Method for producing pluripotent stem cells

Abstract

Provided is a method for producing pluripotent stem cells from somatic cells, the method comprising: (1) a step of seeding somatic cells into a container having one or more sections in which two or more somatic cells can be gathered; (2) a step of bringing the somatic cells into contact with an initializing factor; and (3) a step of culturing, in a state in which the two or more somatic cells have been gathered in the sections, the somatic cells having been contacted with the initializing factor.

Description

[Technical Field]

[0001] This application relates to a method for producing pluripotent stem cells from somatic cells, particularly hematopoietic cells. [Background technology]

[0002] iPS cells, which are pluripotent stem cells produced by reprogramming somatic cells, can differentiate into various cell types and are expected to be used in regenerative medicine, including transplantation therapy. Methods for producing pluripotent stem cells using somatic cells of various origins are known.

[0003] The production of pluripotent stem cells from hematopoietic cells (specifically peripheral blood mononuclear cells) has the excellent advantages of being less invasive and less burdensome for patients, but its reprogramming efficiency is very low, so there is a strong desire for methods to improve this efficiency. In addition, hematopoietic cells can be cultured in suspension without adhering to the bottom surface of plastic petri dishes, and they also have the advantage of being easy to handle in cultures using automated culture equipment. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2018-23401 [Patent Document 2] Japanese Patent Publication No. 2016-135102 [Patent Document 3] Japanese Patent Publication No. 2021-035399 [Patent Document 4] Japanese Patent Publication No. 2020-036608 [Patent Document 5] Re-table 2018 / 143243 [Patent Document 6] Retable 2017 / 110724 [Overview of the project] [Problems that the invention aims to solve]

[0005] This application aims to provide a method for producing pluripotent stem cells.

Means for Solving the Problems

[0006] This application provides the following embodiments. [1] A method for producing pluripotent stem cells from somatic cells, comprising: (1) A step of seeding somatic cells in a container having one or more compartments capable of aggregating two or more somatic cells; (2) A step of contacting the somatic cells with reprogramming factors; (3) A step of culturing the somatic cells contacted with the reprogramming factors in a state where two or more of the somatic cells are aggregated within the compartment. The method as described above. [2] The compartment has a cell low-adhesion aggregation part capable of aggregating two or more somatic cells, and an aggregation step of aggregating two or more somatic cells into the aggregation part. The method according to [1] above, further comprising the above. [3] The aggregation step in the method according to [2] above involves at least one of the operations of rotation, rocking, and vibration of the container. [4] The container has a plurality of surfaces, and the rotation operation of the container is performed around a rotation axis parallel or intersecting with the first surface having the largest area among the plurality of surfaces. The method according to [3] above. [5] The angle of the rotation axis with respect to the first surface is a right angle. The method according to [4] above. [6] Two or more somatic cells are aggregated in a region closer to the rotation axis than the radial center with respect to the rotation axis of the container. The method according to [5] above. [7] Two or more somatic cells are aggregated in a region farther from the rotation axis than the radial center with respect to the rotation axis of the container. The method according to [5] above. [8] The compartment is It has a recess including an opening provided on one surface of the container and a bottom with low cell adhesiveness. The recess is an aggregation part capable of aggregating two or more somatic cells, and a guiding part for guiding the somatic cells flowing into the interior of the container to the aggregation part and has the method according to any one of [1] to [7] above. [9] The method according to [8] above, wherein the guiding part is an inclined surface inclined with respect to one surface of the container.

[10] The method according to [9] above, wherein the angle formed by the inclined surface and one surface of the container is 15 to 60 degrees.

[11] The method according to [9] or

[10] above, wherein the inclined surface is composed of a flat surface.

[12] The method according to [9] or

[10] above, wherein the inclined surface is composed of a curved surface.

[13] The method according to any one of [9] to

[12] above, wherein the guiding part has two or more of the inclined surfaces.

[14] The method according to any one of [9] to

[11] above, wherein the inclined surface is composed of a flat surface with a polygonal shape.

[15] The recess is composed of three or more of the inclined surfaces arranged adjacent to each other, and has a polygonal pyramid shape that tapers from the opening toward the bottom, the bottom constitutes the aggregation part, and the side surface of the recess constitutes the guiding part, according to the method of

[14] above.

[16] The method according to

[14] above, wherein the inclined surface is a flat surface with a substantially triangular shape.

[17] The recess has a substantially conical shape that tapers from the opening toward the bottom, the bottom constitutes the aggregation part, and the side surface of the recess constitutes the guiding part, according to the method of

[12] above.

[18] The method according to any one of the [1] to

[17] , wherein the container has two or more compartments.

[19] The method according to any one of the above [1] to

[18] , wherein the somatic cells are floating somatic cells.

[20] The method according to

[19] , wherein the floating somatic cells are hematopoietic cells. [twenty one] The method according to

[20] , wherein the hematopoietic cells are peripheral blood mononuclear cells. [twenty two] The method according to

[20] , wherein the hematopoietic cells include hematopoietic progenitor cells. [twenty three] The method according to

[20] , wherein the hematopoietic cells are CD34-positive cells. [twenty four] The method according to any one of the

[20] to

[23] , wherein the hematopoietic cells are derived from peripheral blood or umbilical cord blood. [twenty five] The method according to any one of the above [1] to

[24] , wherein in step (3), somatic cells are concentrated at one point within the compartment.

[26] The method according to

[25] , wherein somatic cells are seeded at a rate of 50 to 400 cells per section.

[27] The method according to

[26] , wherein somatic cells are seeded at a rate of 100 to 300 cells per section.

[28] The method according to any one of the above [1] to

[13] and

[18] to

[24] , wherein somatic cells are aggregated linearly in step (3).

[29] The method according to

[28] , wherein somatic cells are seeded such that the theoretical value calculated for how many somatic cells are accumulated in a cross section perpendicular to the linear portion is approximately 100 to 1,000.

[30] The method according to any one of the above [1] to

[29] , comprising the step of expanding the culture of somatic cells before contacting the somatic cells with a reprogramming factor.

[31] The method according to any one of the above [1] to

[30] , wherein in step (2), a reprogramming factor is brought into contact with somatic cells using a Sendai virus vector.

[32] The method according to any one of the above [1] to

[31] , wherein the reprogramming factor is RNA.

[33] The method according to any one of the above [1] to

[32] , wherein the somatic cells are human somatic cells.

[34] The method according to any one of the items [1] to

[33] , wherein step (2) is performed after step (1).

[35] The method according to any one of the items [1] to

[33] , wherein step (1) is performed after step (2).

[0007]

[36] A container for use in the method of any one of the items [1] to

[35] , having the compartment capable of aggregating two or more somatic cells.

[37] A container body capable of containing somatic cells inside, The container body comprises at least one compartment located inside it, Equipped with, The aforementioned section is A low-adhesion cell aggregation area capable of aggregating two or more somatic cells, A guide section that guides somatic cells that have flowed into the container to the aggregation section. A container having

[38] The container according to

[37] , further comprising a circulation section that connects the inside of the container body to the outside of the container body, enabling the circulation of somatic cells.

[39] The aforementioned section is The container body has a recess that includes an opening that opens to the inner surface and a bottom with low cell adhesion, The aforementioned guide section The container according to

[37] or

[38] , wherein a part of the recess is an inclined surface that is inclined with respect to the inner surface of the container body.

[40] The aforementioned section is The container body has an opening on its inner surface and a recess including a cell-low adhesion bottom, The aforementioned guide section The container according to

[38] , wherein the protrusion is located outside the recess, adjacent to the opening, and further away from the flow portion than the opening, and protrudes from the inner surface.

[41] The container according to any one of the above

[36] to

[40] , wherein the compartment contains an initialization factor.

[0008]

[42] A container as described in any one of the above items

[36] to

[41] , A seeding device that supplies somatic cells into the container and seeds them, A recovery device for discharging somatic cells that have come into contact with the reprogramming factor from the inside of the container to the outside of the container for recovery, A control device that controls the seeding device and the harvesting device. A system equipped with these features.

[43] The system according to

[42] , further comprising a tilting device capable of tilting part or all of the container with respect to a reference plane to concentrate two or more somatic cells inside the container into the compartment.

[44] The container has a flexible bottom surface, The system according to

[43] , wherein the tilting device deforms the bottom surface to tilt it with respect to the reference surface.

[45] The system according to

[44] , wherein the tilting device tilts the container by rotating it around a rotation axis extending in a direction perpendicular to the reference plane, or by pressing a part of the bottom surface of the container to tilt part or all of the container with respect to the reference plane.

[46] The system according to any one of the claims

[42] to

[45] , comprising a vibrating device capable of shaking or vibrating the container to concentrate two or more somatic cells inside the container into the compartment.

[47] The system according to any one of the claims

[42] to

[46] , comprising a rotating device capable of rotating the container around a rotation axis to concentrate two or more somatic cells inside the container into the compartment by centrifugal force.

[0009]

[48] A kit for producing pluripotent stem cells from somatic cells, A cell-low adhesion container having one or more compartments capable of accumulating two or more cells, and A kit containing initialization factors.

[0010]

[49] A program that causes a computer to perform any of the methods described in any one of the above items [1] to

[35] .

[0011]

[50] A method for producing differentiated cells A step of preparing pluripotent stem cells produced by the method described in any one of the above items [1] to

[35] , and The step of culturing the prepared cells in a culture medium for differentiation induction, Methods that include...

