WO2005040361A1

WO2005040361A1 - Method of simply preparing stem cell and feeder cell to be used therein

Info

Publication number: WO2005040361A1
Application number: PCT/JP2004/015854
Authority: WO
Inventors: Norio Nakatsuji; Hirofumi Suemori
Original assignee: Kyoto University
Priority date: 2003-10-28
Filing date: 2004-10-26
Publication date: 2005-05-06
Also published as: JPWO2005040361A1

Abstract

It is aimed at providing a technique by which a stem cell (in particular, a primate ES cell) can be established at such a high efficiency that cannot be achieved by the existing techniques. Namely, it is intended to provide a feeder cell preparation for preparing stem cells containing a normal cell and a cell line. It is also intended to provide a method of preparing a stem cell which comprises the step of culturing a stem cell on a feeder cell preparation containing a normal cell and a cell line. Moreover, it is intended to provide a method of preparing an implant for the regeneration of an organ, a tissue or a cell which comprises: A) the step of providing a stem cell capable of differentiating into a desired organ, tissue or cell; B) the step of culturing the stem cell on a feeder cell preparation for preparing stem cells containing a normal cell and a cell line; and C) the step of differentiating the stem cells into an implant for the regeneration of the desired organ, tissue or cell.

Description

Specification

Simple method for preparing stem cells and feeder-one cells used for them

Technical field

[0001] The present invention is in the field of stem cells. More specifically, the present invention relates to a method for simple preparation and storage of stem cells (particularly, primate embryonic stem cells (hereinafter, also referred to as ES cells)).

Background art

[0002] Disease treatment by regenerative medicine has recently attracted attention. Regenerative medicine is expected to play a role as an alternative to organ transplantation and the use of auxiliary systems in medical devices. However, this has not been routinely applied to many patients with organ or tissue dysfunction.

[0003] Stem cells, especially ES cells, which are at the center of regenerative medicine, have recently gained importance due to their potential in medical applications (Non-Patent Document 1). Human ES cells are particularly important among them, but their establishment or stable subculture maintenance is difficult, and they hinder efficient use. Primate ES cells, including humans, require a very small supply of blastocysts to establish them. A small number of blastocysts need to efficiently establish ES cell lines. According to the conventional method, the efficiency of the establishment from the blastocyst is at most about 10 to 30%.

[0004] Production output of transgenic animals This is made possible by using ES cells. ES cells are obtained by culturing cells of the inner cell mass (ICM), which is an undifferentiated cell group that will be future animal individuals and is present in embryos at the developmental stage, usually called blastocysts. The resulting cell line. ES cells were established in 1981 by M. J. Evans and M. H. Kaufman (Non-Patent Document 2), followed by G. R. Martin (Non-Patent Document 3) as a cell line possibly having a dangling ability in mice.

[0005] With the emergence of ES cells in primates and increasing expectations for their applications, there is increasing hope for regenerative medicine, especially stem cell therapy and its applications, to overcome these problems. Is growing.

[0006] Non-Patent Document 4 is a paper using fibroblasts of human skin as a feeder, and is found to be 1 The force that establishes one strain from two inner cell masses. Its efficiency is quite low. Non-Patent Document 5 is the first paper on human ES cells. Its establishment efficiency is 5 cell lines inside Z14. In Non-Patent Document 6, human ES cells are established using STO cells. However, the efficiency of the establishment is still very low, such as the internal cell mass of three strains Z14. In this way, in terms of the establishment efficiency, it is expected that 50% will be a downsized force and 30% will be achieved.

[0007] In Patent Document 1, the present inventors have studied the optimal conditions for a medium. However, optimizing the medium alone has a limitation in increasing the establishment efficiency, and differentiated cells are significantly present in established ES cells.

[0008] Other groups have also developed methods for stem cell maintenance (eg, Patent Document 2, Non-Patent Documents 5 and 7). However, in these methods, only one kind of feeder cell is used, and differentiating cells are always contaminated. In particular, in Non-Patent Document 7, about 50% is a partially divided cell population.

[0009] Therefore, there is an increasing demand for a technique used to reduce the contamination of differentiated cells and further enhance the maintenance of an undifferentiated state.

[0010] (Prior art documents)

Patent Document 1: JP 2003-116527A

Patent Document 2: US Patent No. 6200806

Non-patent document 1: Stem cell 'clone research protocol Nakatsuji ed., Yodosha (2001) Non-patent document 2: M.J.Evans & M.H.Kaufman: Nature, 292, 15

4, 1981

Non-Patent Document 3: G.R.Martin: Proc. Natl. Acad. Sci. USA, 78, 7634, 1981

Non-Patent Document 4: Hovatta O., et al., Human Reproduction Vol. 18, No. 7 pp. 1404-1409, 2003

Non-Patent Document 5: Thomson A., et al., Science.282: 1145-1147, 1998.

Non-Patent Document 6: Park JH et al., Biology of Reproduction, 2003 onl ine Aug. 20.

Non-Patent Document 7: Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A, Nat Biotechnol. 2000 Apr; 18 (4): 399-404

Disclosure of the invention

Problems to be solved by the invention

[0011] The present invention provides a technique capable of establishing stem cells (especially ES cells of primates including humans) with an efficiency (particularly, reducing contamination of differentiated cells) that cannot be achieved by conventional techniques. The task is to provide

Means for solving the problem

[0012] The present invention provides an unexpectedly efficient use of a conventionally used feeder cell by mixing a plurality of feeder cells (eg, primary cultured fibroblast cells, fibroblast cell lines, etc.) and using them as a feeder cell. The above problem was solved by finding that stem cells could be established.

Therefore, the present invention provides the following.

[0014] In one aspect, the present invention provides a feeder-one cell preparation for preparing a stem cell, comprising a normal cell and a cell line.

[0015] In one embodiment, the normal cells include primary cultured cells.

[0016] In one embodiment, the normal cells are non-immortalized cells.

[0017] In one embodiment, the normal cells include fibroblasts.

[0018] In one embodiment, the normal cells are primary cultured fibroblast cells.

[0019] In one embodiment, the normal cells are derived from a mouse.

[0020] In one embodiment, the normal cells are derived from a fetus.

[0021] In one embodiment, the normal cells have a passage number of 5 or less.

[0022] In one embodiment, the normal cells are fibroblasts obtained by enzymatic treatment of mouse 11-16 days old fetal power.

In one embodiment, the normal cells are primary cultured cells having a passage number of 5 or less.

[0024] In one embodiment, the cell line is immortalized. [0025] In one embodiment, the cell line is a fibroblast cell line.

[0026] In one embodiment, the cell line has been subcultured for 2 months or less.

[0027] In one embodiment, the cell line has been passaged for one month or less.

[0028] In one embodiment, the cell line is derived from a mouse.

[0029] In one embodiment, the cell line is derived from a fetus.

[0030] In one embodiment, the cell line is an STO line.

[0031] In one embodiment, the cell line is neo-resistant.

[0032] In one embodiment, the cell line is the SL10 line.

[0033] In one embodiment, the ratio of the normal cells to the cell line is about 1:10 to 10: 1.

[0034] In one embodiment, the normal cells and the cell line are about 1: 3-3: 1.

[0035] In one embodiment, the normal cells and the cell line are present in approximately equal amounts.

[0036] In one embodiment, the stem cells are embryonic stem cells.

[0037] In one embodiment, the stem cells are human embryonic stem cells.

[0038] In one embodiment, the normal cells are mouse fibroblast primary culture cells, and the cell line is SL10 strain.

[0039] In one aspect, the present invention provides a method for preparing a stem cell, comprising a step of culturing the stem cell on a feeder-one-cell preparation containing a normal cell and a cell line. .

[0040] In one embodiment, the feeder-one-cell preparation can take any form of the embodiment described above.

[0041] In one embodiment, the culture dish on which the feeder-cell preparation is placed is gelatin-coated.

[0042] Te it!, In one embodiment, the feeder single cell preparations is plated at about 1 X 10 ⁴ cells / cm ² one about 1 X 10 ⁵ cells / cm ^2.

[0043] In one embodiment, the feeder-one cell preparation is mixed and seeded. Used within 5 days.

[0044] In one embodiment, the normal cells in the feeder-one-cell preparation are mouse fibroblast primary culture cells, and the cell line is SL10 strain.

[0045] In one embodiment, the stem cells are cultured in a medium containing a knockout serum replacement additive (KSR).

[0046] In one embodiment, the method further includes a sub-passing step, wherein collagenase is used in the sub-passing step.

[0047] In another aspect, the present invention provides a stem cell preparation prepared by the method of the present invention.

[0048] In another aspect, the present invention provides an ES cell preparation prepared by the method of the present invention.

[0049] In another aspect, the present invention provides a primate ES cell preparation prepared by the method of the present invention.

[0050] In another aspect, the present invention provides a human ES cell preparation prepared by the method of the present invention.

[0051] In another aspect, the present invention provides a method for preparing a transplant for regenerating an organ, tissue or cell, comprising: A) a stem cell capable of differentiating into a desired organ, tissue or cell. B) culturing the stem cells on a feeder cell containing normal cells and a cell line; and C) transforming the stem cells into a transplant for regenerating a desired organ, tissue or cell. Differentiating.

[0052] In one embodiment, the feeder-cell preparation can take any form of the embodiments described in the feeder-cell preparation.

[0053] In one embodiment, the desired organ, tissue or cell is a nerve, blood cell, bone, cartilage, heart, pericardium, blood vessel, muscle, eye, liver, spleen, intestine, stomach, Lung, trachea, hair and skin.

[0054] In one embodiment, the desired organ, fibrous tissue or cell and the feeder cell are of the same species.

[0055] In one embodiment, the desired organ, tissue or cell is a primate. In addition, the feeder cells are derived from a mouse.

[0056] In one embodiment, the culturing is performed ex vivo.

[0057] In one embodiment, the stem cell is a force that is also obtained immediately after the removal of the test subject, or is a cryopreserved one.

[0058] In one embodiment, the culturing is performed at 37 ° C in a saturated humidity under 5% CO.

2

[0059] In one embodiment, the differentiation is performed by using a DNA demethylating agent, a histone deacetylating agent, a nuclear receptor ligand, a cell growth factor, a cytokinin, hexamethylene bisacetamide. Dimethylacetamide, dibutyl cAMP, dimethylsulfoxide, eododexperidine, hydroxylurea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyperidine, polyprene and selenium. It is performed in a culture solution containing at least one selected factor.

[0060] In another aspect, the present invention provides an organ, tissue or cell prepared by the method for preparing a transplant for regenerating an organ, tissue or cell of the present invention.

[0061] In one aspect, the present invention provides a system for regenerating an organ, tissue or cell, comprising: A) a container; and B) a normal cell and a cell line seeded on the container. And a feeder cell containing the same.

[0062] In one embodiment, the feeder-cell preparation can take any form of the embodiment described in the feeder-cell preparation.

[0063] In one aspect, the present invention provides a mixture of a normal cell and a cell line.

[0064] In one embodiment, the mixture comprises primary fibroblast cells and a fibroblast cell line.

[0065] In one embodiment, the primary cultured cells are mouse primary cultured fibroblasts.

[0066] In one embodiment, the fibroblast cell line is an STO line.

[0067] In one embodiment, the normal cells are mouse fibroblast primary culture cells, and the cell line is the SL10 strain. [0068] In one aspect, the present invention relates to a method for regenerating an organ, fibrous tissue or cell, comprising: A) providing a stem cell capable of dividing into a desired organ, tissue or cell; B) a step of culturing the stem cells on a feeder cell containing normal cells and a cell line; and C) a step of transplanting the cultured stem cells to a site of the subject to be treated. A method is provided.

[0069] In one embodiment, the feeder-cell preparation can take any form of the embodiments described in the feeder-cell preparation.

[0070] In one embodiment, the present invention further includes D) a step of differentiating the stem cells.

[0071] In one aspect, the present invention provides use of a cell mixture containing primary fibroblast cells and a fibroblast cell line as a feeder cell.

[0072] In one aspect, the present invention provides use of a cell mixture containing a primary fibroblast cell line and a fibroblast cell line for producing a medicament containing a feeder cell.

[0073] In another aspect, the present invention provides the use of a cell mixture containing normal cells and a cell line as a feeder-cell preparation.

[0074] In one embodiment, the feeder-cell preparation can take any form of the embodiment described in the feeder-cell preparation.

[0075] In one embodiment, the feeder cell preparation is treated so as not to proliferate.

[0076] In one embodiment, the feeder cell preparation is treated with mitomycin C.

[0077] In another aspect, the present invention provides a feeder comprising a normal cell and a cell line in the manufacture of a medicament containing a stem cell for treating or preventing a disease, disorder or condition in which the use of the stem cell is appropriate. The use of a single cell preparation is provided.

[0078] In one embodiment, the feeder-cell preparation can take any form of the embodiments described in the feeder-cell preparation.

[0079] In one embodiment, the feeder-cell preparation is treated so as not to proliferate! It is.

[0080] In one embodiment, the feeder-cell preparation is treated with mitomycin C.

[0081] Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, with reference to the accompanying drawings.

The invention's effect

[0082] The present invention provides a technique for efficiently establishing stem cells in particular. In particular, in primate cells, it has become possible to establish a cell line with a high efficiency of 50% or more (preferably 80% or more) of the present invention, which has not been achieved conventionally. In particular, it is clear that the stem cell preparation prepared using the feeder cell of the present invention contains less than 50% of differentiated cells, and can achieve a low rate of less than 10% or less than about 1%. Became. Thus, the present invention provides a powerful and highly pure preparation of pluripotent cells that could not be achieved conventionally.

Brief Description of Drawings

FIG. 1A is an explanatory photograph of a monkey ES cell line established by the method of the present invention. No. 1 indicates 100 / zm.

[FIG. 1B] FIG. 1B is an explanatory photograph of a monkey ES cell line established by this method. Bars indicate 50 μm.

FIG. 2A is a photograph of human ES cells. Bars indicate 275 μm.

FIG. 2B is a photograph of human ES cells. The bar indicates 70 m.

FIG. 3 shows undifferentiated state-specific marker expression.

