

[0069] Figure 28 HE-stained images of cell constructs 30 days after osteoblast differentiation induction under different conditions are shown (left column for each condition) and von Kossa and methylene blue double-stained images (right column for each condition). Number of cells seeded: The number of cells seeded in one well of a 24-well Elplasia (trademark) culture plate during the embryoid formation step. 1 to 8 wells: Represents the number of wells transferred to one flask for mesodermal cell culture at the start of the osteoblast differentiation induction step.

[0070] Figure 29 The images show HE staining (left column for each condition) and von Kossa and methylene blue double staining (right column for each condition) images of osteoblast constructs generated using human iPS cells cultured in a feeder-free environment, with or without 1 μM retinoic acid supplementation in mesodermal differentiation induction medium and osteoblast differentiation induction medium. Detailed Implementation

[0071] Method for producing osteoblast constructs

[0072] This invention provides a method for producing osteoblast constructs from iPS cells, the method comprising the following steps:

[0073] (1) Inducing embryoid formation by subjecting undifferentiated iPS cells to non-adherent culture;

[0074] (2) Inducing iPS cells to differentiate into mesodermal cells by subjecting the embryoids of iPS cells obtained in step (1) to non-adherent culture; and

[0075] (3) The iPS cells obtained in step (2) were induced to differentiate into osteoblasts by undergoing non-adherent culture of mesodermal cells.

[0076] Steps (1) and (2) are performed using a culture container that includes a bottom surface and an annular sidewalls arranged vertically on the bottom surface, the bottom surface having multiple recesses arranged independently of each other.

[0077] In this invention, "non-adherent culture" refers to culture in a state in which cell adhesion to the bottom surface, etc. (e.g., bottom and wall surfaces) of the culture container is inhibited. In this invention, non-adherent culture encompasses, for example, shaking culture and static culture using non-adherent culture flasks (such as non-adherent culture dishes, non-adherent wells, non-adherent bottles, three-dimensional culture plates, or cell construct generation containers) in which cell adhesion to the bottom surface, etc., of the culture container is inhibited. As a non-adherent culture container, for example, a container with a low-adherence surface treatment by means of phospholipid gel, hydrogel, microfabrication, etc., can be used. Furthermore, in this invention, for shaking culture, the aforementioned non-adherent culture container can be used, or a culture container that is not non-adherent can be used. When performing shaking culture, the method used is not particularly limited; for example, methods such as... Figure 22 The seesaw-type bioreactor shown is used for oscillating culture. Furthermore, the tilt angle of the oscillation is not particularly limited, but preferably 1° to 40° relative to the horizontal direction, more preferably 5° to 35°, and even more preferably 10° to 30°. The amplitude of the oscillation is not particularly limited, but can be, for example, from about 0.1 cm to about 20 cm. The period of oscillation is not particularly limited, but can be, for example, from about 0.01 Hz to about 1.00 Hz. Figure 22 The tilt angle is 10° and the amplitude is 5.5 cm. There are no particular restrictions on the width of the platform (a component of the seesaw-type bioreactor) for placing the culture flasks; platforms with widths in the range of, for example, 20 cm to 50 cm, or 30 cm to 40 cm, can be used. There are no particular restrictions on the benchtop culture flasks, but a culture area (growth area) of, for example, 5 cm² can be used. 2 Up to 200cm 2 10cm 2 Up to 60cm 2 Or 15cm 2 up to 35cm 2 Bottles within a certain range. In the case of shaking culture, there are no particular restrictions on the initial cell concentration at the start of the culture, but when using bottles with a growth area of ​​25 cm²... 2 When using culture flasks, the cell concentration is preferably about 1 × 10⁻⁶. 2Cells / flask up to 1×10 8 Cells / flask, more preferably 1×10 6 Cells / flask up to 7×10 6 Cells / flask.

[0078] Microspatial culture flask

[0079] The present invention is characterized in that both the steps of inducing differentiation into germinal cells (step (1)) and inducing differentiation into mesodermal cells (step (2)) are performed using a culture vessel, which includes one or more bottom surfaces and one or more annular sidewalls vertically arranged on the bottom surface, the bottom surface having a plurality of recesses arranged independently of each other. In the present invention, the culture vessel including the bottom surface and the annular sidewalls vertically arranged on the bottom surface, and the bottom surface having a plurality of recesses arranged independently of each other, is sometimes referred to as a "microspace culture vessel". Similarly, the culture using this microspace culture vessel is sometimes referred to as "microspace culture".

[0080] Typical embodiments of the present invention are described below with reference to the accompanying drawings. First, in Figure 16 The diagram illustrates a microspace culture container 1 according to a typical embodiment of the present invention. The microspace culture container 1 includes a bottom surface 2 and an annular sidewall 3 vertically arranged on the bottom surface. Therefore, it can also be said that the microspace culture container 1 includes holes 5 formed by the bottom surface 2 and the annular sidewall 3 vertically arranged on the bottom surface. The annular sidewall 3 is not limited to... Figure 16 Those shown have a near-circular shape at each of the openings and the bottom, and may have, for example, polygons (triangles, quadrilaterals (e.g., rectangles, such as squares or rectangles, parallelograms or trapezoids), pentagons, hexagons, etc.).

[0081] Figure 17 It shows Figure 16 A cross-sectional view of the plane perpendicular to the microspace culture container 1, including the dashed line AB. Figure 17 In the preferred embodiment shown, the materials forming the wall surface and the materials forming the bottom surface can be different from each other. Figure 17 In this case, the bottom surface is made of a transparent material, while the walls are made of an opaque material (e.g., black). Furthermore, Figure 18 It shows Figure 16 An enlarged view of region 4 on the bottom surface 2. Additionally, Figure 19 It shows Figure 18 The area shown, region 4, is a cross-sectional view of the plane containing the dashed line CD and which is approximately perpendicular to the plane formed by the microspace culture container 1. (See diagram below.) Figure 18 As shown, Figure 16The bottom surface 2 of the microspace culture container 1 has a plurality of recesses 6 arranged independently of each other. Here, the recesses formed on the bottom surface of the microspace culture container (and the pores of the microspace culture container) are sometimes referred to as micropores. In this invention, the shape of each recess 6 is not particularly limited, as long as the shape has a bottom surface; examples include those with a near-circular shape (e.g., a near-circular shape). Figure 18 The opening 7 (as shown in the diagram) is circular or has a polygonal shape (triangle, quadrilateral (e.g., rectangle, such as a square or rectangle, parallelogram or trapezoid), pentagon, hexagon, etc.). In this invention, "opening 7" means the portion formed by connecting the highest point of the sidewall 9 in a recess 6 (in... Figure 19 In this case, the opening is parallel to an approximately plane formed by the main body of the microspace culture container 1. In this invention, each recess 6 forms a microspace for culturing cells. In this invention, at least one of the plurality of recesses 6 (preferably more than 80%, more preferably more than 90%, particularly all) has an equivalent circle diameter "l" of preferably 200 μm to 900 μm, more preferably 400 μm to 700 μm, and even more preferably 450 μm to 550 μm. In this invention, the equivalent circle diameter "l" means the diameter of the inscribed circle of a planar figure formed by the point where the planes intersect the generally parallel plane formed by the microspace culture container 1 and the sidewall 9 (e.g., the equivalent circle diameter "l" in the case where the planar figure is a regular hexagon is as follows). Figure 20 (As shown). In this invention, when the "planar pattern formed by the points intersecting the plane that is approximately parallel to the plane formed by the microspace culture container 1 and the sidewall 9" changes with the depth of the recess, the equivalent circle diameter "l" refers to its maximum value.

[0082] Figure 19 It shows Figure 18 A cross-sectional view including the dashed line CD, perpendicular to the plane approximately formed by the microspace culture container 1. In this invention, in each recess 6, the bottom surface 8 may have, as... Figure 19 The shape of the curved surface shown (such as a round bottom) can also be a plane. Furthermore, the sidewall 9 can be as follows: Figure 19 The parallel or conical shape shown may be (e.g., a cone shape that is wider on the opening side and narrower on the bottom side). In this invention, at least one of the plurality of recesses 6 (preferably more than 80%, more preferably more than 90%, particularly all) has a depth “d” of preferably 200 μm to 1000 μm, more preferably 300 μm to 800 μm, and even more preferably 400 μm to 700 μm. In this invention, when the surface of the opening 7 forming the recess is not parallel to the surface forming its bottom surface 8 and / or when the bottom surface is curved, the “depth” of each recess 6 is the distance from the opening to the opening on the bottom surface of the recess.

[0083] In the present application, the volume of the space formed by each recess 6 (the space defined by the opening 7, the bottom surface 8, and the side wall 9) is not particularly limited, but is preferably 0.02 mm 3 to 0.55 mm 3 , more preferably 0.04 mm 3 to 0.35 mm 3 , still more preferably 0.07 mm 3 to 0.15 mm 3 . Preferably, at least 1 of the plurality of recesses 6 (preferably more than 80%, more preferably more than 90%, particularly all of the number of recesses 6) forms a space having the above-mentioned volume. In the present application, the recesses preferably have a size such that each recess accommodates one or two (preferably one) iPS cell constructs (such as, Figure 21 ).

[0084] The material used to form the micro-space culture vessel 1 is not particularly limited, and the vessel can be made of, for example, a resin such as an acrylic resin, a polystyrene-based resin, an acrylic / styrene-based resin, a polycarbonate-based resin, a melamine resin, polyglycolic acid, polylactic acid, a polyester-based resin, a polyimide, a polyvinyl alcohol-based resin, an ethylene / vinyl alcohol-based resin, a thermoplastic elastomer, a vinyl chloride-based resin, or a silicone resin or a combination thereof.

