JP2007061026A - Specific induction of stem cell and cell obtained thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare various differentiated materials such as a differentiated cell and a differentiated tissue from a stem cell as one likes and develop a technique for preparing a blood island (hematopoietic stem cell) from an undifferentiated cell. <P>SOLUTION: The method for preparing the hematopoietic stem cell comprises (A) a step for providing an undifferentiated cell, (B) a step for dissociating the undifferentiated cell and (C) a step for culturing the undifferentiated cell after dissociation in a culture medium containing activin for a time enough for differentiating into the hematopoietic stem cell. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to induction of stem cell differentiation.

  Disease treatment by regenerative medicine (regenerative medicine) has recently attracted attention. However, it has not yet been routinely applied to many patients with organ or tissue dysfunction. To date, treatment of such patients has been only applied to patients with limited use of assistive systems in medical devices in addition to organ transplantation. However, these therapies have problems such as donor shortage, rejection, infection, and service life. In particular, the shortage of donors is a serious problem, and in the case of bone marrow transplantation, it is difficult to provide a limited sample to many patients even though the bone marrow or cord blood bank is gradually enriched in Japan and overseas. Therefore, in order to overcome these problems, there is an increasing expectation for regenerative medicine centered on stem cell therapy and its application.

  Amplification and differentiation of hematopoietic stem cells is one of the important issues in regenerative medicine, and is considered to play a very important role in understanding the stem cells and treating the disease with an understanding of the mechanism, The research is progressing in recent years.

  Therefore, the identification of factors that regulate the differentiation of stem cells (particularly hematopoietic stem cells) is one of the unsolved issues that have not been studied and are awaited by those skilled in the art.

  In regenerative medicine, organ reconstruction is the most important. In this case, there are a method of roughly constructing an organ in vitro and using it as an artificial organ, and a method of reconstructing an organ in vivo. Once stem cells are obtained, their application is limited unless a controlled regeneration method is available.

  On the other hand, attempts have been made to perform gene therapy by introducing genes into stem cells and transplanting the stem cells. For example, it has been reported that clinical symptoms are improved by transplanting a stem cell (CD34 positive bone marrow cell) into which an IL-2 receptor γ chain has been introduced into a patient with X-linked severe combined immunodeficiency (non-patent document). 1 = Cavazzana-Calvo, M. et al. Science, 288: 669-672, 2000). However, when a gene is introduced into a stem cell, various harmful effects are considered to occur, so that usable hematopoietic stem cells cannot be produced.

  Various proteins play an important role as a mechanism for regulating stem cell differentiation. For example, stem cell factor (steel cell factor or steel factor; also referred to as SCF), thrombopoietin (TPO), interleukin (IL) -3, IL-6, IL-11, granulocyte colony stimulating factor (G-CSF) Other cytokines such as are factors attracting attention in hematopoietic stem cell differentiation.

  Xenopus laevis is early in development because of its advantages such as early development, easy judgment of results, easy egg collection, observation of the development process outside the body, and easy analysis of gene functions. Has played an important role in research. So far, methods have been developed for producing various organs such as heart, pancreas, eye and kidney from Xenopus undifferentiated cells.

  However, until now there has been no technique for inducing blood islands (hematopoietic stem cells) from undifferentiated cells. The absence of such technology not only causes blood formation research in the early stages of vertebrates to be in the early stages, but also causes blood disorders such as human anemia, abnormal blood cells, and leukemia. It was a serious obstacle to the development of detailed research and therapeutic methods and drugs.

There are several reports on activin (Patent Document 1 = Japanese Patent Laid-Open No. 2005-095138), but differentiation into blood islands is not known.
Cavazzana-Calvo, M.C. et al. Science, 288: 669-672,2000. JP2005-095138

  An object of the present invention is to produce various differentiated cells, differentiated tissues and the like from stem cells as desired. Another object of the present invention is to develop a technique for producing blood islands (hematopoietic stem cells) from undifferentiated cells. If blood islands (hematopoietic stem cells) can be created from undifferentiated cells, such blood islands (hematopoietic stem cells) are specifically lacking or abnormally expressing proteins involved in the formation of these hematopoietic stem cells. There is a possibility of providing useful materials for the purpose of diagnosing diseases such as humans and treating abnormalities and diseases such as humans caused by defects or defects in hematopoietic stem cells such as leukemia. It is another object of the present invention to provide good materials for research on such abnormalities and diseases, and to provide materials for assay systems useful in the development of drugs for treating those abnormalities and diseases.

  The present invention has solved the above problems by finding that various differentiated cells or differentiated tissues can be produced and differentiated by slightly varying the concentration of activin or its equivalent.

  Starting from the undifferentiated cells of Xenopus, which is a good model for studying early development, a dissociated and reassembled body is formed, and the resulting dissociated and reassembled body is converted into two sheets of undifferentiated cells. Blood islands (hematopoietic stem cells) can be induced by sandwiching and culturing with (FIG. 1). Blood islands (hematopoietic stem cells) induced in this way have an appearance unique to blood islands (hematopoietic stem cells), and a protein SCL (Stem cell leukemia protein) that is a protein (marker protein) unique to blood islands (hematopoietic stem cells). It was revealed that it is a blood island (hematopoietic stem cell). The important point in the present technology is that, when activin is treated, activin is not directly applied to undifferentiated cells, but activin is treated when dissociated undifferentiated cells are reassembled.

  FIG. 1 shows a typical method scheme of the present invention. FIG. 1 shows a schematic diagram of the induction of blood islands (hematopoietic stem cells) from undifferentiated cells. Animal caps were excised from Xenopus late blastocysts, cells were dissociated in Steinberg's solution excluding calcium and magnesium ions, and treated with activin prepared in Steinberg's solution containing calcium and magnesium ions for 3 and 5 hours. . Tissue differentiation was examined when this activin-treated dissociated reassortant was cultured alone and when it was sandwich-cultured with an untreated animal cap. As a result, dissociated reassemblies that were cultured alone after 3 hours of activin treatment were irregularly shaped epidermis at an activin concentration of 0 ng / ml, blood cells and mesenchyme at 1 ng / ml, and muscle at 10 ng / ml. At ng / ml, they differentiated into endoderm cells.

  On the other hand, when activin-treated dissociated reassemblies were sandwich-cultured, an activin-treated concentration of 0 ng / ml gave an irregular epidermis, 1 ng / ml a blood island, 10 ng / ml a prorenal tube, 100 ng / ml In ml, differentiation into cement glands was observed. According to this experiment, explants obtained by sandwich culture of dissociated reassemblies treated with activin showed differentiation depending on the concentration and treatment time of activin, and sandwiched dissociated reassemblies treated with 1 ng / ml of activin for 3 hours. It was found that blood islands were induced at a high rate by culturing.

  In many of the organ formations using animal caps reported so far, activin treatment was performed on sheet-shaped animal caps, and single culture or sandwich culture was performed. In this method, cells that receive the activin signal and cells that do not receive the activin signal are mixed and non-uniform.

  In this study, dissociation and activin treatment were performed to make each cell uniformly receive activin signal, and single culture and sandwich culture were performed.