[FIG. 4] FIG. 4 is a diagram illustrating the division of nerve cells.

[FIG. 5] FIG. 5 is a proof of the supposedly terrifying ability by teratoma formation.

FIG. 6 shows an example of an ES cell establishment scheme.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Therefore, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of the plural unless specifically stated otherwise. . Also used in this specification It should be understood that the terms used are used in the meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . In case of conflict, the present specification, including definitions, will control.

[0085] The definitions of terms used particularly in the present specification are listed below, necessary techniques are explained, and the present invention is described.

[0086] As used herein, "cell" is defined in the same broadest sense as used in the art, and is a constituent unit of a tissue of a multicellular organism, wrapped in a membrane structure that isolates the outside world. Rarely, it refers to an organism that has a self-renewal ability inside and has genetic information and its expression mechanism. In the present invention, any cell can be targeted. The number of "cells" used in the present invention can be counted through a light microscope. When counting through an optical microscope, count by counting the number of nuclei. The tissue is used as a slice of a tissue section, and extracellular matrix (for example, elastin or collagen) and nuclei derived from cells are stained with a dye by hematoxylin-eosin (HE) staining. This tissue section can be examined under an optical microscope, and the number of nuclei per specific area (for example, 200 m × 200 m) can be counted by estimating the number of cells. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or a source of cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line. Such cell mixtures include, but are not limited to.

[0087] The cells used as the feeder cells used in the present invention include normal cells and cell lines, and preferably, cells derived from any organism as long as fibroblasts or their counterparts are present. (E.g., mammals (e.g., monotremes, marsupials, oligodonts, skin wings, winged fins, carnivores, carnivores, long nose, odd ungulates, artiodactyla, tubulars, Cells derived from squamous species, marine species, cetaceans, whales, primates, rodents, apogonidae, etc.) may be used. In one embodiment, cells from primates (eg, chimpanzees, macaques, humans), particularly cells from humans, Cells are used, but are not limited thereto. In another embodiment, preferably, the feeder cell is a rodent cell, and more preferably, a mouse cell is used as the feeder cell.

[0088] The cells used as stem cells used in the present invention include cells derived from any organism (eg, mammals (eg, monopores, marsupials, oligodentates, Skin wings, winged hands, carnivores, carnivores, longnoses, hoofed horses, artiodactyla, tube teeth, scales, squids, cetaceans, primates, primates, rodents, etc. ) -Derived cells) may be used. More preferably, cells derived from primates (for example, chimpanzees, Japanese macaques, and humans) are used as stem cells. Most preferably, human-derived cells are used as the stem cells.

[0089] As used herein, the term "counterpart", for example, when used for a cell, refers to a cell in a certain organism, and refers to a cell of another species having similar properties and Z or function. Such similar properties can be identified by secreted cytokins, growth factors, expressed cellular markers, and the like.

[0090] As used herein, the term "stem cell" has a self-renewal ability and a pluripotency (that is,

("Pluripot _e ncy"). Stem cells can usually regenerate tissue when it is damaged. As used herein, a stem cell can be, but is not limited to, an ES cell or a tissue stem cell (also a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell). In addition, artificially created cells (eg, reprogrammed cells, etc.) can also be stem cells, as long as they have the above-mentioned ability.

[0091] As used herein, "embryonic stem cells" and "ES cells" are used interchangeably and refer to any pluripotent stem cells derived from an early embryo. Generally, ES cells are considered to be totipotent or nearly totipotent. When the chimera was prepared by introducing the ES cells into a normal host blastocyst and returning to the foster parent uterus, a germline chimera (a chimeric mouse having functional germ cells derived from ES cells) having high chimera-forming ability was obtained. ) Was obtained (A. Bradley et al .: Nature, 309, 255, 1984). Various gene transfer methods (for example, a calcium phosphate method, a retrovirus vector method, a ribosome method, an electoral poration method, etc.) can be applied to the ES cell line in culture. We also devised a method to select cells into which genes have been incorporated. However, it is also possible to obtain a clone of a cell that has been modified (substitution, deletion, insertion) by targeting homologous gene recombination (homologous recombination). Since ES cell lines treated in this way retain their ability to germline in vitro, studies to examine the function of certain genes at the individual level are currently being actively conducted (MR Capecchi : Science, 244, 1288, 1989). The transgenic mouse production method using ES cells has many advantages over the transgenic animal production method using microinjection in that it makes it possible to obtain individuals in which only certain genes have been arbitrarily modified. Benefits are conceivable. In particular, a knockout animal in which a specific gene has been inactivated can be produced, and the function of the gene can be elucidated, or only the exogenous gene can be expressed. Therefore, if the establishment of ES cells is facilitated, the effect is immeasurable.

[0092] As used herein, the term "tissue stem cell" refers to a cell that has a limited direction of differentiation, unlike ES cells. Normally, tissue stem cells are present at specific positions in a tissue and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells are poor in organelles with a high nuclear Z cytoplasmic ratio. Tissue stem cells generally have a probable ability to maintain their proliferative capacity over the life of an individual whose cell cycle is slow. As used herein, ES cells are preferably used as stem cells, but tissue stem cells may also be the object of maintenance.

[0093] ES cell lines are extremely important in producing chimeric mice, knockout mice, and the like, and the analysis of gene function has been dramatically advanced by these techniques. The development of a system for inducing specific fibrous tissue from ES cells has been progressing, and the system has been applied to transplantation medicine. Considering clinical application using human ES cells, preclinical research using ES cells from appropriate model animals such as monkeys is considered to be almost as important as essential. Standing is also important.

[0094] Establishment of an ES cell line is performed using a single feeder cell. Usually, ES cells are established by culturing a blastocyst-separated inner cell mass (ICM) on a single feeder cell. Heretofore, mouse fetal fibroblasts or cell line STO derived therefrom have conventionally been used alone. However, it has been reported that these cells have a low establishment efficiency, especially primate stem cells, and contaminated cells with a remarkably low level of about 10% to 30%. Ma In addition, this efficiency varies depending on the stage of the blastocyst used, the ICM separation method, and the like. Therefore, in the current system with low establishment efficiency, several trials and errors were necessary to succeed in establishment. Using the present invention, the efficiency is over 80%, the contamination of differentiated cells is drastically reduced, and the need for such trial and error is eliminated.

Next, a general method for establishing ES cells that can be used in the present invention will be described.

In terms of mice, embryos usually develop into blastocysts 3.5 days after fertilization. The inner cell mass, which is an undifferentiated stem cell forming the embryo body, is separated from the blastocyst by immunosurgery. This ICM has the ability to differentiate into trophectoderm, a cell made by the placenta and the like. Therefore, if ICM is directly isolated from 3.5-day embryos, undifferentiated cell strength S is often lost due to the differentiation of ICM into trophectoderm. 3. After culturing the 5-day embryo for 1 day, 4.0-4.5 days, the blastocyst escapes the zona pellucida. At this stage, the ICM no longer differentiates into trophectoderm. From such a state, if the ICM is separated and culture is started, the ratio of undifferentiated cells can be increased. Therefore, even when stem cells are established in the present invention, undivided cells having such a number of days can be used. In addition, in the case of human ES cells, the number of days can be used as a reference and used as appropriate. In the case of humans, those that are 5-8 days after fertilization (excluding the freezing period) are often used, but are not limited thereto.

[0096] When establishing ES cells, blastocysts can be cultured directly on feeder cells, but in those cases, blastocysts adhere and nutritive ectoderm extends. Exposure of the ICM is also less favorable. A culture obtained by separating and culturing this may be used, but immunosurgery is preferably used. Fig. 6 shows a schematic ES cell line establishment scheme. Briefly, ICM isolated by immunosurgery is cultured on a single feeder cell, and as the passage is continued, undifferentiated stem cell colonies appear in the mixed cells. By separating and subculturing this, an ES cell line that can be stably maintained can be established. According to the present invention, since the establishment rate exceeds 80%, the efficiency of obtaining ES cells that can be stably maintained is significantly improved.

[0097] In order to establish the ES cells, the feeder cell of the present invention was used to prepare the cells of the feeder, PBS (containing no Ca and Mg), 0.25% trypsin 'ImM EDTA in PBS, a medium for ES cells (DMEM ( Noigram course) 400ml, fetal calf serum 100ml, non-essential amino acid solution (Gibco) 4ml, Nucle Oside solution (nucleoside, guanosine, cytidine, peridine 3 mM each, thymidine ImM aqueous solution, dissolve by heating at 40 ° C, filter sterilize, and store at 20 ° C) 4 ml, 2-mercaptoethanol 4 1 (0. ImM), LIF 100 ^ l (2000 U / ml)), Μ2 medium (available from Sigma etc.), M16 medium (available from Sigma etc.), acidic Tyrode's solution (available from Sigma etc.), anti-mouse serum (5 x 10 ⁸ mouse spleen cells or lymphocytes are immunized intravenously three times every 2 weeks into a egret, and blood is collected 2 weeks after the final immunization. This is inactivated as an antiserum and stored at 80 ° C ), Guinea pig complement, and liquid paraffin (light; available as nacalitesk) are necessary. As an experimental device, CO ink

It is advisable to prepare a 2 incubator, a stereo microscope, a capillary, and a multi-well plate (for example, 4-well, 12-well, NUNC, etc. are available).

[0098] An exemplary protocol for establishing ES cells is described below. This protocol is based on the protocol described in the Human ES Cell Line Establishment Program compiled at the Institute of Regenerative Medicine at Kyoto University. In this specification, the protocol is not limited to this particular protocol, but may be any protocol. Can also be used.

[0099] (1) Thawing of frozen embryos and culture up to the blastocyst stage

Cryopreserved human fertilized eggs or early embryos up to the blastocyst stage are sequentially thawed and cultured. Since the substance that can identify the donor is removed from each frozen embryo container, the origin of the human fertilized embryo used in each thawing and culture experiment cannot be identified. However, in order to prevent the handling of each human fertilized embryo from being neglected, the human fertilized embryos stored in one cryocontainer from the time of their acceptance as frozen embryos were identified as individual entities during the establishment research. And record what progress has been made.

(2) Separation of inner cell mass and establishment of cell line

Embryos that have reached the blastocyst stage (the development period after fertilization is about 14 days or less) are separated from the inner cell mass by immunosurgery with anti-human serum, and then cultured on the feeder cell layer I do. The feeder cell of the present invention is used as the feeder cell. The remainder after collecting the inner cell mass is also handled with courtesy. The cells grown on a single feeder cell are dissociated in a timely manner, divided and subcultured, and cell cultures that are likely to be ES cells are established while performing selective culture of cell colonies that are thought to be stem cells. During this time, We will conduct research aimed at improving the culture maintenance method and cell cryopreservation method.

[0101] (3) Stem cell marker expression and chromosome assay

Detection of stem cell markers (alkaline phosphatase activity and specific antigens) is performed to confirm that the cells are ES cells. In addition, karyotype analysis is performed to test whether the number and morphology of chromosomes are normal.

[0102] (4) Differentiation ability test

In order to test the ability to divide cells in culture, induction of cell differentiation by changing culture conditions and creation of cell aggregates and analysis of the ability to differentiate into various functional cells are performed. In addition, transplantation into an immunodeficient mouse or the like is performed to analyze the tissue division ability by teratoma formation.

[0103] (5) Ensuring safety and preventing accidents

Temporary storage of human frozen embryos will use a dedicated liquid nitrogen tank to prevent contamination by viruses and microorganisms derived from cells and animal embryos other than those used for the establishment plan. In addition, by using a dedicated carbon dioxide incubator for cell culture experiments, mixing with other types of cells is prevented, and the possibility of contamination by viruses and microorganisms is reduced. The culture medium and culture equipment used for cell culture are subjected to autoclaving in a laboratory and then discarded. Strictly control the entry to the laboratory where human fertilized embryos are stored and cell lines are established.

[0104] In the present specification, "pluripotency" or "probably dandelion" is used interchangeably and refers to a property of a cell, and is divided into one or more, preferably two or more various tissues or organs. The ability to dagger. Therefore, “pluripotency” and “pluripotency” are used interchangeably with “undifferentiated” in this specification unless otherwise specified. Normally, cell pluripotency is limited as development progresses, and in adults, the constituent cells of one tissue or organ rarely change into cells of another. Thus pluripotency is usually lost. In particular, epithelial cells are unlikely to change into other epithelial cells. When this happens, it is usually a pathological condition and is called metaplasia. Forced mesenchymal cells have a high degree of pluripotency because metastasis is likely to occur in other mesenchymal cells with relatively simple stimulation. ES cells have pluripotency. Tissue Stem cells are pluripotent. In the present specification, among pluripotency, the ability to divide into all kinds of cells constituting a living body such as a fertilized egg is called totipotency, and pluripotency is totipotency. It can encompass concepts. Whether a certain cell has pluripotency includes, but is not limited to, for example, formation of an embryoid body (Embryoid Body) in an in vitro culture system, culture under conditions for inducing induction, and the like. Assays for the presence or absence of pluripotency using living organisms include the formation of teratomas by transplantation into immunodeficient mice, the formation of chimeric embryos by injection into blastocysts, and the transfer into living tissues. And proliferation by infusion into ascites, but are not limited thereto.

[0105] In the present specification, among the pluripotency, the ability to divide into all kinds of cells constituting a living body such as a fertilized egg is called "totipotency", and pluripotency is a concept of totipotency. May be included. However, when the distinction is made clearly, totipotency and pluripotency can be distinguished, the former refers to the ability to divide into any cell, and the latter has multiple directions, Has the ability to divide in all possible directions. The ability to divide in only one direction is also referred to as singularity.