[0085] In order to perform culture while maintaining the state in which the cells or cell constructs are placed in the recesses, culture using the micro-space culture vessel 1 is generally performed by static culture. Therefore, in order to prevent the cells or cell constructs from adhering to the bottom surface 2 and / or the side wall 3, particularly the bottom surface 2, of the micro-space culture vessel 1, at least a portion thereof is preferably non-cell-adhesive. The method of making the wall surface non-cell-adhesive is not particularly limited, but examples thereof are conceivable methods involving the application of a non-cell-adhesive agent to form a hydrophilic phase on at least a portion of the wall surface. Examples of the non-cell-adhesive agent include agents each containing a hydrophilic polymer such as polyethylene glycol, a polymer having a betaine structure, a phospholipid-containing polymer, or a poly(hydroxyethyl) methacrylate polymer.

[0086] The present application is described above with reference to the drawings, in which typical embodiments are shown, but the present application is not limited to the culture vessel shown in the drawings. For example, the micro-space culture vessel 1 is not limited to Figure 16 the rectangular plate shown, but can be, for example, a circular plate.

[0087] (1) Induction of embryoid body formation

[0088] The method of the present application includes the step of inducing the formation of embryoid bodies by subjecting undifferentiated iPS cells to non-adherent culture.

[0089] As the iPS cells used as the starting material, iPS cells produced by introducing a nuclear reprogramming substance into a somatic cell can be used. Examples of the iPS cells include iPS cells derived from mammals such as humans, mice, rats, monkeys, dogs, pigs, cows, cats, goats, sheep, rabbits, guinea pigs, and hamsters. Among them, iPS cells derived from humans, mice, rats, monkeys, dogs, and the like are preferred, and iPS cells derived from humans or mice are more preferred.

[0090] The somatic cells that can be used as the starting material for producing iPS cells can be any cell other than germ cells, and examples thereof include oral mucosal cells (e.g., gingival fibroblasts, buccal mucosal fibroblasts, gingival epithelial cells, and buccal mucosal epithelial cells), keratinized epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., tongue epithelial cells), exocrine gland epithelial cells (e.g., mammary cells), hormone-secreting cells (e.g., adrenal medulla cells), metabolic and storage cells (e.g., liver cells), luminal epithelial cells constituting an interface (e.g., type I alveolar cells), luminal epithelial cells in the closed circulatory system (e.g., vascular epithelial cells), cells having motile cilia (e.g., airway epithelial cells), extracellular matrix-secreting cells (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), hematopoietic and immune cells (e.g., T lymphocytes), sensory cells (e.g., rod cells), autonomic neurons (e.g., cholinergic neurons), supporting cells of peripheral neurons of sensory organs (e.g., satellite cells), neural cells and glial cells in the central nervous system (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and precursor cells thereof (e.g., tissue precursor cells). The differentiation level of the cells is not particularly limited, and both undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be similarly used as the source of somatic cells in the present application. In the present context, examples of the undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0091] In the present application, the "nuclear reprogramming substance" can include any substance (a group of substances) capable of inducing iPS cells from somatic cells, such as a protein factor or a nucleic acid encoding the protein factor (including a form introduced into a vector), or a low-molecular-weight compound. When the nuclear reprogramming substance is a protein factor or a nucleic acid encoding the protein factor, preferred examples thereof include the following combinations (only the names of the protein factors are given in the following description)

[0092] [1] Oct3 / 4, Klf4, and c-Myc

[0093] [2] Oct3 / 4, Klf4, c-Myc, and Sox2 (here, Sox2 can be replaced by Sox1, Sox3, Sox15, Sox17, or Sox18. In addition, Klf4 can be replaced by Klf1, Klf2, or Klf5. Further, c-Myc can be replaced by T58A (active mutant), N-Myc, or L-Myc)

[0094] [3] Oct3 / 4, Klf4, c-Myc, Sox2, Fbx15, Nanog, Eras, ECAT15-2, Tcf1, and β-catenin (active mutant S33Y)

[0095] [4] Oct3 / 4, Klf4, c-Myc, Sox2, TERT, and SV40 large T antigen (hereinafter referred to as SV40LT)

[0096] [5] Oct3 / 4, Klf4, c-Myc, Sox2, TERT, and HPV16 E6

[0097] [6] Oct3 / 4, Klf4, c-Myc, Sox2, TERT, and HPV16 E7

[0098] [7] Oct3 / 4, Klf4, c-Myc, Sox2, TERT, HPV6 E6, and HPV16 E7

[0099] [8] Oct3 / 4, Klf4, c-Myc, Sox2, TERT, and Bmil (for the combinations described above, see WO 2007 / 069666 Al (provided that in the above combination [2], Sox18 is used instead of Sox2 or Klf1 or Klf5 is used instead of Klf4, see Nature Biotechnology, 26, 101-106 (2008)). The combination of "Oct3 / 4, Klf2 (or Klf5), c-Myc, and Sox2" also see Nat. Cell Biol., 11, 197-203 (2009). The combination of "Oct3 / 4, Klf4, c-Myc, Sox2, hTERT, and SV40LT" also see Nature, 451, 141-146 (2008))

[0100] [9] Oct3 / 4, Klf4, and Sox2 (see Nature Biotechnology, 26, 101-106 (2008))

[0101]

[10] Oct3 / 4, Sox2, Nanog, and Lin28 (see Science, 318, 1917-1920 (2007))

[0102]

[11] Oct3 / 4, Sox2, Nanog, Lin28, hTERT and SV40LT (see Stem Cells, 26, 1998-2005 (2008))

[0103]

[12] Oct3 / 4, Klf4, c-Myc, Sox2, Nanog and Lin28 (see Cell Research (2008) 600-603)

[0104]

[13] Oct3 / 4, Klf4, c-Myc, Sox2 and SV40LT (see also Stem Cells, 26, 1998-2005 (2008))

[0105]

[14] Oct3 / 4 and Klf4 (see Nature 454:646-650 (2008), Cell Stem Cell, 2:525-528 (2008))

[0106]

[15] Oct3 / 4 and c-Myc (see Nature 454:646-650 (2008))

[0107]

[16] Oct3 / 4 and Sox2 (see Nature, 451, 141-146 (2008), WO 2008 / 118820 A2)

[0108]

[17] Oct3 / 4, Sox2 and Nanog (see WO 2008 / 118820 A2)

[0109]

[18] Oct3 / 4, Sox2 and Lin28 (see WO 2008 / 118820 A2)

[0110]

[19] Oct3 / 4, Sox2, c-Myc and Esrrb (herein, Essrrb can be replaced by Esrrg. See Nat. Cell Biol., 11, 197-203 (2009))

[0111]

[20] Oct3 / 4, Sox2 and Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))

[0112]

[21] Oct3 / 4, Klf4 and L-Myc

[0113]

[22] Oct3 / 4 and Nanog

[0114]

[23] Oct3 / 4

[0115]

[24] Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28, and SV40LT (see Science, 324: 797-801 (2009))

[0116]

[25] Oct3 / 4, Klf4, Sox2, and a member of the GLIS family (herein given, for example, GLIS1, GLIS2, and GLIS3, and suitably given GLIS family zinc finger 1 (GLIS1). See WO 2010 / 098419 Al and WO 2011 / 102531 A2)

[0117]

[26] Oct3 / 4, Klf4, Sox2, and a member of the IRX family (herein given, for example, IRX1, IRX2, IRX3, IRX4, IRX5, and IRX6, and suitably given Iroquois homeobox protein 6 (IRX6). See WO 2010 / 098419 Al)

[0118]

[27] Oct3 / 4, Klf4, Sox2, and a member of the PTX family (herein given, for example, PITX1, PITX2, and PITX3, and suitably given paired-like homeodomain transcription factor 2 (PITX2). PITX2 is known to have three isoforms (isoform a, b, and c) and any of these isoforms can be used, with isoform b being particularly preferred. See WO 2010 / 098419 Al)

[0119]

[28] Oct3 / 4, Klf4, Sox2, and a member of the DMRT-like family B with proline-rich C-terminus 1 (DMRTB1, see WO 2010 / 098419 Al)

[0120] In the above combinations [1] -

[28] , any other Oct family member (such as Oct1A or Oct6) can be used in place of Oct3 / 4. In addition, any other Sox family member (such as Sox7) can be used in place of Sox2 (or Sox1, Sox3, Sox15, Sox17, or Sox18). In addition, any other Lin family member (such as Lin28b) can be used in place of Lin28.

[0121] In addition, a combination which is not completely identical to any one of the above-described combinations [1] to

[28] but contains all the components in any one of the above-described combinations [1] to

[28] and further contains any other substance can also be included in the category of "nuclear reprogramming substance" of the present application. In addition, a combination of only components other than the above-described components can also be included in the category of "nuclear reprogramming substance" of the present application, under the condition that some of the components in any one of the above-described combinations [1] to

[28] are endogenously expressed at a level sufficient for nuclear reprogramming within the somatic cell subjected to nuclear reprogramming.

[0122] Among these combinations, a preferred example of the nuclear reprogramming substance is at least one, preferably at least two or more, more preferably at least three or more, selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin28, and SV40LT.

[0123] The human cDNA sequence information of the above-described nuclear reprogramming substance can be obtained with reference to the NCBI accession number described in WO 2007 / 069666 Al or WO 2010 / 098419 Al (Nanog is described in these publications under the name "ECAT4"). The human cDNA sequence information of Lin28, Lin28b, Esrrb, Esrrg, and L-Myc can be obtained with reference to the corresponding NCBI accession number shown in Table 1 below). The skilled person can easily isolate the cDNA thereof.

[0124] Table 1

[0125] Gene name NCBI Accession No. Lin28 NM_024674 Lin28b NM_001004317 Esrrb NM_004452 Esrrg NM_001438 L-Myc NM_001033081

[0126] In addition, the human cDNA sequence information of GLIS family members, IRX family members, PTX family members, and DMRTB1 can be obtained with reference to the corresponding NCBI accession number shown in Table 2 below.

[0127] Table 2

[0128]

[0129]

[0130] In addition, a natural or artificial mutant protein having 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more identity with any of the above-described amino acid sequences and having a nuclear reprogramming ability equivalent to that of the wild-type protein as a substitute for Klf4, and a nucleic acid encoding the mutant protein can also be used as the nuclear reprogramming substance of the present application in place of Klf4.