Accordingly, the present invention provides the following.
(1) A method for preparing hematopoietic stem cells comprising:
A) providing undifferentiated cells;
B) dissociating the undifferentiated cells;
C) culturing the undifferentiated cells after dissociation in a medium containing activin for a time sufficient to differentiate into hematopoietic stem cells;
Including the method.
(2) The method according to Item 1, wherein the undifferentiated cells include cells derived from late blastocysts.
(3) The method according to Item 1, further comprising the step of contacting the cell obtained by the step D) C) with an undifferentiated cell.
(4) The method according to Item 3, wherein the contact is achieved by sandwiching the cells obtained in the step C) with a sheet of undifferentiated cells.
(5) The method according to Item 3, wherein the undifferentiated cells include animal cells of late blastula.
(6) The method according to Item 3, wherein the contact with the undifferentiated cell is a time sufficient to contact the undifferentiated cell from the initial contact to the final differentiation.
(7) The method according to Item 3, wherein the contact with the undifferentiated cells is performed for at least one day.
(8) The method according to Item 3, wherein the contact with the undifferentiated cells is performed within 1 day to 3 days.
(9) The method according to Item 1, wherein the activin is an activin selected from the group consisting of activin-A, activin-B, inhibin and activin-C.
(10) The method according to Item 1, wherein the activin comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12.
(11) The method according to Item 1, wherein the activin is applied at a concentration of 0.5 to 1 ng / ml per animal cap.
(12) The method according to Item 1, wherein the hematopoietic stem cell expresses a stem cell leukemia protein (SCL) marker.
(13) The method according to Item 1, wherein the dissociation is achieved by culturing in a medium containing neither Ca ²⁺ nor Mg ²⁺ .
(14) The method according to Item 1, wherein the dissociation is achieved by culturing in a medium containing neither Ca ²⁺ nor Mg ^{2+ for} at least 10 minutes.
(15) The method according to Item 1, wherein the dissociation is achieved by culturing in a medium containing neither Ca ²⁺ nor Mg ^{2+ for} 10 to 20 minutes.
(16) The method according to item 1, wherein a dissociated and reassembled body of the cells is formed in the step C).
(17) The method according to item 16, wherein the formation of the dissociated reassemblies in the step C) is achieved by culturing in the medium for at least 30 minutes.
(18) The method according to item 1, wherein the activin treatment in step C) is performed for at least 3 hours.
(19) The method according to Item 1, wherein the undifferentiated cell is provided by an animal cap.
(20) The method according to Item 19, wherein 5 to 10 animal caps are used.
(21) The method according to Item 1, further comprising the step of culturing the dissociated reassemblies together with the undifferentiated cells in Step C, wherein dissociated reassemblies are formed.
(22) The method according to Item 1, further comprising the step of sandwich-culturing the dissociated reassemblies with the undifferentiated cells in Step C, wherein dissociated reassemblies are formed.
(23) The method according to item 1, wherein the cell is a vertebrate cell.
(24) A method according to item 1, wherein the cell is an amphibian animal cell.
(25) The method according to Item 1, wherein the cell is a frog cell.
(26) The method according to Item 1, wherein the cell is a Xenopus cell.
(27) A cell prepared by the method according to any one of items 1 to 26.
(28) A blood island prepared by the method according to any one of items 1 to 28.
(29) A medium used in the step of dissociating undifferentiated cells in the method for inducing hematopoietic stem cells,
A medium characterized in that it contains neither Ca ²⁺ nor Mg ²⁺ .
(30) A medium according to item 29, wherein the medium contains a composition of 58 mM NaCl, 0.67 mM KCl, 3.0 mM hydroxyethylpiperazinylethanesulfonic acid and 100 mg / L kanamycin sulfate, pH 7.4.
(31) A medium used in the step of differentiating undifferentiated cells into hematopoietic stem cells in the method for inducing hematopoietic stem cells,
A medium comprising an effective amount for inducing activin into hematopoietic stem cells.
(32) A medium according to Item 31, wherein the activin is present at a concentration of 0.5 to 1 ng / ml.
(33) A medium according to item 31, wherein the activin is present at a concentration of about 1 ng / ml.
(34) A medium combination used in a method for inducing hematopoietic stem cells, wherein the combination does not contain Ca ²⁺ or Mg ²⁺ ,
A medium combination comprising a medium comprising an effective amount for inducing activin into hematopoietic stem cells.
(35) A kit for preparing hematopoietic stem cells, comprising:
a) a dissociation means for dissociating the undifferentiated cells;
b) differentiation means for differentiating the undifferentiated cells into hematopoietic stem cells;
c) instructions for instructing to differentiate the undifferentiated cells by means of time differentiation sufficient to differentiate the undifferentiated cells after dissociation into hematopoietic stem cells;
A kit comprising:
(36) The kit according to item 35, wherein the differentiation means is a medium containing activin.
(37) Use of activin to prepare hematopoietic stem cells.
(38) A composition for preparing hematopoietic stem cells, the composition comprising an effective amount for inducing activin into hematopoietic stem cells.
(39) A composition according to Item 38, wherein the activin is present at a concentration of 0.5 to 1 ng / ml.
(40) A composition according to Item 38, wherein the activin is present at a concentration of about 1 ng / ml.
(41) A method for preparing a desired differentiated cell comprising:
A) providing undifferentiated cells;
B) dissociating the undifferentiated cells;
C) culturing the undifferentiated cells after dissociation in a medium containing activin under conditions sufficient to differentiate into desired differentiated cells;
Including the method.

  The present invention provides a method for producing hematopoietic stem cells by a simple method. This was achieved with a specific concentration of activin. It was also found that changing the concentration of activin differentiates into another tissue form. Therefore, the present invention has an effect of enabling subtle differentiation regulation by activin. In both experimental systems with 5 or 10 animal caps, blood islands are not induced even when the activin treatment is performed at 1 ng / ml / sandwich culture when the activin treatment time is 5 hours. However, since blood cells are formed, expression of SCL (gene marker) is observed. The number of cells for activin treatment is not always universal. In both experimental systems with 5 or 10 animal caps, blood islands are induced by treatment with activin 1 ng / ml for 3 hours and sandwich culture. If we focus only on blood island formation, the number of cells may be universal.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, 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.

(Definition)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “stem cell” refers to a cell having a self-replicating ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells may preferably be embryonic stem cells, although tissue stem cells can also be used depending on the circumstances.

As used herein, “differentiation” or “cell differentiation” refers to two or more morphologically and / or functionally qualitative differences among daughter cell populations derived from the division of one cell. A phenomenon in which types of cells are generated. Therefore, the process in which a cell population (cell lineage) derived from cells that cannot originally detect special characteristics exhibits distinct characteristics such as production of a specific protein is also included in differentiation. At present, it is common to consider cell differentiation as a state in which a specific gene group in the genome is expressed, and cell differentiation by searching for intracellular or extracellular factors or conditions that cause such gene expression state. Can be identified. The result of cell differentiation is in principle stable, especially in animal cells, which differentiates into other types of cells only in exceptional cases. When referring to hematopoietic stem cells, “differentiation” means that the surface antigen molecule represented by CD is changed to become the next stage cell. Specifically, the phenotype of the differentiation antigen is changed from pluripotent stem cells (CD34 ⁺ CD38 ⁻ ) to lymphoid stem cells (CD34 ⁺ CD38 ⁺ ) or myeloid stem cells (CD34 ⁺ CD38 ⁺ CD33 ⁺ ). , Lymphoid stem cells to T precursor cells or B precursor cells, myeloid stem cells to BFU-C, CFC-MEG, EO-CFC or CFU-GM, BFU-E to CFU-E, T precursor cells to T cells, B precursor It means cell to B cell, CFU-E to red blood cell, CFC-MEG to platelet, EO-CFC to eosinophil, CFU-GM to monocyte, neutrophil or basophil.

  In the present specification, among pluripotency, the ability to differentiate into all types of cells constituting a living body like a fertilized egg is referred to as “totipotency”, and pluripotency can encompass the concept of totipotency. However, when clearly distinguishing, totipotency and pluripotency can be distinguished, the former refers to the ability to differentiate into any cell, the latter has multiple directions, but organisms are possible Having the ability to differentiate in all directions. The ability to differentiate in only one direction is also referred to as unipotency.

  In this specification, totipotency and pluripotency can be determined by, for example, the number of days after fertilization. For example, in the case of a mouse, it can be distinguished on the basis of about 8 days after fertilization. Without being bound by theory, it is normal for mice to follow the following course after fertilization. At 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 longitudinal axis of the embryo. The primitive streak represents the future posterior end of the embryo and extends across the ectoderm to the distal end of the cylinder. The original stripe is an area where cell movement is performed, and as a result, future endoderm and mesoderm are formed. By E7.5, a head process appears in front of the nodule, and a notochord and a future endoderm surrounding the lower part of the nodule are formed in this part, and a neural plate is formed in the upper part. The nodules appear around E6.5 days and move backwards, forming the shaft structure from front to back. By day E8.5, the embryo is somewhat longer, with a large head fold consisting mostly of the anterior neural plate at the front end. The somites begin to form from front to back at a rate of one in 1.5 hours from day E8. Cells that have passed this period will no longer exhibit totipotency and will not form individuals unless they are dedifferentiated, even if they are returned to the placenta. Before this, it can be said that this is a branch point of totipotency because it can show totipotency without special treatment. This is because it is difficult to establish ES cells from subsequent embryos, and cells called EG (germ cell-derived) cells are usually established from later embryos. It can be said that it is a point.