[0106] In the present specification, totipotency and pluripotency can be determined, for example, by the number of days after fertilization. For example, in the case of mice, they can be distinguished on the basis of about 8 days after fertilization. Without being bound by theory, mice typically follow the following course after fertilization. 6.5 days after fertilization (also referred to as E6.5), the primitive streak (also referred to as the original streak) appears on one side of the epiblast, revealing the future anterior-posterior axis of the embryo. The original streak indicates the future posterior end of the embryo, extending across the ectoderm to the distal end of the cylinder. The original streak is the area where cell motility takes place, resulting in the formation of future endoderm and mesoderm. By E7.5, a head process appears in front of the nodule, which forms the notochord, surrounding it, the future endoderm in the lower layer and the neural plate in the upper layer. The nodules appear around E6.5 and move backwards, forming an axial structure from front to back. E8.5 By day 5, the embryos have grown somewhat longer, and large anterior folds are formed at their anterior ends, most of which also have anterior neural plate strength. The somites begin to evolve backwards at a rate of one every 1.5 hours at E8. Cells beyond this time will no longer exhibit totipotency and will not form an individual, even if they are returned to the placenta, unless dissociated. Before this point, it is possible to show totipotency without any special treatment, so this point can be said to be a branch point of totipotency. This means that it is difficult for ES cells to establish subsequent embryonic power, and subsequent embryonic power is usually EG (germ cell-derived) cells In this sense, it is a divergence point because a cell called と is established.

[0107] As used herein, the term "fetus" or "fetus" is used interchangeably and refers to a period in which the offspring of a mammal complete differentiation of each organ primordium and enter the anagen phase until power delivery. A certain organism.

[0108] As used herein, an "established" or "established" cell refers to a cell that maintains a specific property (eg, pluripotency) and proliferates stably under culture conditions. It is the state that has started to continue. Therefore, established stem cells maintain pluripotency. Established differentiated cells have certain defined functions. Established differentiated cells are often cancerous, but are not limited thereto.

[0109] As used herein, the term "differentiation" as used herein generally refers to the separation of one system into two or more qualitatively different systems. When used for, it means that the function and Z or form are special. With differentiation, pluripotency usually decreases or disappears.

[0110] As used herein, the term "divided cells" refers to cells whose functions and morphology are specialized (for example, muscle cells, nerve cells, and the like). No or little potential. The differentiated cells include, for example, epidermal cells, spleen parenchymal cells, splenic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, and pigments Cells, smooth muscle cells, fat cells, bone cells, chondrocytes and the like. When used in the present invention, the sorting cells may be in the form of a population or a tissue. Incorporation of shunt cells into a stem cell preparation is often detrimental in the use of stem cells. It has been difficult in the prior art to reduce such shunt cells.

[0111] In the present specification, "somatic cells" are cells other than germ cells such as eggs and sperm, and refer to all cells that do not directly transfer their DNA to the next generation. Somatic cells usually have a limited or pluripotent force or disappear. As used herein, somatic cells may be naturally occurring or genetically modified.

[0112] As used herein, "normal cells" refers to any cells that are not in a pathological state. Therefore, such normal cells often have a finite number of divisions. Such finite split times The number is usually 50-80 times.

[0113] As used herein, "immortalization" refers to the fact that cells do not die even after a finite number of divisions (for example, 50 to 80 times).

[0114] As used herein, the term "cell line" or "cell line" refers to a cell that survives the above-mentioned finite number of divisions. Such cell lines are obtained by sequentially subculturing passaged cells, such as primary cultured cells, and selecting cells that survive after passage beyond the number of passages at which normal cells usually die. Can be established. Such establishment methods are known in the art.

[0115] As used herein, the term "fibroblast" refers to a cell that supplies a fiber component of a supporting tissue and forms an important component of a fibrous connective tissue. Tissue section diagrams often have a flat, long outline and irregular projections. The cytoplasm contains mitochondria, Golgi apparatus, centrosomes, small fat globules, etc., but does not show any special restriction. The nucleus is elliptical and often lies close to collagen fibers.

[0116] In the present specification, "feeder layer" or "feeder cell" (feeder layer or feeder cell) is used interchangeably and is a cell provided on a culture substrate and which cannot be maintained alone by culture. Refers to a feeder layer with other cell types that allows the growth of the species and the expression of the Z or sliver trait. It is said that there are many types of tissue cells that cannot express the shunting phenotype or even proliferate under normal cell culture conditions. Such cells include, for example, stem cells (especially, ES cells, neural cells). Stem cells, mesenchymal stem cells, hematopoietic stem cells, etc.), embryonic germ cells (EG cells), germ stem cells (GS cells), and the like, but are not limited thereto. These cell types generally require specific auxotrophic growth and differentiation inducing factors. By utilizing a specific cell layer formed by supporting cells of the cell type or cells similar to the cell type in the living body as a culture substrate, the factors required by the cell type to be cultured and Z Or, by being supplied with a nutrient source, they grow and divide. The cell type to be used as a feeder cell is selected depending on the target cell type, but it is often used by suppressing cell proliferation by a method such as UV irradiation. The ability to culture germ cells, ES cells, hematopoietic stem cells, etc., is largely dependent on the further utilization of feeders. Conventionally, as a feeder cell, Mouse fibroblasts (primary cultured cells or cell lines) are used, and the efficiency of stem cell establishment is said to be about 10%. In the present specification, a preparation containing such a feeder-cell is referred to as a “feeder-cell preparation”. Further, in the present invention, when a cell is a mixture of two or more cells and is used as a single feeder cell, it may be referred to as “a single feeder cell mixture” or “a single cell mixture”.

[0117] As used herein, the term "STO cell" refers to an established mouse fibroblast cell line, and includes a cell deposited as CRL-1503 in the ATCC. STO cells are derived from the SIM fibroblast cell line, are resistant to 6-thioguanine and ゥ abain, are HG PRT— (HPRT—), and are said to be sensitive to HAT. Conventionally, STO cells have been used as feeder cells, and their targets include the strength of ES cells, teratocarcinoma, and hybridomas. The ability to use any culture conditions as appropriate to maintain STO cells.For example, add 4 mM L-glutamine to DMEM, add 1.5 gZL sodium bicarbonate and 4.5 gZL glucose, and It can be cultured using 10% serum (FCS). For passage of STO cells, remove the medium, rinse with 0.25% trypsin, 0.03% EDTA solution, remove the solution, and add 1-2 ml of trypsin-EDTA solution. Then, move the container to room temperature (or 37 ° C) to detach the cells. Then fill with fresh medium, aspirate, and then dispense into new culture vessels to complete the passage. The culture vessel may be coated with gelatin. It is recommended, but not limited to, passage at 1: 3—1: 10. As the STO cells, those deposited with the ATCC may be used, or irradiated STO cells (eg, deposited with ATCC as 56-X) may be used.

[0118] In the present specification, "SL10 cell" is a sub-established cell line of STO cell line, and its establishment method is described in Kawase E., et al., Int. J. Dev. Biol. 38: 385-390 (1994), preparation of feeder cells can be performed according to the method of Kawase et al. [Kawase et al., Experimental Medicine, Vol. 10. No. 13 (extra), 1575-1580, 1992]. . Robertson E. J. (ed) Teratocarsmomas and Emryonic Stem Cells, IRL Press, New York (1987), which is incorporated herein by reference. To do. Briefly, transfected Hue Tato the STO cells with the vector containing the neo ¹ gene. Thereafter, the colonies that have become neomycin resistant are amplified, and their suitability as feeder cells is confirmed. In this case, the ES cell line CCE can be used for the test, but is not limited thereto. SL10 cells are selected for good maintenance of undifferentiated stem cell colonies of CCE. SL10 cells are said to be suitable as a feeder because of their good adhesion to gelatin.

[0119] As used herein, the term "primary cultured cell" refers to a cell that has been cultured until the first passage, in which cells, tissues, organs, and the like separated from the body are implanted. It is understood in this document that passages performed up to about 5 times fall within the definition of primary cultured cells herein. As the primary culture cells, it is usually preferable to use cells without any modification, but it is also possible to use cells inactivated with mitomycin C or X-rays.

[0120] The cells that have been passaged herein are also referred to as "passaged cells".

[0121] As the cells of the present invention, any culture solution can be used as long as the cells are maintained or differentiated into desired differentiated cells. As such a culture solution, for example, DMEM, M2, M16, P199, MEM, HBSS, Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), M199, MEMa, DMEM / F12 = 1: 1, L-15 and the like, but are not limited thereto. Such cultures include adrenocortical steroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate 2-phosphate, ascorbic acid and its derivatives, glycemic phosphate, Estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, growth factors such as aFGF, bFGF, EGF, IGF, TGF jS, ECGF, BMP, PDGF, pituitary extract, pineal extract, retinoic acid, vitamin D , Thyroid hormone, calf serum, calf serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (such as sodium selenite), linolenic acid, 3-isobutyl-1-methylxanthine Demethylating agents such as 5-azancytidine; Histone deacetylating agents such as costatin, activin, sitin such as'1-2'-1'-6, hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (db cAMP) , Dimethylsulfoxide (DMSO), eododexperidine (IdU), hydroxy ゥ Rare (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, etc. may be contained as one or a combination thereof.

[0122] As used herein, the term "diversion factor" refers to both a "differentiation promoting factor" and a factor known to promote diversion to differentiated cells (for example, a chemical substance, Temperature, etc.). Such factors include, for example, various environmental factors, such as temperature, humidity, pH, salt concentration, nutrition, metals, gases, organic solvents, pressure, and chemicals. (Eg, steroids, antibiotics, etc.) and any combination thereof, but are not limited thereto. Representative of such factors are DNA demethylating agents (such as 5-azacytidine), histone deacetylating agents (such as trichostatin), nuclear receptor ligands (such as retinoic acid (ATR A), vitamin D, T3, etc.), cell growth factors (activin, IGF-1, FGF, PDGF, TG

Three

F-β, BMP2 / 4, etc.), cytotoxicity (LIF ゝ IL2, IL-6, etc.), hexamethylenebisacetamide, dimethylacetamide, dibutyl cAMP, dimethylthiol sulfoxide, iododexiridine, hydroxyl urea, Examples include, but are not limited to, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyperidine, polypropylene, selenium, and the like.

[0123] Specific examples of the diversion factor include the following.

[0124] A) Nerve: NGF, CNTF, EGF, FGF;

B) Blood cells: VEGF, EPO, interleukins and interferons (eg, interleukin 2, etc.);

C) Liver: HGF

D) Bone, cartilage: BMP.

[0125] As used herein, "in vivo" or "in vivo" (in vivo) refers to the inside of a living body.

In a particular context, "in vivo" refers to the location where the tissue or organ of interest is to be located.

[0126] As used herein, the term "in vitro" refers to a state in which a part of a living body is extirpated or released (for example, in a test tube) for various research purposes. Say. A term that contrasts with in vivo.

[0127] As used herein, "ex vivo" (ex vivo) refers to the extraction of a target cell for gene transfer from a subject, the introduction of a therapeutic gene in vitro, and then the same subject again. When returning, a series of actions is called ex vivo.

[0128] In this specification, the term "self" or "self" refers to an individual or a part thereof (eg, a cell, a tissue, an organ, etc.) derived from the individual. As used herein, the term self may broadly include a transplant from another genetically identical individual (eg, an identical twin).

[0129] In the present specification, the allogeneic (allogeneic) refers to an individual who is the same species but is genetically different from another individual to be transplanted or a part thereof (eg, cells, tissues, organs, etc.). Say. Due to their genetic differences, allogenes can elicit an immune response in the transplanted individual (recipient). Examples of such cells include, but are not limited to, cells derived from the parent.

[0130] In the present specification, the term "heterologous" refers to a substance which is transplanted into a heterologous individual. Thus, for example, where a human is the recipient, a transplant from a pig is referred to as a xenograft.

[0131] As used herein, the term "recipient" (recipient) refers to an individual that receives transplanted cells and the like, and is also referred to as a "host". On the other hand, an individual that provides cells or the like to be transplanted is called a “donor” (donor). Recipient and donor may be the same or different

[0132] The cells used in the present invention may be derived from syngeneic (autologous (autologous)), allogeneic (from another individual (heterologous)), or heterologous. Autologous cells are preferred because rejection is likely, but if rejection is not an issue, it may be allogeneic.

[0133] In the present specification, "transplantation" means that the cells, compositions, medicaments, and the like of the present invention are transferred into the body alone or in combination with other therapeutic agents. The present invention can use the following methods, forms and amounts of introduction into a treatment site (eg, bone, etc.): And then insert it. Combinations of the adipose stem cells of the present invention and the sorting cells may be, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. May be administered. This includes presentation in which the combined drug power is administered together as a therapeutic mixture, and also includes procedures in which the combined drugs are administered separately but simultaneously (eg, a differentiation promoting factor). "Combination" administration or transplantation further includes separately administering one of the cells, drugs, compounds or agents given first, followed by the second.

[0134] As used herein, "maintenance", when used for a cell, tissue or organ, refers to substantially retaining its function and Z or morphology. For example, maintaining the cornea means having the function and Z or morphology of the cornea that the subject should normally have without substantially damaging the subject. The function includes maintaining the visual acuity and the form includes maintaining the appearance. Listed, but not limited to.

[0135] As used herein, "regeneration" refers to a phenomenon in which, when a part of an individual's tissue or organ is lost, the missing tissue is supplemented and restored. Depending on the species between animals or tissue in the same individual, the extent and mode of regeneration will vary. Many human tissues have a limited ability to regenerate, and complete loss cannot be expected if they are severely lost. In a major injury, incomplete regeneration can occur in which a highly proliferative tissue that is different from the lost tissue proliferates, incompletely regenerating the tissue and ending in a state in which function cannot be restored. In this case, by using a structure that also has a bioabsorbable material strength, the proliferation power to the tissue defect is prevented, and the invasion of the tissue is prevented, thereby securing a space in which the original tissue can proliferate. Regenerative medicine is being used to increase the ability of native tissues to regenerate by supplementing growth factors. An example of this is regenerative medicine for cartilage, bone and peripheral nerves. Alternatively, if the stem cells established by the method of the present invention are used, any tissue can be regenerated in principle, and the organs, tissues and cells of the present invention thus prepared can be regenerated. Provided as an implant for Cells can be classified according to their origin into ectoderm, mesoderm and endoderm-derived stem cells. The cells derived from the ectoderm mainly exist in the brain, and include neural stem cells and their cells. Mesodermal-derived cells are mainly present in bone marrow, and include vascular stem cells and their differentiated cells, hematopoietic stem cells and their differentiated cells, mesenchymal stem cells and their cells, and the like. Endoderm-derived cells are mainly present in organs, and include hepatic stem cells and their cells, knee stem cells and their cells. Honmei In the text, the cells can be from any germ layer.