[51] The method according to

[50] , wherein the differentiated cells are cardiomyocytes. [Effects of the Invention]

[0012] This invention provides an efficient method for producing pluripotent stem cells. It also provides containers, systems, kits, and programs for producing pluripotent stem cells. [Brief explanation of the drawing]

[0013] [Figure 1] Morphology of cells immediately after seeding on an Aggrewell® plate. [Figure 2] Morphology of control cells on Day 4 (left) and cells seeded on an Aggrewell® plate (right). [Figure 3]Morphology of control cells on Day 10 (left) and cells seeded on an Aggrewell® plate (right). [Figure 4] Fluorescence images of cells seeded on an Aggrewell™ plate on Day 10. The upper left image shows the GFP fluorescence image. The upper right image shows the immunostaining image using TRA-1-60 antibody. The lower left image shows a merged image of the GFP fluorescence image, the immunostaining image using TRA-1-60 antibody, and the bright-field image. The lower right image is a magnified view of the lower left image. [Figure 5] Morphology and fluorescence images of cells that were subculturified on Day 11 and seeded on an Aggrewell™ plate on Day 16. The upper left image shows the GFP fluorescence image. The upper right image shows the immunostaining image using TRA-1-60 antibody. The lower left image shows a merged image of the GFP fluorescence image and the immunostaining image using TRA-1-60 antibody. The lower right image shows the bright-field image. [Figure 6] Bright-field images of cell cultures on Day 9 in a 24-well plate (control) (left image) and in an Aggrewell® plate (right image). [Figure 7] Relationship between the number of PBMC cells seeded per microwell and the number of viable cells on Day 14. [Figure 8] Relationship between the number of PBMC cells seeded per microwell and the number of TRA-1 positive cells on Day 14. [Figure 9] Relationship between the number of PBMC cells seeded per microwell and the TRA-1 positivity rate on Day 14. [Figure 10] A schematic diagram illustrating the aggregation step included in the manufacturing method of the first embodiment of this disclosure. [Figure 11] A schematic diagram illustrating the aggregation step included in the manufacturing method of the second embodiment of this disclosure. [Figure 12] A schematic diagram showing a container according to the first embodiment of this disclosure. [Figure 13] A schematic diagram showing a container according to a second embodiment of the present disclosure. [Figure 14] A schematic diagram showing a container according to a third embodiment of the present disclosure. [Figure 15] A schematic diagram showing a container according to a fourth embodiment of this disclosure. [Figure 16] A schematic diagram showing a container according to a fifth embodiment of this disclosure. [Figure 17] A schematic diagram showing a container according to the sixth embodiment of this disclosure. [Figure 18] A schematic diagram showing a container according to the seventh embodiment of this disclosure. [Figure 19] A schematic diagram showing a container according to the eighth embodiment of this disclosure. [Figure 20] A schematic diagram showing a container according to the ninth embodiment of this disclosure. [Figure 21] A schematic diagram showing a container according to the tenth embodiment of this disclosure. [Figure 22] A schematic diagram showing a container according to the eleventh embodiment of this disclosure. [Figure 23] A schematic diagram showing a container according to the twelfth embodiment of this disclosure. [Figure 24] A block diagram showing a system of one embodiment of the present disclosure. [Figure 25] Flow cytometry analysis showing the percentage of troponin-positive cells after induction of cardiomyocyte differentiation of iPS cells produced by the present invention. [Modes for carrying out the invention]

[0014] In this specification and the claims, when a number is accompanied by the term “approximately,” it is intended to include a range of ±10% of that value. For example, “approximately 20” includes “18 to 22.” A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. The “approximately” in relation to a range applies to both endpoints of that range. Thus, for example, “approximately 20 to 30” includes “18 to 33.”

[0015] [Method for producing pluripotent stem cells] This application relates to a method for producing pluripotent stem cells from somatic cells, (1) A process of sowing somatic cells in a container having one or more compartments capable of accumulating two or more somatic cells. (2) Steps to bring somatic cells into contact with reprogramming factors (3) A step of culturing somatic cells that have come into contact with the reprogramming factor in a state in which two or more of the somatic cells are aggregated in the compartment. The present invention provides the method including the above.

[0016] (somatic cells) In this disclosure, somatic cells are not particularly limited and any somatic cells can be used. For example, keratinizing epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the tongue surface), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic and storage cells (e.g., hepatocytes), luminal epithelial cells that constitute the interface (e.g., type I alveolar cells), luminal epithelial cells of the inner chain canal (e.g., vascular endothelial cells), ciliated cells with transporting ability (e.g., airway epithelial cells), and extracellular matrix-secreting cells (e.g., fibroblasts). Examples include cells, contractile cells (e.g., smooth muscle cells), blood and immune system cells (e.g., peripheral blood mononuclear cells, umbilical cord blood cells, T lymphocytes), sensory cells (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., accompanying cells), central nervous system neurons and glial cells (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and their progenitor cells (tissue progenitor cells). There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected; both undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as the origin of somatic cells in this application. Examples of undifferentiated progenitor cells include neural stem cells, hematopoietic progenitor cells, mesenchymal stem cells, dental pulp stem cells, and other tissue stem cells (somatic stem cells). In one embodiment, the somatic cells may be floating cells such as hematopoietic cells (e.g., peripheral blood mononuclear cells, hematopoietic progenitor cells, and CD34-positive cells). The hematopoietic cells may be derived, for example, from peripheral blood or umbilical cord blood.

[0017] In this application, there are no restrictions on the animals from which somatic cells are derived. Examples include mammals such as mice, rats, hamsters, guinea pigs, cattle, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, birds, and humans, with primates being preferred and humans more preferred.

[0018] (pluripotent stem cells) In this application, "pluripotent stem cells" refers to cells that possess both the ability to differentiate into various cell types and the ability to proliferate. A method of inducing pluripotent stem cells by reprogramming somatic cells using factors called reprogramming factors is well known.

[0019] (culture medium) In this application, the culture medium can be prepared by appropriately adding necessary factors to the basal medium used for culturing animal cells. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, MEM Zinc Option medium, IMEM Zinc Option medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, insulin, transferrin, selenium, KnockOut Serum Replacement (KSR) (Invitrogen) (serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or one or more substances such as lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), antioxidants, pyruvate, buffers, inorganic salts, cytokines, and their equivalents. A commercially available medium may be used as the basal medium, for example, StemSpan ACF (STEMCELL Technologies) or StemFit® AK03N medium (Ajinomoto Co., Inc.).

[0020] ·Process (1) In step (1), somatic cells are seeded in a container having one or more compartments capable of aggregating two or more somatic cells.

[0021] (Somatic cell aggregation) "Aggregation" of somatic cells means a state in which a somatic cell is in contact with at least one other somatic cell, and multiple somatic cells are gathered in one place. A compartment capable of aggregating two or more somatic cells is not particularly limited as long as it can aggregate two or more somatic cells, but for example, it is a compartment in which two or more somatic cells can be gathered in one place by physical force (including force generated by their own weight). Specifically, examples include a compartment in which two or more somatic cells can be gathered by applying actions such as rotation, shaking, and vibration to the container, or a compartment having an inclined surface from the opening toward the bottom, in which two or more somatic cells can be gathered toward the bottom by the inclined surface, for example by their own weight. In the compartment having an inclined surface from the opening toward the bottom, actions such as rotation, shaking, and vibration may be further applied to the container. Within the compartment, somatic cells may be gathered in a point-like manner (one or more points) or in a linear manner.

[0022] In this embodiment, the container has one or more compartments capable of aggregating two or more somatic cells. The number of compartments in the container is not particularly limited and can be determined as appropriate by those skilled in the art. The container may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compartments, or 1 to 100,000 or more compartments. For example, if the container is a 24-well plate with 1,200 compartments in each well, the number of compartments is 28,800. In one embodiment, the container has two or more of the above-mentioned compartments.

[0023] In this embodiment, the compartment may have low cell adhesion. Examples of low cell adhesion compartments include, but are not limited to, compartments in commercially available cell culture vessels that have not been artificially treated (e.g., coated with extracellular matrix) for the purpose of improving adhesion to cells, or compartments that have been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylic acid (poly-HEMA) or a polymer of 2-methacryloyloxyethyl phosphorylcholine (Lipidure)).

[0024] The number of somatic cells seeded in step (1) can be determined as appropriate by a person skilled in the art and is not particularly limited, but for example, the number of cells should be determined so that almost all somatic cells can come into contact with at least one other somatic cell in step (3). If the somatic cells are concentrated at one point within a compartment in step (3), the number of cells may be 2-500 cells / compartment, 2-400 cells / compartment, or 3-300 cells / compartment, for example, 5-500 cells / compartment, 10-500 cells / compartment, 50-400 cells / compartment, or 100-300 cells / compartment, for example, about 200 cells / compartment. If the somatic cells are concentrated linearly in step (3), the number of somatic cells to be seeded can be determined according to the length of the part where the somatic cells are concentrated or have the potential to be concentrated (also called the linear part). The shape of the linear part is not particularly limited and can be determined depending on the shape of the container. For example, the linear part may be straight or curved. In one embodiment, somatic cells are seeded such that the theoretical value of how many somatic cells accumulate in a cross section perpendicular to the linear portion is approximately 1 or more, approximately 10 or more, approximately 100 or more, approximately 1,000 or more, or approximately 10,000 or more, for example, approximately 1 to 10,000 or approximately 100 to 1,000. Such a theoretical value is calculated by the following formula: (average diameter of somatic cells seeded in the compartment) × (number of somatic cells seeded in the compartment) / (length of the linear portion where cells are aggregated). For example, if the average diameter of somatic cells is 10 μm and the length of the linear portion of the compartment is 10 cm, the number of cells is approximately 10 4 More than 10 cells / compartments 5 More than 10 cells / compartments 6 More than 10 cells / compartments7 cells / compartments or more, or about 10 8 cells / compartments or more, for example about 10 4 ~10 8 cells / compartments or about 10 6 ~10 7 cells / compartments may be.

[0025] · Step (2) In step (2), a somatic cell is brought into contact with a reprogramming factor.

[0026] (Reprogramming factor) In the present application, the reprogramming factor means a substance used alone or in combination with a plurality of factors to induce the differentiation state of a certain cell into a more undifferentiated state. The reprogramming factor includes a factor essential for nuclear reprogramming and an auxiliary factor (co-factor) that increases the efficiency of nuclear reprogramming. The reprogramming factor can be, for example, a gene (DNA, RNA), a gene product (mRNA, miRNA, protein, etc.), a low molecular compound, and a combination thereof.

[0027] When the reprogramming factor is a gene or its gene product, at least one selected from the group consisting of genes of the Oct gene family, genes of the Sox gene family, genes of the Klf gene family, genes of the Myc gene family, genes of the Lin gene family, and the Nanog gene, and their gene products can be mentioned (International Publication No. 2007 / 69666; Patent No. 5696282; Science, 2007, 318: 1917-1920). Among these, preferably, at least one selected from the group consisting of genes of the Oct gene family, genes of the Sox gene family, genes of the Klf gene family, genes of the Myc gene family, and gene products of these genes can be mentioned.

[0028] Specific examples of genes of these families and their combinations are listed below. In the following, only the names of the genes are described, but the case of using their gene products is also included.