  As used herein, “pluripotency” or “pluripotency” is used interchangeably, refers to the nature of a cell, and refers to the ability to differentiate into one or more, preferably two or more, various tissues or organs. . Accordingly, “pluripotency” and “multipotency” are used interchangeably with “undifferentiated” in this specification unless otherwise specified. In general, cell pluripotency is limited as development progresses, and in adults, the constituent cells of one tissue or organ rarely change into another cell. Therefore, 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. However, mesenchymal cells have a high degree of pluripotency because they tend to undergo metaplasia instead of other mesenchymal cells with a relatively simple stimulus. Embryonic stem cells are pluripotent. Tissue stem cells are pluripotent. In the present specification, among pluripotency, the ability to differentiate into all types of cells constituting a living body like a fertilized egg is called totipotency, and pluripotency can encompass the concept of totipotency. Whether or not a certain cell has pluripotency includes, but is not limited to, formation of embryoid bodies and culture under differentiation-inducing conditions in an in vitro culture system. In addition, assay methods for the presence or absence of pluripotency using living organisms include the formation of teratomas by implantation into immunodeficient mice, the formation of chimeric embryos by injection into blastocysts, and transplantation into living tissues. Examples include, but are not limited to, proliferation by injection into ascites.

  As used herein, “undifferentiated cell” refers to a cell having multipotency as described above. Preferably, cells derived from late blastocysts are used.

As used herein, “hematopoietic stem cell” refers to a stem cell having the ability to differentiate into cells such as blood cells (blood cells) such as red blood cells, white blood cells, and platelets. Preferably, the blood cell line has some degree of differentiation but retains pluripotency. It is a cell that has the ability to differentiate into all types of blood cells and has the ability to reconstruct hematopoiesis, and is mainly present in bone marrow, umbilical cord blood, spleen, or liver, and is also present in peripheral blood in a small amount. These hematopoietic stem cells are CD34 strongly positive cells, and high-proliferative potential colony-forming cells (HPP-CFC) are also included in the present invention. “Stem cells” mean pluripotent hematopoietic stem cells and lymphoid stem cells and myeloid stem cells (CFU-GEMM) differentiated therefrom. These cells are CD34 positive cells.
“Progenitor cell” means a cell in which blood cells of each lineage cannot be identified from pluripotent hematopoietic stem cells, but can only differentiate into unidirectional blood cells such as erythroid. Specifically, platelet colony forming cells (CFC-MEG), eosinophil colony forming cells (EO-CFC), granulocyte monocyte colony forming cells (CFU-GM), erythropoiesis cells (BFU-E, CFU-E). ), T precursor cells, B precursor cells, and the like. These are all CD34 positive cells.

Hematopoietic cells are made in the bone marrow, differentiate into erythrocytes, platelets, leukocytes, etc. and flow through the peripheral blood. Looking at the differentiation of myeloid cells, the largest source is pluripotent stem cells, followed by hematopoietic stem cells specialized in hematopoietic cells, which differentiate into pluripotent progenitor cells, and then myeloid cells Differentiating into progenitor cells and lymphoid progenitor cells In the myeloid system, pluripotent stem cells differentiate into cells called CFU-GEMM. The cells differentiate from CFU-GEM cells to CFU-GM cells, followed by myeloblasts, promyelocytes, and myelocytes. These are cells present in the bone marrow, and when they differentiate, they become neutrophils and flow in the peripheral blood. When going to the next line, it goes from the cell called CFU-GM toward monocytes and differentiates into monocytes, promonocytes, and monocytes. These monocytes appear in the peripheral blood. In the third line, CFU-GEMM cells are differentiated into BFU-E cells, and then differentiated into pre-erythroblasts, erythroblasts, and erythrocytes. There is also a megakaryocyte system, which differentiates into CFU-Meg (abbreviation for megacaryosite), megakaryocyte, megakaryocyte, and platelet.

  In this differentiation process, leukemia results from abnormal pluripotent stem cells. Therefore, the present invention can be applied to the treatment of leukemia in the sense of improving such abnormalities.

  In the lymphocyte lineage, CML differentiates from pluripotent stem cells into lymphoid stem cells, which are divided into B cell lines and T cell lines. Separately, there is a line that separates towards NK cells. The B cell line is differentiated into precursor B cells, precursor precursor B cells (pre-pre-B-cell), early B cells (early-B-cell), etc., and intermediate B cells (intermediate-B cells). -Cell), mature B cells (mature B-cell), plasmacytoid-B-cells, and plasma cells (plasma-cell). As T cell lines, thymic progenitor cells, immature thymocytes, common thymocytes, and mature thymocytes are differentiated. There are other routes to go to helper / inducer T cells and to differentiate from mature thymocytes into suppressor / cytotoxic T cells. For a more detailed explanation of such differentiation, see Koichi Akashi, Modern Medicine 56 (2), 15-23, 2001. This document is incorporated herein by reference.

  The cells used in the present invention may be cells derived from any organism (for example, any kind of multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.)). ) Etc.)). Preferably, cells derived from vertebrates (eg, eel eels, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, more preferably mammals (eg, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, rabbit eyes, etc.). More preferably, amphibian (eg, Xenopus) cells are used.

  The organ targeted by the present invention may be any organ, and the tissue or cell targeted by the present invention may be derived from any organ or organ of an organism. In the present specification, the term “organ” or “organ” is used interchangeably, and a function of an organism individual is localized and operated in a specific part of the individual, and that part has morphological independence. Is a structure. In general, in a multicellular organism (eg, animal, plant), an organ is composed of several tissues having a specific spatial arrangement, and the tissue is composed of many cells. Such organs or organs include organs or organs associated with the vascular system. In one embodiment, organs targeted by the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, peripheral limbs, retina and the like. It is not limited to them. In the present specification, cells differentiated from the pluripotent cells of the present invention include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, and skeletal myoblasts. , Nerve cells, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells, chondrocytes and the like.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / or morphology. Therefore, when a tissue is regenerated using the stem cells of the present invention, a cell population having two or more different origins can constitute one tissue. Usually, tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis. Plants are roughly classified into meristems and permanent tissues according to the stage of development of the constituent cells, and are classified into single tissues and composite tissues according to the types of constituent cells.

  In the present specification, “activin” refers to a protein hormone that is secreted from ovarian granulosa cells and the like and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Representative activins include, for example, activin-A, activin-B, activin-C, and inhibin, which are well conserved among animals, and humans act on Xenopus and vice versa. Is also expected to be true. SEQ ID NO: 1 and SEQ ID NO: 9 (accession number: NM002192 (SEQ ID NO: human inhibin βA) and accession number: X68250 (Xenopus activin A) = nucleic acid sequence) and SEQ ID NO: 2 and SEQ ID NO: 10 (human inhibin βA, respectively) And the corresponding factor (ortholog) in other species of animals, including but not limited to those having the sequence shown in SEQ ID NO: and Accession No. X68250 (Xenopus activin A) = amino acid sequence). Xenopus has a registration of inhibin βB (accession number: S61773) (SEQ ID NOs: 11 and 12). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin is a protein hormone that is secreted from granulosa cells of the ovary and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Isolated from follicular fluid in the course of purifying inhibin (1986). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin gene expression has been reported in various vertebrate organs, promotes the synthesis of FSH receptors in ovarian granulosa cells, suppresses proliferation of erythroid progenitor cells in friend cells and bone marrow, and induces hemoglobin synthesis, It has many physiological activities such as promotion of insulin secretion from the pancreas and mesoderm induction in amphibian embryos. For activin, reference can be made to: Nakamura et al. , Isolation and charactarization of native activin B. J Biol. Chem. 1992, 267, 16385-9; Uchiyama and Asashima, Specific erythroid differentiation of the unwanted erythroleukemia cells by activities and its sensitization. Biochem Biophys. Res. Commun. 1992, 187, 347-52; Fukui et al. , Isolation and characterization of Xenopus follistatin and activins. Dev. Biol. 1993, 159, 131-9; Fukui et al. , Identification of activins A, AB, and B and follistatin proteins in Xenopus embryos. Dev. Biol. 1994, 163, 279-81; Nakano et al. , Comparison of mesoderm-inducing activity with monometic and dimeric inhibin alpha and beta-A subunits on Xenopus ectoderm. Horm. Res. 1995, 44, Suppl. 2, 15-22.