In the present invention, stem cells can be genetically modified. Since stem cells are cultured cells, various gene transfer methods (eg, calcium phosphate method, ribosome method, microinjection method, electoral poration method, etc.) can be used just like other cultured cells. The gene that can be introduced is not limited, and examples thereof include genes derived from bacterial, animal or human chromosomes, but are not limited thereto. Similarly, modification by homologous gene recombination of an endogenous gene using a targeting vector used as a method of gene targeting using an ES cell line is also possible.

[0137] The disease, disorder, or condition that can be targeted by the method of the present invention includes any disease, disorder, or condition in which regeneration of an organ or tissue is desired. The present invention is particularly advantageous for diseases, disorders and conditions related to organs, fibrils or cells requiring stem cells which cannot be regenerated without feeder cells.

[0138] Such diseases and disorders that can be targeted by the present invention include: diseases or disorders of the circulatory system (such as blood cells); diseases or disorders of the nervous system; diseases or disorders of the immune system; Diseases or disorders of the skin; diseases or disorders of the skin; diseases or disorders of the endocrine system; diseases or disorders of the respiratory system; diseases or disorders of the digestive system; diseases or disorders of the urinary system; Obstacles, but are not limited to them.

[0139] The animal targeted by the present invention is any animal that has stem cells (for example, metal eel, scallop, cartilaginous fish, teleost, amphibian, reptile, bird, mammal, etc.). It may be. Preferably, such an animal is a mammal (e.g., monotremes, marsupials, oligodentates, dermis, winged fins, carnivores, carnivores, longnoses, snouts, even Hoofs, tube rodents, squamata, sponges, cetaceans, primates, rodents, egrets). Exemplary subjects include, but are not limited to, animals such as, for example, horses, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, power-quisars, diplomatic monkeys, humans) are used. Most preferably, it is intended for humans.

[0140] When the present invention is used as a medicament, such a medicament may further include a pharmaceutically acceptable carrier and the like. A pharmaceutically acceptable carrier contained in the medicament of the present invention; For example, any substance known in the art can be used.

[0141] Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents , Buffering agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the invention will be administered in the form of a composition comprising the cells of the invention together with one or more physiologically acceptable carriers, excipients or diluents. For example, suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.

[0142] As used herein, an acceptable carrier, excipient, or stabilizer is non-toxic to the recipient, and is preferably inert at the dosages and concentrations employed. Eg, phosphate, citrate or other organic acids; ascorbic acid, a-tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polybutyl) Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA); For example, mannitol or sorbitol); salt-forming counterion (Eg, sodium); and Z or non-ionic surfactants (eg, Tween, pluronic or polyethylene glycol (PEG)), and the like.

[0143] Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizate using suitable excipients (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired.

[0144] If necessary, the medicament of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener, and the like, and other agents.

[0145] The amount of the drug used in the treatment method of the present invention is determined in consideration of the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, medical history, cell morphology or type of the patient, etc. Then, those skilled in the art can easily determine. The frequency of applying the treatment method of the present invention to a subject (or patient) also depends on the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, medical history, and course of treatment of the patient. A person skilled in the art can easily determine this in consideration of such factors. The frequency includes, for example, administration once every few months (for example, once a week, once a month). It is preferable to administer once a week once a month while observing the progress. The amount to be administered can be determined by estimating the amount required by the site to be treated.

[0146] In the present specification, the "instruction" refers to a method of administering or diagnosing the medicament or the like of the present invention for a physician, a patient or the like who administers or diagnoses (possibly a patient). It is described. This instruction describes a word indicating a procedure for administering the diagnostic agent, the medicine and the like of the present invention. This instruction is prepared in accordance with the format prescribed by the competent authority of the country where the present invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) and is issued by the competent authority. The approval is clearly stated. Instructions are so-called package inserts, which are usually provided on paper media, but are not limited to, for example, electronic media (eg, homepages (websites) provided on the Internet, emails, etc.). ) Can also be provided.

[0147] Judgment of the termination of treatment by the method of the present invention may be based on the results of standard clinical tests using commercially available atseys or devices or the above-mentioned diseases (eg, neurological diseases, cardiac diseases, etc.). Can be supported by the disappearance of various clinical symptoms. Treatment can be resumed upon recurrence of the disease (eg, neurological disease, heart disease, etc.).

[0148] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components (eg, stem cells such as ES cells) of the medicament of the present invention. A notice in the form of a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products. Or a government approval for sale.

[0149] When the present invention is used as a research reagent, the stem cells of the present invention are desirably distributed according to ethical rules set by the authorities. Such rules include, but are not limited to, those stipulated by the Japanese government. The following is an example of the rules. The

[0150] In accordance with the tasks and duties of the institution, as stipulated in government guidelines, etc., and in response to a request from the institution having a research plan approved in accordance with the guidelines on the establishment and use of human ES cells, Distribute cell lines that have undergone post-mortem characterization. The timing of the start of the distribution is probably from the time when the cell lines that have been tested and confirmed for normal karyotype and the number of frozen cell samples necessary for cell line preservation are obtained. It is contemplated to begin distribution.

[0151] At the start of establishment and distribution, the distributor is responsible for the distribution. Distributors should have clean rooms and equipment necessary for cell culture and a liquid nitrogen tank for cell cryopreservation. It is used by the human ES cell line establishment team, and its staffing can be, for example, five researchers (two of whom are preferably doctor's license holders). At the start of cell distribution, about two additional assistants for experimentation and one assistant clerk for office work may be assigned to work. It is preferable to increase the number of personnel and facilities in response to the increasing demand for cell distribution and training.

[0152] Examples of the distribution method and conditions include the following: The priority should be given to securing a cell stock at an institution required for maintaining and growing the ES cell line.

[0153] If the donor of stem cells is a human, it is usually necessary to obtain informed consent

[0154] For human fertilized embryo donor candidates, it is determined whether or not informed consent is given when requesting a frozen embryo for ES cell research according to the following procedure ^! Confirm. Provides an exemplary overview of the process from initiation of fertility treatment to the decision to dispose of frozen embryos, as well as related documentation, followed by a process for submission to ES cell research

[0155] (1) Until it is decided that human fertilized embryos created and cryopreserved as a result of fertility treatment should be discarded without being transferred to the uterus, fertility treatment completely unrelated to ES cell research The process is a clinical problem with the patient and the fertility physician.

[0156] (2) The fertility treatment physician is responsible for neutralizing frozen embryos that have been decided to be discarded. Ask the candidate to receive an explanation of ES cell research while keeping the field.

[3] (3) For the donors who indicated their willingness to receive explanations, the explainer of the institution (other than the chief of establishment) asked the ES cell what is ES cell and how to apply it to future medical treatment. A thorough explanation of the possibilities, an overview of the research, that the establishment of the ES cell line does not benefit or detriment the donor, and that the privacy of the donor will be protected, will be fully explained. These explanations are given in a completely neutral way.

[0158] (4) After receiving the explanation, the providing candidate responds to the head of the providing medical institution whether or not he / she agrees with the providing. Consent requires the signing of informed 'consent' documents, which will be kept securely by the provider. The results of consent or disagreement by a particular candidate will not be communicated to the relevant parties.

[5] (5) The signing ability of the consent of the donor is also given a grace period of at least one month, and if there is no change in the intention of the donor, the frozen embryo is transferred to the institution. At that time, multiple frozen embryos from the donor are delivered at the same time, and the label for identifying the donor is completely removed from the frozen embryo container. Therefore, the instructor of the institution will meet with multiple donors, but will not know the personal information such as their names, nor will they know who has agreed to become a donor. Since there is no such thing, the anonymity of the provider can be guaranteed. In addition, since only a part of the embryonic cell lines are established using frozen embryos from multiple donors, it is important to determine which donor's embryos actually established the ES cell lines between Do not know both.

[0160] In the present specification, the "kit" refers to a unit to which a part to be provided (eg, a reagent, a particle, or the like) is provided, usually divided into two or more sections. This kit form is preferred when it is intended to provide a composition such that it should preferably be mixed and used shortly before use, which should be provided as a mixture. Advantageously, such kits will preferably include instructions describing how to process the provided components (eg, reagents, particles, etc.). Such instructions may be in any medium. Examples of such medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Transmission media include, for example, the Internet, intranets, , LAN, etc., but are not limited to them. Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.

[0161] (Transgenic organisms)

The stem cells of the present invention can be used to generate transgenic mice. General techniques for producing transgenic mice are described in International Publication WO 01Z13150 (Ludwig Inst. Cancer Res.). U.S. Pat.No. 4,873,191 (Wagner et al.) Teaches mammals having exogenous DNA obtained by microinjection of DNA into mammalian zygotes; The description is incorporated herein. .

[0162] In addition, various methods for producing transgenic organisms are described, for example, in M. Markkula et al., Rev. Reprod., 1, 97-106 (1996); RT Wall et al., J. Dairy S. Ci., 80, 2213-2224 (1997); JC Dalton, et al., Adv. Exp. Med. Biol., 411, 419-428 (1997); Pu and H. Lubon et al., Transfus. Med. Rev., 10 , 131-143 (1996), but are not limited thereto. Each of these documents is incorporated herein by reference.

[0163] Under such circumstances, in the last decade or so, analysis of transgenic (including knockout and knockin) animals through homologous recombination of embryonic stem (ES) cells has been conducted for the purpose of analyzing gene function. It has become an important tool.

[0164] In higher organisms, efficient selection of recombinants is performed by, for example, positive selection using a neomycin resistance gene and negative selection using the thymidine kinase gene or diphtheria toxin gene of HSV. Homologous recombinants are selected by PCR or Southern blotting. In other words, a part of the target gene is replaced with a neomycin resistance gene for positive selection, etc., and a targeting vector having the HSVTK gene, etc. for negative selection ligated to its end, and introduced into ES cells by electoporation. , G418 and ganciclovir, and the resulting colonies are isolated and homologous recombinants are selected by PCR or Southern blot.

[0165] As described above, the force or the loss of function by replacing or destroying the endogenous target gene The method of producing a transgenic (target transgenic) mouse having an altered mutation is useful for analyzing the function of the gene because the mutation is introduced only into the targeted gene.

[0166] After selecting a desired homologous recombinant, the obtained recombinant ES cells are mixed with normal embryos by a scutellum injection method or an assembly chimera method to produce a chimeric mouse of ES cells and a host embryo. . In blastocyst injection, ES cells are injected into blastocysts with a glass pipette. In the assembly chimera method, a mass of ES cells is adhered to an 8-cell stage embryo from which the zona pellucida has been removed. The blastocyst into which the ES cells have been introduced is transplanted into the uterus of a surrogate mother who has been pseudopregnant to obtain a chimeric mouse. Since ES cells have totipotency, they can be divided into all kinds of cells including germ cells in vivo. When a chimeric mouse having germ cells derived from ES cells is crossed with a normal mouse, a mouse having a heterologous ES cell chromosome is obtained. A mouse is obtained. To obtain transgenic mice homozygous for the modified chromosome from the resulting chimeric mice, male chimeric mice and female wild-type mice are bred to produce F1 generation heterozygous mice. And breeding female heterozygous mice to select F2 generation homozygous mice. Whether or not the desired gene mutation has been introduced into each generation of F1 and F2 can be determined by methods commonly used in the art, such as Southern blotting, PCR, and decoding of the base sequence, as in the case of recombinant ES cells. Can be analyzed using

[0167] As a next-generation technology that overcomes the inability to selectively analyze various gene functions, a technology that combines cell type-specific expression of Cre recombinase and site-specific recombination of Cre- CreοχΡ has attracted attention. Being done. A transgenic mouse using Cre-ΙοχΡ inserts a neomycin resistance gene at a position that does not inhibit the expression of the target gene, and inserts a targeting vector that inserts the する οχΡ sequence with an exon to be deleted later. The cells are introduced, and then the homologous recombinant is isolated. A chimeric mouse is obtained from the isolated clone, and a genetically modified mouse is produced. Next, when this mouse is crossed with a transgenic mouse that expresses Cre, a site-specific recombination enzyme derived from the P1 phage of Escherichia coli, the gene is disrupted only in the tissue that expresses Cre. In the Cre, the ΙοχΡ sequence (34bp) is specifically recognized, and the sequence sandwiched between two ΙοχΡ sequences To cause recombination, which is destroyed). Cre can be expressed in adults using the ability to breed with a transgenic mouse having a Cre gene linked to an organ-specific promoter, or a virus vector having a Cre gene.

[0168] As a method for analyzing a specific gene, a gene trap (gene trap) method has attracted attention. In the gene trap method, a reporter gene without a promoter is introduced into a cell, and when that gene is accidentally inserted into the genome, the novel gene is isolated by utilizing the expression of the reporter gene ( Trap). The gene trap method is a method for efficient insertion mutation and identification of unknown genes based on mouse early embryo manipulation, ES cell culture, and gene targeting by homologous recombination (Stanford WL., Et al.). al., Nature Genetics 2: 756—768 (2001)). In the gene trap method, introduction of a gene, selection of an inserted mutant, and phenotyping thereof are relatively easy.

[0169] In the gene trap method, for example, a gene trap vector in which β geo, a fusion gene of lacZ and neo, is linked between a splicing Z receptor sequence and a poly-A-added signal is introduced into ES cells. When selecting with G418, only clones that accidentally trap the gene expressed in ES cells are selected.

When the clonal chimeric embryo thus obtained is produced, various X-gal staining patterns are exhibited depending on the expression pattern of the trapped gene. Thus, in the gene trap method, an unknown gene is isolated, its gene expression pattern is analyzed, and the gene is destroyed.

[0171] As described above, the present invention can be used for production of various transgenic organisms.

[0172] (General technology)

The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Loning: A Laboratory Manual, Cold pring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocols in Molecular Biology, Greene Pub. AssociatES and Wiley- Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley—Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992) .Short Protocols in Molecular Biology: A Compendium of Methods. from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995) PCR Strategies, Academic Press; Ausubel, FM (1999) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al.