[0029] (a) A type of reprogramming factor consisting of genes from the Oct gene family; (b) A combination of two reprogramming factors consisting of a gene from the Oct gene family and a gene from the Sox gene family; (c) A combination of two reprogramming factors consisting of a gene from the Oct gene family and a gene from the Klf gene family; (d) A combination of two reprogramming factors consisting of a gene from the Oct gene family and the Nanog gene; (e) A combination of three reprogramming factors consisting of genes from the Oct gene family, genes from the Sox gene family, and genes from the Klf gene family; (f) A combination of three reprogramming factors consisting of genes from the Oct gene family, the Klf gene family, and the Myc gene family; (g) A combination of four reprogramming factors consisting of genes from the Oct gene family, genes from the Sox gene family, genes from the Klf gene family, and genes from the Myc gene family; and (h) A combination of four reprogramming factors consisting of genes from the Oct gene family, the Sox gene family, the Lin gene family, and the Nanog gene.

[0030] More specifically, the following combinations are exemplified, but are not limited to them. In the following combinations, the Sox2 gene can be replaced with the Sox1, Sox3, Sox15, Sox17, or Sox18 gene. The Klf4 gene can be replaced with the Klf1, Klf2, or Klf5 gene. The c-Myc gene can be replaced with the T58A (active mutant) gene, the N-Myc gene, or the L-Myc gene. (1) Oct3 / 4 gene, Klf4 gene, c-Myc gene (2) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene (3) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, Fbx15 gene, Nanog gene, Eras gene, ECAT15-2 gene, TclI gene, β-catenin (active mutant S33Y) (4) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, SV40 Large T antigen (hereinafter, SV40LT) gene (5) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, HPV16 E6 gene (6) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, HPV16 E7 gene (7) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, HPV6 E6 gene, HPV16 E7 gene (8) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, Bmil gene (For the combinations (1) to (8) above, see International Publication No. 2007 / 069666 (however, for the substitution of Sox2 gene to Sox18 gene and Klf4 gene to Klf1 gene or Klf5 gene in the combination of (2) above, see Nature Biotechnology, 26, 101-106 (2008)). For the combination of "Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene", also see Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007), etc. For the combination of "Oct3 / 4 gene, Sox2 gene, Klf2 (or Klf5) gene, c-Myc gene", see Nat. Cell Biol., 11, 197-203 (2009) See also: For combinations of "Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, hTERT gene, SV40LT gene," see Nature, 451, 141-146 (2008). (9) Oct3 / 4 gene, Sox2 gene, Klf4 gene (see Nature Biotechnology, 26, 101-106 (2008)) (10) Oct3 / 4 gene, Sox2 gene, Nanog gene, Lin28 gene (see Science, 318, 1917-1920 (2007)) (11) Oct3 / 4 gene, Sox2 gene, Nanog gene, Lin28 gene, hTERT gene, SV40LT gene (see Stem Cells, 26, 1998-2005 (2008)) (12) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, Nanog gene, Lin28 gene (see Cell Research (2008) 600-603) (13) Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene, SV40LT gene (see also Stem Cells, 26, 1998-2005 (2008)) (14) Oct3 / 4 gene, Klf4 gene (See Nature 454:646-650 (2008), Cell Stem Cell, 2:525-528 (2008)) (15) Oct3 / 4 gene, c-Myc gene (see Nature 454:646-650 (2008)) (16) Oct3 / 4 gene, Sox2 gene (See Nature, 451, 141-146 (2008), International Publication No. 2008 / 118820) (17) Oct3 / 4 gene, Sox2 gene, Nanog gene (see International Publication No. 2008 / 118820) (18) Oct3 / 4 gene, Sox2 gene, Lin28 gene (see International Publication No. 2008 / 118820) (19) Oct3 / 4 gene, Sox2 gene, c-Myc gene, Esrrb gene (Essrrb gene can be replaced with Esrrg gene. See Nat. Cell Biol., 11, 197-203 (2009)) (20) Oct3 / 4 gene, Sox2 gene, Esrrb gene (see Nat. Cell Biol., 11, 197-203 (2009)) (21) Oct3 / 4 gene, Klf4 gene, L-Myc gene (22) Oct3 / 4 gene, Nanog gene (23) Oct3 / 4 gene (24) Oct3 / 4 gene, Klf4 gene, c-Myc gene, Sox2 gene, Nanog gene, Lin28 gene, SV40LT gene (see Science, 324: 797-801 (2009))

[0031] In the combinations (1) to (24) above, other member genes of the Oct gene family (e.g., Oct1A, Oct6, etc.) can be used instead of the Oct3 / 4 genes. Other member genes of the Sox gene family (e.g., Sox7, etc.) can be used instead of the Sox2 gene (or Sox1, Sox3, Sox15, Sox17, Sox18 genes). Other member genes of the Lin gene family (e.g., Lin28b, etc.) can be used instead of the Lin28 gene.

[0032] A combination that does not fall under any of the above combinations (1) to (24), but includes all the components of any one of them, and also includes any other substance, may also be included in the category of "reprogramming substances" in this invention. Under conditions in which the somatic cells to be reprogrammed endogenously express some of the components of any of the above combinations (1) to (24) at a sufficient level for reprogramming, a combination of only the remaining components, excluding those components, may also be included in the category of "reprogramming factors" in this invention.

[0033] In addition to the reprogramming factors listed above, one or more reprogramming factors selected from the group consisting of the Fbx15 gene, ERas gene, ECAT15-2 gene, Tcl1 gene, and β-catenin gene may be combined, and / or one or more reprogramming factors selected from the group consisting of the ECAT1 gene, Esg1 gene, Dnmt3L gene, ECAT8 gene, Gdf3 gene, Mybl2 gene, ECAT15-1 gene, Fthl17 gene, Sall4 gene, Rex1 gene, UTF1 gene, Stella gene, Stat3 gene, and Grb2 gene may be combined. These combinations are described in detail in International Publication No. 2007 / 69666.

[0034] Examples of preferred reprogramming factors include at least one selected from the group consisting of the Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene (or L-Myc gene), Lin28 gene, Nanog gene, and the gene products of these genes. Preferably, it is a combination of two or more, more preferably three or more, selected from the group consisting of the Oct3 / 4 gene, Sox2 gene, Klf4 gene, c-Myc gene (or L-Myc gene), Lin28 gene, Nanog gene, and their gene products. In particular, among these, combinations of reprogramming factors that are preferably introduced include: (1) Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, and Klf4 gene or its gene product; (2) Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and c-Myc gene or its gene product; and (3) Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and L-Myc gene or its gene product. Among these, combinations of Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, and Klf4 gene or its gene product, as well as combinations of Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and L-Myc gene or its gene product are preferred. Among these, the combination of the Oct3 / 4 gene, the Sox2 gene, and the Klf4 gene, as well as the combination of the Oct3 / 4 gene, the Sox2 gene, the Klf4 gene, and the L-Myc gene, are more preferred.

[0035] When the reprogramming factor is a gene or its gene product, there are no particular restrictions on the species from which such a gene originates; it can be appropriately selected according to the origin of the cells to be reprogrammed. It may be of human origin, or of other mammalian origin, such as primates like mice, rats, rabbits, pigs, or monkeys, but it is preferably of human origin.

[0036] The cDNA sequence information for each of the above-mentioned reprogramming factors can be obtained from publicly known databases. For example, one may refer to the accession number in GenBank listed in International Publication No. 2007 / 069666. The Nanog gene is referred to as "ECAT4" in the aforementioned publication.

[0037] The mouse and human cDNA sequence information for four particularly preferred genes (Oct3 / 4 gene, Sox2 gene, Klf4 gene, and L-Myc gene) among the above-mentioned reprogramming factors is described below. Gene name Mouse Human Oct3 / 4 NM_013633 NM_002701 Sox2 NM_011443 NM_003106 Klf4 NM_010637 NM_004235 L-Myc NM_008506 NM_001033081

[0038] The cDNA of each of the above-mentioned reprogramming factors can be easily isolated from the cells of the organism from which the sequence originates, based on the above-mentioned cDNA sequence information or sequence information registered in a known database, using known methods such as PCR.

[0039] The sequences of the genes and gene products of the aforementioned reprogramming factors do not necessarily have to be wild-type sequences and may have any mutations as long as they can induce reprogramming. For example, genes or mRNAs that encode amino acid sequences in which one or a few (e.g., a few, up to three, up to five, up to ten, up to fifteen, up to twenty, or up to twenty-five) amino acids are added, deleted, substituted, and / or inserted, and that can induce reprogramming, can be used in this application. The same applies to proteins encoded by such genes or mRNAs. Furthermore, as long as they maintain biological activity (ability to induce reprogramming), polypeptides in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) of amino acids are deleted or added at the N-terminus and / or C-terminus, and polypeptides in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15, or 20 residues) of amino acids are substituted, and the genes or mRNAs that encode them can also be used. Potential variants include, for example, fragments, analogs, derivatives, and fusion proteins with other polypeptides (e.g., those with heterologous signal peptides or antibody fragments attached). Specifically, these include polypeptides that have sequences in which one or more amino acids are substituted, deleted, and / or added to the wild-type amino acid sequence, and that have equivalent biological activity (e.g., activity to induce reprogramming) to the wild-type protein. When using fragments of the wild-type protein, they typically contain 70% or more, preferably 80% or more, 85% or more, more preferably 90% or more, 95% or more, or 98% or more of a continuous region of the wild-type polypeptide (or the mature form in the case of secreted proteins).

[0040] There are no particular restrictions on the number of amino acids to be modified, but for example, it is within 30% of the total amino acids of the natural mature polypeptide, preferably within 25%, more preferably within 20%, more preferably within 15%, more preferably within 10%, 5%, or within 3%, and for example, within 15 amino acids, preferably within 10 amino acids, more preferably within 8 amino acids, more preferably within 5 amino acids, and more preferably within 3 amino acids. When substituting amino acids, it is expected that the protein activity will be maintained by substituting them with amino acids that have similar side chain properties. Such substitutions are referred to as conservative substitutions in this application. Conservative substitutions include substitutions between amino acids within each group, such as basic amino acids (e.g., lysine, arginine, histidine), acidic amino acids (e.g., aspartic acid, glutamic acid), non-charged amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (e.g., threonine, valine, isoleucine), and aromatic amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0041] Modified proteins exhibit high homology to the amino acid sequence of the wild-type protein. High homology refers to amino acid sequences with, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 96% or more identity. Amino acid sequence identity can be determined using, for example, the BLASTP program (Altschul, SF et al., J. Mol. Biol. 215: 403-410, 1990). For example, searches can be performed using default parameters on the NCBI (National Center for Biotechnology Information) BLAST webpage (Altschul SF et al., Nature Genet. 3:266-272, 1993; Madden, TL et al., Meth. Enzymol. 266:131-141, 1996; Altschul SF et al., Nucleic Acids Res.25:3389-3402, 1997; Zhang J. & Madden TL, Genome Res. 7:649-656, 1997). For example, the blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999) can be used to create alignments of two sequences and determine their identity. Gaps are treated similarly to mismatches, and for example, the identity value for the entire amino acid sequence of a native cytokine (the mature form after secretion) is calculated. Specifically, the proportion of matching amino acids in the total number of amino acids of the wild-type protein (or the mature form in the case of secreted proteins) is calculated.