Activins are known to have homologues in mammals including humans, rats, mice and Xenopus, as well as in Drosophila. Therefore, in this specification, activin usually refers to activin which exists not only in mammals but also in living organisms in general. Activin-A is a dimer of inhibin βA (human inhibin βA accession number NM002192; SEQ ID NOs: 1 and 2 (nucleic acids and amino acids)). Activin-AB is a dimer of inhibin βA and inhibin βB (human inhibin βB accession number NM002193; SEQ ID NOs: 3 and 4 (nucleic acids and amino acids)). Activin-B is a dimer of inhibin βB. Activin-C is a dimer of inhibin βC (human inhibin βC accession number NM005538; SEQ ID NOs: 5 and 6 (nucleic acids and amino acids)). Inhibin is a dimer of inhibin α (human inhibin α accession number NM002191; SEQ ID NOs: 7 and 8 (nucleic acids and amino acids)).
Examples of such activin genes include:
(A) (a) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12 or a fragment thereof;
(C) a variant having one or more amino acids having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 A polynucleotide, which encodes a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11;
(E) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to A polynucleotide comprising a nucleotide sequence having at least 70% identity to any one polynucleotide of (e) or a complementary sequence thereof and encoding a polypeptide having biological activity;
including,
A nucleic acid molecule; or (B) (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a fragment thereof;
(B) in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10 or 12, wherein at least one amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion; and A polypeptide having biological activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12; or (e) against any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence with at least 70% identity and having biological activity;
including,
Examples include, but are not limited to, a nucleic acid molecule that encodes a polypeptide, or a polypeptide that it encodes.

  As used herein, “ligand” refers to a substance that specifically binds to a protein. Examples of ligands include lectins, antigens, antibodies, hormones, neurotransmitters, and the like that specifically bind to various receptor protein molecules present on the cell membrane. If activin is the ligand, ligands with similar (equivalent) action are intended to be within the scope of the present invention.

  In the activin used in the present invention, as a moiety to which a sugar chain can be added, an N-glucoside-bondable moiety to which N-acetyl-D-glucosamine or the like binds, and an N-acetyl-D-galactosamine O-glycoside bond (Parts where serine or threonine residues frequently appear). The activin used in the present specification is not particularly affected by the presence or absence of a sugar chain, but proteins with these sugar chains are usually stable against degradation in vivo. , May have strong physiological activity. Therefore, polypeptides to which these sugar chains are added are also within the scope of the present invention.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is done. The gene product of the present invention usually takes the form of a polypeptide. The gene product of the present invention in such a polypeptide form is useful as a composition for diagnosis, prevention, treatment or prognosis of the present invention.

  As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”.

  As used herein, the term “oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as explicitly indicated sequences. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98). (1994)). The gene of the present invention usually takes this polynucleotide form.

  As used herein, “nucleic acid molecule” is also used interchangeably with nucleic acids, oligonucleotides and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules encoding certain gene sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition. Therefore, in the present specification, for example, a gene (activin gene) that can be used in the present invention may include a splice variant thereof.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Therefore, a gene such as activin usually includes both a structural gene of the gene of the present invention and a transcriptional and / or translational regulatory sequence such as its promoter. In the present invention, it is understood that in addition to structural genes, regulatory sequences such as transcription and / or translation are also useful for nerve regeneration, diagnosis, treatment, prevention, prognosis, etc. of nerve diseases. As used herein, “gene” refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”. Sometimes. Also herein, a “gene product” is a “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligo” expressed by a gene. Includes "peptide" and "peptide". A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In this specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences with respect to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes have homology. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In the present specification, the similarity, identity and homology of amino acid sequences and nucleotide sequences are compared, and the search for identity is performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12). be able to. In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is taken as the identity value. When identity is evaluated in a plurality of regions, the highest value among them is set as the identity value.

  In the present specification, the “amino acid” may be natural or non-natural as long as the object of the present invention is satisfied. “Amino acid derivative” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art.

  The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, the term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  As used herein, “amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As activin in this specification, in addition to those containing natural amino acids, those containing such amino acid analogs or amino acid derivatives can be used.

  In the present specification, the “nucleotide” may be natural or non-natural. A “derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA). .

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one letter code.

  As used herein, a “corresponding” amino acid or nucleic acid has the same action as a predetermined amino acid or nucleotide in a polypeptide or polynucleotide to be compared in a certain polypeptide molecule or polynucleotide molecule, or An amino acid or nucleotide that is predicted to have, especially an enzyme molecule, is an amino acid that is present at a similar position in the active site and contributes similarly to catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Therefore, in this specification, a specific amino acid sequence in mouse activin can be associated with a specific amino acid in human activin by analysis such as alignment. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (eg, a polypeptide molecule or a polynucleotide molecule) has or has the same action as a given gene in a species to be compared in a certain species. It refers to a predicted gene (for example, a polypeptide molecule or a polynucleotide molecule), and when there are a plurality of genes having such action, it refers to one having the same evolutionary origin. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, a gene corresponding to a gene such as Xenopus activin can also be found in other animals (human, rat, pig, cow, etc.). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an animal is a sequence database of that animal (eg, human, rat) using the sequence of the gene that serves as a reference for the corresponding gene (eg, a gene such as Xenopus activin) as a query sequence. Can be found by searching.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include a number above and below that number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. Whether the length of a fragment useful in the present specification retains at least one of the functions of a full-length protein that serves as a reference for the fragment (for example, differentiation-regulating action, specific interaction with other molecules) It can be decided by how.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, a protein-lipid interaction, a nucleic acid-lipid interaction, and the like, such as a reaction with a binding site of a transcription factor. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules, but not limited to. As a factor specific to a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and a complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to. As used herein, activin can be used for stem cell differentiation, but it is understood that any factor having the same biological activity as activin can be used interchangeably with activin in the present invention. Such factors can be identified by screening using technical common sense in the field based on the disclosure in the present specification, and it is understood that these are within the scope of well-known and conventional techniques. .

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, the organic small molecule has a molecular weight of about 1000 or less, but it may be higher. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, low molecular ligands). It is not limited.

  As used herein, “contacting” refers to physically contacting a compound, either directly or indirectly, with an activin or the like (eg, a polypeptide or polynucleotide) of the present invention. It means close proximity. The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof. Typically, the contact can be achieved by placing a contact object (for example, a cell) in a solution (for example, a medium) containing a factor (for example, activin) to be contacted.

  As used herein, “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such complex molecules include, but are not limited to, glycolipids and glycopeptides. In this specification, activin can also be used in the form of such a complex molecule.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  As used herein, “biological activity” refers to the activity that a factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). Activity is included. For example, when two factors interact (eg, activin and its specific factor bind), the biological activity is the binding between the two molecules and the resulting biological change, eg, When two molecules are co-precipitated when one molecule is precipitated using an antibody, the two molecules are considered to be linked. Therefore, seeing such coprecipitation is one method of judgment.

  Thus, “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other Similar measures of function are mentioned. Such activity can be measured by assays such as competitive inhibition of binding of specific factors to activin.

  In this specification, the term “interaction” refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

  The term “modulate” or “modify” as used herein means an increase or decrease or maintenance in the quantity, quality or effect of a particular activity or protein.