(1999) .PCR Applications: Protocols for Functional Genomics, Academic Press ¾'J, Experimental Medicine “Experimental Methods for Gene Transfer and Expression Analysis,” Yodosha, 1997, and the like. Portions (which can be all) are incorporated by reference.

[0173] For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical.

Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oliigonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994) .AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press. ; Hermanson, GT (1996).

Bioconjugate Techniques, Academic Press, etc., and the relevant portions are incorporated herein by reference.

(Description of Preferred Embodiment)

Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is apparent that those skilled in the art can appropriately make modifications within the scope of the present invention in view of the description in the present specification.

[0175] (Feeder-one cell preparation) In one aspect, the present invention provides a feeder-one cell preparation for preparing a stem cell, including a normal cell and a cell line. There have been no examples in which a mixture of feeder cells having such a plurality of properties has been used as a feeder cell preparation.Furthermore, conventionally, several cell types that can be used as feeder cells have been known. No substantial difference was expected between the two, and it was anticipated that combining cells would significantly improve feeder-cell function. The mechanism by which the combination of cells as in the present invention greatly improves the efficiency of stem cell line establishment and maintenance! /, And the functions of feeder cells are elucidated at the molecular level! Therefore, it is not clear that the above-mentioned factors are not completely supplied by using a single cell. Therefore, it is considered that an unexpected effect was obtained by combining a plurality of feeder cells. Thus, it is understood that, in one aspect, the invention can employ a mixture of any plurality of feeder-cells. It is understood that the feeder cell used in the present specification can be any cell that is conventionally used as a feeder cell.

[0176] It is understood that the normal cell used in the present specification can be used as long as the cell is appropriately used as a feeder cell. Examples of such normal cells include, but are not limited to, mouse primary cultured fibroblasts, rat primary cultured fibroblasts, monkey primary cultured fibroblasts, and human primary cultured fibroblasts. No.

[0177] As a method for preparing such primary cultured fibroblasts, for example, a fetus from which a head, internal organs, and the like are removed from an 11-16-day-old fetus is shredded, and the fibroblast is mainly extracted by enzymatic treatment. Then, it can be performed by using a method.

[0178] Specifically,

1. Prepare a mouse on day 12 (preferably day 11 to 16) after fertilization.

2. Remove the fetus, remove the amniotic membrane's placenta without damaging the fetus, and transfer to a new Petri dish o

3. Remove the internal organs such as the liver and intestine of the fetal head and abdomen with tweezers as much as possible under a stereoscopic microscope. Rinse excess organs from the fetus with PBS (-), ,Move.

4. Dispense 10 ml of medium into 100 mm cell culture dishes in a clean bench. Prepare a 21G needle and syringe. Put one fetus in a syringe, slowly suck up the medium in the dish, and slowly return the medium to the same dish. Repeat this operation 2-3 times.

5. Make sure that the fetus is in pieces.

6. Incubate at 37 ° C in a CO incubator.

2

t, Fibroblasts can be prepared according to the method.

[0179] Preferably, the normal cells are not immortalized. While not wishing to be bound by theory, it is believed that non-immortalization provides some of the factors necessary to maintain stem cells without differentiation.

[0180] The normal cells used in the present invention may be fibroblasts, but need not be. Preferably, fibroblasts are used. While not wishing to be bound by theory, it is important to note that fibroblasts, when given some of the factors necessary to maintain stem cells without differentiation, often do not confer suitability as feeder cells It is a conceivable force.

[0181] In a more preferred embodiment, the normal cells used in the present invention are fibroblast primary culture cells. More preferably, the normal cells used in the present invention are mouse-derived. While not wishing to be bound by theory, a mouse is preferred because of its ease of preparation. Therefore, any mammal can be used, and if another simple preparation method exists, it can be preferably used by using cells of another mammal.

. While not wishing to be bound by theory, empirical evidence suggests that the use of allogeneic fibroblasts in ES-establishment studies of Escherichia coli and Egret does not seem to be as effective as mice. Mouse cells are generally considered to be good.

[0182] In a preferred embodiment, the normal cells used in the present invention may be derived from a fetus (fetus). While not wishing to be bound by theory, it is believed that cells from the fetus provide some of the factors necessary to maintain stem cells without differentiation and are more suitable as feeder cells. is there. In addition, the cell line used in the present invention is considered to have the most different properties from the viewpoint of cytology, and therefore, cannot be provided by a cell line. This is because it is considered to have the ability to supply factors necessary for the maintenance of cells.

[0183] Therefore, in the present invention, any passage number of cells can be used as long as the ability as a feeder cell can be provided, but it is preferable that primary culture cells have a low passage number. Cells (about 4 or 5 times) are recommended, and cells with 5 or less passages, 4 or less, 3 or less, 2 or less, 1 passage, or no passage are used.

[0184] In a preferred embodiment, the normal cells used in the present invention are fibroblasts obtained by enzymatic treatment from a mouse 11-16 day old fetus.

[0185] It is understood that any cell line can be used as long as it has the ability as a feeder cell as used herein. Such cell lines are typically immortalized. Although not wishing to be bound by theory, the use of immortalized cells does not provide for normal cells such as primary cell lines, but does not provide other factors necessary to maintain stem cells without fragmentation. It is considered that some of this will be supplied.

[0186] Preferably, the cell line used in the present invention is a fibroblast cell line.

[0187] The cell line used in the present invention may have any passage period. Preferably, the passage period is 2 months or less, and more preferably, the passage period is not more than 2 months. It may be advantageous to use cells that have been for a period of less than one month. Without wishing to be bound by theory, it is possible to stably supply cells secreting the initial factor of interest by using such a cell line with a short passage time, and to obtain stem cells without differentiation. It is not necessary that the passage period be short, because some of the other factors necessary to maintain the force will be supplied adequately.

[0188] The cell line used in the present invention may be derived from a mouse, but is not limited thereto. While not wishing to be bound by theory, a mouse is preferred because of its ease of preparation. Therefore, any mammal can be used, and if another simple preparation method exists, it can be preferably used by using cells of another mammal. While not wishing to be bound by theory, it has been empirically shown that the use of allogeneic fibroblast cell lines in ES-establishment studies in Escherichia coli and egrets does not appear to be as effective as in mice. Generally, mouse cells are considered to be good. [0189] Preferably, in embodiments, the cell line used in the present invention may be derived from a fetus (fetus). While not wishing to be bound by theory, it is believed that fetal-derived cells provide some of the factors needed to maintain stem cells without differentiation and are more suitable as feeder cells It is. The normal cells used in the present invention are considered to have the most different properties from a cytological point of view, and therefore have the ability to supply factors necessary for maintaining stem cells that normal cells cannot provide. Because it is possible.

[0190] In a preferred embodiment, the cell line used is an STO line. Preferably, this cell line has the properties of neomycin resistance (neo resistance). Most preferred! In the ヽ embodiment, the cell line used in the present invention is the SL10 strain.

[0191] Normal cells (for example, primary fibroblast cells) and cell lines (for example, fibroblast cell lines) contained in the feeder-cell preparation of the present invention are typically about 1: 10- Can exist at 10: 1. Without wishing to be bound by theory, outside the range of 1:10 to 10: 1, there is little effect of using multiple feeder cells, but within the range of 1:10 to 10: 1. This is because the effect is significantly observed in the maintenance of stem cells.

[0192] Preferably, in embodiments, normal cells (eg, primary fibroblast cells) and cell lines

(Eg, a fibroblast cell line) can be about 1: 4 to 4: 1. While not wishing to be bound by theory, the use of a feeder-one cell preparation containing multiple types of cells significantly improves the effects of maintaining stem cells and possibly maintaining dangling ability. It is. A more preferred cell ratio may be a ratio of about 1: 3 to about 3: 1 normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts).

[0193] Most preferred! In embodiments, the ratio of normal cells (eg, primary fibroblasts) to cell lines (eg, fibroblasts) is approximately equal. Without wishing to be bound by theory, it is because using approximately equal amounts of multiple types of cells results in sufficient components of each other to maintain stem cells to complement each other. Therefore, those skilled in the art understand that, when referring to substantially equivalent amounts in this specification, means any ratio that is close to equivalent amounts but is optimal as a feeder cell.

[0194] In one embodiment, the cell of interest in the feeder-one-cell preparation of the present invention is an embryonic stem cell, more preferably a human embryonic stem cell. Cells like human embryonic stem cells It can be grown while maintaining pluripotency and has less than 50% differentiated cell contamination (preferably less than 30%, more preferably less than 10%, and most preferably less than 1%). It can be said that this is a powerful effect that cannot be achieved with the technology described above.

[0195] (Stem cell preparation method)

In one aspect, the present invention provides a process for culturing a stem cell on a feeder-one cell preparation containing a normal cell (eg, a primary fibroblast cell) and a cell line (eg, a fibroblast cell line). There is provided a method for preparing a stem cell, comprising: Normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts) are each normal cells (eg, primary cells) and, as long as they are established, Anything can be used. As the feeder cell, any cell may be used as long as it contains these two types of cells, but it is advantageous that the cells are uniformly mixed. Such mixing ratios are preferably about 10: 1 to 1:10, about 9: 1 to 1: 9, about 8: 1 to 1: 8, about 7: 1 to 1: 7, about 6: 1: 1: 6, approx. 5: 1—1: 5, approx. 4: 1: 1-1: 4, approx. 3: 1-1—1: 3, approx. 2: 1-1—1: 2, approx. 1: 1 (almost equivalent And the like). Preferably, approximately equal amounts are mixed. Without being bound by theory, normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts) are factors that have mutually synergistic effects (eg, stem cell maintenance). It is considered that the unexpected effect of the present invention has been achieved by complementarily secreting (required).

[0196] In one embodiment, the normal cells (eg, primary fibroblasts) used in the feeder cell of the present invention are advantageously derived from a mouse, but are not limited thereto. Therefore, any animal can be used in the present invention. Mice are technically well established experimental animals, and methods for obtaining normal cells such as primary cultured cells are also well known to those skilled in the art.

[0197] Preferably, in the embodiments, the normal cells (eg, primary cultured fibroblast cells) used in the present invention are advantageously derived from a fetus, but are not limited thereto. Conventionally, for example, primary fibroblast cells have been used as feeder cells, but those derived from the fetus are mainly used. Even when primary cultures of fetal fibroblasts are used alone, the efficiency of stem cell establishment is not so high. About 10-30%. In turn, with the feeder-cell preparations and cell mixtures of the present invention, the establishment efficiency is well over 50%, and in some cases as high as 80%. Therefore, it can be said that such a high rate of establishment was beyond imagination from the prior art. Here, a normal cell is preferably a primary cultured cell in a strict sense, but even if it is a subcultured cell, if it has a small number of passages, it has properties similar to those of the primary cultured cell. Since it can have, it can be used advantageously in the present invention. Examples of such a passage number include, but are not limited to, about 5 passages or less, 4 passages or less, 3 passages or less, 2 passages or less, 1 passage, and the like.

[0198] In one preferred embodiment, the cell line (eg, fibroblast cell line) contained in the feeder cell of the present invention is advantageously an STO line. Conventionally, the STO strain has been used as a feeder cell for stem cells, but the establishment efficiency of stem cells is still as low as 30% or less, and the cell mixture and the cell preparation of the present invention can be prepared using a feeder cell. When used as a product, the efficiency of stem cell establishment is well over 50% and can be as high as 80%. Therefore, it can be said that such a high rate of establishment was beyond imagination from the prior art.

[0199] In a more preferred embodiment, a more preferable cell line can be established by using a method for separating cells having a high ability to support an ES cell in an undivided state from STO cells. The following is a brief description of such a method.

[0200] 1. Dissociate STO cells into individual cells according to a standard method.

[0201] 2. Seed 100 to 500 dissociated STO cells in a culture dish.

[0202] 3. After culturing for several days, a colony is formed from each of the seeded cells. Therefore, culture is continued until a colony of a sufficient size is formed.

[0203] 4. Collect cells from each colony, culture them separately, grow to a sufficient amount, and establish a subline of about 100 clones.

[0204] 5. One feeder cell is used using the cells of each subclone, and ES cells are cultured using the cells to select a subline having a high ability to support an undivided state.

[0205] In one preferred embodiment, the STO strain used in the present invention is advantageously neo-resistant. Without being bound by theory, such neo-resistant Choosing a cell strain also has the power to make it easier to select one with the highest feeder effect. To neo resistance, neo ¹ gene any known gene transfer techniques in the art (e.g., transformation, full Ekushi Yung, etc.) you to achieved by introducing with the leave in.

[0206] In one preferred embodiment, the cell line used in the present invention is advantageously the SL10 strain. The SL10 strain is a cell strain isolated based on the above method. It is understood that the SL10 strain is available in Meiji Dairy Power, distributed at Kyoto University's Institute of Regenerative Medicine, and readily available to third parties.

[0207] The stem cells supported by the present invention include any stem cells, and include, but are not limited to, ES cells, tissue stem cells, and the like. In a preferred embodiment, the stem cells supported by the present invention are ES cells. ES cells are a feeder cell with high establishment efficiency The present invention is noted for achieving an establishment efficiency of 80%, far exceeding the previously achievable upper limit of 30% for ES cells Should.

[0208] In another embodiment, the stem cells supported by the present invention are tissue stem cells. Examples of such tissue stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and the like. Conventional techniques, which also require a single feeder cell for tissue stem cells, do not have a technology that achieves so much satisfaction and a particularly high establishment efficiency. Therefore, it is understood that the present invention is also applicable to tissue stem cells.

[0209] In a more preferred embodiment, the stem cells supported by the present invention are primate cells, more preferably human stem cells. Primate cells, including humans, have only recently been developed as ES cells, and research on feeder cells is still under development. In particular, it is not an exaggeration to say that feeder cells that provide satisfactory establishment efficiency do not exist at all with respect to primate stem cells, more preferably human stem cells. The height is particularly high for primate stem cells, more preferably human stem cells, and the significance is high.