[0042] Furthermore, silent mutations can be introduced into genes or mRNA without altering the encoded amino acid sequence. In particular, in AT(U)-rich genes, stable high gene expression can be achieved by substituting five or more consecutive A or T(U) bases with G or C without changing the encoded amino acid sequence.

[0043] In another embodiment, the reprogramming factor may be a small molecule compound (e.g., a combination of VPA, CHIR99021, 616452, tranylcypromine, forskolin, and DZNep) or a miRNA used to induce dedifferentiation of differentiated cells. If the reprogramming factor is a small molecule compound, it may be added to the culture medium (Science 09 Aug 2013: Vol. 341, Issue 6146, pp. 651-654).

[0044] (Contact of reprogramming factors with somatic cells) In step (2), the reprogramming factor is brought into contact with somatic cells. If the reprogramming factor is a gene, contact with somatic cells can be achieved by introducing an expression vector encoding the reprogramming factor into the somatic cells using a known method. The type of expression vector is not particularly limited, and any known expression vector can be used. Examples of expression vectors include episomal vectors, artificial chromosome vectors, plasmid vectors, and viral vectors.

[0045] Episomal vectors are vectors capable of autonomous replication outside of chromosomes. Specific methods for using episomal vectors are disclosed in Yu et al., Science, 324, 797-801 (2009). For example, an episomal vector can be used in which loxP sequences are positioned in the same direction on the 5' and 3' ends of the vector elements necessary for replication. Because episomal vectors can autonomously replicate outside of chromosomes, they can provide stable expression within host cells even without integration into the genome. However, once iPS cells are produced, it is desirable to promptly remove the vector. By sandwiching the vector elements necessary for replication between two loxP sequences and cleaving them with Cre recombinase, the autonomous replication ability of the episomal vector can be lost, allowing for early removal of the vector from iPS cells.

[0046] Examples of episomal vectors include vectors that contain sequences necessary for autonomous replication derived from EBV, SV40, etc. Specifically, the vector elements necessary for autonomous replication include the replication origin site and genes encoding proteins that bind to the replication origin site and control replication. For example, in the case of EBV, these include the replication origin site oriP and the EBNA-1 gene, and in the case of SV40, the replication origin site ori and the SV40LT gene.

[0047] Examples of artificial chromosome vectors include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, PAC (P1-derived artificial chromosome) vectors, and HAC (Human artificial chromosome) vectors.

[0048] The plasmid vector is not particularly limited as long as it is a plasmid vector that can be expressed in the somatic cells to be introduced. If the somatic cells to be introduced are mammalian, a plasmid vector commonly used for animal cell expression can be used. Examples of plasmid vectors for animal cell expression include pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0049] Examples of viral vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpesvirus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, sylvisvirus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. In this specification, a viral vector means a vector having a genomic nucleic acid derived from the virus, and capable of expressing a gene by incorporating the introduced gene into the nucleic acid.

[0050] As a viral vector, the Sendai virus vector can be suitably used. The Sendai virus is a virus belonging to the order Mononegavirales and the family Paramyxoviridae (which includes genera such as Paramyxovirus, Morbillivirus, Rubulavirus, and Pnemovirus), and contains a single negative-sense RNA strand (the antisense strand relative to the sense strand that codes for viral proteins) as its genome. The Sendai virus vector is a non-chromosomal-integrated viral vector, and the vector is expressed in the cytoplasm. Therefore, since the introduced gene is not integrated into the host chromosome, it is highly safe, and the vector can be removed from the introduced cells after the objective has been achieved.

[0051] Sendai virus vectors include complexes consisting of infectious virus particles, viral cores, complexes of viral genome and viral proteins, or non-infectious virus particles, which have the ability to express the genes they carry when introduced into cells. For example, a ribonucleoprotein (the core portion of the virus, i.e., RNP) consisting of the Sendai virus genome and the Sendai virus proteins (NP, P, and L proteins) that bind to it can express the introduced gene within cells when introduced into those cells (International Publication No. 00 / 70055). Introduction into cells can be carried out using common methods such as electroporation, lipofection, and microinjection. Therefore, such ribonucleoproteins (RNPs) are also included in Sendai virus vectors.

[0052] Examples of known methods for introducing expression vectors into somatic cells include infection methods for viral vectors (e.g., retroviral vectors (Cell 2007 Nov 30;131(5):861-72, Sendai virus vector (Patent No. 5963309))) and calcium phosphate methods, lipofection methods, retronectin methods, or electroporation methods for plasmid vectors (Nat Methods. 2011 May;8(5):409-12).

[0053] When the reprogramming factor is mRNA or miRNA, examples of methods for bringing the reprogramming factor into somatic cells include gene transfer methods using synthetic mRNA (e.g., calcium phosphate method, lipofection method (Cell Stem Cell. 2010 Nov 5; 7(5): 618-630), or electroporation).

[0054] If the reprogramming factor is a protein, it may be brought into contact with somatic cells by direct injection methods (e.g., needle injection, lipofection, or electroporation) of the protein (e.g., a cell membrane-permeable recombinant protein) (Cell Stem Cell, 4, 8 May 2009, 381-384).

[0055] Step (2) may be performed before or after step (1). In one embodiment, step (2) is performed after step (1). In another embodiment, step (2) is performed before step (1).

[0056] In one embodiment, the method of the present invention includes a step of expanding the culture of somatic cells before step (2). The culture conditions can be appropriately determined by those skilled in the art and are not particularly limited. For example, if the somatic cells are peripheral blood mononuclear cells (PBMCs), they may be cultured in a CD34-positive cell medium to increase the number of CD34-positive cells. Any known CD34-positive cell medium can be used as appropriate, for example, a medium containing IL-6, SCF, TPO, Flt-3L, IL-3 and G-CSF.

[0057] The culture temperature is not limited to the following, but is approximately 30-40°C, for example, approximately 37°C. The culture is carried out in an atmosphere containing CO2, O2, and N2, with a CO2 concentration of approximately 0.05-15%, preferably approximately 3-7%, more preferably approximately 4-6%, and most preferably approximately 5%. The O2 concentration is approximately 0.05-100%, preferably 2-25%. The N2 concentration is approximately 0.05-100%, preferably 30-75%.

[0058] The duration of the extended culture is not particularly limited and can be determined as appropriate by those skilled in the art, but it can be 1 to 20 days, 3 to 10 days, or, for example, 4 to 7 days, while changing the culture medium as needed. Furthermore, by using an automated culture device, it is possible to perform continuous medium changes (perfusion culture).

[0059] In the expansion culture process, the somatic cells may be cultured in suspension. In this application, "suspension culture" means that the cells are cultured in a state in which they do not adhere to the culture substrate. Although not particularly limited, this can be done using commercially available cell culture vessels that have not been artificially treated (e.g., coated with an extracellular matrix) for the purpose of improving adhesion to cells, or that have been treated to artificially suppress adhesion (e.g., coated with polyhydroxyethyl methacrylic acid (poly-HEMA) or a polymer of 2-methacryloyloxyethyl phosphorylcholine (Lipidure)). For example, commercially available products such as 96-well low-adhesion plates (Sumitomo Bakelite Co., Ltd.) and 35mm low-adhesion dishes (Sumitomo Bakelite Co., Ltd.) may be used. If the somatic cells are suspension-type somatic cells (e.g., hematopoietic cells such as peripheral blood mononuclear cells, hematopoietic progenitor cells, and CD34-positive cells), suspension-type somatic cells do not adhere to the culture vessel, so culture vessels for adherent culture can also be used. For example, commercially available products such as the 24F independent well type adherent cell culture plate (Sumitomo Bakelite Co., Ltd.) may be used.

[0060] ·Process (3) In step (3), somatic cells that have come into contact with the reprogramming factor are cultured in the compartment in a state where two or more of the somatic cells are aggregated. The somatic cells can be cultured in suspension.

[0061] Step (3) is performed when two or more somatic cells are aggregated within the compartment. For example, step (3) is performed when 2 to 500, 2 to 400, or 3 to 300 somatic cells are aggregated at a single point within the compartment, for example, 5 to 500, 10 to 500, 50 to 400, or 100 to 300, for example, about 200 somatic cells. If somatic cells are aggregated in a linear portion within the compartment in step (3), the number of somatic cells aggregated can be determined according to the length of the linear portion. In one embodiment, the theoretical value calculated for how many somatic cells are aggregated in a cross section perpendicular to the linear portion is about 1 or more, about 10 or more, about 100 or more, about 1,000 or more, or about 10,000 or more, for example, about 1 to 10,000 or about 100 to 1,000. Somatic cells may be aggregated by applying actions such as rotation, shaking, and vibration to the container, or by using a compartment with a sloping surface from the opening to the bottom to aggregate the somatic cells at the bottom.

[0062] In step (3), the culture temperature is not limited to the following, but is approximately 30-40°C, for example, approximately 37°C. The culture is carried out in an atmosphere containing CO2, O2, and N2, with a CO2 concentration of approximately 0.05-15%, preferably approximately 3-7%, more preferably approximately 4-6%, and most preferably approximately 5%. The O2 concentration is approximately 0.05-100%, preferably 2-25%. The N2 concentration is approximately 0.05-100%, preferably 30-75%.

[0063] In step (3), the culture period for somatic cells is not particularly limited and can be appropriately determined by a person skilled in the art based on the type of somatic cells, the type of reprogramming factor, the means of contacting the reprogramming factor, etc., but may be 3 to 30 days, 5 to 20 days, 8 to 15 days, or, for example, about 10 days. The generation of pluripotent stem cells can be confirmed by the expression of pluripotent stem cell markers such as SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct3 / 4, Rexl, and Nanog. The expression of pluripotent stem cell markers may be visually confirmed under a microscope by immunostaining, or confirmed using flow cytometry or FACS (fluorescence-activated cell sorting).