  In the present specification, the “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide obtained under well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing a filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the polynucleotide capable of hybridizing is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide (for example, activin) having the amino acid sequence specifically shown in the present invention. A polynucleotide having a homology of 80% or more is preferable, and a polynucleotide having a homology of 95% or more is more preferable.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands with a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA with significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + in the hybrid Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20 % Formamide, 37-50 ° C. As an example, a “moderately stringent” condition of 50 ° C. in 0.015 M sodium ion tolerates a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is
Tm = (2 ° C. per AT base) + (4 ° C. per GC base pair)
Provided by. The sodium ion concentration in 6 × sodium citrate salt (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

Natural nucleic acids encoding proteins such as activin or variants or fragments thereof can be readily obtained from a cDNA library having PCR primers and hybridization probes comprising, for example, a portion of a nucleic acid sequence such as SEQ ID NO: 1 or a variant thereof. To be separated. A nucleic acid encoding a preferred activin or variant or fragment thereof is essentially a high containing 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; 7% SDS at a temperature of 42 ° C. More preferably essentially under low stringency conditions defined by a hybridization buffer and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C. Hybridization buffer containing 1% bovine serum albumin (BSA) at a temperature of 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS, and 1 × essentially 50 ° C. SSC (300 mM NaCl; 30 mM Sodium citrate); 1% bovine serum albumin (BSA) under low stringent conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS and essentially 0.5 × SSC at 65 ° C. (150 mM NaCl; 15 mM sodium citrate); wash containing 0.1% SDS It can hybridize to one or a portion of the sequence shown in SEQ ID NO: 1 or 3 under low stringency conditions as defined by the buffer.

  In the present specification, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that activin and the like used in the present invention should also include corresponding genes identified by such electronic and biological searches.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In particularly preferred embodiments, the Basic Local Alignment Tool (BLAST) (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990, well known in the prior art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high score segment pairs” between an amino acid query sequence or nucleic acid query sequence and a test sequence, preferably derived from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Strategic Stratecence. thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).

(Modification of genes, protein molecules, nucleic acid molecules, etc.)
In certain protein molecules (eg, activin), certain amino acids included in the sequence can be found in other protein structures such as, for example, the cationic region or the binding site of a substrate molecule, without any apparent loss or loss of interaction binding capacity. Of amino acids. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be substituted with another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  The hydrophilicity index is also useful for modifying the amino acid sequence of the present invention. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be replaced with another that has a similar hydrophilicity index and still can provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present specification, the “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid substituted with the original amino acid are similar as described above. Examples of conservative substitutions include those having a hydrophilicity index or hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and Examples include, but are not limited to, isoleucine.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such variants include one or several substitutions, additions and / or deletions, or one or more substitutions, additions and / or deletions relative to a reference nucleic acid molecule or polypeptide. But are not limited thereto. Alleles refer to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations),” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, and site-specific base substitution using synthetic oligonucleotides (specification). Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other modifications may also be made by methods usually used in the field of molecular biology. it can.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. Addition of an amino acid means adding 1 or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, shortening, lipidation, phosphorylation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg acetylation), etc. Including, but not limited to. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” or “peptide derivative” refers to a compound that is different from a peptide but is equivalent to at least one chemical or biological function of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives are added or substituted to the original peptide. Peptide analogs have functions similar to those of the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so that they are substantially similar to (eg, similar sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  Chemically modified polypeptide compositions in which a polypeptide of the invention is bound to a polymer are within the scope of the invention. The polymer can be water soluble and can prevent precipitation of the protein in a water soluble environment (eg, a physiological environment). Suitable aqueous polymers can be selected, for example, from the group consisting of: polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymer, poly (N-vinyl pyrrolidone) polyethylene glycol, Polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol. The selected polymer is usually modified and has a single reactive group (eg, an active ester for acylation or an aldehyde for alkylation) so that the degree of polymerization can be controlled. The polymer can be of any molecular weight, and the polymer can be branched or unbranched, and mixtures of such polymers can also be used. This chemically modified polymer of the present invention is selected for use by a pharmaceutically acceptable polymer when determined for therapeutic use.

  If the polymer is modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is modified by reductive alkylation, the polymer should have a single reactive aldehyde group. Preferred reactive aldehydes are polyethylene glycol, propionaldehyde (which is water-soluble) or mono-C1-C10 alkoxy or aryloxy derivatives thereof (eg, US Pat. No. 5,252,714). No., which is hereby incorporated by reference in its entirety).

  Pegylation of the polypeptides of the invention can be performed by any pegylation reaction known in the art, for example as described in the following references: Focus on Growth Factors 3,4- 10 (1992); EP 0 154 316; and EP 0 401 384, each of which is hereby incorporated by reference in its entirety. Preferably, this pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer). A preferred water soluble polymer for pegylation of polypeptides of the invention (eg, activin, Mel-18, M33, Mph-1 / Rae28, etc.) is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any form of PEG, where the PEG is another protein (eg, a mono (C1-C10) alkoxy polyethylene. Glycol or mono (C1-C10) aryloxypolyethylene glycol).

  Chemical derivatization of the polypeptides of the present invention can be performed under suitable conditions used to react biologically active substances with activated polymer molecules. A method for preparing a PEGylated polypeptide of the invention generally comprises the following steps: (a) Polyethylene glycol (eg, of PEG) under conditions such that activin is attached to one or more PEG groups. Reactive ester or aldehyde derivative) and the polypeptide, and (b) obtaining the reaction product. It is easy for those skilled in the art to select the optimal reaction conditions or acylation reaction based on known parameters and the desired result.

  PEGylated polypeptides of the invention can generally be used to treat conditions that can be alleviated or modulated by administering a polypeptide described herein, but herein The chemically derivatized polypeptide molecules of the present invention disclosed in (1) have additional activity, increased or decreased biological activity, or other characteristics (eg, increased) compared to their non-derivative molecules. Reduced half-life). The polypeptides, fragments, variants and derivatives thereof of the invention can be used alone, in combination, or in combination with other pharmaceutical compositions. These cytokines, growth factors, antigens, anti-inflammatory agents and / or chemotherapeutic agents are suitable for treating symptoms.

  Similarly, “polynucleotide analog” or “nucleic acid analog” refers to a compound that is different from a polynucleotide or nucleic acid but is equivalent to at least one chemical or biological function of the polynucleotide or nucleic acid. . Thus, polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer and Expression Analysis”, Yodosha, 1997, etc., which are related parts in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T. T. et al. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

(Genetic engineering)
Activins and the like and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for nucleotide transcription. Among vectors, a vector suitable for cloning is called “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra). Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (ie, fragments that can be integrated into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenovirus, baculovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus and retrovirus.

  One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) can replicate autonomously in the host cell into which they are introduced. Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell, and are thereby replicated along with the host genome. Furthermore, certain vectors may direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors”.

  Therefore, in the present specification, the “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory elements used can vary depending on the host cell.

“Recombinant vectors” for prokaryotic cells that can be used in the present invention include pcDNA3 (+), pBluescript-SK (+/−), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY ( Invitrogen) and the like.

  Examples of “recombinant vectors” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], a retroviral expression vector based on murine stem cell virus (MSCV), pEF-BOS, pEGFP, and the like.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  In this specification, “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base sequence that initiates transcription upon binding of RNA polymerase. . Therefore, in this specification, a part having a promoter function of a gene is referred to as a “promoter part”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.

  As used herein, “replication origin” refers to a specific region on a chromosome where DNA replication begins. The origin of replication can either be provided by constructing the vector to include an endogenous origin, or it can be provided by the host cell's chromosomal replication machinery. The latter may be sufficient if the vector is integrated into the host cell chromosome. Alternatively, rather than using a vector containing a viral origin of replication, one of skill in the art can transform mammalian cells by a method that co-transforms a selectable marker and the DNA of the present invention. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase (see US Pat. No. 4,399,216).

  For example, recombinant mammalian expression vectors can preferentially direct expression of a nucleic acid in a particular cell type by expressing the nucleic acid using tissue-specific regulatory elements. Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include developmentally regulated promoters (eg, mouse hox promoter (Kessel and Gruss (1990) Science 249, 374-379) and α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546)), albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymph specific promoter (Calame and Eaton (1988) Adv. Immunol. 43, 235-275), in particular T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) and immunoglobulins. (Banerji et al. (1983) Cell 33, 729-740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (eg, nerve fiber promoters; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86, 5473-5477), pancreas specific promoter (Edrund et al. (1985) Science 230, 912-916), and mammary gland specific promoter (eg, whey promoter; US Pat. No. 4,873,3). 316 and European Application Publication No. 264,166), but are not limited thereto.