[0210] The culture dish in which the feeder cells of the present invention are arranged is coated with gelatin. However, the coating is not limited thereto, and any coating may be applied as long as it enhances the culture efficiency. Such coatings include, but are not limited to, for example, any extracellular matrix (eg, fibronectin, collagen, vitronectin, etc.). The culture dish may be made of any material.For example, it is desirable that the material of the container is biocompatible, but unless a material that gives toxicity to the living body is used. It is understood that anything can be used. Examples of the material of such a container include glass, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinyl chloride, polyvinyl acetate, and polyvinyl acetate. Bull alcohol, polybutyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene 'acrylonitrile copolymer, acrylonitrile Organic materials such as butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone, or those coated with silane, poly-L-lysine, and the like can be used.

[0211] In one preferred embodiment, the feeder single cell of the present invention, about 1 X 10 ⁴ cells Z cm ² - seeded at approximately 1 X 10 ⁵ cells / cm ^2, preferably, 2-5 X 10 ⁵ It is seeded in a cell / cm ^2. Without being bound by theory, it is considered that such an appropriate cell density results in an optimal state of the secreted cell bioactive substance.

[0212] In the method of the present invention, the feeder-cell of the present invention is usually used within 5 days after being mixed and inoculated, preferably within 3 days, more preferably within 2 days, and still more preferably. Used within one day. However, it is preferable to set a fixed time after mixing. Without being bound by theory, after mixing, a certain period of time (eg, 1 hour) is a force that establishes sufficient conditions for the components necessary for stem cell maintenance to emerge.

[0213] In one embodiment, the stem cells used in the present invention are advantageously cultured in a medium containing knockout serum replacement supplement (KSR). KSR is also available in power, such as Invitrogen. When KSR is used, the ability to generally show a tendency for cell growth to be slow can be used, as long as the object of the present invention is satisfied. Or Preferably, the stem cells used in the present invention are established in a liquid medium containing fetal bovine serum. It is understood that fetal serum can be used without any problem as long as ES cells can be stably maintained for a long period of time.

[0214] The method of the present invention further includes a sub-passing step, in which collagenase is used. It is thought that the use of collagenase facilitates cell recovery and contributes to improving the establishment efficiency.

[0215] (Stem cells)

In another aspect, the present invention provides a stem cell preparation prepared by the method for preparing a stem cell of the present invention. Such stem cells have an advantageous effect different from stem cells prepared by the conventional method in that they are considered to remain in a more undivided state than stem cells obtained by the conventional method. It is thought that. The cells are preferably ES cells, more preferably primate ES cells, and even more preferably human ES cells. Such ES cells (particularly primates such as humans) have an advantageous effect that differs from stem cells prepared by conventional methods in that they remain in an undivided state at an earlier stage of development. It is considered to have Therefore, the stem cell preparation of the present invention can: 1) maintain a more undivided state than stem cells obtained by conventional methods; 2) reduce stem cells that are more normal and support tissue. 3) It is found that it has advantages such as being closer to a normal tissue, having properties, and being more normal. Compared with the conventional method (for example, Non-Patent Documents 5 and 7), the method of the present invention allows the culture to be performed more easily and stably while maintaining the undivided state. It has been shown that compared to the contamination with cells, the method of the present invention shows almost no shunt cells. In conventional reports, there are few reports with specific numerical values, but in the group of Pera (NBt; Non-Patent Document 7), about 50% are cell populations that are partially divided, In the present invention, as exemplified in the examples, it is shown that a numerical value of less than 50% and a force of about 1% or less can be achieved. It is understood that such a stem cell preparation is novel because the preparation itself is novel because it has been difficult to prepare conventionally.

[0216] (Preparation of regenerated transplant)

In another aspect, the present invention provides a transplant for regenerating an organ, tissue or cell. A method for preparing is provided. The method comprises the steps of: A) providing a stem cell capable of differentiating into a desired organ, tissue or cell; B) converting the stem cell into a normal cell (eg, a primary fibroblast cell) and a cell line (eg, a fiber Culturing on a feeder-cell preparation containing the blast cell line); and C) separating the stem cells into a transplant for regenerating a desired organ, tissue or cell. . Here, as the feeder-cell, any of the forms described in detail in the “Stem cell preparation method” and “Feeder-cell preparation” section can be used. Any stem cells can be used as long as they can be separated into desired organs, tissues or cells. Whether it can be differentiated into a desired organ, tissue or cell can be confirmed using a technique known in the art. As such a technique, after exposing the stem cells to shading conditions and then culturing for a certain period of time, it is checked whether or not the shading is performed as desired, for example, with the naked eye or by observing with a microscope. Alternatively, examples include, but are not limited to, observations by molecular biology and identification of cell surface markers.

[0217] In the preparation of the regenerative transplant of the present invention, any technique known in the art can be used for the sorting step. In one preferred embodiment, the dangling factor can be removed for culturing in the method of the present invention. Such differentiation factors include, for example, DNA demethylating agents (eg, 5-azacytidine), histone deacetylating agents (eg, trichostatin), nuclear receptor ligands (eg, retinoic acid (ATRA), vitamin D

3,

T3), cell growth factors (activin, IGF-1, FGF, PDGF, TGF-j8, BMP2 / 4, etc.), cytodynamics (LIF, IL-2, IL-6, etc.), hexamethylene bisacetate Amide, dimethyl acetoamide, dibutyl cAMP, dimethyl sulfoxide, ododeoxyperidine, hydroxyl urea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyperidine, polyprene, selenium And the like, but are not limited thereto. In order to divide the nerve, NGF and FGF can be added. In order to divide blood cells, interleukin and EPO can be added.

[0218] Desired organs, tissues or cells for the purpose of preparing the regenerative transplant of the present invention include nerves, blood cells, bones, cartilage, heart, pericardium, blood vessels, muscles, eyes, liver, spleen, intestine, Stomach, lungs, trachea, Hair, skin, and the like, but are not limited thereto.

[0219] In one embodiment, the desired organ, fibrous tissue or cell and the feeder cell are of the same species. By being the same species, it is also possible to minimize the immune response, which is preferable in terms of compatibility.

[0220] In another embodiment, the above-mentioned desired organ, tissue or cell is a primate, and the feeder cell is preferably derived from a mouse. This combination is a powerful factor in achieving a high rate of stem cell establishment efficiency of 80% or more. However, it should be understood that combinations of species other than this combination may also be preferred in the present invention. Therefore, it is understood that feeder cells derived from primates such as humans may be used.

[0221] In another embodiment, the culture of the present invention is performed ex vivo (ie, for the purpose of returning to self).

[0222] In one embodiment, the stem cells of the present invention may be those that have just been removed from a subject or those that have been cryopreserved. It is preferable to use it as soon as it is removed. This is because the establishment efficiency is high.

[0223] In another embodiment, the cultivation in the method of the present invention may be performed at 37 ° C under a saturated humidity of 5% CO under a 5% CO concentration. , DNA removal

2

Tilating agents (eg, 5-azacytidine), histone deacetylating agents (eg, trichostatin), nuclear receptor ligands (eg, retinoic acid (ATRA), vitamin D)

3, T3, etc.), cell growth factors (activin, IGF-1, FGF, PDGF, TGF-j8, BMP2Z4, etc.), cytodynamics (LIF, IL 2, IL-6, etc.), hexamethylene bisacetamide , Dimethylacetamide, dibutyl cAMP, dimethyl sulfoxide, eododexperidine, hydroxyl urea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyperidine, polyprene, selenium, etc. Preferably, one or more are included.

[0224] (Organs, etc.)

In another aspect, the present invention provides an organ, tissue or cell prepared by the method for preparing a regenerative transplant of the present invention. Such an organ, tissue or cell is similar to a normal tissue and has properties in that it can be a regenerated organ or tissue prepared by a conventional method. It is thought to have a different beneficial effect than tissue or cells.

[0225] (Organ regeneration system)

In another aspect, the present invention provides a system for regenerating an organ, tissue or cell. The system comprises: A) a container; and B) feeder cells seeded on the container, comprising normal cells (eg, primary fibroblasts) and a cell line (eg, a fibroblast cell line). Preparation. Here, it is understood that the feeder-one-cell preparation can take any form described in the above “feeder-one-cell preparation” and “stem cell preparation method”. It is understood that the container can also utilize any material, for example, take any of the forms described in the section "Preparing a Regenerated Implant".

[0226] (Cell mixture)

The present invention provides a mixture comprising normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts). This mixture can be used as a stem cell feeder cell, as demonstrated in the present invention. Normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts) can be any form as described in “Stem cell preparation method” and “Feeder-one cell preparation”. It is understood that it can be taken. Thus, it can be appreciated that substantially the cell mixture may be the same as the feeder-cell preparation.

[0227] In a preferred embodiment, the primary cultured cells in the mixture of the present invention are advantageously mouse fetal primary cultured fibroblasts. This form is advantageous because stem cells can be established with unprecedented efficiency by supplying factors such as STO cells and other factors that cannot be obtained, and stem cells supporting more normal tissues can be established. This is because it is possible to do so.

[0228] In another preferred embodiment, the fibroblast cell line in the mixture of the present invention is advantageously an STO line. The advantage of this form is that the factor supplied from mouse fetal fibroblasts can be obtained, so that stem cells can be established with unprecedented efficiency, and that stem cells that support more normal tissues can be established. It is a force that can be considered as an effect that can be done. [Methods for Regenerating Organs, etc.]

In another aspect, the present invention provides a method for regenerating an organ, tissue or cell. This method comprises the steps of: A) providing a stem cell capable of dividing into a desired organ, tissue or cell; B) converting the stem cell into a normal cell (eg, a primary fibroblast cell) and a cell line (eg, , A fibroblast cell line) and C) transplanting the cultured stem cells to a site to be treated in a subject. It is understood that the feeder cell used herein can take any of the forms described in the above "feeder cell preparation" and "stem cell preparation method". It is understood that the stem cells can also take any of the forms described in the “Feeder Cell Preparations” and “Stem Cell Preparation Methods” above. Transplantation techniques can also be administered using any technique known in the art. Such techniques include, but are not limited to, stem cell transplantation into the myocardium using a catheter, for example.

[0230] In a preferred embodiment, the method of the present invention further comprises D) a step of differentiating the stem cell. For such a dividing process, a method known in the art can be applied. It is understood that such conditions can vary depending on the target organ, tissue or cell. It is understood that those skilled in the art can, in consideration of such variations, appropriately determine and select appropriate fragmentation conditions.

[0231] (Use of mixture as feeder-cell)

In another aspect, the invention provides the use of a cell mixture comprising normal cells (eg, primary fibroblasts) and cell lines (eg, fibroblasts) as a feeder-cell preparation. provide. Alternatively, there is provided the use of a cell mixture comprising primary fibroblast cells and a fibroblast cell line as feeder cells. Alternatively, a feeder cell containing a primary cultured fibroblast cell and a fibroblast cell line in the manufacture of a medicament containing a stem cell for the treatment or prevention of a disease, disorder or condition for which the use of a stem cell is appropriate. Use of the preparation is provided.

[0232] It is understood that the feeder cell used herein can take any of the forms described in the above "stem cell preparation method" and "feeder cell preparation". Fee Dar cells are intended to support any cells, preferably stem cells. Here, it is understood that the stem cells can also take any of the forms described in the above “feeder-one cell preparation” and “stem cell preparation method”.

[0233] In another aspect, the present invention relates to a normal cell (for example, a primary fibroblast cell) and a cell line (for example, a fibroblast cell line) for producing a medicament containing a feeder cell. The use of a cell mixture comprising: It is understood that the feeder cells used herein can take any of the forms described in the “Stem cell preparation method” and “Feeder cell preparation” above. The feeder cell can support any cells, but preferably aims to support stem cells. Here, it is understood that the stem cells can also take any of the forms described in the above “Stem cell preparation method” and “Feeder-one cell preparation”. As a technique for producing a medicament, a technique known in the art can be used. A pharmaceutically acceptable carrier can be appropriately added to the medicament.

[0234] Preferably, the feeder-cell preparation is treated so as not to proliferate (for example, with mitomycin C). This allows the feeder-cell preparation to be sold in a package.

Hereinafter, the present invention will be described based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor to the following Examples, but only by the appended claims.

Example

[0236] The reagents used in the examples shown below were Wako Pure Chemical unless otherwise specified.

Sigma obtained. Animal breeding Puma, created by the National Society for Medical Researches, `` Principles of Laboratory Animal Care J and the Institute of Laboratory Animal Resource S '', published by the National Institute of Health S, `` Guide for the Care and Use of Laboratory Animals ”(NIH Publication, No. 86-23, 1985, revised), in compliance with the standards stipulated by Kyoto University and the Japanese government, and in accordance with the spirit of animal protection. In the case of human subjects, conduct experiments with the prior consent of the Ministry of Health, Labor and Welfare or equivalent governmental authorities. That The consent method is as described herein.

(Example 1: Preparation of primary cultured cells (MEF) of mouse fetal fibroblasts)

Primary embryonic fibroblast cells (also referred to as MEFs) were prepared for mouse fetal power. Specifically, it is as follows.

[0238] A part of the fetus was removed from a pregnant mouse (ICR, CLEA Japan), the cells were separated, and seeded in a medium called DMEM + 10% fetal serum (FBS). The removed cells were maintained in DMEM + 10% FBS.

(Example 2: Pretreatment of MEF cells)

Next, in order to increase the efficiency as a single feeder cell, the MEF cells prepared in Example 1 were pretreated. The following were used for the pretreatment.

[0240] Culture solution: DMEM + 10% FCS

PBS (without Ca, Mg)

0.05% Trypsin + lmM EDTA

2mgZml mitomycin C aqueous solution

0.1% gelatin aqueous solution.

[0241] Pretreatment was performed as follows.

(1) Mitomycin C was added to the confluent cells at a concentration of 10 μg / ml in the medium, and the cells were cultured for 1.5 to 12 hours.

[0243] (2) The medium was removed from the culture dish treated with mitomycin C and washed three times with PBS. Add medium

And cultured for several hours to overnight.

[0244] (3) The medium was removed, washed three times with PBS, the cells were dissociated by trypsin treatment, the medium was added, the cells were well suspended, and the mixture was centrifuged to remove the supernatant.