[0131] When the protein factor itself is used as the nuclear reprogramming agent, the protein factor can be prepared by inserting the obtained cDNA into a suitable expression vector, introducing the expression vector into a host cell, culturing the cell, and collecting the recombinant protein factor from the resulting culture. Meanwhile, when a nucleic acid encoding the protein factor is used as the nuclear reprogramming agent, an expression vector is constructed by inserting the obtained cDNA into a viral vector, a plasmid vector, an episomal vector, or the like, and subjected to the nuclear reprogramming step.

[0132] For introduction of the nuclear reprogramming agent into the somatic cell, a method used in the technical field to which the present application pertains, such as the method of Patent Literature 1 (WO 2015 / 64705 Al), can be appropriately used.

[0133] Further, iPS cells can be obtained by using factors such as C4ORF51, HHLA1, ABHD12B, or ZNF541 as an index of differentiation resistance, and selecting a cell line in which the factor is not significantly expressed (see WO 2013 / 014929 Al).

[0134] Further, the iPS cells used can be obtained by, for example, culture with feeder cells that provide soluble factors required for survival, proliferation, and undifferentiated maintenance of cells and serve as a scaffold for cell adhesion, and the feeder cells are appropriately removed before induction of embryoid body formation. In another embodiment of the present application, iPS cell culture without feeder cells is superior to the above-described method involving the use of feeder cells from the viewpoint of improving the efficiency of production of osteoblast constructs (a larger number of osteoblast constructs can be obtained).

[0135] This step can be performed by subjecting those undifferentiated iPS cells to non-adherent culture in a liquid medium for induction of embryoid body formation using the above-described micro-space culture vessel.

[0136] For the medium, a medium for culturing primate ES / iPS cells can be appropriately used. In the present application, a medium for culturing ES cells is also sometimes simply referred to as an ES medium. Examples of the medium for culturing primate ES / iPS cells include: RCHEMD001 produced by ReproCELL Inc.; NutriStem produced by Biological Industries; Essential 6 Medium produced by ThemoFisher Scientific; StemFit AK02N Medium produced by Ajinomoto Co., Inc.; and StemFlex Medium produced by ThemoFisher Scientific. Growth factors such as fibroblast growth factor (FGF) can be incorporated in such media. These growth factors can be used alone or in combination. Furthermore, an additive useful for cell culture of stem cells and the like can be optionally mixed in the medium. Specific examples of such additives include fetal bovine serum, amino acids such as L-glutamine, a ROCK inhibitor such as Y-27632, and antibiotics such as penicillin, streptomycin, and amphotericin B. In the present application, it is preferable to use a ROCK inhibitor and the like as an additive. By this step, the undifferentiated iPS cells are first grown into embryoid bodies, rather than being directly cultured in mesoderm cell-inducing medium as in Non-Patent Literature 1, with the result that cell death in the next step of inducing mesoderm cells is inhibited. Therefore, in the method of the present application, the purpose of the embryoid body formation step as step (1) is to substantially not cause mesoderm cell induction, and to not go beyond the formation of embryoid bodies. Therefore, a medium not containing a mesoderm cell-inducing agent is generally used as the medium used in step (1).

[0137] In a typical embodiment of the present application, this step is performed by placing and keeping a suspension of undifferentiated iPS cells in the above-mentioned medium in the recess of the culture vessel. There is no limitation on the number of undifferentiated iPS cells in suspension when they are placed in the recess of the culture vessel, but from the viewpoint of improving the production efficiency of the osteoblast construct, for example, the cell concentration is preferably 0.5 x 10 5 cells / ml to 7.5 x 10 5 cells / ml, more preferably 1.5 x 10 5 cells / ml to 3.5 x 10 5 cells / ml. The preferred cell concentration is generally shown as the value when 2 ml of cell suspension is added per well in the experimental examples. In addition, there is no limitation on the number of undifferentiated iPS cells added per well when they are placed in the recess of the culture vessel, but from the viewpoint of improving the production efficiency of the osteoblast construct, for example, it is preferably 1 x 10 5cells to 15 x 10 5 cells, more preferably 3 x 10 5 cells to 7 x 10 5 cells. The number of the above-mentioned preferred undifferentiated iPS cells is generally shown as a value in the case of using a 24-well microspatial low attachment plate [Elplasia (trademark) (Corning, catalog number 4441: having a micro-well size of 500 μm in diameter and 400 μm in depth, and having 554 to 580 depressions / well] size plate used in Experimental Example 3. Further, the number of undifferentiated iPS cells placed in each depression of the culture vessel when the undifferentiated iPS cells are placed in the depressions of the culture vessel is not limited, but from the viewpoint of improving the production efficiency of the osteogenic cell construct, for example, 100 cells to 3000 cells, more preferably 500 cells to 1200 cells are preferred. The culture time of this step is, for example, preferably about 0.5 days to about 3.5 days, more preferably 0.625 days to 2.5 days, even more preferably 0.875 days to 1.25 days. Further, the culture time of this step is, for example, preferably about 12 hours to about 84 hours, more preferably about 15 hours to about 60 hours, even more preferably about 21 hours to about 30 hours. It is preferred that the culture time in this step be set within the above-mentioned range, because the final osteogenic cell construct can be obtained in a non-bag-like solid shape. The culture temperature of this step is not particularly limited, and is, for example, preferably 30°C to 42°C, more preferably 35°C to 39°C. The culture in this step is preferably performed under an atmosphere of 3% to 10% CO2. In the present application, since the culture in step (1) uses a microspatial culture vessel, this step is generally performed by static culture in order to keep the cells or cell constructs in a state in the wells of the microspatial culture vessel.

[0138] (2) Induction of mesoderm differentiation

[0139] The method of the present application includes inducing differentiation of the iPS cells into mesoderm cells by performing non-adherent culture of the embryoid bodies of the iPS cells obtained in the above-mentioned step (1).

[0140] As the medium used in the present step, a medium suitable for induction of differentiation into mesoderm cells can be appropriately used. Examples of such a medium include: DMEM medium produced by Nacalai Tesque Inc.; DMEM / F12 medium produced by ThermoFisher Scientific; Neurobasal medium produced by Thermo Fisher Scientific; RPMI 1640 medium produced by Thermo Fisher Scientific; and Stemline (trademark) II hematopoietic stem cell growth medium produced by Sigma-Aldrich Corporation. These media can be used alone or in combination.

[0141] From the viewpoint of promoting induction of differentiation into mesoderm, the medium used in the present step is preferably admixed with a Wnt signal activator. The Wnt signal activator is not particularly limited, but examples thereof include CHIR99021, 6-bromoindirubin-3'-oxime (BIO), Kenpaullone, SB-216763, SKL2001, deoxycholic acid, WAY-316606, NSC-693868, ricinine, 7-oxo-β-sitosterol, IM-12, HLY78, and retinoic acid (such as all-trans-retinoic acid (manufactured by FUJIFILM Wako Pure Chemical Corporation). Among these Wnt signal activators, CHIR99021 or the like is preferred. These Wnt signal activators can be used alone or in combination. In a preferred embodiment of the present application, retinoic acid is preferably used in combination with a Wnt signal activator other than retinoic acid. When retinoic acid is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in the present step, it is, for example, preferably 0.01 μM to 10 μM, more preferably 0.1 μM to 5 μM.

[0142] When a Wnt signal activator is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in the present step, it is, for example, preferably 1 μM to 100 μM, more preferably 10 μM to 50 μM.

[0143] From the viewpoint of promoting induction of differentiation into mesoderm, it is preferable that the medium used in the present step is admixed with a hedgehog signal inhibitor. The hedgehog signal inhibitor is not particularly limited, and examples thereof include cyclamates, AY9944, GANT58, GANT61, jervine, SANT-1, SANT-2, U18666A, veratramine, vismodegib, Cur-61414, Robotnikinin, JK184, and HPI-4. Among these hedgehog signal inhibitors, cyclamates and the like are preferable. These hedgehog signal inhibitors can be used alone or in combination.

[0144] When the hedgehog signal inhibitor is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in the present step, it is, for example, preferably 1 μM to 100 μM, more preferably 1 μM to 10 μM.

[0145] In the present application, it is preferable that a Wnt signal activator and a hedgehog signal inhibitor are used simultaneously.

[0146] As described above in the "Solution to the Problem", the present application was accomplished by further improvement on the basis of the following new findings in order to solve the problems involved in the findings: the cell construct obtained by the method described in Patent Literature 1, which involves culturing in ES medium for 2 days, further adding retinoic acid and culturing for 2 days, and culturing in an osteoblast differentiation induction medium, has a hollow bag-like shape. Therefore, the method of performing the culturing step using a medium admixed with only retinoic acid as a component capable of inducing differentiation into mesoderm after the step of inducing formation of embryoid bodies was excluded from the method of the present application.

[0147] In addition, the medium can be optionally admixed therein with an additive useful for cell culture. Specific examples of such an additive include fetal bovine serum, amino acids (e.g., L-glutamine), antibiotics (e.g., penicillin, streptomycin, amphotericin B).

[0148] Further, the medium can be supplemented with, for example, commercially available supplements for cell culture (used as additives for cell culture). Examples of such supplements include: N-2 supplement produced by Thermo Fisher Scientific; B-27 supplement produced by Thermo Fisher Scientific; insulin, transferrin, selenium solution produced by Thermo Fisher Scientific; Wnt 3a produced by R&D Systems; Activin A produced by R&D Systems; and BMP4 produced by PeproTech. These supplements can be used alone or in combination. When the step of inducing differentiation into mesoderm cells is implemented between (1) the above-described step of inducing blastema formation and (3) the step of inducing differentiation into osteoblasts described below, a solid cell construct can be obtained, particularly in the case where human iPS cells are used as a starting material.

[0149] The method of the present application is a method of producing a cell construct of osteoblasts. Therefore, the mesoderm cell differentiation induction step is also performed by non-adherent culture. In the present application, the culture of step (2) uses a micro-space culture vessel, and therefore this step is generally performed by static culture in order to keep the cells or cell construct in a state in the well of the micro-space culture vessel.