[0064] The produced pluripotent stem cells can be passaged every 3 to 30 days, every 4 to 20 days, every 5 to 10 days, or, for example, every 7 days. The number of passages is not particularly limited, but may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. The passage period is not particularly limited and may be 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, or 1000 or more. As containers for passage, for example, the suspension culture containers described above can be used. When using a closed-type automated culture system, the concept of subculturing does not exist; proliferation and differentiation of pluripotent stem cells can be achieved by increasing the volume of culture medium and / or perfusion of the medium.

[0065] (Consolidation process) In one embodiment, the method of the present invention further includes an aggregation step of aggregation two or more somatic cells, wherein the compartment has a cell-low adhesion aggregation section capable of aggregating two or more somatic cells. For example, the aggregation step aggregates two or more somatic cells with at least one of the following actions relative to the container: rotation, shaking, and vibration. If the method of the present invention includes an aggregation step, step (3) is performed after two or more somatic cells have been aggregated in the compartment by the aggregation step. In other words, two or more somatic cells that have come into contact with the reprogramming factor can be cultured in a state in which they are aggregated more reliably.

[0066] Referring to Figures 10 and 11, an example of an aggregation process involving rotation of a container will be described. The container 100 in Figures 10 and 11 is a roughly rectangular dish containing one compartment and having multiple surfaces, including a first surface 101. The first surface 101 is the surface with the largest area among the multiple surfaces of the container 100 and faces the opening surface 102. With multiple somatic cells (not shown) contained inside the container 100, the multiple somatic cells contained in the container 100 are aggregated by rotating the container 100 around a rotation axis L1 that is parallel to or intersects the first surface 101.

[0067] The container 100 in Figure 10 is configured to rotate around a rotation axis L1 parallel to the first surface 101. Multiple somatic cells contained in the container 100 are concentrated in a region 103 near the rotation axis L1 of the container 100. For example, if the container 100 is placed on a horizontal plane P, and the container 100 is rotated to a position above the horizontal plane P, then region 103 will be located at the edge of the container 100 closest to the horizontal plane P. Note that if the rotation axis L1 is not connected to a bearing mechanism at both ends, but rather, for example, at one end on the near side in Figure 10, and the bearing mechanism is a ball joint specification or the like that allows the rotation axis L1 to rotate while also tilting in the elevation direction, then region 103 will be located at the corner of the container 100 closest to the horizontal plane P.

[0068] The container 100 in Figure 11 is configured to be rotatable about a rotation axis L1 that intersects the first surface 101. Multiple somatic cells contained in the container 100 are concentrated in a region 104 that is far from the rotation axis L1 of the container 100. For example, if the container 100 is placed on a horizontal plane P, when the container 100 is rotated about a rotation axis L1 that is perpendicular to the horizontal plane P, region 104 will be located at the edge of the container 100 furthest from the rotation axis L1.

[0069] Referring to Figure 12, an example of an aggregation process involving rocking or vibration will be described. The container 100 in Figure 12 is a roughly rectangular plate with a recess 110. The recess 110 has a roughly circular opening 111 that opens on one side 105 of the container 100. With multiple somatic cells contained inside the recess 110, the container 100 is rocked or vibrated in the direction of arrow A in Figure 12, causing the multiple somatic cells inside the recess 110 to aggregate.

[0070] The container 100 in Figures 10 and 11 may have recesses in regions 103 and 104. The recesses may, for example, have a cell-low adhesion bottom and be configured to allow two or more somatic cells to be aggregated at this bottom. Furthermore, multiple recesses may be provided within regions 103 and 104 depending on the amount of somatic cells to be seeded, in which case the required amount of somatic cells can be efficiently aggregated.

[0071] The containers shown in Figures 13 to 21, which will be described later, can also be used in the aggregation process of this embodiment.

[0072] [container] This application is, A container body capable of containing somatic cells inside, The container body includes at least one compartment arranged inside it, Equipped with, The aforementioned section is A low-adhesion cell aggregation area capable of aggregating two or more somatic cells, A guide section that guides somatic cells that have flowed into the container to the aggregation section. The present invention provides a container having the following characteristics: By using such a container, two or more somatic cells can be aggregated without rotating, shaking, or vibrating the container.

[0073] The container of this disclosure only needs to have at least one compartment, and may be, for example, plate-shaped, pack-shaped, or dish-shaped.

[0074] The container of this disclosure may further include a circulation section that connects the inside of the container body with the outside of the container body, enabling the circulation of somatic cells.

[0075] The guide section is not limited to a pre-defined shape, but may also be a path or means that enables the guidance of somatic cells to the aggregation section by external force.

[0076] Examples of containers according to this disclosure are shown in Figures 13 to 23. Figures 13 to 18 show examples of dish-shaped containers, Figures 19 to 21 show examples of plate-shaped containers, and Figures 22 and 23 show examples of pack-shaped containers.

[0077] The container 200 in Figures 13 to 17 is a roughly rectangular dish shape and includes at least one compartment 210. The compartment 210 has a collection section 220 and a guide section 230. The container 200 has a roughly rectangular opening surface 201 with an opening 203 and a roughly rectangular bottom section 202 facing the opening surface 201. The collection section 220 and the guide section 230 are located on the bottom section 202.

[0078] The container in Figure 13 includes one compartment 210. In the container 200 of Figure 13, the aggregation section 220 is located approximately in the center of the bottom 202 in the longitudinal direction (hereinafter referred to as the longitudinal direction) of the container 200 and extends approximately linearly in the short direction (hereinafter referred to as the short direction) of the container 200. The bottom 202 of the container 200 is composed of two inclined surfaces that extend from each end in the longitudinal direction toward the center in the longitudinal direction. The two inclined surfaces are inclined from the opening surface 201 toward the bottom 202 as they move toward the center in the longitudinal direction from each end, and constitute a guide section 230. In the container 200 of Figure 13, somatic cells that flow into the interior of the container 200 are aggregated linearly by the multiple inclined surfaces, each composed of a plane. The angle between each inclined surface constituting the guide section 230 and one surface of the container 200 (for example, the opening surface 201) is preferably between 15 and 60 degrees.

[0079] The container in Figure 14 includes one compartment 210. In the container 200 of Figure 14, the aggregation section 220 is located at one end of the longitudinal direction of the bottom 202 and extends substantially linearly in the transverse direction. The bottom 202 of the container 200 is composed of a single inclined surface extending from the other end in the longitudinal direction toward the one end in the longitudinal direction. The inclined surface slopes from the opening surface 201 toward the bottom 202 as it moves from the other end in the longitudinal direction toward the one end in the longitudinal direction, and constitutes a guide section 230. In the container 200 of Figure 14, somatic cells flowing into the interior of the container 200 are aggregated linearly by the inclined surface composed of planes. The angle between the inclined surface constituting the guide section 230 and one surface of the container 200 (e.g., the opening surface 201) is preferably between 15 and 60 degrees.

[0080] The container 200 in Figure 15 differs from the container 200 in Figure 14 in that the inclined surface constituting the guide portion 230 is composed of a curved surface that protrudes in the direction from the opening surface 201 toward the bottom portion 202. In the container 200 of Figure 15, somatic cells that flow into the interior of the container 200 are aggregated in a curved shape by the inclined surface composed of a curved surface.

[0081] The container 200 in Figure 16 includes one compartment 210. In the container 200 of Figure 16, the aggregation section 220 is located at one end in the longitudinal direction of the bottom 202 and approximately in the center in the short direction, and is provided as a point. The bottom 202 of the container 200 is composed of an inclined surface 231 extending from the other end in the longitudinal direction toward the aggregation section 220, and a groove 232 located approximately in the center of the first inclined surface 231 in the short direction. The inclined surface 231 and the groove 232 constitute the guide section 230. The inclined surface 231 is inclined from the opening surface 201 toward the bottom 202 as it moves from the other end in the longitudinal direction toward one end in the longitudinal direction, and is also inclined from the opening surface 201 toward the bottom 202 as it moves from each of the short ends toward the center in the short direction. The groove 232 opens into the inclined surface 231 and has a groove width that decreases from the other end in the longitudinal direction to the one end in the longitudinal direction (in other words, the dimension of the groove 232 in the short direction). In the container 200 of Figure 16, somatic cells that flow into the inside of the container 200 are concentrated at a single point by multiple inclined surfaces, each composed of a plane. The groove 232 can also be omitted.

[0082] The container 200 in Figure 17 includes two compartments 210 arranged adjacent to each other in the longitudinal direction. In the container 200 of Figure 17, the aggregation portion 220 of each compartment 210 is located at the longitudinal end of the bottom 202 and extends substantially linearly in the transverse direction. The bottom 202 of the container 200 is composed of a first inclined surface 231 extending from approximately the center in the longitudinal direction toward one end in the longitudinal direction, and a second inclined surface 232 extending from approximately the center in the longitudinal direction toward the other end in the longitudinal direction. Each of the first inclined surface 231 and the second inclined surface 232 constitutes a guide portion 230 for each compartment 210. Each of the first inclined surface 231 and the second inclined surface 232 is inclined from the opening surface 201 toward the bottom 202 as it moves from the longitudinal center toward one or the other end in the longitudinal direction. In the container 200 shown in Figure 17, somatic cells that flow into the container 200 are guided by two inclined surfaces, each composed of a plane, to one of the two aggregation sections 220 and aggregated in a linear fashion. The angle between each of the first inclined surface 231 and the second inclined surface 232 and one surface of the container 200 (for example, the opening surface 201) is preferably between 15 and 60 degrees.

[0083] The container 200 in Figure 18 contains one compartment 210. The container 200 in Figure 18 is a roughly circular dish shape and has a roughly circular opening surface 201 and a bottom 202. The aggregation section 220 is located at the end of the bottom 202 that is radially farther from the center line L2 and extends in a circular linear shape. The bottom 202 of the container 200 has a roughly conical shape with its apex located on the center line L2 that passes through the center of the container 200 and is perpendicular to the opening surface 201 and the bottom 202, and is composed of an inclined surface that extends radially from the center line L2. The inclined surface slopes from the opening surface 201 toward the bottom 202 as it moves away from the center line L2, and constitutes a guide section 230. In the container 200 of Figure 18, somatic cells that flow into the interior of the container 200 are aggregated in a circular linear shape by the inclined surface which is composed of a curved surface. The angle between each of the first inclined surface 231 and the second inclined surface 232 and one surface of the container 200 (for example, the bottom 202) is preferably between 15 and 60 degrees.