[0245] (4) was suspended in a concentration of cells lxl0 ⁵ / ml.

[0246] The MEF cells thus prepared were used alone or in a mixture with other cells as feeder cells in the following experiments.

(Example 3: Pretreatment of STO cells)

As STO cells, subclone SL10 cells of STO cells were used. SL10 cells were obtained from Meiji Dairies (Tokyo, Japan). In the case of SL10, 2 weeks is one feeder cell It is the limit of use, and it was subcultured in 7-10 days. DMEM + 10% FCS was used as the medium.

[0248] The STO cells were pretreated for use as feeder cells. The procedure is as follows. The following were used for the pretreatment.

[0249] Culture solution: DMEM + 10% FCS

PBS (Ca, Mg free)

0.05% Trypsin + lmM EDTA

2mgZml mitomycin C aqueous solution

0.1% gelatin aqueous solution.

[0250] Pretreatment was performed as follows.

[0251] (1) Mitomycin C was added to the confluent cells to a medium at 10 gZml, and the cells were cultured for 1.5 to 2 hours.

(2) The culture medium was removed from the culture dish treated with mitomycin C and washed three times with PBS. Add medium

And cultured for several hours to overnight.

(3) The medium was removed, washed three times with PBS, the cells were dissociated by trypsinization, the medium was added, the cells were well suspended, and the mixture was centrifuged to remove the supernatant.

(4) The cells were suspended at a concentration of 1.5 × 10 ⁵ / ml.

(Example 4: Preparation of another STO cell)

As another example, STO cell strength also subclones suitable clones. The procedure is as follows.

[0256] Establishment of ES cell line in mice. STO cells used for maintaining the passage were cell lines and were considered to be a uniform population. Among them, it was particularly excellent in establishing and maintaining ES cell lines. It has been shown to contain cell populations of the nature!

[0257] A subline of STO cells with similar properties can be isolated as follows.

[0258] (1) Dissociate STO cells into individual cells according to a standard method.

(2) About 100 to 500 dissociated STO cells are seeded on a culture dish.

[0260] (3) After culturing for several days, a strong colony is formed for each of the seeded cells. Therefore, the culture is continued until a colony of a sufficient size is formed. [0261] (4) Cells are also collected from each colony, and cultured separately to grow to a sufficient amount to establish a subline of about 100 clones.

[0262] (5) One feeder cell is used by using the cells of each subclone, and ES cells are cultured using the cells to select a subline having a high ability to support an undivided state.

[0263] The cell line subcloned in this manner can also be used as a feeder cell.

. Such subclones were pre-treated as described in Example 3.

(Example 5: Preparation of feeder single cell)

In this example, one feeder cell was prepared. As a single feeder cell of the present invention, a case where a fibroblast primary culture cell and a fibroblast cell line were used alone and a case where they were mixed were prepared. Preparation of feeder cells followed a similar protocol as shown below.

(1) A culture dish for preparing a feeder was gelatin-coated in advance. The gelatin coating was performed by covering the surface of the culture dish with a gelatin solution and incubating at 37 ° C for 1 hour or more.

(2) Pretreated STO cells and pretreated MEF cells were used alone or as a 1: 1 mixture of suspensions.

[0267] (3) The gelatin dish was removed from the culture dish coated with gelatin, and a cell sample was added. When 2 ml of the cell suspension was added to a 35 mm culture dish, the feeder cells covered the surface of the culture dish without gaps. The cell density was approximately ^2.5 × 10 ⁴ / cm ² .

[0268] The prepared feeder cells were used within 4 days.

(Example 6: Establishment of stem cells)

Next, stem cells were established using the feeder cells prepared in Example 5. The culture solutions used are as follows.

[0270] (Culture for ES cells) Medium composition:

D M E M / F 1 2 (sig ma a D 6 4 2 1) 80m

Non-essential amino acids (Gibc0) 0.8 m

20 O mM L glutamine

K S R (G i b c o) 20 m

2 1 M E 0.8 μ

Human LIF (10, ug / 1) 00 μI (1 ng / ml) Basic fibroblast growth factor (1 _mg / m1) ϋ .4 / i1 (4 ng / ml).

[0271] (Cell Dissociation Solution)

0.25% trypsin (PBS)

ImM CaCl

2

20% KSR.

[0272] (Dish)

The culture dish for making the feeder should be gelatin-coated in advance. 0.1 Cover with 1% gelatin solution (swine skin, Type A: Sigma) and incubate at 37 ° C for 1 hour or more. Remove the gelatin solution from the culture dish and add the cell suspension. After several hours, it can be used as a feeder cell.

[0273] ES cells were established using a single feeder cell as follows.

[0274] 1. The culture solution of one feeder cell was replaced with a culture solution for ES cells.

[0275] 2. The inner cell mass of the blastocyst was isolated by immunosurgery or mechanical operation. Briefly, the immunosurgical procedure is as follows.

[0276] The zona pellucida was removed by enzyme treatment or the like, and the cells were incubated in an antibody solution that reacts with the mouse surface antigen. The outermost cells, vegetative ectoderm, were then removed by incubation in complement solution, and the inner cell mass was separated.

[0277] In the case of human ES cells, the above procedure was performed while keeping in mind that an antibody solution that reacts with human cell surface antigens was used. In the case of Riki-Quisal (also available from Cary Power Co., Ltd.), the above procedure was performed while keeping in mind that an antibody solution that reacts with the monkey cell surface antigen was used.

[0278] 3. The separated inner cell mass was cultured on one feeder cell.

[0279] 4. Check that the inner cell mass adheres to the feeder cell the next day, and exchange the culture solution. Changed. Thereafter, the medium was changed every day.

[0280] After 5 to 10 days, ES cell-like cells in which the inner cell mass also proliferated became evident. Subculture was performed when the cell mass reached 100 to 200 m.

[0281] 6. Except for the culture solution, the cell dissociation solution was added.

[0282] After treatment for 7.5 to 10 minutes, ES cell-like cells came off the culture dish. The cells were picked up with a thinly drawn glass tube, raised and removed several times to divide the cell mass into several pieces. .

[0283] 8. The cell dissociation solution was washed away in the culture solution, and the cell mass was transferred onto a new feeder.

[0284] 9. The next day, the culture solution was replaced. Thereafter, the medium was changed every day.

[0285] 10. When individual cell clusters grew to about 100 to 200 µm on the feeder, subculture was performed by the same operation.

[0286] 11. If the cells were able to proliferate sufficiently (the number of cells increased to one per 35 mm culture dish), the cells could be passaged in the same manner as the normal passage operation of monkey ES cells. It is confirmed that stable culture is possible after this stage is reached.

[0287] The cells established in this example are shown in Figures 1 and 2 (Figure 1 shows monkeys, Figure 2 shows humans)

[0288] This example shows that DMEM / F12 as a basic medium can be used without any problem even with DMEM or another basic medium.

[0289] In the present example, the strength of using KSR as a supplementing component It seems that instead of KSR, commonly used fetal serum may be used. However, KSR seemed to give better results than fetal fetal serum.

[0290] Further, if necessary, 1-2 mg / ml collagenase was added thereto. With the addition of collagenase, it appears to be slightly upright.

(Example 7: Staining with marker)

Next, the undifferentiated state of the cells established in Example 6 was examined by staining with a specific marker. Embryonic stem cells were fixed with 4% PFA and immunostained in standard cultured cells (Willingham, MC et.al., 1985.An Atlas of Immunofluorescence in Cultured Cell, Academic Press, Orlando, FL, pp. 1-13. Staining was performed. Blocking was performed using 0.1% Triton X / PBS / 2% skim milk for 1 hour at room temperature, and washing was performed 4 times using 0.1% Triton XZPBS for 5 minutes at room temperature. The primary antibody was a 200 μg / ml (CL ONTECH) 1Z100 diluted monoclonal antibody of SSEA-4 and TRA-1-60, respectively, and the secondary antibody was FITC-labeled goat anti-mouse IgG (H + L ) (ZYMED LABORATORIES, INC) diluted 1Z200 was used. After the reaction with the secondary antibody, staining was performed in the order of rhodamine phalloidin (Molecular Probe) and DAPI (SIGMA) to detect a signal.

[0292] ALP (alkaline phosphatase) histologic staining was performed as follows. The culture tissue was washed twice with Dulbecco's PBS (-), fixed with cold 95% ethanol at 4 ° C for 30 minutes or more, and then dehydrated with absolute ethanol at 4 ° C for 30 minutes or more. After removing the fixative, the dishes were washed three times for 5 minutes at room temperature with 0.1 M Tris-HCl (pH 9.0-9.5), and then 1.5--2 ml of the staining solution (Naphthol AS-BI phosphate ( Sigma, Catalog No. N-2125) 2. 5 mg and fast red TR salt (Sigma) 15 mg [or fast purple B salt 6 mg (Sig ma), fast blue BB salt 12.5 mg (Sigma)] in 0.1 M Tris — Add 25 ml of HC1 buffer, dissolve with stirring for 3-5 minutes in the dark, then filter) and stain with ALP for 15-30 minutes at room temperature in the dark. After washing twice with PBS (-), glycerol was added, and a red-brown stained EG cell colony was examined using a phase contrast microscope.

(Shi / ko.

[0293] The results are shown in FIG. Fig. 3 shows ALP, SSEA-4, and TRA-1-60 from left power.

[0294] As is clear from Fig. 3, it was confirmed that the stem cells established using the single feeder cell of the present invention normally retained pluripotency.

(Example 8: Reproducibility with multiple cells)

The experiments performed in Examples 6 and 7 were performed on force-quiz monkey ES cells and human ES cells,

Tested with MEF feeder alone, STO stock feeder alone, and mixed feeder alone

[0296] The results are shown in the following table. Si Force-Quisal ES J¾

Samburu «ί use 敫« standing »»)

MEFtWt: 32 8 25%

STO ¾: 4 0 0%

«Mixed feeder: 5 4 80% 2 human

HEMBIS * W cubes for Samburu

EF ^ Ut: N. Λ.

STOHUt: N. Λ.

Combined feeder: 3 100%

[0297] As is clear from this table, when each feeder cell was used alone, only a stem cell establishment efficiency of 25% or less was achieved, whereas in the mixed feeder single cell, An excellent effect was demonstrated in which a stem cell establishment efficiency of 80% or more, and in some cases, 100% stem cell establishment efficiency could be achieved. The efficiency of the present invention is considered to be surprising, since human ES cells have been hardly established by MEF or STO alone.

(Example 9: Differentiation into nerve cells)

The ES cells prepared in the above Examples of the present invention were washed three times with a serum-free medium to completely remove serum. Undifferentiated cells were cultured on PA6 feeder cells for 8 to 11 days using ES cell culture medium containing KSR (Knockout Serum Replacement; GIBCO / Invitrogen Cat. No. 10828-028) (Kawasaki et al., 2000, Neur on 28; 31-40; see Tada et al., 2003; Dev. Dyn., 227; 504-510.

) o This differentiated into neurons.

[0299] The results are shown in Figure 4.

[0300] As is clear from the results in Fig. 4, it was revealed that the ES cells established by the method of the present invention retained the ability to differentiate into nerves.

(Example 10: Teratoma formation)

Next, the teratoma-forming ability of the ES cells of the present invention was confirmed. SCID mouse (Clear Japan, Approximately 10 ⁷ ES cells of the present invention were transplanted subcutaneously and intraperitoneally in (Tokyo) and observed for 13 months.

[0302] Teratoma obtained after one to three months was collected and histologically analyzed. Figure of an example

See Figure 5. As apparent from FIG. 5, various tissues derived from the ectoderm, endoderm, mesoderm and the like were observed in the ES cells of the present invention. Therefore, the ES cells established in the present invention

Probably, it was proved that it had retained the ability.

(Example 11: Differentiation into hematopoietic cells)

Next, differentiation of the ES cells of the present invention into hematopoietic cells is observed. Add 10% fetal calf serum to MEM (Gibco BRL, Cat # 11900-016) as a

Using broth plus 10- ⁵ M mercaptoethanol.

[0304] First, ES cells were differentiated into mesodermal cells. Undifferentiated ES cells are suspended in the above-mentioned differentiation-inducing culture medium, and seeded on a type IV collagen-coated 6 plate at a density of 1 × 10 ⁴ Z-well. Incubate this at 37 ° C, 5% CO for 4 days.

2

[0305] The culture supernatant is collected, and the wells are washed once with PBS (-). Next, add a cell dissociation solution (Cell Dissociation Buffer (Gibco BRL, # 13150-016) and let stand for 10 minutes (37 ° C, 5% CO 2) to detach and collect the cells. If you can't loose it, remove it with a mesh,

2

Count the number of cells. After centrifugation at 1,200 rpm for 5 minutes, the supernatant was removed.

Next, the cells were suspended in 10 ml of HBSSZBSA, and centrifuged at 1,200 rpm for 5 minutes to remove the supernatant. The cells were suspended in 0.1 ml of normal mouse serum per 1 × 10 ⁷ cells and allowed to stand for 10 minutes. Appropriate amounts of E-force doherin antibody and anti-FLK1 antibody were placed on ice water for 20 minutes. After washing the cells twice with HBSSZBSA, the FLK1 + E-forcedherin monomesoderm cells are sorted by FACS.

[0307] The mesodermal cells selected here are suspended in a culture medium for inducing differentiation, and seeded on a type IV collagen-coated plate at a density of 3 x 10 ⁵ . The culture solution is 3ml per liter, 37 ° C, 5%

Incubate for 3 days in CO.

2

[0308] The cells are collected and blocked. Recovery and blocking are performed as described above. Add an appropriate amount of VE-doherin antibody and allow to stand at ice temperature for 20 minutes. After washing cells with HBSS ZBSA, VE-forcedherin + vascular endothelial cells were sorted by FACS Prepare endothelial cells.

[0309] In this way, it is demonstrated that the ES cells of the present invention are also divided into hematopoietic cells.

(Example 12: Induction of differentiation into myocardium)

Embryoid bodies are formed by suspending and culturing the ES cells prepared in the above Examples in a basic medium (eg, DMEM) containing KSR. Four to seven days after the formation of embryoid bodies, the embryoid bodies are collected and seeded on a culture dish under gelatin coating. 1 in a basic medium containing KSR

By culturing for 3 weeks, the cardiomyocytes are divided.