[0150] In a typical embodiment of the present application, this step is performed by placing and keeping the above-described medium containing the blastema obtained in step (1) in the recess of the culture vessel. The culture time of this step is, for example, preferably 0.125 days to 10 days, more preferably 1 day to 8 days, and even more preferably 3 days to 6 days. Further, the culture time of this step is, for example, preferably about 3 hours to about 240 hours, more preferably about 24 hours to about 192 hours, and even more preferably about 72 hours to about 144 hours. The culture temperature in this step is not particularly limited, and is, for example, preferably 30°C to 42°C, and more preferably 35°C to 39°C. The culture in this step is preferably performed under an atmosphere of 3% to 10% CO2.

[0151] (3) Induction of osteoblast differentiation

[0152] The method of the present application includes inducing differentiation into osteoblasts by performing non-adherent culture of the mesoderm cells of the iPS cells obtained in step (2).

[0153] As the medium used in this step, a medium suitable for induction of differentiation into osteoblasts can be appropriately used. Examples of such a medium include: a DMEM medium (such as a sodium pyruvate-free DMEM medium produced by Nacalai Tesque); and an aMEM medium (such as an aMEM medium produced by Nacalai Tesque). These media can be used alone or in combination.

[0154] In this step, a medium can be doped with an osteoblast differentiation induction promoter or the like. Examples of the osteoblast differentiation induction promoter include ascorbic acid, β-glycerophosphate, dexamethasone, BMP-2, hydrocortisone hemisuccinate, and retinoic acid (such as all-trans-retinoic acid). The ascorbic acid can be ascorbic acid-2-phosphoric acid or a salt thereof. Furthermore, as the differentiation inducer, hydrocortisone succinate can be used instead of or in combination with dexamethasone. These osteoblast differentiation induction promoters can be used alone or in combination.

[0155] When ascorbic acid is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in this step, it is, for example, preferably 50 μM to 300 μM, more preferably 150 μM to 200 μM. When β-glycerophosphate is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in this step, it is, for example, preferably 1 mM to 100 mM, more preferably 5 mM to 15 mM. When dexamethasone is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in this step, it is, for example, preferably 0.001 μM to 10 μM, more preferably 0.01 μM to 1.0 μM. When retinoic acid is incorporated, the amount of incorporation thereof is not particularly limited, but in terms of the final concentration in the medium used in this step, it is, for example, preferably 0.01 μM to 10 μM, more preferably 0.1 μM to 5 μM.

[0156] From the viewpoint of mineralization of osteoblasts, it is preferable that the medium in this step is further doped with a hypoxia mimetic compound. Examples of the hypoxia mimetic compound include deferoxamine (DFX) and cobalt chloride (CoCl2). These hypoxia mimetic compounds can be used alone or in combination.

[0157] From the viewpoint of mineralization of osteoblasts, it is preferable that the medium in this step is further doped with a statin compound. Examples of the statin compound include atorvastatin, fluvastatin, simvastatin, lovastatin, pitavastatin, pravastatin, and rosuvastatin. These statin compounds can be used alone or in combination.

[0158] Further, the medium can optionally be mixed therein with an additive useful for cell culture. Specific examples of such an additive include fetal bovine serum, amino acids (e.g., L-glutamine), antibiotics (e.g., penicillin, streptomycin, amphotericin B).

[0159] The method of the present application is a method of producing a cell construct of osteoblasts. Therefore, the mesoderm cell differentiation induction step is also performed by non-adherent culture. Specific modes of non-adherent culture include those described above. In this step (induction of differentiation into osteoblasts), from the perspective of promoting differentiation by mechanical stimulation, oscillation culture or the like is preferred.

[0160] In a typical embodiment of the present application, this step is performed by placing the suspension of mesoderm cells of iPS cells obtained in step (2) in the above-described medium in a culture vessel (e.g., 25 cm 2 This step is performed by placing the suspension of mesoderm cells of iPS cells obtained in step (2) in the above-described medium in a culture vessel (e.g., 25 cm 2The step (3) is preferably performed in a low-attachment bottle, and the medium is changed to 10 ml of the above-mentioned osteoblast differentiation-inducing medium. The culture time of this step is, for example, preferably from about 1 day to about 90 days, more preferably from 7 days to 60 days, even more preferably from 21 days to 50 days. Further, the culture time of this step is, for example, preferably from about 24 hours to about 2160 hours, more preferably from about 168 hours to about 1440 hours, even more preferably from about 504 hours to about 1200 hours. The culture temperature of this step is not particularly limited, and is, for example, preferably from 30°C to 42°C, more preferably from 35°C to 39°C. The culture in this step is preferably performed under an atmosphere of 3% to 10% CO2.

[0161] According to the present application, due to the above-described structural features, an osteoblast construct having a high degree of mineralization can be obtained. Further, as described above, according to the present application, an osteoblast construct having a high bone regenerative capacity can be obtained. Therefore, for example, when the osteoblast construct obtained in the present application is used as a raw material, is subjected to an inactivation treatment as appropriate, and is then implanted into a bone defect site, new bone can be formed to fill such a defect site (void), and thus the present application is extremely useful. Therefore, the present application also provides methods such as a method of promoting mineralization of iPS cell-derived osteoblasts (or cell constructs), a method of enhancing the bone inductive capacity of iPS cell-derived osteoblasts (or cell constructs), and a method of applying a stimulus for inducing self-assembly of osteoblasts (spatial environment around the cells) to iPS cells in culture. The culture vessel, materials, and various conditions in each of these methods are similar to those described above.

[0162] Osteoblast constructs

[0163] When the method of the present application is implemented using human iPS cells as a raw material, an osteoblast construct having a diameter of 0.5 mm to 5 mm can be obtained. Such an osteoblast construct derived from human iPS cells and having such a diameter has not been reported previously, and thus it is a novel osteoblast construct. Further, as described above, an osteoblast construct having such a diameter has a high bone regenerative capacity, and thus is extremely useful. The diameter of the osteoblast construct is preferably in the range of 0.5 mm to 5 mm, more preferably in the range of 1 mm to 4 mm, even more preferably in the range of 1 mm to 3 mm.

[0164] Method for producing bone regenerative agents

[0165] The present application also provides a method of producing a bone regenerative agent, the method comprising the steps of: producing an osteoblast construct from iPS cells by the above-described method; and subjecting the osteoblast construct to an inactivation treatment.

[0166] The iPS cells used as a raw material in the production step of the osteoblast construct, the treatment required in the production step of the osteoblast construct, and the like are as described above.

[0167] The method of the present application includes a step of subjecting the osteoblast construct obtained by the above step to inactivation treatment.

[0168] The method for inactivation is not particularly limited, but examples thereof include freeze-drying, heat treatment, high-pressure treatment, acid or alkali solution treatment, high-pressure steam sterilization, radiation sterilization, gas sterilization, and electromagnetic wave treatment. The conditions for freeze-drying are not particularly limited, and known methods can be used. Furthermore, for example, preliminary freezing can be performed before freeze-drying. The temperature for preliminary freezing is not particularly limited, but is preferably, for example, about -20°C to about -12°C. The temperature for freeze-drying is not particularly limited, but is preferably, for example, about -100°C to about -5°C. In addition, the pressure for freeze-drying is not particularly limited, but is preferably, for example, 600 Pa or less, more preferably 50 Pa or less. As a specific freeze-drying condition, there is provided, for example, a method involving gradually reducing the pressure from 5 Pa to 20 Pa at a fixed temperature of -10°C, starting freeze-drying at the same time as the start of pressure reduction. According to the present application, a bone regenerative agent having a high degree of mineralization and a high bone regenerative ability can be obtained. In one embodiment, the present application also provides a bone regenerative agent obtained by the above production method.

[0169] The present application will now be described more specifically by way of examples and comparative examples. However, the present application is not limited thereto.

[0170] Example

[0171] Experiment 1: Screening of optimal microspatial size for inducing iPS cells into osteoblast constructs

[0172] <Method>

[0173] 1.1. Culture of mouse iPS cells

[0174] For this experiment, an iPS cell line established from mouse gingival fibroblasts [PLoS ONE, 5(9): e12743, 2010] was used. The cells were cultured by using an ES medium [containing 15% fetal bovine serum (Gibco / Life Technologies, Grand Island, NY, USA), 2 mM L-glutamine (Wako Pure Chemical, Osaka), 1 x 10 -4 M non-essential amino acids (Life Technologies, Grand Island, NY, USA), 1 x 10 -4M 2-mercaptoethanol (Life Technologies, Grand Island, NY, USA), 50 U of penicillin and 50 μg / ml of streptomycin (Wako Pure Chemical) in Dulbecco's modified Eagle's medium (DMEM: containing 4.5 g / L of glucose and no sodium pyruvate; Nacalai Tesque, Kyoto) were maintained and cultured as iPS cell constructs on SNL P76.7-4 feeder cells (treated with mitomycin C).

[0175] 1.2. Generation of osteoblast constructs of mouse iPS cells using microspatially shaped low attachment plates

[0176] The above cultured mouse iPS cell constructs were dissociated into single cells by treatment with 0.25% trypsin (Wako Pure Chemical) containing 1 mM EDTA, and used for the present experiment as a cell suspension (3.9 x 10 6 cells / ml) adjusted with ES medium. The method for generating osteoblast cell constructs from mouse iPS cells in the present experiment is shown in Figure 1 .

[0177] As a culture vessel, a micro space low attachment plate Elplasia (trademark) (Kuraray, Japan) having a micro space (micro well) of a different concave size on the bottom surface of each well of a 6-well plate was used: Figure 1

[0178] Elp400; Cat. # RB 400 560NA6: having a diameter of 400 μm and a depth of 560 μm, Elp500; Cat. # RB 500 700NA6: having a diameter of 500 μm and a depth of 700 μm, or Elp900; Cat. # RB 900 700NA6: having a diameter of 900 μm and a depth of 700 μm. 2 ml of the above cell suspension was inoculated per well, and cultured for 2 days to induce embryoid body formation. Figure 2

[0179] After 2 days of culture, the medium in the well was replaced with ES medium containing 1 μM of all-trans retinoic acid (RA; Wako Pure Chemical), and further cultured for 3 days.