  As used herein, “enhancer” refers to a sequence used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique for introducing a nucleic acid molecule into a cell may be used, and examples thereof include transformation, transduction, transfection, and the like. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject. The cell used in the present invention may be a transformant.

  When a prokaryotic cell is used in the present invention for genetic manipulation or the like, the prokaryotic cell includes, for example, a prokaryotic cell belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, animal cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, and human leukemia cells. HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1 and Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

  As used herein, any method for introducing DNA can be used as a method for introducing a recombinant vector. For example, a calcium chloride method, an electroporation method [Methods. Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

(Method for producing polypeptide)
A transformant derived from a microorganism, an animal cell, or the like carrying a recombinant vector incorporating a DNA encoding the polypeptide of the present invention (for example, activin or a variant or fragment thereof) is cultured according to a normal culture method. Then, the polypeptide of the present invention can be produced by accumulating the polypeptide of the present invention and collecting the polypeptide of the present invention from the culture of the present invention.

  The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.

  Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing substances, and peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin or tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used for culturing a microorganism transformed with an expression vector using the trp promoter. An acid or the like may be added to the medium. Cells or organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention includes RPMI1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122). , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum or the like was added to these media A medium or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  In order to isolate or purify the polypeptide of the present invention from the culture of the transformant transformed with the nucleic acid sequence encoding the polypeptide of the present invention, isolation of conventional enzymes well known and commonly used in the art Alternatively, a purification method can be used. For example, when the polypeptide of the present invention is secreted outside the transformant for producing the polypeptide of the present invention, the culture is treated with a technique such as centrifugation to obtain a soluble fraction. Get minutes. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei), etc. Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography A purified sample can be obtained by using a technique such as a chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide of the present invention (for example, activin or a variant or fragment thereof) accumulates in a dissolved state in the cells of the transformant for producing the polypeptide of the present invention, the culture is centrifuged, After collecting the cells in the culture and washing the cells, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei) Anion exchange chromatography using a resin, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenyl sepharose A purified sample can be obtained by using a technique such as a chromatography method, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide of the present invention is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted and centrifuged from the precipitate fraction obtained by the usual method. After recovering the polypeptide, the insoluble body of the polypeptide is solubilized with a polypeptide denaturant. The solubilized solution is diluted or dialyzed into a dilute solution that does not contain the polypeptide denaturing agent or the concentration of the polypeptide denaturing agent does not denature the polypeptide, and forms the polypeptide of the present invention into a normal three-dimensional structure. Then, a purified sample can be obtained by the same isolation and purification method as described above.

  Further, a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. In addition, the polypeptide of the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having affinity for the fused protein [Akio Yamakawa, Experimental Medicine ( (Experimental Medicine), 13, 469-474 (1995)]. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227-8231 (1989), Genes Develop. , 4, 1288 (1990)], the polypeptide of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

  In addition, the polypeptide of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)]. In such fusion proteins, in the expression vector, the proteolytic cleavage site joins the fusion moiety to the recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Introduced to the department. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include pGEX (Pharmacia Biotech; Smith and Johnson (1988) Gene 67, which fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein, respectively. , 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).

  Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself of the present invention. The polypeptide of the present invention can be produced by known methods [J. Biomolecular NMR, 6,129-134, Science, 242, 1162-1164, J. Biol. Biochem. , 110, 166-168 (1991)], can be produced using an in vitro transcription / translation system.

  The polypeptide of the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) based on the amino acid information. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  The structural analysis of the purified polypeptide of the present invention can be carried out by a method usually used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Kagaku Dojin, 1993). is there. The physiological activity of the polypeptide of the present invention can be measured according to a known measurement method.

  Production of soluble polypeptides useful in the present invention can also be accomplished by various methods known in the art. For example, a polypeptide can be derived from an intact transmembrane activin polypeptide molecule by proteolysis by using certain endopeptidases in combination with exopeptidase, Edman degradation, or both. This intact activin polypeptide molecule can be purified from its natural source using conventional methods. Alternatively, intact activin polypeptides can be produced by recombinant DNA techniques utilizing well known techniques for cDNA, expression vectors and recombinant gene expression.

  Preferably, soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for the entire activin polypeptide as starting material. This can be accomplished by conventional chemical synthesis techniques, or by well-known recombinant DNA techniques where only the DNA sequence encoding the desired peptide is expressed in the transformed host. For example, a gene encoding the desired soluble activin polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble activin polypeptide. The specific DNA sequence encoding the desired peptide can also be derived from the full length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of specific regions from cDNA.

  Deletion, substitution or addition (including fusion) of amino acids of the polypeptide of the present invention (for example, activin) can be carried out by site-directed mutagenesis which is a well-known technique. Such deletion, substitution or addition of one or several amino acids may be performed by Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in BioJ. 1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5562 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.

(Cell culture)
It has been found that the cell culture characteristics of stem cell differentiation of a wide variety of media formulations known in the art can be dramatically altered by adjusting the amount of activin or equivalents disclosed herein. .

  Many media formulations have been developed over the years to maximize cell growth, cell viability, and / or biologic production for a given cultured cell or cell line. These media compositions differ, for example, in the number, type, and concentration of growth factors, antibiotics, and amino acid supplements added to the media, and components such as protease inhibitors, and particularly cells in suspension. When grown, it contains one or more anti-foaming agents. Other personalized ingredients include additives that enhance recombinant protein production. For example, if the host cell was developed for gene amplification of a recombinant protein, the selectable marker DHFR (dihydrofolate reductase) typically constitutes part of the host transfected as a selectable marker, And methotrexate is contained in the culture medium. Insulin or other growth factors such as factors that play a role in the energy source cascade, such as IGF, are also often included to enhance cell proliferation.

  The activins disclosed herein or equivalents thereof are expected to have an effect on various media formulations as well as in standard formulations.

For example, all serum-free media formulations contain essential ingredients that allow cell growth, and (1) an energy source, typically glucose or glutamine, or other sugars such as fructose, galactose, mannose, etc .; (2) contains a nitrogen source (typically obtained by inclusion of one or more amino acids); and (3) vitamins (cofactors in enzymatic reactions). Also includes a wide range of inorganic salts including Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ⁻ , HPO ₃ ^{2− and the} like, as well as fatty acids (preferably conjugates), cholesterol, phospholipids, and their precursors. A wide range of fat and fat-soluble ingredients are also essential. The components of a standard serum-free medium formulation are listed in Table 1. This includes GrandIsland Biological Co. (GIBCO), Grand Island, N .; Y. Ingredients found in “DMEM / F-12” obtained from media manufacturers such as For a review of animal cell culture media, see Mizrahi and Lazar, Cytotechnology , 1 : 199-214 (1988). Media considerations for insect cell culture are described in Goodwin, R .; H. (1990) Nature 347 : 209-210. An example of a defined serum-free medium that is individualized for hybridoma culture is described in Kovar, J. et al. (1987) Folia Biology 33: 377-384. Imagawa et al. (1989) PNAS86 : 4122-4126 describe a medium developed to maximize cell proliferation in mouse mammary epithelial cells and Miyazaki et al. (1991) Res. Exp. Med. 191 : 77-83 describes a medium to enhance the viability of rat hepatocytes in vitro. For Xenopus, see Sive, Granger, and Harland. Early Development of Xenopus laevis ColdSpring Harbor Laboratory Press (2000).

  As used herein, “nutrient medium” includes natural medium, semi-synthetic medium, synthetic medium, solid medium, semi-solid medium, liquid medium, etc. Alternatively, any medium may be used as long as it is used for storage and is usually used for cell culture. Examples include Steinberg medium, α-MEM medium, RPMI-1640 medium, or MEM basic medium. As basic components, sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, sugars, or other chemical components or biological components such as serum may be contained depending on the purpose.