[0084] Figures 13 to 18 show examples where the bottom surface of each container has a pre-molded slope or gradient. However, the bottom surface of the container may also be formed from a flexible material such as silicone to create the slope or gradient. In this case, for example, a tilt mechanism controlled by MEMS (Micro Electro Mechanical Systems) can be installed below the bottom surface of the container, and by activating this tilt mechanism, a slope or gradient can be created on the bottom surface of the container. Alternatively, in another embodiment, a member having a surface corresponding to the slope or gradient shown in Figures 13 to 18 may be installed on the underside of the bottom surface formed from a flexible material, and the predetermined slope or gradient can be created on each container by pushing this member upward from the bottom surface in a vertical upward direction.

[0085] The container 300 in Figure 19 includes nine compartments 302. Each compartment 302 has one recess 310. The container 300 in Figure 19 is a roughly rectangular plate shape and has a roughly rectangular opening surface 301 with nine recesses 310. Each recess 310 has an opening 311 that opens into the opening surface 301 and a bottom 312 opposite the opening 311. Each recess 310 is roughly frustoconical in shape, tapering from the opening 311 towards the bottom 312, with the bottom 312 forming an aggregation section 320, and the side surface 313 connected to the opening 311 and bottom 312 forming a guide section 330. In the container 200 in Figure 19, somatic cells that flow into the interior of each recess 310 are aggregated in one place by an inclined surface composed of a curved surface. The angle between the side surface of the recess 310 constituting the guide portion 330 and one surface of the container 200 (for example, the opening surface 301) is preferably between 15 and 60 degrees.

[0086] The recess 310 can employ any configuration that allows for the aggregation of two or more somatic cells that have flowed into the recess 310. For example, the recess 310 may be a roughly frustoconical shape with curved side surfaces 313 projecting toward the center of the recess 310, as shown in Figure 20, or it may be a roughly frustopyramidal shape, as shown in Figure 21. In the recess 310 of Figure 20, somatic cells that have flowed into each recess 310 are aggregated in one place by an inclined surface composed of a curved surface, similar to Figure 19. In the recess 310 of Figure 21, somatic cells that have flowed into each recess 310 are aggregated in one place by an inclined surface composed of a polygonal (for example, roughly triangular) plane.

[0087] In Figures 10 to 21, the opening surface does not need to be an opening across the entire surface; it may be an opening in a part of the wall surface, and furthermore, the opening may be opened and closed by a cap mechanism or the like. Also, as long as the concept of the invention remains the same, the opening surface does not necessarily have to face the bottom surface; it may be provided on the side of the container.

[0088] The container 400 in Figure 22 includes a container body 410 capable of containing somatic cells, at least one compartment 420, and a circulation section 430 that allows for the circulation of somatic cells. The container body 410 is pack-shaped, with the compartment 420 located inside. The circulation section 430 connects the inside of the container body 410 to the outside of the container body 410. The compartment 420 has a cell-low adhesion aggregation section 440 capable of containing two or more somatic cells, and a guide section 450 that guides the somatic cells that have flowed into the container 400 to the aggregation section 440. In Figure 22, the compartment 420 has a recess 421. The recess 421 has an opening 422 that opens into the inner surface 411 of the container body 410, and a cell-low adhesion bottom 423 facing the opening 422. The bottom portion 423 has an aggregation portion 440 located at one end in the longitudinal direction, and an inclined surface that slopes from the opening 422 towards the bottom portion 423 as it moves from the other end in the longitudinal direction towards the one end in the longitudinal direction. This inclined surface constitutes a guide portion 450. The recess 421 can employ any configuration that can aggregate two or more somatic cells that have flowed into the interior of the container body 410. For example, the recess 310 shown in Figures 19 to 21 may be used as the recess 421.

[0089] The container 400 in Figure 23 differs from the container 400 in Figure 22 in that the aggregation section 440 is composed of a recess 421, and the guide section 450 is composed of a projection that protrudes from the inner surface 411 of the container body 410. The recess 421 has a cell-low adhesion bottom 423 that is substantially parallel to the inner surface 411 of the container body 410. The projection that constitutes the guide section 450 is located outside the recess 421, adjacent to the opening 422, and further away from the flow section 430 than the opening 422. Thus, the guide section 450 is not limited to being a part of the recess 421.

[0090] The distribution unit 430 may be configured to perform all of the supply and discharge of somatic cells and the supply of reprogramming factors. Alternatively, the distribution unit 430 may consist of a first distribution unit that supplies and discharges somatic cells and a second distribution unit that supplies reprogramming factors, or it may consist of a first distribution unit that supplies somatic cells, a second distribution unit that discharges somatic cells, and a third distribution unit that supplies reprogramming factors.

[0091] The shape and configuration of the recess 421 of container 400 are not limited to the shapes shown in Figures 22 and 23, but may be the same as, for example, the same shape and configuration as the containers 100, 200, and 300 illustrated in Figures 12 to 21.

[0092] The container of this disclosure may contain a reprogramming factor within the compartment. The reprogramming factor can be any of the above-mentioned factors, such as the above-mentioned proteins, small molecules, or miRNAs. The amount of reprogramming factor contained in the compartment can be determined as appropriate by those skilled in the art and is not particularly limited.

[0093] [system] This application is, The aforementioned container, A seeding device that supplies somatic cells into the container and seeds them, A recovery device for discharging somatic cells that have come into contact with the reprogramming factor from the inside of the container to the outside of the container for recovery, A control device that controls the seeding device and the harvesting device. We provide a system that includes the following features. This system allows for the production of pluripotent stem cells from somatic cells.

[0094] System 1 of this disclosure comprises a container 2, a control device 10, a seeding device 20, and a harvesting device 30, as shown in Figure 24. System 1 in Figure 24 further comprises, as an example, an initialization factor supply device 40, a tilting device 50, a vibrating device 60, a rotating device 70, and a culture device 80.

[0095] Container 2 can be any container having one or more compartments with low cell adhesion. For example, containers 100, 200, 300, and 400 shown in Figures 10 to 23 can be used.

[0096] The control device 10 includes, for example, a CPU that performs calculations and a memory device that stores the program necessary for the calculations and the calculation results. In system 1 of this embodiment, the control device 10 controls the seeding device 20 and the harvesting device 30, as well as the initialization factor supply device 40, the tilting device 50, the vibration device 60, the rotation device 70, and the culture device 80 to produce pluripotent stem cells from somatic cells. The control device 10 monitors the operating status of various devices electrically connected to the control device 10, and for example, when using the container 100 shown in Figure 10 as the container 2, it transmits a control signal related to the tilt angle to the tilting device 50 at any timing after seeding somatic cells. When the tilting device 50 receives the control signal from the control device 10, it tilts the first surface 101 of the container 100 according to the tilt angle. This makes it possible to create a guiding situation inside the container 100 equivalent to the guide section 230 of the container 200 in Figure 14. The tilting device 50 preferably has the function of adjusting the tilt angle in multiple stages or steplessly, which allows for more precise control of the aggregation of somatic cells compared to when a guide is provided in the container beforehand. Furthermore, when using the container 100 shown in Figure 11, the control device 10 transmits control signals to the rotating device 70 at any timing after seeding the somatic cells, relating to the rotation angle, rotation speed, rotational speed, acceleration, angular velocity, torque, etc., relative to the rotation axis L1 of the container 100. When the rotating device 70 receives the control signals from the control device 10, it aggregates the somatic cells in the container 100 into region 104 by centrifugal force based on the rotation. In this case, if the container 100 is made of an optically visible material, the aggregation status monitoring device 90 monitors the aggregation status of the somatic cells in the container 100 and outputs the result as a monitor signal to the control device 10. The control device 10 receives the monitor signals from the aggregation status monitoring device 90 sequentially and can generate the optimal rotation of the container 100 according to the aggregation status of the somatic cells by inverter control. This makes it possible to produce pluripotent stem cells without subjecting somatic cells and pluripotent stem cells to excessive stress. Furthermore, the control mechanism, which is composed of the control device 10 and the tilting device 50 or rotating device 70 described above, can be effectively utilized even when reprogramming factors are brought into contact with somatic cells.

[0097] The seeding device 20 introduces somatic cells into the container 2 before they come into contact with the reprogramming factor, thereby seeding the somatic cells into the container 2. In this embodiment, the seeding device 20 includes, for example, a storage tank in which somatic cells are stored before they come into contact with the reprogramming factor, and a drive device (e.g., a pump) for sending the somatic cells in the storage tank to the container 2.

[0098] The recovery device 30 recovers somatic cells that have come into contact with the reprogramming factor by discharging them from inside the container 2 to outside the container 2. In this embodiment, the recovery device 30 includes, for example, a drive device that discharges somatic cells that have come into contact with the reprogramming factor from inside the container 2 to outside the container 2, and a storage tank that temporarily stores the somatic cells discharged by the drive device. The somatic cells stored in the storage tank are sent to the culture device 80 by the drive device.

[0099] The initialization factor supply device 40 includes, for example, a storage tank in which initialization factors are stored, and a drive device that delivers the initialization factors in the storage tank to the container 2.

[0100] The tilting device 50 is configured to tilt part or all of the container 2 with respect to a reference plane (for example, the horizontal plane P shown in Figures 10 and 11) so that two or more somatic cells inside the container 2 can be concentrated into a compartment of the container 2. For example, as shown in Figure 10, the tilting device 50 is configured to tilt the container 2 by rotating it around a rotation axis L1 parallel to the reference plane, or, if the container 2 has a flexible bottom surface, to deform a part of the bottom surface of the container 2.

[0101] The vibration device 60 is configured to shake or vibrate the container 2, thereby concentrating two or more somatic cells inside the container 2 into a compartment of the container 2.

[0102] The rotating device 70 is configured to rotate the container 2 around the rotation axis L1, thereby allowing two or more somatic cells inside the container to be concentrated into the compartment by centrifugal force.

[0103] The culture device 80 cultures somatic cells that have come into contact with the reprogramming factors recovered by the recovery device 30.

[0104] The aggregation status monitoring device 90 has an optical element such as a CCD (charge-coupled device) provided, for example, on a part of the rotation axis L1 or near the outer periphery of the container 2. The aggregation status monitoring device 90 is configured to monitor the aggregation status of somatic cells when the container 100 is made of an optically visible material.