[0180] ​​After that, the medium was changed to an osteoblast differentiation induction medium [an α-MEM medium (Nacalai Tesque) containing 15% FBS (Gibco / Life Technologies), 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich), and 50 μg / ml ascorbic acid-2-phosphate (Sigma-Aldrich), 1% antibiotic-antimycotic (100 units / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B (Gibco / Life Technologies)], and cultured for 35 days. The medium was changed every 2 days. The time point at which the culture in the osteoblast differentiation induction medium was started was defined as "day 0 of osteoblast differentiation induction".

[0181] <Results>

[0182] 1.3. Effect of microspatial diameter on the size of osteoblast constructs

[0183] The size (Feret's diameter) of the cell constructs after 5 days of culture from the time when the iPS cell suspension was added to each microspace size well (day 0 of osteoblast differentiation induction) was calculated using the Image J image analysis software. As a result, the average Feret's diameter of the samples of the Elp400 group, the Elp500 group, and the Elp900 group cultured in the respective microspaces was about 187.6, about 242.5, and about 328.8 μm, respectively, and the size of the Elp500 group and the Elp900 group was significantly larger than that of the Elp400 group. The size of the cell constructs of the Elp400 group and the Elp500 group gradually increased over the 35 days after transfer to the osteoblast differentiation induction medium. Figure 3 : left).

[0184] The cell constructs of each group in osteoblast differentiation induction were observed under a phase-contrast microscope, and as a result, during the period from day 21 to day 28 of induction, it was observed that the cell constructs of the Elp400 group and the Elp900 group collapsed, and their contours gradually became less smooth. Meanwhile, the cell constructs of the Elp500 group existed as cell constructs until day 35 of induction before collapsing Figure 3 : right).

[0185] Cell survival and death in each cell construct were studied using a live / dead cell simultaneous staining kit (LIVE / DEAD (trademark) Viability / CytotoxicityKit, Molecular Probes / Thermo Fisher Scientific, Eugene, OR, USA). Results showed that in the Elp400 and Elp900 groups 14 days after osteoblast differentiation induction, a large number of red cells representing dead cells were found. In particular, the central portion of the Elp900 group cell construct was red, indicating that most of the cells inside the cell construct had died. Meanwhile, from the start of induction to 14 days after induction, the Elp500 group cell construct consisted of green cells representing live cells, with almost no red dead cells observed. Figure 4 ).

[0186] 1.4. Effect of microspatial diameter on osteoblast differentiation of cell constructs

[0187] The expression of osteoblast-specific marker genes (Runx2, Osterix, collagen 1a1, osteosialin, osteopontin, and osteocalcin) in cell constructs 10 days after osteoblast differentiation induction was analyzed using the SYBR Green real-time RT-PCR method (Thunderbird SYBR qPCR Mix, TOYOBO). The primer sequences used in the SYBR Green real-time RT-PCR method are shown below. 18S rRNA was used as an internal control.

[0188] Runx2 forward primer: 5'-CGGGCTACCTGCCATCAC-3'

[0189] Runx2 reverse primer: 5'-GGCCAGAGGCAGAAGTCAGA-3'

[0190] Osterix forward primer: 5'-CTCGTCTGACTGCCTGCCTAG-3'

[0191] Osterix reverse primer: 5'-GCGTGGATGCCTGCCTTGTA-3'

[0192] Collagen 1a1 forward primer: 5'-TGTCCCAACCCCCAAAGAC-3'

[0193] Collagen 1a1 reverse primer: 5'-CCCTCGACTCCTACATCTTCTGA-3'

[0194] Osteocalcin forward primer: 5'-CCGGGAGCAGTGTGAGCTTA-3'

[0195] Osteocalcin reverse primer: 5'-CCGGGAGCAGTGTGAGCTTA-3'

[0196] Osteopontin forward primer: 5'-TCTCCTTGCGCCACAGAATG-3'

[0197] Osteopontin reverse primer: 5'-TCCTTAGACTCACCGCTCTT-3'

[0198] Osteosialoprotein forward primer: 5'-CGGAGGAGACAACGGAGAAG-3'

[0199] Osteosialoprotein reverse primer: 5'-GTAAGTGTCGCCACGAGGCT-3'

[0200] 18s rRNA forward primer: 5'-GTAACCCGTTGAACCCCATT-3'

[0201] 18s rRNA reverse primer: 5'-CCATCCAATCGGTAGTAGCG-3'

[0202] As a result, the expression of all these genes in the Elp500 group was significantly higher than those in the Elp400 group and the Elp900 group. Figure 5 ).

[0203] In addition, the cell constructs of each group were sectioned after 35 days of osteoblast differentiation induction, and HE staining or von Kossa and methylene blue double staining were performed for histological observation.

[0204] For the results of HE staining, the following images were observed: most of the cells in the Elp400 group and the Elp900 group lost their nuclei and died, and the cell constructs collapsed. At the same time, it was observed from the images that in the cell constructs of the Elp500 group, layered cell structures were found, and osteoblasts formed bone-like tissue on the outer layer. For the results of von Kossa and methylene blue double staining, in the Elp400 group and the Elp900 group, mineralization stained black was observed on the entire cell construct, but showed a brittle and collapsed image, and no cell components were observed, suggesting the possibility of mineralization accompanied by cell death. At the same time, in the Elp500 group, abundant extracellular matrix was found in the inner layer of the cell structure accompanied by mineralization, and bone-like tissue formation accompanied by partial mineralization was also found in the outer layer Figure 6 ).

[0205] 1.5. Conclusion of Experiment 1

[0206] The above results reflect that the microspatial low-attachment plate Elplasia (trademark) with recesses each having a diameter of 500 μm (Elp500) can effectively produce three-dimensional osteoblast constructs from mouse iPS cells. In addition, when Elp400 or Elp900 having recesses each having a diameter of 400 μm or 900 μm is used, the results indicate that it is difficult to produce osteoblast constructs containing living cells from mouse iPS cells.

[0207] Experiment 2: Effect of concave microspaces (Elp500) on osteoblast construct induction of human iPS cells <Method>

[0208] 2.1. Culture of human iPS cells

[0209] In the experiment, a human skin fibroblast-derived iPS cell line (409B2: RIKEN BRC CELL BANK) was used. SNLP76.7-4 cells (provided by Dr. Allan Bradley of the Sanger Institute, UK) were used as feeder cells.

[0210] The SNLP76.7-4 feeder cells were seeded in a 10 cm cell culture plate (coated with 0.1% gelatin) and cultured using DMEM medium containing 7% fetal bovine serum (FBS: Japan Bio Serum, Lot #JBS-011501), 2 mM L-glutamine (Thermo Fisher Scientific), 50 U of penicillin, and 50 μg / ml of streptomycin (Thermo Fisher Scientific) (without sodium pyruvate: Nacalai Tesque). The medium was replaced every 2 days. Before iPS cell culture, the SNLP76.7-4 feeder cells were treated with 12 μg / ml of mitomycin C (Nacalai Tesque) for 2.5 hours, and then seeded in a 10 cm cell culture plate (coated with 0.1% gelatin) at a concentration of 1.5 x 105cells / dish.

[0211] 1.5 x 105 6 cells / dish.

[0212] The iPS cells were seeded on the SNLP76.7-4 feeder cells and cultured using primate ES medium (ES medium: REPROCELL) containing 4 ng / ml of human basic FGF (REPROCELL). The medium was replaced every day.

[0213] The method of generating osteoblast constructs from human iPS cells in this experiment is shown in Figure 7 .

[0214] 2.2. Formation of embryoid bodies

[0215] The iPS cells were washed with phosphate buffered saline (PBS) and treated with 1 ml of CTK solution (0.25% trypsin, 0.1 mg / ml collagenase IV, 10 mM CaCl2, 20% KSR) at 37°C for 1 minute. Thereafter, the CTK solution was removed by aspiration, and 1 ml of PBS was added. The PBS was removed, and the feeder cells peeled off from the culture plate were removed as much as possible only by aspiration. Thereafter, 4 ml of ES medium was used to collect the attached iPS cells remaining in the culture plate. 2 ml of the cell suspension (number of iPS cells in the suspension: 6.25 x 105cells / ml) was transferred to one well of a microspatial low-attachment plate Elplasia (trademark) (Elp500: Kuraray, Cat. # RB 500 700NA24) having a recess of 500 μm in diameter on the bottom surface of each well of a 24-well plate (554 to 580 recesses per well) (number of iPS cells placed per well: 12.5 x 105cells), and cultured for 1 day to induce the formation of embryoid bodies. 5 The cells were washed with phosphate buffered saline (PBS) and treated with 1 ml of CTK solution (0.25% trypsin, 0.1 mg / ml collagenase IV, 10 mM CaCl2, 20% KSR) at 37°C for 1 minute. Thereafter, the CTK solution was removed by aspiration, and 1 ml of PBS was added. The PBS was removed, and the feeder cells peeled off from the culture plate were removed as much as possible only by aspiration. Thereafter, 4 ml of ES medium was used to collect the attached iPS cells remaining in the culture plate. 2 ml of the cell suspension (number of iPS cells in the suspension: 6.25 x 105cells / ml) was transferred to one well of a microspatial low-attachment plate Elplasia (trademark) (Elp500: Kuraray, Cat. # RB 500 700NA24) having a recess of 500 μm in diameter on the bottom surface of each well of a 24-well plate (554 to 580 recesses per well) (number of iPS cells placed per well: 12.5 x 105cells), and cultured for 1 day to induce the formation of embryoid bodies. 5 As a control, a group in which both the embryoid body culture and the mesoderm induction were performed using a low-attachment culture dish (Nunc untreated multi-dish, Thermo Fisher Scientific) instead of Elp500 was set.