(Example 13: Experiment with other stem cells (eg, tissue stem cells))

Remove tissue containing tissue stem cells (neural stem cells, mesenchymal stem cells, etc.) and culture on mixed feeder cells. By isolating the proliferating stem cell population and culturing it using the feeder cell prepared as described in Example 5 of the present invention, a cell line that can be stably maintained and maintained is established.

(Example 14: Regenerative treatment in monkeys)

Next, ES cells are collected from the force-quisar, and the same treatment as in Example 9 is performed to divert the cells to nerve cells. Transplanting this transplant of nerve cells into a force-quisar subject with neurological disease shows that the transplant can function.

(Example 15: Regenerative treatment in human)

Next, ES cells were collected from humans with consent from the patient, as described in Example 6.

The processing is performed according to the same procedure as in Example 9 as described in Example 14. It can be seen that transplantation of this transplant of nerve cells into a human subject having a neurological disease after obtaining consent in advance has enabled the transplant to function.

(Example 16: Examination of feeder-one effect)

Next, the primary cultured cells prepared in Example 1 and the cell line prepared in Example 3-4 were appropriately mixed to prepare a feeder-cell preparation, and whether or not a difference in the feeder effect was exhibited. Verify that

[0315] The ratios used are shown below. Primary ^ cultured cells (%) Cell line (%)

1 00 0

95 5

90 10

80 20

75 2 δ

70 0

60 40

50 50

40 60

30 70

25 7 δ

20 80

1 0 90

5 9 δ

0 100

[0316] Of the above conditions, up to 10:90 to 90:10, an improved effect is shown as compared to the case of using the cells alone as a feeder cell. At 5:95 and 95: 5, the same effect is observed as when used alone. In particular, at 20: 80-80: 20, the number of contaminating differentiated cells is reduced to less than 30%, and when observed in almost the same amount, the amount of contaminating differentiated cells is reduced to about 1%.

[0317] Therefore, it is advantageous to use, for example, a preferable ratio of 1: 4 to 4: 1, or 1: 3 to 3: 1, and more preferably, an approximately equivalent amount (for example, 3: 7—7: 3, 6: 4—4: 6, etc.).

(Example 17: Examination with other stem cells)

Next, an experiment similar to that in Example 16 is performed using force-quisar as a stem cell.

[0319] The protocol is the same as in Example 16.

[0320] As a result, as in Example 16, the effect improved from 10:90 to 90:10 as compared to the case where the feeder cell was used alone was shown. At 5:95 and 95: 5, the same degree of effect is observed as when used alone. In particular, at 20: 80—80: 20, contaminating differentiated cells When observed at about the same amount, it is observed that the number of mixed differentiated cells is reduced to about 1%.

[0321] Therefore, as a preferable ratio, for example, it is advantageous to use 1: 4 to 4: 1, or 1: 3 to 3: 1, and more preferably, approximately equal amounts (eg, 3: 7—7: 3, 6: 4—4: 6, etc.).

(Example 18: Verification in human cells)

Next, an experiment similar to that in Example 16 is performed using human cells as stem cells.

The protocol is according to Example 16. However, human ES cells are used with the consent of the provider as described above.

[0324] As a result, as in Example 16, up to 10:90-90:10, an improved effect was obtained as compared to the case where the single feeder cell was used alone. At 5:95 and 95: 5, the same degree of effect is observed as when used alone. In particular, at 20: 80-80: 20, the amount of contaminated differentiated cells is reduced to less than about 30%, and when observed in almost equal amounts, the amount of contaminated differentiated cells is reduced to about 1%.

[0325] Therefore, as a preferable ratio, for example, it is advantageous to use 1: 4 to 4: 1, or 1: 3 to 3: 1, and more preferably, to an approximately equivalent amount (for example, 3: 3). 7—7: 3, 6: 4—4: 6, etc.).

(Example 19: Verification with another feeder cell)

Next, the same experiment as in Example 16 was performed using human cells as stem cells, and as primary feeder cells, mouse fibroblast primary cultured cells and human epidermal fibroblasts (Clonetics, a division of BioWhittaker® USA; Cambrex, USA) using SL10 and MRC5 (ATCC number CCL-171) as cell lines.

[0327] The protocol is according to Example 16. However, human ES cells are used with the consent of the provider as described above.

[0328] As a result, as in Example 16, up to 10:90-90:10, an improved effect was exhibited as compared to the case where the single feeder cell was used alone. At 5:95 and 95: 5, the same degree of effect is observed as when used alone. In particular, at 20: 80—80: 20, contaminating differentiated cells When observed at about the same amount, it is observed that the number of mixed differentiated cells is reduced to about 1%.

[0329] Therefore, as a preferable ratio, for example, it is advantageous to use 1: 4 to 4: 1, or 1: 3 to 3: 1, and more preferably, approximately equal amounts (for example, 3: 1: 7—7: 3, 6: 4—4: 6, etc.) may be advantageous.

[0330] Accordingly, it can be seen that the feeder effect when mixed as well with other normal cells and other cell lines used as feeder cells is significantly enhanced.

(Example 20: Packaging of feeder-cell preparation)

It is confirmed below whether the feeder-cell preparation prepared in Example 5 or the like can maintain the activity even through a marketable route.

[0332] The preparation to be used as a feeder cell is treated with mitomycin C. Mitomycin C is available from Sigma. By treating with mitomycin C, feeder cells do not proliferate, can be stored for a long time, and can be sold. Such mitomycin C-treated cells can be cryopreserved and sold. For such cryopreservation, the same method as used in conventional primary culture cells can be used. For example, such a method is described in “Genetic Manipulation of ES Cells” Molecular Biology Protocol (Nan-Edo) p 471 — It is described in 479 (In addition, refer to Non-Patent Document 1.) o

[0333] Specifically, the processing can be performed as follows.

[0334] The package at the time of sale of the feeder cells prepared in this example is provided, for example, in the form of a cryotube. The processing is as follows.

[0335] Up to the treatment with mitomycin C, the procedure is the same as that for normal preparation (see Examples 2 and 3), and then the following operation is performed.

[0336] The medium is removed from the culture dish treated with mitomycin C, washed three times with PBS, the medium is cultivated, and cultured for several hours to overnight.

[0337] Remove the medium, wash three times with PBS, dissociate the cells by trypsinization, add the medium, suspend the cells well, and centrifuge to remove the supernatant.

The cells are suspended in, for example, 5 × 10 ⁶ Zml in a cell freezing medium (90% FCS + 10% DMSO). [0339] Add the cell suspension to a cryotube (NUNC 377224) and place the lid tightly.

[0340] Put the freezing tube in a cell freezing container (Nulgene 5100), and put the container in a -80 ° C freezer.

[0341] After 4 hours to overnight, transfer the cryotube to a liquid nitrogen container.

[0342] The feeder cell preparation for sale prepared in this manner is provided to the seller, preferably in a frozen state. At the provider, the cells can be thawed in the same manner as normal frozen cells and used as a single feeder cell.

As described above, the present invention which has exemplified the present invention using the preferred embodiment of the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the appended claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and common technical knowledge from the description of the specific preferred embodiments of the present invention. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Is understood.

Industrial applicability

[0344] The present invention has tremendous utility in regenerative medicine. The usefulness of the preferred embodiment is particularly high, since stem cells (especially ES cells of primates including humans) can be established with a high efficiency of establishment that was previously impossible. Therefore, the present invention has applicability in the field of manufacturing regenerative medicine and pharmaceuticals for treating it.

Claims

The scope of the claims

[I] A feeder-one cell preparation for preparing a stem cell, comprising a normal cell and a cell line.

[2] The feeder-one cell preparation according to claim 1, wherein the normal cells include primary cultured cells.

[3] The feeder-one cell preparation according to claim 1, wherein the normal cells are cells that have not been immortalized.

[4] The feeder-one cell preparation according to claim 1, wherein the normal cells include fibroblasts.

[5] The feeder-cell preparation according to claim 1, wherein the normal cells are primary cultured fibroblast cells.

[6] The feeder-one cell preparation according to claim 1, wherein the normal cells are derived from a mouse.

[7] The feeder-one cell preparation according to claim 1, wherein the normal cells are derived from a fetus.

[8] The feeder-one cell preparation according to claim 1, wherein the normal cells have a passage number of 5 or less.

[9] The feeder-one cell preparation according to claim 1, wherein the normal cells are fibroblasts obtained by enzymatic treatment from a mouse 11-16 days old fetus.

[10] The feeder cell preparation according to claim 1, wherein the normal cell is a primary cultured cell having a passage number of 5 or less.

[II] The feeder-one cell preparation according to claim 1, wherein the cell line is immortalized.

[12] The feeder-one cell preparation according to claim 1, wherein the cell line is a fibroblast cell line.

[13] The feeder-cell preparation according to claim 1, wherein the subculture period of the cell line is 2 months or less.

[14] The feeder-cell preparation according to claim 1, wherein the cell line has been passaged for one month or less.

[15] The feeder-one cell preparation according to claim 1, wherein the cell line is derived from a mouse.

[16] The feeder-one cell preparation according to claim 1, wherein the cell line is derived from a fetus.

[17] The feeder-one cell preparation according to claim 1, wherein the cell line is an STO line.

[18] The feeder-cell preparation according to claim 1, wherein the cell line is neo-resistant.

[19] The feeder-one cell preparation according to claim 1, wherein the cell line is a SL10 line.

[20] The feed according to claim 1, wherein the normal cells and the cell line are about 1:10 to 10: 1. Dar cell preparation.

[21] The feeder one-cell preparation according to claim 1, wherein the normal cell and the cell line are about 1: 3-3: 1.

[22] The feeder cell preparation according to claim 1, wherein the normal cell and the cell line are present in approximately equal amounts.

[23] The feeder-one cell preparation according to claim 1, wherein the stem cells are embryonic stem cells.

[24] The feeder-one cell preparation according to claim 1, wherein the stem cells are human embryonic stem cells.

[25] culturing stem cells on a feeder-one cell preparation containing normal cells and a cell line,

A method for preparing a stem cell.

26. The method according to claim 25, wherein the feeder-cell preparation is the feeder-cell preparation according to any one of claims 1 to 24.

[27] The method according to claim 25, wherein the culture dish in which the feeder cells are arranged is coated with gelatin.

[28] The method of claim 25, wherein the feeder single cell preparation is seeded at about 1 × 10 ⁴ cells / cm ^{2 to} about 1 × 10 ⁵ cells / cm ² .

[29] The method according to claim 25, wherein the feeder-one cell preparation is used within 5 days after being mixed and inoculated.

[30] The method according to claim 25, wherein the stem cells are cultured in a medium containing a knockout serum replacement additive (KSR).

31. The method according to claim 25, wherein the method further comprises a step of subculturing the stem cell, wherein collagenase is used in the subculturing step.

[32] A stem cell preparation prepared by the method according to claim 25.

[33] An embryonic stem cell preparation prepared by the method according to claim 25.

[34] A primate embryonic stem cell preparation prepared by the method according to claim 25.

[35] A human embryonic stem cell preparation prepared by the method according to claim 25.

[36] A method for preparing a transplant for regenerating an organ, tissue or cell, comprising:

A) a step of providing a stem cell that can be divided into a desired organ, tissue or cell; B) culturing the stem cells with the feeder-cell preparation of claim 1; and

C) a step of differentiating the stem cells into a transplant for regenerating a desired organ, tissue or cell,

A method comprising:

[37] The desired organ, tissue or cell also includes nerve, blood cell, bone, cartilage, heart, pericardium, blood vessel, muscle, eye, liver, spleen, intestine, stomach, lung, trachea, hair and skin power. 37. The method of claim 36, wherein the method is selected from a group.

38. The method of claim 36, wherein the desired organ, tissue or cell and the feeder-cell preparation are of the same species.

[39] The method according to claim 36, wherein the desired organ, tissue or cell is a primate, and the feeder-cell preparation is derived from a mouse.

[40] The method according to claim 36, wherein the culturing is performed ex vivo.

[41] The method according to claim 36, wherein the stem cell is a force that is immediately obtained after the subject force has been removed, or has been cryopreserved.

42. The method according to claim 36, wherein the culturing is performed at 37 ° C. in a saturated humidity under 5% CO.

[43] The differentiation is carried out by DNA demethylating agent, histone deacetylating agent, nuclear receptor ligand, cell growth factor, cytokin, hexamethylenebisacetamide, dimethylacetamide, dibutyl cAMP, dimethoate Including at least one factor selected from the group consisting of rusulfoxide, ododeoxyperidine, hydroxyl urea, cytosine arabinoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyperidine, polypropylene and selenium. 37. The method according to claim 36, wherein the method is performed on a culture solution.

[44] An organ, tissue or cell prepared by the method of claim 36.

[45] A system for regenerating an organ, tissue or cell, comprising:

A) container; and

B) a feeder-one cell preparation comprising a fibroblast primary culture cell and a fibroblast cell line, seeded on the container; A system comprising:

[46] A method for regenerating an organ, tissue or cell, comprising:

A) a step of providing a stem cell that can be divided into a desired organ, tissue or cell;

B) culturing said stem cells with the feeder-cell preparation of claim 1; and

C) transplanting the cultured stem cells to a site to be treated in a subject.

[47] The method according to claim 46, further comprising: D) separating the stem cells.

[48] Use of a cell preparation containing normal cells and a cell line as a feeder-cell preparation.

[49] The use according to claim 48, wherein the feeder-cell preparation is treated so as not to proliferate.

[50] The use according to claim 48, wherein the feeder-cell preparation has been treated with mitomycin C.

[51] Use of a feeder-cell preparation according to claim 1 in the manufacture of a medicament comprising stem cells for the treatment or prevention of a disease, disorder or condition for which use of stem cells is appropriate.

[52] The use of claim 51, wherein the feeder-cell preparation is not proliferated!

[53] The use of claim 51, wherein the feeder-cell preparation has been treated with mitomycin C.

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Figure 1A