[0105] System 1 may comprise five drive units that individually drive each of the seeding device 20, the collection device 30, the initialization factor supply device 40, the tilting device 50, and the vibration device 60, or it may comprise one to four drive units that drive some or all of the seeding device 20, the collection device 30, the initialization factor supply device 40, the tilting device 50, and the vibration device 60.

[0106] The system in this application includes, for example, an automated system for producing pluripotent stem cells from somatic cells. In this case, in addition to the aforementioned devices, the system may further include devices for efficiently realizing the automation. When producing pluripotent stem cells according to GMP (Good Manufacturing Practice) standards, it is desirable that all the devices constituting the system in this application be housed as a single system in the same enclosure with dustproofing and the like. However, in this case, the system in this application may be configured to share some processes with other devices, or to have the operator of the system in this application perform some processes themselves. The devices constituting the system in this application do not need to be installed in the same facility; for example, only the control device 10 may be installed in a separate facility and remotely connected to various devices such as seeding devices via the internet.

[0107] The initialization factor supply device 40, tilting device 50, vibrating device 60, rotating device 70, culture device 80, and aggregation status monitoring device 90 can be omitted. If the initialization factor supply device 40 is omitted, for example, container 2 can be configured so that the initialization factors are pre-filled in the compartments. If the tilting device 50 and vibrating device 60 are omitted, for example, containers 200, 300, and 400 shown in Figures 13 to 23 can be used.

[0108] [kit] This invention also relates to a kit for producing pluripotent stem cells from somatic cells, A cell-low adhesion container having one or more compartments capable of accumulating two or more cells, and A kit containing reprogramming factors is provided. In this case, the kit may be configured such that the reprogramming factors are pre-sealed within the container or sealed in a separate ampoule or similar. In particular, if the kit is provided in the former configuration, the kit user does not need to separately introduce the reprogramming factors after seeding somatic cells; the reprogramming of somatic cells can be achieved simply by seeding the somatic cells in the container, thus enabling the production of multi-potency stem cells under sterile conditions more easily.

[0109] Examples of containers and initialization factors used in this embodiment are as described above. The kit of this application may also include buffer solutions, culture media, instructions for use, etc.

[0110] [program] The present invention also provides a program for causing a computer to execute a method for producing pluripotent stem cells from somatic cells. For example, in the system 1 shown in Figure 24, the method for producing pluripotent stem cells is realized by the CPU of the control device 10 executing a predetermined program.

[0111] Programs can be stored and supplied to a computer using various types of non-temporary computer-readable media. These non-temporary computer-readable media include, for example, magnetic recording media (e.g., flexible disks, magnetic tapes, hard disk drives), magneto-optical recording media (e.g., magneto-optical disks), CD-ROMs, CD-Rs, CD-R / Ws, and semiconductor memory (e.g., mask ROMs, PROMs, flash ROMs, RAMs). Programs can also be supplied to a computer via wired communication channels such as electric wires and optical fibers, or wireless communication channels.

[0112] [Method for producing differentiated cells] This application also relates to a method for producing differentiated cells. A step of preparing pluripotent stem cells produced by the method of the present invention, and The step of culturing the prepared cells in a culture medium for differentiation induction, The present invention provides a method for producing differentiated cells, including [specific details omitted].

[0113] Differentiated cells are not particularly limited, but include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neurons, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, hematopoietic cells such as blood cells and bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, organ cells such as hepatocytes, pancreatic β cells, and kidney cells, chondrocytes, germ cells, and other cells, as well as progenitor cells and somatic stem cells that differentiate into these cells (e.g., mesenchymal stem cells, hematopoietic stem cells, neural stem cells, etc.). In one embodiment, the differentiated cells are cardiomyocytes.

[0114] Differentiation induction of pluripotent stem cells can be carried out using any known method. The conditions for differentiation induction are not particularly limited and can be appropriately set according to the desired differentiated cell type, etc. For example, differentiation can be induced by culturing pluripotent stem cells in a differentiation induction medium in which a predetermined concentration of a predetermined cytokine, growth factor, or other compound is added to the culture medium. If the differentiated cells are cardiomyocytes, pluripotent stem cells may be differentiated into cardiomyocytes according to the method described in WO2021 / 172542. [Examples]

[0115] The present invention will be further described with reference to the following examples, but the present invention is not limited in any way by these examples. In the following examples, the day on which somatic cells are exposed to the reprogramming factor (or the day on which the exposure began if it lasted for several days) is designated as Day 0, and the number of days that have passed thereafter is designated as Day X. Therefore, for example, two days after the day on which somatic cells are exposed to the reprogramming factor is designated as Day 2.

[0116] [Example 1] 1. Implementation Method 1-1. Reagents Used Table 1 shows the details of the reagents used in this example. [Table 1]

[0117] The SRV(trademark) iPSC-2 Vector is a stealth RNA vector optimized for gene expression levels to generate iPS cells from peripheral blood monocytes, peripheral blood mononuclear cells, and CD34-positive cells. It carries four human-derived reprogramming genes: human OCT3 / 4, human KLF4, human SOX2, and human c-MYC, as well as the EGFP gene derived from jellyfish Aequorea victoria and the puromycin (Puro) resistance gene derived from Streptomyces alboniger. The introduction of the SRV(trademark) iPSC-2 Vector into cells can be confirmed by observing the fluorescence of EGFP using a fluorescence microscope. The SRV(trademark) iPSC-2 Vector is automatically eliminated in response to the expression of miR-302, a microRNA specifically expressed in pluripotent stem cells. The SRV(trademark) iPSC-4 Vector may also be used.

[0118] The AggreWell® plate has 1200 microwells with a diameter of 400 μm in each well (see Figure 6, right). By seeding and culturing cells on the AggreWell® plate, a large number of cell aggregates of uniform size can be created.

[0119] 1-2. Equipment used Table 2 shows the details of the equipment used in this embodiment. [Table 2]

[0120] 2. Derived cells Table 3 shows the details of the cells (PBMC, ZenBio) used in this example. The PBMCs, which had been frozen, were thawed and used in this example. [Table 3]

[0121] 3. Procedure A. Expanded culture of CD34-positive cells A medium for CD34-positive cells was prepared by adding 2 μL each of cytokines, adjusted to the following concentrations, to 2 mL of A1 StemSpan ACF. [Table 4] 1 mL of PBS was added to each of the outer wells of the A2 24-well plate for humidification. The A3 PBMCs were gently thawed in a 37°C water bath, and approximately 1 mL of CD34-positive cell culture medium was added to completely dissolve them. A4 was centrifuged at 1500 rpm for 5 minutes. A5 Remove the supernatant, loosen the cell clumps by tapping, and then suspend in 1 mL of CD34-positive cell medium. A6 cell suspension was seeded in a 24-well plate and cultured statically in a 37°C 5% CO2 incubator for 5 days.

[0122] B. Production of iPS cells (adherent culture: control) B1 Following procedure A1, 5 mL of culture medium for CD34-positive cells was prepared. B2 The entire volume of PBMCs after culture was collected in a 1.5 mL tube and the cells were counted. The cell count results are shown in Table 5. [Table 5] B3 5×10 5 A cell sample (467 μL) was taken and centrifuged at 300 G for 5 minutes at 4°C. The vector quantity was calculated so that B4 MOI = 3. (TITER 4.3 × 10 7 CIU / mL, 150×10 4 / 4.3×10 7 = 34.88 (μL) 34.88 μL of B5 vector and 965.12 μL of CD34-positive cell culture medium were mixed to prepare a total of 1 mL of vector solution. B6 After centrifugation, the supernatant was removed from the cell suspension, the cell clumps were loosened by tapping, and the cells were suspended in 1 mL of vector solution to bring them into contact with the reprogramming factor (=Day 0). The cell density at the time of infection was 0.5 × 10⁶. 6 The value was cells / mL. The samples were incubated in a B7 37°C 5% CO2 incubator for 2 hours. The tubes were gently tapped every 30 minutes. The cell suspension was transferred to a 15 mL B8 tube, 1 mL of StemSpan ACF was added, and the mixture was centrifuged at 300 G for 5 minutes at 4°C. B9 The supernatant was removed, and the cell clumps were loosened by tapping. Steps B8 and B9 were repeated in B10. 400 μL of CD34-positive cell medium and 1.8 μL of iMatrix were placed in a B11 24-well plate, and the plate was shaken to mix the contents. B12 2×10 4 Cells were seeded to achieve a cell / well ratio (40 μL). Plate B13 was placed in a 37°C 5% CO2 incubator, and cultivation was started. 400 μL of AK03 medium was added to each of the following B14 days: Day 2 and Day 4. On Day 7 and Day 9 of B15, the culture medium was changed with 400 μL of AK03 medium. B16 Day 10 passed to a 6-well plate.

[0123] C. Production of iPS cells (suspension culture: AggreWell® plate) To remove air bubbles from the C1 microwells, 2 mL of Anti-Adherence Rinsing Solution was added to one well of an AggreWell™ plate and centrifuged at 2000 G for 5 minutes. The C2 rinsing solution was removed with an aspirator, and 2 mL of StemSpanACF was added and then removed with an aspirator. 500 μL of CD34-positive cell culture medium was added to each well in C3, and the cells were placed in an incubator. From the cell suspension obtained in step B10 of procedure C4, 1.2 × 10 5 The cells were separated (240 μL). 760 μL of CD34-positive cell medium was added to the cell suspension so that the total volume of medium in the C5 well was 1.5 mL. The AggreWell® plate was removed from the C6 incubator and the entire contents were used for sowing seeds. C7 The cells in the wells were homogenized by pipetting several times. The cells were centrifuged at C8 100G for 3 minutes to settle them into microwells. It was placed in a C9 37℃ 5%CO2 incubator. The culture medium was changed on Day 2, Day 4, Day 7, and Day 9 of C10. This was done by removing 750 μL of medium and adding 750 μL of AK03 medium. Immunostaining with TRA-1-60 was performed on Day 10 of C11, and the samples were observed under a fluorescence microscope (the method is shown in Procedure D). I passed the AggreWell(trademark) plate on Day 11 of C12.