[0216] 2.3. Induction of mesoderm differentiation

[0217] After the formation of embryoid bodies for 1 day, the ES medium was replaced with 2 ml of mesoderm differentiation induction medium [1:1 mixed medium of DMEM / F12 (Thermo Fisher Scientific) and Neurobasal medium (Thermo Fisher Scientific) containing 2% B-27 supplement (Thermo Fisher Scientific), 1% N-2 supplement (Thermo Fisher Scientific), 30 μM CHIR99021 (Wako Pure Chemical Industries), and 5 μM cyclopamine (Enzo Life science)], and cultured for 5 days. The medium was replaced every 2 days.

[0218] 2.4. Induction of osteoblast differentiation

[0219] After the mesoderm differentiation induction, the cell constructs corresponding to 8 wells (corresponding to 16 ml of culture solution) were removed from Elplasia (trademark) and resuspended in 10 ml of osteoblast differentiation induction medium [DMEM medium (sodium pyruvate-free: Nacalai Tesque, Kyoto) containing 15% FBS (Thermo Fisher Scientific), 0.1 μM dexamethasone (Sigma Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich), and 50 μg / ml ascorbic acid-2-phosphate (Sigma Aldrich), 100 units / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B (Thermo Fisher Scientific)]. The cell suspension was subjected to low-attachment culture for 30 days using low-attachment bottles (Greiner bio-one, growth area 25 cm 2 ) while being shaken on a seesaw-type bioreactor (10° inclination, cycle: 0.33 Hz, platform width: 32 cm, amplitude: 5.5 cm) (BC-700: BIOCRAFT). The medium was replaced every 7 days.

[0220] 2.5. Evaluation of induction of osteoblast differentiation

[0221] After 30 days of osteoblast differentiation induction, the expression of osteoblast differentiation-specific genes (Osterix, Collagen 1a1, Runx2, and Osteocalcin) was analyzed by SYBR Green real-time RT-PCR method (Thunderbird (trademark) SYBR (trademark) qPCR Mix, TOYOBO).

[0222] In addition, the expression of undifferentiated marker genes (Nanog), osteoblast progenitor marker genes (Brachyury and Runx2), and osteoblast marker genes (Osterix, Collagen 1a1, Runx2, and Osteocalcin) until 60 days after osteoblast differentiation induction was analyzed by SYBR Green real-time RT-PCR method (Thunderbird (trademark) SYBR (trademark) qPCR Mix, TOYOBO). The base sequences of the primers used in the SYBR Green real-time RT-PCR method are shown below. GAPDH was used as an internal control.

[0223] Runx2 forward primer: 5'-CAGACCAGCAGCACTCCATA-3'

[0224] Runx2 reverse primer: 5'-CAGCGTCAACACCATCATTC-3'

[0225] Osterix forward primer: 5'-AAGCTGATCTGGTGGTGCAT-3'

[0226] Osterix reverse primer: 5'-GACTCCACAAAGGGCATGAT-3'

[0227] Collagen 1a1 forward primer: 5'-GTGCTAAAGGTGCCAATGGT-3'

[0228] Collagen 1a1 reverse primer: 5'-CTCCTCGCTTTCCTTCCTCT-3'

[0229] Osteocalcin forward primer: 5'-CACTCCTCGCCCTATTGGC-3'

[0230] Osteocalcin reverse primer: 5'-CCCTCCTGCTTGGACACAAAG-3'

[0231] Nanog forward primer: 5'-AACTGGCCGAAGAATAGCAA-3'

[0232] Nanog reverse primer: 5'-TGCACCAGGTCTGAGTGTTC-3'

[0233] Brachyury forward primer: 5'-CAGTCAGTACCCCAGCCTGT-3'

[0234] Brachyury reverse primer: 5'-ACTGGCTGTCCACGATGTCT-3'

[0235] GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGTCA-3'

[0236] GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3'

[0237] In addition, the resulting samples were subjected to HE staining or von Kossa and methylene blue double staining for histochemical observation.

[0238] Fourier transform infrared (FT-IR) spectroscopy was used to analyze the components of the iPS cell constructs. All iPS cell constructs were collected, fixed with a 10% neutral buffered formalin solution, washed with distilled water, and then gradually dehydrated with ethanol (30%, 70%, 90%, 100%). The ethanol was again replaced with fresh ethanol (100%), and then all of them were placed in a desiccator at 37°C for 12 hours. FT-IR analysis was performed on the dried cell samples using a potassium bromide (KBr) plate method. An FT-IR measuring device, FT / IR-6300ST (JASCO Corporation), was used for the analysis, and the infrared absorption spectrum obtained by scanning 1000 times at a resolution of 2 cm -1 in the range of 650 cm -1 to 4000 cm -1 was analyzed.

[0239] 2.6. Production of iPS cell-derived bone substitute material

[0240] The osteoblast cell constructs after 120 days of osteoblast cell differentiation induction were washed with PBS, and then immersed in 10 ml of PBS at 4°C overnight. The next day, the osteoblast cell constructs were removed and transferred to a 6 cm cell culture dish, and initially frozen at -80°C in a freezer overnight. Thereafter, the culture dish was placed in a freeze dryer (VD-250R; Taitec) and freeze-dried overnight to inactivate the cells. Thus, iPS cell-derived bone substitute materials were obtained. The culture dish in which the bone substitute materials were placed was capped with a lid, sealed with a seal, and stored in a moisture-proof storage (glass desiccator).

[0241] 2.7. Transplantation into a rat calvarial defect model

[0242] Ten-week-old SD rats (Slc:SD; Japan SLC, Inc.) were placed under general anesthesia. Thereafter, the scalp was peeled to form a periosteal flap, and a 5 mm diameter defect was formed through the sagittal suture of the skull. The formation of the skull defect was performed under flowing water using an animal surgery engine and a trephine rod (Implatex, Tokyo). The freeze-dried iPS cell constructs were implanted into the skull defect, the periosteum was covered, and the scalp was sutured. Thereafter, the rats were raised under specific pathogen-free and free water and contact conditions.

[0243] RESULTS

[0244] 2.8. Generation efficiency of osteoblast constructs

[0245] Using the methods described in 2.2 to 2.4 above, the number of osteoblast constructs obtained from 80% confluent iPS cells in a 10 cm cell culture plate on day 30 of induced differentiation was calculated. The result obtained using low-attachment culture dishes was 5.7 ± 0.77, and the result obtained using Elp500 was 46.2 ± 8.8, indicating that using Elp500 to generate embryoids significantly increased the generation efficiency. Figure 8 ).

[0246] 2.9. Evaluation of three-dimensional osteoblast constructs generated using Elp500

[0247] After inducing embryoid and mesodermal cells with Elp500, section specimens were prepared from human iPS cell constructs induced by osteoblast differentiation for 30 days and stained with von Kossa and methylene blue. Histological observation revealed partial mineralization within the cell structure 30 days after osteoblast differentiation induction. Figure 9 (See upper figure). Furthermore, when osteoblast differentiation induction was performed for 60 and 90 days, the number of mineralized sites inside the cell construct increased ( Figure 9 (Middle and lower figures). Meanwhile, no mineralization was observed in human iPS cell constructs induced into embryoids and mesoderms using low-attachment culture dishes and subjected to 30 days of osteoblast differentiation induction, but partial mineralization was finally observed after 60 days of osteoblast differentiation induction. Therefore, the results indicate that using Elp500 can shorten the time required for mineralization and calcification in human iPS cell constructs.

[0248] Furthermore, it was found that the expression of osteoblast-specific marker genes Runx2, SP7, collagen 1a1, and osteocalcin in cell constructs generated using Elp500 on day 30 of osteoblast differentiation induction was significantly higher than that in those using low-attachment culture dishes. Figure 10 ).

[0249] The differentiation of human iPS cells into germ cells, mesoderm, and osteoblasts was analyzed using real-time RT-PCR. Figure 7 The expression of undifferentiated marker genes (Nanog), marker genes indicating mesoderm (mesenchymal stem cells), and osteoblast progenitor cells (Brachyury, Runx2, and Osterix) and osteoblast-specific genes (collagen 1a1 and osteocalcin) during the process. Figure 11). The results showed that the expression of Nanog, which was highly expressed in iPS cells before the embryoid body culture, was significantly reduced by mesoderm induction and almost disappeared at day 5 of the induction. It was found that the expression of Brachyury, Runx2, and Osterix genes showed a peak of expression during the initial stage of the mesoderm induction to osteoblast differentiation induction, and then the expression of Osterix, Collagen 1a1, and Osteocalcin genes increased over time until 60 days after the differentiation induction. The human iPS cell constructs that were induced into embryoid bodies and mesoderm cells using Elp500 and subjected to osteoblast differentiation induction for 0 to 60 days were dried, and then subjected to compositional analysis using FTIR analysis. As a result, it was found that the human iPS cell construct samples that underwent osteoblast differentiation induction for more than 30 days had FTIR spectral peaks similar to those of natural bone tissue (human freeze-dried allogeneic bone graft) ( Figure 12 ).

[0250] The Feret's diameter of the human iPS cell constructs that were induced into embryoid bodies and mesoderm cells using Elp500 and subjected to osteoblast differentiation induction for 3 to 30 days was measured, and the results showed that in the cell construct group that underwent osteoblast differentiation induction for 30 days, cell constructs of different sizes were found, with Feret's diameters ranging from 0.66 mm to 2.68 mm ( Figure 13 A). The cell construct group that underwent osteoblast differentiation induction for 30 days was divided into sizes of 0.5 mm to less than 1 mm, 1 mm to less than 2 mm, and 2 mm to 3 mm according to the Feret's diameter, and sections were generated from these cell construct groups and evaluated for the degree of mineralization by using von Kossa staining. The results showed that the cell constructs of 2 mm to 3 mm showed the most significant mineralization ( Figure 13 B), indicating that more mature osteoblast constructs were formed as the size of the cell constructs increased.

[0251] 2.10. Evaluation of bone regenerative ability of osteoblast constructs generated using Elp500

[0252] The cell constructs generated at day 120 of osteoblast differentiation induction using Elp500 were subjected to freeze-drying treatment by the method described in 2.6 ( Figure 14 ).