  The media components can typically be added in any order, and the final combination can be sterilized by standard methods, such as filter sterilization.

  Utilizing the media of the present invention, a wide range of cell stem cells (both vertebrates and invertebrates) can be differentiated in the desired direction. The culture medium of the present invention can be used to improve cell culture properties in many different cell culture systems.

  In the case of animals, serum-free media such as Iskov media, RPMI media, Dulbecco MEM media, MEM media, and F12 media that are usually used for culturing animal cells can be used. In addition, factors other than serum known to be effective for cell growth and maintenance, such as lipid and fatty acid sources, cholesterol, pyruvate, glucocorticoid, DNA and RNA synthetic nucleosides, etc. are added according to known literature. May be.

  As used herein, the term “culture vessel” refers to a vessel used when proliferating desired cells, for example, undifferentiated cells. Feeder cells such as stromal cells can be maintained and survived as needed, and undifferentiated. Any material or shape may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, or self-replication of cells. Specifically, the material of the culture vessel includes glass, synthetic resin, natural resin, metal, plastic, and the shape is specifically a polygonal prism such as a triangular prism, a cube, a rectangular parallelepiped, a triangular pyramid, a quadrangular pyramid, etc. Polygonal pyramids, arbitrary shapes such as gourds, spheres, hemispheres, cylinders (including a round bottom, ellipse or semicircle) etc. can be mentioned, and for example during culturing from hemisphere to sphere The shape may be changed as necessary. Cultivation may be under open conditions or under closed (sealed) conditions.

  The cell culture method in the present invention is a culture dish having a different matrix coating, a two-dimensional culture method using a culture dish having a different presence or absence of matrix coating, a three-dimensional culture method using a soft gel such as Matrigel, a collagen sponge, Alternatively, a method using them in combination may be mentioned, but preferably a two-dimensional culture method using a culture dish having a different matrix coating or a culture dish having a different presence or absence of a matrix coating, such as a gelatin-coated culture dish or type I collagen. It is a two-dimensional culture method using two of a coated culture dish or a laminin coated culture dish. In addition, when a human-derived cell is used as the multipotent cell, it is preferable to use a type I collagen-coated culture dish.

The culture conditions of the method of the present invention are not limited to specific conditions such as Examples, and generally acceptable conditions can be taken. For example, as the number of cells at the start of differentiation induction, a range of 5.0 × 10 ^{3 to} 5.0 × 10 ⁶ cells / culture dish can be exemplified. The differentiation induction period is, for example, 2 to 10 days (preferably 5 days), or 1 to 4 days (preferably 2 days), and 2 to 5 days (preferably 3 days) in the preculture step. In the case of human mesenchymal cells, the differentiation induction period is 12 to 21 days (preferably 14 days).

  In culturing, physical environmental conditions such as temperature, osmotic pressure, and light, and chemical environmental conditions such as oxygen, carbon dioxide, pH, and oxidation-reduction potential are maintained and maintained by undifferentiated cells before and after differentiation. Any environmental condition may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, and self-replication of undifferentiated cells. Preferred conditions are shown below.

  Specifically, the temperature is 30 ° C. to 40 ° C., preferably 37 ° C. The osmotic pressure is specifically an osmotic pressure under physiological conditions, and preferably an osmotic pressure equal to that of physiological saline.

The light may be as dark as a dark room, or as bright as the brightness outside in sunny weather.
Specifically, as for the oxygen concentration, the culture system is in contact with the gas phase having the oxygen concentration in the gas phase in contact with the gas phase having the oxygen concentration in the gas phase of 10% to 30% in the gas phase. The oxygen concentration may be the oxygen concentration in a state of being in contact with the gas phase, preferably in the state of being in contact with the gas phase having a concentration of oxygen in the gas phase of 20%. Concentration.

  In general, the pH for controlling the pH in the culture system is specifically pH 6.0 to pH 8.0, preferably the pH equivalent to physiological conditions. Carbon dioxide may be used to control the pH, or any other buffer may be used. Specifically, the concentration of carbon dioxide is the concentration of dissolved carbon dioxide in a state where the culture system is in contact with the 5% gas phase.

  As used herein, the term “colony” refers to a visible clump formed from a single cell in a solid medium.

  As used herein, the term “kit” refers to a unit in which parts to be provided (eg, reagents, particles, etc.) are usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably include instructions that describe how to treat the provided parts (eg, reagents, particles, etc.). Such a manual may be any medium. Examples of such a medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN. 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.

(screening)
As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, agents (eg, antibodies), polypeptides or nucleic acid molecules of the invention can be used. For the screening, a system using an actual substance such as in vitro or in vivo may be used, or a library generated using an in silico (computer system) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing a computer modeling drug based on the disclosure of the present invention.

  In one embodiment, the present invention provides an assay for screening candidate or test compounds that bind to or modulate the protein of the present invention (activin), or a biologically active portion thereof. I will provide a. Test compounds of the invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; spatially Accessible parallel solid phase or solution phase library; synthetic library method requiring deconvolution; “one bead one compound” library method; and synthetic library method using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12). 145).

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. MoI. Med. Chem 37: 2678; Cho et al. (1993) Science 2611: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. MoI. Med. Chem 37: 1233.

  The library of compounds can be in solution (eg, Houghten (1992) BioTechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwilla et al. (1990) Proc. Natl. Acad. ci.U.S.A.87: 6378~6382; Felici (1991) J Mol Biol 222: 301~310; Ladner above) can be shown in.

(disease)
In one aspect, the present invention provides a method for treating any disease, disorder or condition where stem cell amplification may be applied, eg, a hematopoietic related disease, disorder or condition.

  Examples of hematopoiesis-related diseases include circulatory diseases, and can be, for example, circulatory systems (blood cells and the like). Such diseases or disorders include, for example, anemia (eg, aplastic anemia (especially severe aplastic anemia), renal anemia, cancerous anemia, secondary anemia, refractory anemia, etc.), cancer or tumor (Eg, leukemia) and hematopoietic failure after chemotherapeutic treatment, thrombocytopenia, acute myeloid leukemia (especially the first remission phase (High-risk group), the remission phase after the second remission phase), acute lymphatic Leukemia (especially the first remission phase, remission phase after the second remission phase), chronic myeloid leukemia (especially the chronic phase, transition phase), malignant lymphoma (especially the first remission phase (High-risk group), 2) Remission period after remission), multiple myeloma (especially early after onset), congenital immune deficiency syndrome, and the like.

  As used herein, “preventing” refers to reducing the likelihood that an organism will contract or develop an abnormal condition.

  As used herein, “treating” refers to having a therapeutic effect and at least partially alleviating or suppressing an abnormal condition in an organism.

  As used herein, “therapeutic effect” refers to an inhibitor or activator that causes or contributes to an abnormal condition. The therapeutic effect alleviates one or more of the symptoms of the abnormal condition to some extent. With respect to the treatment of abnormal conditions, a therapeutic effect may refer to one or more of: (a) an increase in cell proliferation, growth, and / or differentiation; (b) inhibition of cell death. (I.e. delay or stop); (c) inhibition of degeneration; (d) some relief of one or more symptoms associated with an abnormal condition; and (e) enhance the function of the affected cell population. thing. Compounds that exhibit efficacy against abnormal conditions can be identified as described herein.

  As used herein, an “abnormal condition” refers to a function in a cell or tissue of an organism that deviates from its normal function in the organism. The abnormal condition can be associated with cell proliferation, cell differentiation, cell signaling, or cell survival. Abnormal conditions may also include hematopoietic disorders, obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and irregularities in fatty acid uptake and metabolism.

  Examples of abnormal cell growth include cancer, neoplasm, tumor and inflammation.

  Examples of abnormal differentiation include malformation and cancer.

  An example of an abnormal cell signaling state is abnormal cell differentiation.

  Abnormal cell viability is also associated with conditions in which the apoptotic (programmed cell death) pathway is activated or arrested. A number of protein kinases are associated with the apoptotic pathway. Abnormalities in the function of any one of the protein kinases can result in cell immortality or premature cell death.

  In another aspect, the present invention provides both prophylactic and therapeutic methods for treating a subject having (or suspected of having) a hematopoietic related disease, disorder or condition, or a subject having the disorder. I will provide a.