[0124] D. Immunostaining (TRA-1-60) 100 μL of D1 FACS Buffer was mixed with 2 μL of TRA-1-60 antibody. To avoid aspirating the D2 sphere, the culture medium was removed using a micropipette, leaving approximately 5 mm of medium at the bottom of the well. D3 1 mL of FACS Buffer was added little by little from the edge of the well. The FACS buffer was removed using the method described in Procedure D2. On D5, 100 μL of TRA-1-60 antibody, diluted in step D1, was added. D6: Covered with aluminum foil to block out light and incubated at room temperature for 30 minutes. 1 mL of D7 FACS Buffer was added, and the culture medium was removed using a micropipette, leaving approximately 5 mm of medium at the bottom of the well. Step D8: Performed step D7 again. 300 μL of D9 FACS Buffer was added. D10 was observed using a Keyence fluorescence microscope.

[0125] 4. Results form Immediately after seeding, clusters of cells were observed at the bottom of the microwells in AggreWell® (Figure 1). On Day 4, aggregates of some cells were observed at the bottom of each microwell in the AggreWell® plate (Figure 2, right). On Day 10, numerous cell aggregates, not round spheres but clusters of cells, were observed within the microwells of the AggreWell® plate (Figure 3, right). In Figure 3, right, cell aggregates were observed in all 12 microwells within the field of view. Numerous colonies were also observed in the control adherent culture (Figure 6, left).

[0126] TRA-1-60 positive cells The expression of TRA-1-60, a pluripotent stem cell marker, was confirmed by immunostaining using the TRA-1-60 antibody. By Day 10, TRA-1-60-positive cells were confirmed in almost all of the formed cell aggregates (Figure 4, upper right). This indicates that iPS cell production in suspension using the AggreWell® plate is possible. Furthermore, a group of GFP-positive vector-residual cells was also confirmed within the same cell aggregate (Figure 4, upper left). This suggests that the cell aggregate may be a chimeric state of cells that have undergone transformation into iPS cells and cells in the process of transformation. This state was maintained even in cell aggregates that had undergone one passage (Figure 5).

[0127] summary The results of Example 1 are shown in Table 6. As shown in Table 6, by agglutinating PBMCs in the early stages of the reprogramming process, it became possible to efficiently obtain iPS cells. [Table 6] Number of seeded cells: 1.2×10 4 cells / well Reprogramming period: 9 days Reprogramming reagent: Tokiwa Bio Co., Ltd. SRV (trademark) - iPSC-2 vector

[0128] [Example 2] Relationship between the size of the initial cell aggregate (number of constituent cells) and the reprogramming efficiency. To investigate the relationship between the size of the initial cell aggregate (number of constituent cells) and the reprogramming efficiency, the number of viable cells, the number of TRA-1 positive cells, and the TRA-1 positivity rate on Day 14 were measured relative to the number of PBMC cells seeded per microwell (1-500 cells / microwell). The results are shown in Table 7 and Figures 7-9. [Table 7]

[0129] The highest total number of iPS cells was obtained when PBMCs were aggregated at 300 cells / microwell. Furthermore, the most efficient aggregation was achieved when PBMCs were aggregated at 5-100 cells / microwell.

[0130] [Example 3] Flow cytometry analysis (undifferentiated markers) iPS cell lines were established using the conventional method (scaffold-dependent culture) and the present invention's method (suspension conditions) according to the method described in Example 1. The number of PBMCs seeded per microwell was 200. iPS cells were established using blood collected from three donors (n=3). The established iPS cells were collected and treated with fixative (4% PFA). After staining with SSEA4 antibody (Alexa Fluor® 647 Mouse anti-SSEA-4, BD Biosciences) or Oct3 / 4 antibody (Oct-4A (C30A3) Rabbit mAb, Cell Signaling Technology), they were measured by flow cytometry (SA3800 Spectral Cell Analyzer, Sony). However, membrane permeabilization was performed before Oct3 / 4 antibody staining.

[0131] Table 8 shows the average results for iPS cells derived from three donors. These results demonstrate that the undifferentiated state of iPS cells established by the present invention method is superior to that of the establishment method used in this invention. [Table 8]

[0132] [Example 4] Reprogramming efficiency when cells are aggregated in a linear fashion In Examples 1-3, cells were seeded in a container (AggreWell® plate) with an inverted pyramidal microwell shape. As a result, the cells concentrated at a single point at the bottom, specifically at the very front of the microwell's guide surface (a roughly triangular inclined surface). Therefore, we investigated the initialization efficiency when the cells were concentrated in a linear manner, rather than at a single point.

[0133] Reprogramming factors were introduced into PBMCs using the same method as in Example 1, and the cell suspension obtained in step B10 was seeded into a low-adhesion cell culture plate having four microwells (compartments) with a rectangular base of 80 × 27.9 mm. The number of cells seeded in each compartment was 2.79 × 10⁶. 3 cells ~2.79×10 6 There are four types of cells.

[0134] After seeding, the culture plate was tilted at approximately 45 degrees to concentrate the cells linearly along one side (short side) of the bottom of each compartment, and culture was performed in this state. The number of iPS cell colonies that had formed in each compartment on Day 14 is shown in Table 9. The "accumulated cell count" in Table 9 is a theoretical value calculated by assuming a cell diameter of 10 μm, representing the number of cells that accumulated vertically in each compartment at the start of culture (Day 0) (e.g., 2.79 × 10⁶ cells with a diameter of 10 μm). 3 When placed side by side in a row, the dimensions are 27.9 mm, therefore 2.79 × 10 3 The number of accumulated cells in the section where cells were seeded becomes 1. [Table 9]

[0135] As shown in Table 9, iPS cell colonies were obtained in all cases from 1 to 1000 accumulated cells. However, as the number of accumulated cells increased tenfold from 10 to 100 and from 100 to 1000, the number of colonies increased fourfold and 230fold, respectively. Therefore, it was shown that, at least up to 1000 accumulated cells, the reprogramming efficiency increases with a larger number of accumulated cells. In Example 2, where cells were aggregated at a single point, iPS cells were not obtained when culture was started with one cell (Table 7). However, in the linear aggregation with one accumulated cell, it is thought that iPS cells were obtained because cells were present on both sides of the cell in question.

[0136] [Example 5] Induction of differentiation into cardiomyocytes iPS cells (four strains derived from different colonies) prepared using the method of Example 1 were lysed using a 100 μM mesh, and then differentiated into cardiomyocytes according to a standard method (for example, the method described in WO2021 / 172542). Specifically, embryoid bodies were formed over three days, and then on the third day from the start of differentiation induction, the culture medium was changed to 1% GlutaMAX (Invitrogen), 4 × 10⁶ cells. -4 The medium was replaced with StemPro-34 medium containing M monothioglycerol (MTG), 50 μg / ml ascorbic acid (AA), 150 μg / ml transferrin, 10 ng / ml vascular endothelial growth factor (VEGF; R&D Systems), 1 μM IWP3 (Stemgent), 0.6 μm dorsomorphin, and 5.4 μm SB431542. On day 7, the medium was diluted with 1% GlutaMAX, 4 × 10⁶. -4 The culture medium was replaced with 2 ml of StemPro-34 medium supplemented with M MTG, 50 μg / ml AA, 150 μg / ml transferrin, and 5 ng / ml VEGF. Thereafter, the medium was changed to a medium of the same composition every 3-4 days. The cells were cultured under a hypoxic environment (5% O2) from the start of differentiation induction until day 12, and thereafter under a normal oxygen environment (20% O2).

[0137] Cells were harvested 15 days after the start of differentiation induction, and the percentage of troponin T-positive cells, a marker for cardiomyocytes, was analyzed using a flow cytometer (anti-Troponin T antibody: Troponin T Ab-1(13-11) (Thermo Fisher Scientific, MS-295-P) was used). The troponin-positive cell rate in the four cell lines ranged from 6% to 30%. The results for the cell line with a troponin-positive rate of 30% are shown in Figure 25.

[0138] Therefore, it has been shown that differentiated cells (e.g., cardiomyocytes) can be obtained by directly inducing differentiation of iPS cells produced by the method described in this application.

Claims

1. A method for producing pluripotent stem cells from floating somatic cells, (1) A step of seeding floating somatic cells into a container having one or more compartments inside which there are low cell adhesion aggregation parts capable of aggregating two or more floating somatic cells, wherein the aggregation part is located at the bottom of the compartment, and the external shape of the container is composed of multiple surfaces. The aforementioned multiple surfaces are The opening surface of the container and The first surface facing the opening surface, The external side surface located between the opening surface and the first surface and It has, (2) Aggregation step of aggregating two or more floating somatic cells from the floating somatic cells seeded in step (1) into the aggregation unit. (3) A step of bringing a reprogramming factor into contact with floating somatic cells, (4) A step of culturing the floating somatic cells that have come into contact with the reprogramming factor in a state in which two or more of the floating somatic cells are concentrated in the compartment. The method, which includes, wherein steps (1) to (4) are performed in this order, or in the order of steps (1), (3), (2), and (4).

2. The method according to claim 1, wherein the aggregation step involves at least one of the following actions with respect to the container: rotation, swinging, and vibration.

3. The method according to claim 2, wherein the rotational movement of the container is performed around a rotation axis parallel to or intersecting the first surface.

4. The angle of the axis of rotation with respect to the first surface is perpendicular, The aforementioned rotational motion is a rotation that generates centrifugal force such that the two or more floating somatic cells are concentrated in a peripheral region away from the axis of rotation by centrifugal force. The method according to claim 3.

5. The axis of rotation is parallel to the first surface, The aforementioned rotational motion is an action of tilting the container so that the two or more floating somatic cells concentrate in a predetermined area within the compartment. The method according to claim 3.

6. The aforementioned section is The container has an opening on its opening surface and a recess including a cell-low adhesion bottom, In the recess, The bottom portion constitutes an aggregation section capable of aggregating two or more floating somatic cells, The side surface connecting the opening and the bottom constitutes a guide portion that guides the floating somatic cells that have flowed into the interior of the recess to the aggregation portion. The method according to claim 1.

7. The method according to claim 6, wherein the recess has a substantially conical shape that tapers towards the bottom from the opening.

8. The method according to claim 1, wherein the floating somatic cells are hematopoietic cells.

9. The method according to claim 1, wherein the somatic cell is a human somatic cell.

10. The method according to claim 1, wherein steps (1) to (4) are performed in this order.

11. A method for producing differentiated cells A step of producing pluripotent stem cells by the method of claim 1 or 2, and A step of culturing the manufactured cells in a culture medium for differentiation induction, Methods that include...

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