[0253] The freeze-dried osteoblast constructs or human freeze-dried allogeneic bone grafts (FDBA) used as a comparative control were implanted into the site of a 5 mm diameter bone defect created in the rat skull. After 4 weeks of implantation of the iPS cell-derived freeze-dried osteoblast constructs, tissue images were observed by HE staining of tissue sections. The results showed that new bone formation filled the bone defect, and images of bone remodeling with a line of adhesion were found around the implanted osteoblast structure. In addition, the results of micro-CT analysis showed that images were observed in which the bone defect site was completely filled with new bone continuous with the surrounding existing bone Figure 15 A).

[0254] Meanwhile, in the HE staining images 4 weeks after FDBA implantation, the FDBA was surrounded by immature fibrous tissue, and no mature new bone formation was found. In addition, in the micro-CT analysis, almost no new bone formation was found at the defect site, and the defect was essentially left as is Figure 15 B).

[0255] 2.11. Conclusion

[0256] Thus, it was revealed that in the generation of three-dimensional osteoblast constructs from human iPS cells, the use of recessed microspaces, particularly Elp500 with a specific recess size, not only improved the generation efficiency in terms of time and quantity compared to the use of conventional low-attachment culture dishes, but also enabled the induction of more mature osteoblast constructs during the process of inducing to embryoid bodies and mesoderm cells. In addition, in the cell constructs generated by this process, the cell constructs with a Feret's diameter size of 2 to 3 mm were particularly advanced in mineralization at the 30th day of osteoblast differentiation induction, indicating that the osteoblast constructs were more mature. Furthermore, the freeze-dried human iPS cell-derived osteoblast constructs showed superior bone regenerative ability compared to the existing bone replacement material (FDBA), indicating that they can be used as a bone replacement material for promoting repair of bone defect sites.

[0257] Experiment 3: Effect of iPS cell culture in a feeder cell-free environment on osteoblast construct formation

[0258] <Method>

[0259] 3.1. Human iPS cells

[0260] In this experiment, a human skin fibroblast-derived iPS cell line (409B2: RIKEN BRC CELL BANK) was used.

[0261] 3.2. Culture of human iPS cells on feeder cells (using feeder cell group)

[0262] Using the same culture method as in Experiment 2, SNLP76.7-4 feeder cells were used as feeder cells, human iPS cells were seeded on these cells treated with mitomycin C, and cultured with 4 ng / ml of human basic FGF (REPROCELL) primate ES medium (ES medium: REPROCELL) containing. The medium was changed every day.

[0263] 3.3. Culture of human iPS cells in a feeder cell-free environment

[0264] The human iPS cell colonies maintained and cultured on feeder cells were collected in a culture fluid obtained by supplementing StemFit (trademark) AK02N medium (ES medium: Ajinomoto Co., Inc.) with 10 μM Y-27632 (Wako Pure Chemical) and seeded at a concentration of 8.0 x 10 4 cells / dish on a 10 cm cell culture plate coated with laminin; iMatrix-511 silk (nippi). The next day, the medium was changed to ES medium to maintain and culture the cells. Feeder-free culture was performed by this. The medium was changed every two days.

[0265] 3.4. Generation of osteoblast constructs of human iPS cells using microspatially shaped low attachment plates

[0266] Figure 24 The method of producing osteocyte constructs from human iPS cells in this experiment is shown.

[0267] 3.4.1. Induction of embryoid body formation

[0268] As the culture vessel, a 24-well micro space-shaped low-attachment plate Elplasia (trademark) (Corning, Cat#4441: with a micro-well size of 500 μm in diameter and 400 μm in depth, and with 554 to 580 recesses / well) was used.

[0269] According to the method of Experiment 2, human iPS cells cultured on feeder cells were prepared into a cell suspension (number of iPS cells in the suspension: 6.25 x 10 5 cells / ml) using ES medium (REPROCELL), and 2 ml of this suspension was added to each well, so that the number of cells seeded in each well of the Elplasia (trademark) plate was 12.5 x 10 5 cells (group using feeder cells: condition 0).

[0270] Human iPS cell colonies cultured in a feeder cell-free environment were dissociated into single cells by treatment with Tryple Select (Thermo Fisher Scientific) and their cell suspensions were adjusted to the following three conditions with ES medium (StemFit medium).

[0271] Condition 1 6.25 x 10 5 cells / ml

[0272] Condition 2 3.125 x 10 5 cells / ml

[0273] Condition 3 1.563 x 10 5 cells / ml

[0274] Cell suspensions of cells cultured on feeder cells (Condition 0) and cell suspensions adjusted to Conditions 1 to 3 in a feeder cell-free environment were added to each well of the above culture plate in an amount of 2 ml, and the number of cells per well was set as follows.

[0275]

[0276] Formation of embryoid bodies was induced by culturing for 1 day using ES medium (REPROCELL) for Condition 0 and respective ES medium (StemFit medium) for Conditions 1 to 3.

[0277] 3.4.2. Induction of mesoderm cell differentiation

[0278] After culturing the embryoid bodies for 1 day, for each condition, the medium was changed to mesoderm differentiation induction medium [1:1 mixed medium of DMEM / F12 (Thermo Fisher Scientific) containing 2% B-27 supplement (Thermo Fisher Scientific), 1% N-2 supplement (Thermo Fisher Scientific), 30 μM CHIR99021 (Wako Pure Chemical Industries), and 5 μM cyclopamine (Enzo Life science) and Neurobasal medium (Thermo Fisher Scientific)] in the same manner as in Experiment 2, and then cultured for 5 days. The medium was replaced every two days.

[0279] 3.4.3. Induction of osteoblast differentiation

[0280] After 5 days of mesoderm differentiation induction, the cell constructs contained in the number of wells shown in conditions A to D below were taken out from the Elplasia (trademark) plate under each condition and transferred to low attachment bottles (Greiner bio-one, growth area: 25 cm 2 ).

[0281]

[0282] For the group cultured on feeder cells (condition 0), the cells corresponding to the wells of 8 Elplasia (trademark) plates were transferred to 1 bottle (condition 0 + condition A).

[0283] The medium in each bottle was changed to 10 ml of osteoblast differentiation induction medium [containing 15% FBS (Gibco / Life Technologies), 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich), and 50 μg / ml ascorbic acid-2-phosphate (Sigma-Aldrich), 1% antibiotic-antimycotic (100 units / ml penicillin, 100 μg / ml streptomycin, and 250 ng / ml amphotericin B in α-MEM medium (Nacalai Tesque)], and cultured for 30 days on a seesaw-type bioreactor (10° inclination, cycle: 0.33 Hz, platform width: 32 cm, amplitude: 5.5 cm) (BC-700: BIO CRAFT) with shaking.

[0284] The medium was changed every 3 days. The time point at which the culture was started in the osteoblast differentiation induction medium was defined as "day 0 of osteoblast differentiation induction".

[0285] 3.5. Evaluation of induction of osteoblast differentiation

[0286] After 30 days of osteoblast differentiation induction, the number of cell constructs in each condition group was calculated, and their size (Feret's diameter) was calculated using ImageJ image analysis software (trademark) NIH.

[0287] In addition, the expression of osteoblast-specific marker genes (Runx2 and osteocalcin) in each group of cell constructs after 30 days of osteoblast differentiation induction was analyzed by SYBR Green real-time RT-PCR method (Thunderbird (trademark) SYBR (trademark) qPCR Mix, TOYOBO). GAPDH was used as an internal control. The primers used were the same as those in Experiment 2.

[0288] After 30 days of osteoblast differentiation induction, the cell constructs of each group were sectioned, and HE staining, or von Kossa and methylene blue double staining were performed for histological observation.

[0289] 3.6. Effect of supplementation of retinoic acid in the induction medium on osteoblast construct formation

[0290] In the experimental system using human iPS cells in a feeder cell-free environment, 10 μM Y-27632 was supplemented in the ES medium (StemFit medium) used in the induction of embryoid body formation in 3.4.1 above. In addition, 1 μM retinoic acid (Wako Pure Chemical) was supplemented in the mesoderm differentiation induction medium of 3.4.2 above and the osteoblast differentiation induction medium of 3.4.3, and osteoblast constructs were induced under conditions 2 and condition B, and after 30 days of osteoblast differentiation induction, the mineralization and maturity of the cell constructs were evaluated by HE staining and von Kossa and methylene blue double staining.

[0291] <Results>

[0292] 3.7. Effect of feeder-free culture of iPS cells and number of seeded cells on the number of osteoblastic constructs Figure 25

[0293] After 30 days of osteoblast differentiation induction, the number of cell constructs formed per flask in the feeder cell culture group (condition 0 + condition A) was 56. On the other hand, in the feeder cell-free culture, the number of cell constructs formed per flask was as follows: when the number of cells seeded was 12.5 x 10 5 cells / well (condition 1), 100 constructs in the 8-well / 1-flask group (condition 1 + condition A), and 93 constructs in the 4-well / 1-flask group (condition 1 + condition B); when the number of cells seeded was 6.25 x 10 5 cells / well (condition 2), 139 constructs in the 8-well / 1-flask group (condition 2 + condition A), and 103 constructs in the 4-well / 1-flask group (condition 2 + condition B); and when the number of cells seeded was 3.125 x 10 5 cells / well (condition 3), 103 constructs in the 8-well / 1-flask group (condition 3 + condition A), 112 constructs in the 4-well / 1-flask group (condition 3 + condition B), and 126 constructs in the 2-well / 1-flask group (condition 3 + condition C). Thus, in the feeder cell-free culture groups under these conditions, although the number of cells used in the embryoid body formation step and the osteoblast differentiation induction step was the same as or less than that in the feeder cell culture group (condition 0 + condition A), the number of osteoblast constructs formed was significantly increased 3.8. Effect of feeder-free culture of iPS cells and number of seeded cells on the size of osteoblastic constructs .

[0294] Figure 26 3.9. Effect of feeder-free culture of iPS cells and number of seeded cells on osteoblastic differentiation of cell constructs

[0295] Thirty days after osteoblast differentiation induction, the Feret's diameter of cell constructs in the feeder cell culture group was often larger than that in the non-feeder cell culture group. Meanwhile, in the non-feeder cell culture group, the seeded cell number was 6.25 × 10⁻⁶. 5 The cell / well (condition 2) group and the number of cells seeded were 3.125 × 10⁶. 5 The cell / well (condition 3) group exhibited less size variation and was therefore more homogeneous than the feeder cell culture group. Figure 27 ).

[0296] 3.10. Effect of feeder-free culture of iPS cells and number of seeded cells on mineralization of osteoblastic constructs Figure 28

[0297] Thirty days after osteoblast differentiation induction, compared with cell constructs from the feeder cell culture group (condition 0 + condition A), the expression of osteoblast-specific marker genes (Runx2 and osteocalcin) was higher in cell constructs from the feeder cell culture group. Specifically, during the embryoid formation step, the number of seeded cells (iPS cells cultured in a feeder cell-free environment) was 6.25 × 10⁻⁶. 5 Cells / well (condition 2) and 3.125 × 10 5 In the cell / well (condition 3) group, the expression of these genes was significantly increased during the osteoblast differentiation induction step under all conditions A through D. 3.11. Effect of retinoic acid supplementation in induction medium on osteoblastic construct formation from iPS cells ).

[0298] Figure 29 3.12. Conclusions

[0299] Thirty days after osteoblast differentiation induction, the cell constructs from the feeder cell culture group (condition 0 + condition A) showed a seeding cell count of 6.25 × 10⁻⁶ cells. 5 Cell culture without feeder at 3.125 × 10⁶ wells (condition 2), i.e., 8-well / flask group (condition 2 + condition A) and 4-well / flask group (condition 2 + condition B), with a seeding cell count of 3.125 × 10⁶ cells / well. 5 The cell / well (condition 3) unfed cell cultures, namely the 8-well / flask group (condition 3 + condition A), the 4-well / flask group (condition 3 + condition B), and the 2-well / flask group (condition 3 + condition C), showed significantly larger internal mineralization ranges and exhibited more mature images as osteoblast constructs. ​ ).

[0300] ​

[0301] In the generation of osteoblast constructs using iPS cells in a feeder-free environment, when the ES medium for embryoid culture was supplemented with 10 μM Y-27632, and the mesodermal differentiation induction medium and osteoblast differentiation induction medium were supplemented with 1 μM retinoic acid, significant mineralization was observed in the inner and outer layers of the cell constructs after 30 days of osteoblast induction, and more mature and homogeneous bone-like tissue was obtained compared to the unsupplemented condition. ​ ).

[0302] ​

[0303] Osteoblasts can be efficiently generated from a small number of cells by using feederless cell culture instead of feeder cell culture during the human iPS cell proliferation phase. Specifically, during embryoid formation, 6.25 × 10⁶ cells are seeded per well. 5 Human iPS cells were cultured in a feeder-free environment, and osteoblast differentiation induction was further performed by transferring culture medium containing mesodermal cells corresponding to 8 or 4 wells to one flask, and during embryoid formation, 3.125 × 10⁶ cells were seeded per well. 5 Human iPS cells cultured in a feeder-free environment, and further osteoblast differentiation induction steps by transferring culture medium containing mesodermal cells corresponding to 8, 4 or 2 wells to a flask, can not only obtain a large number of osteoblast constructs, but also more mature osteoblast constructs. sequence list <110> National University Corporation Tohoku University <120> Methods for producing osteoblast constructs using iPS cells <130> FP210145JP <150> JP2019-033277 <151> 2019-02-26 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 1 cgggctacct gccatcac 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 ggccagaggc agaagtcaga 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ctcgtctgac tgcctgccta g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gcgtggatgc ctgccttgta 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 tgtcccaacc cccaaagac 19 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ccctcgactc ctacatcttc tga 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ccgggagcag tgtgagctta 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 ccgggagcag tgtgagctta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 tctccttgcg ccacagaatg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 tccttagact caccgctctt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 cggaggagac aacggagaag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 gtaagtgtcg ccacgaggct 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 gtaacccgtt gaaccccatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 ccatccaatc ggtagtagcg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 cagaccagca gcactccata 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 cagcgtcaac accatcattc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 aagctgatct ggtggtgcat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 18 gactccacaa agggcatgat 20 <210> 19 <211> 20 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> PRIMER <400> 19 gtgctaaagg tgccaatggt 20 <210> 20 <211> 20 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> PRIMER <400> 20 ctcctcgctt tccttcctct 20 <210> 21 <211> 19 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> PRIMER <400> 21 cactcctcgc cctattggc 19 <210> 22 <211> 21 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> PRIMER <400> 22 ccctcctgct tggacacaaa g 21 <210> 23 <211> 20 <212> DNA <213> ARTIFICIAL SEQUENCE <220> <223> PRIMER <400> 23 aactggccga agaatagcaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 tgcaccaggt ctgagtgttc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 cagtcagtac cccagcctgt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 actggctgtc cacgatgtct 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 gaaggtgaag gtcggagtca 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 gaagatggtg atgggatttc 20

Claims

1. A method for producing an osteoblast construct from iPS cells, the method comprising the steps of: (1) inducing formation of embryoid bodies by subjecting undifferentiated iPS cells to non-adherent culture; (2) inducing differentiation of the iPS cells into mesoderm cells by subjecting the embryoid bodies of the iPS cells obtained in step (1) to non-adherent culture; and (3) inducing differentiation into osteoblasts by subjecting the mesoderm cells of the iPS cells obtained in step (2) to non-adherent culture, each of steps (1) and (2) being performed using a culture vessel comprising wells formed of a bottom surface having a plurality of recesses arranged independently of each other and a ring-shaped side wall arranged vertically on the bottom surface, wherein at least one of the plurality of recesses has a circular equivalent diameter of 450 μm to 700 μm, the step (3) being a step of producing an osteoblast construct by transferring the cells cultured in the plurality of recesses to another culture vessel and performing shaking culture.

2. A method for producing an osteoblast construct from iPS cells, the method comprising the steps of: (1) inducing formation of embryoid bodies by subjecting undifferentiated iPS cells to non-adherent culture; (2) inducing differentiation of the iPS cells into mesoderm cells by subjecting the embryoid bodies of the iPS cells obtained in step (1) to non-adherent culture; and (3) inducing differentiation into osteoblasts by subjecting the mesoderm cells of the iPS cells obtained in step (2) to non-adherent culture, each of steps (1) and (2) being performed using a culture vessel comprising wells formed of a bottom surface having a plurality of recesses arranged independently of each other and a ring-shaped side wall arranged vertically on the bottom surface, wherein at least one of the plurality of recesses has a circular equivalent diameter of 450 μm to 700 μm, in step (1), the number of undifferentiated iPS cells placed in each of the recesses of the culture vessel is 100 cells to 3000 cells, the step (3) being a step of producing an osteoblast construct by transferring the cells cultured in the plurality of recesses to another culture vessel and performing shaking culture.

3. The method according to any one of claims 1 to 2, wherein each of the plurality of recesses has a substantially circular shape of an opening.

4. The method according to any one of claims 1 to 3, wherein the iPS cells are human iPS cells or mouse iPS cells. wherein 5. The method according to any one of claims 1 to 4, wherein the culture time of step (1) is 0.625 days to 3.5 days.

6. The method according to any one of claims 1 to 5, wherein the culture in step (2) is performed in the presence of at least one selected from the group consisting of a Wnt signal activator and a hedgehog signal inhibitor. In step (1), the value of the undifferentiated iPS cells at the time of adding 2 ml of the cell suspension per well when placed in the recess of the culture vessel was 0.5 x 10 5 cells / ml to 7.5 x 10 5 cells / ml, 7. The method according to claim 6, wherein the Wnt signal activator is at least one selected from the group consisting of CHIR99021, 6-bromoindirubin-3'-oxime, Kenpaullone, SB-216763, SKL2001, deoxycholic acid, WAY-316606, NSC-693868, ricinine, 7-oxo-β-sitosterol, IM-12, HLY78, and retinoic acid.

8. The method according to any one of claims 1 to 7, wherein the culture in step (3) is performed in the presence of at least one selected from the group consisting of a Wnt signal activator and a hedgehog signal inhibitor. ​ ​ ​ wherein ​ ​ ​ ​ 3. The method of claim 1 or 2, wherein, ​ 4. The method of claim 1 or 2, wherein, ​ 5. The method of claim 4, wherein, ​ 6. The method of claim 1 or 2, wherein, ​ 7. The method of claim 6, wherein, ​ 8. The method of claim 6, wherein, The hedgehog signaling inhibitor is at least one selected from the group consisting of cyclomine, AY9944, GANT58, GANT61, saponareine, SANT-1, SANT-2, U18666A, veratramine, vismodegib, Cur-61414, robotnikinin, JK184, and HPI-4.

9. The method of claim 1 or 2, wherein, The culturing of step (3) is performed in the presence of at least one selected from the group consisting of a hypoxia mimetic compound and a statin.

10. The method according to claim 1 or 2, further comprising, before step (1), a step of culturing the undifferentiated iPS cells without feeder cells.

11. The method of claim 10, wherein, by placing a suspension of undifferentiated iPS cells having a cell concentration of 1.5 x 10 5 cells / ml to 3.5 x 10 5 cells / ml in a culture vessel, and then culturing.

12. The method of claim 1, wherein, Step (1) is performed by placing a suspension of the undifferentiated iPS cells, and When the undifferentiated iPS cells are placed in step (1), the number of the undifferentiated iPS cells placed per the recess is from 100 cells to 3,000 cells.

13. The method according to claim 12, wherein, Step (2) is performed using a culture vessel having at least one well, and wherein step (3) is performed by placing a culture solution corresponding to 1 to 10 wells containing the mesoderm cells obtained in step (2) in a culture vessel, and then culturing.

14. The method according to claim 1 or 2, wherein The culturing of step (1) is performed in the presence of a ROCK inhibitor; and wherein each of the culturing in step (2) and step (3) is performed in the presence of retinoic acid.

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