(Gene therapy)
In certain embodiments, a nucleic acid comprising a normal gene nucleic acid sequence of the invention, a sequence encoding an antibody or functional derivative thereof, is a disease or disorder associated with abnormal expression and / or activity of a polypeptide of the invention. Is administered for the purpose of gene therapy. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

  Any method for gene therapy available in the art can be used in accordance with the present invention. An exemplary method is as follows.

  For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExplorMorL. , Stockton Press, NY (1990).

  Therefore, in the present invention, gene therapy using a nucleic acid molecule encoding activin or a variant or fragment thereof may be useful.

  As used herein and as understood in the art, the terms “synthesis” or “synthesize” are purely chemical as opposed to enzymatic methods. Refers to chemical substances (eg, polynucleotides, polypeptides, etc.) produced in Thus, “globally” synthesized chemicals (eg, polynucleotides, polypeptides, etc.) are generated entirely by chemical means, and “partially” synthesized chemicals (eg, Polynucleotide, polypeptide, etc. includes chemicals (eg, polynucleotides, polypeptides, etc.) in which only a portion of the resulting chemicals (eg, polynucleotides, polypeptides, etc.) are produced by chemical means. To do.

  By the term “region” as used herein is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by a contiguous portion of the amino acid sequence of the protein. The term “domain” is defined herein to refer to the structural portion of a biomolecule that contributes to the known or suspected function of the biomolecule. A domain can have the same extent as a region or portion thereof; a domain can also incorporate a portion of a biomolecule that is distinct from a particular region in addition to all or a portion of the region. Examples of activin domains of the present invention include, but are not limited to, signal peptides, extracellular (ie, N-terminal) domains, leucine-rich repeat domains.

(Administration and composition for regeneration / treatment / prevention)
The present invention provides a method for the treatment, inhibition and prevention of neurological diseases, disorders or abnormal conditions, or hematopoietic related diseases, disorders or abnormal conditions by administration of an effective amount of a compound or pharmaceutical composition of the present invention to a subject. provide. In a preferred aspect, the compound can be substantially purified (eg, in a state substantially free of substances that limit its effect or cause undesirable side effects).

  In the present specification, the “effective amount for diagnosis, prevention, treatment or prognosis” refers to an amount which is recognized as being medically effective in diagnosis, prevention, treatment (or treatment) or prognosis, respectively. Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

  The animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has a nervous system or a similar system. Preferably, it is a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (for example, single holes, marsupials, Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents , Rabbit eyes, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, cells derived from primates (eg, chimpanzee, Japanese monkey, human) are used. Most preferably, human-derived cells are used.

  When the nucleic acid molecule or polypeptide of the present invention is used as a medicament, such a composition may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Examples include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, a medicament of the invention comprises a polypeptide or polynucleotide, such as activin or a variant or fragment thereof, or a variant or derivative thereof, one or more physiologically acceptable carriers, excipients or It is administered in the form of a composition containing with a diluent. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, for example Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone); amino acid (Eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol) Salt formation vs. Io (E.g., sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)) but the like are not limited thereto.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The general preparation method of the pharmaceutical composition of this invention is shown below. In addition, veterinary drug compositions, quasi drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  The polypeptide, polynucleotide and the like of the present invention are mixed with a pharmaceutically acceptable carrier, and are solid preparations such as tablets, capsules, granules, powders, powders, suppositories, or syrups, injections, suspensions. It can be administered orally or parenterally as a liquid preparation such as an agent, solution or spray. As described above, the pharmaceutically acceptable carrier includes an excipient, a lubricant, a binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, an adsorbent, a humectant, a solubilizing agent in a solid preparation, Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like can be mentioned. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than the polynucleotide of this invention, polypeptide, etc. with the composition of this invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

  Examples of the excipient in the solid preparation include glucose, lactose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

  Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal silicic acid, talc, and polyethylene glycol.

  Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, starch solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate , Potassium phosphate, shellac and the like.

  Examples of disintegrants in solid preparations include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Examples include, but are not limited to, esters, sodium lauryl sulfate, starch, stearic acid monoglyceride, lactose, and calcium calcium glycolate.

  Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter and hydrogenated oil.

  Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

  Examples of the adsorbent in the solid preparation include, but are not limited to, starch, lactose, kaolin, bentonite and colloidal silicic acid.

  Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

  Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, aspartic acid and the like.

  Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and lactose.

  When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Tablets include tablets with ordinary coatings as necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When forming into the form of a suppository, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added in addition to the additives listed above, but are not limited thereto.

  Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to them.

  Suitable examples of the suspending agent in the liquid preparation include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, Examples include, but are not limited to, hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate and citrate.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When preparing as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericidal agent, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, if necessary, the pharmaceutical composition may contain coloring agents, preservatives, flavors, flavoring agents, sweeteners, and the like, as well as other agents.

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizer as necessary (Japanese Pharmacopoeia 14th edition, its supplement or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, A R. Gennaro, ed., Mack Publishing Company, 1990, etc.) and a sugar chain composition having a desired degree of purity, and prepared and stored in the form of a lyophilized cake or aqueous solution. Can be done.

  Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, etc.). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through the epithelium or mucosal lining (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.), and others. It can be administered with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the invention may include any suitable route (including intraventricular and intrathecal injection; intraventricular injection, for example, intraventricularly attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce into the central nervous system by a catheter). Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

  In certain embodiments, it may be desirable to administer the polypeptide, polynucleotide or composition of the invention locally to the area in need of treatment (eg, central nervous system, brain, etc.); this limits Although not intended, for example, local injection during surgery, topical application (eg in combination with a post-surgical wound dressing), by injection, by catheter, by suppository, or an implant (the implant is shearable ( a porous, non-porous, or glue-like material), including membranes or fibers such as (sialastic) membranes. Preferably, when administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

  In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious). Disesease and Cancer, Lopez-Berstein and Fidler (eds.), Liss, New York, pages 353-365 (1989); see Lopez-Berstein, pages 317-327; see broadly the same book).

  In yet another embodiment, the compound or composition can be delivered in a controlled sustained release system. In one embodiment, a pump can be used (Langer (supra); Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugDrivable Drug Bioavailability. And Ball (eds.), Wiley, New York (1984); see Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 1985); During et al., Ann. l.25: 351 (1989); see also Howard et al., J. Neurosurg. 71: 105 (1989)).

  In yet another embodiment, the controlled sustained release system can be placed near the therapeutic target, i.e., the brain, and therefore requires only a portion of the systemic dose (e.g., Goodson, Medical Applications of Controlled Release). , (Supra), Vol. 2, pages 115-138 (1984)).

  Other controlled controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  The amount of the composition used in the treatment method of the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. Can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  The dose of the polypeptide, polynucleotide and the like of the present invention varies depending on the age, weight, symptom or administration method of the subject, and is not particularly limited, but is usually 0.01 mg to 10 g in the case of oral administration per day for an adult. Preferably, it may be 0.1 mg to 1 g, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like. In the case of parenteral administration, it is 0.01 mg to 1 g, preferably 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like.

  As used herein, “administering” refers to a polypeptide, polynucleotide, factor or the like of the present invention or a pharmaceutical composition containing the same, alone or in combination with other therapeutic agents, to cells or tissues of an organism. It means capturing. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This also includes a procedure in which the combined drugs are administered together as a therapeutic mixture, and the combined drugs are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  An abnormal condition can also be prevented or treated by administering a compound (such as an agent identified by the present invention) to a group of cells having an abnormality in a signal transduction pathway to an organism. The effect of administering the compound on the biological function can then be monitored. This organism is preferably a laboratory animal such as a mouse, rat, rabbit, guinea pig, goat or monkey (monkey or ape), and most preferably a human.

  In the present specification, the “instruction” describes a method for administering or diagnosing the pharmaceutical of the present invention to a doctor, a patient or other person who performs administration, or a person to be diagnosed (which may be the patient). It is. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

  Determination of the end of treatment according to the method of the present invention is based on clinical symptoms characteristic of diseases (eg, hematological disorders) associated with standard clinical laboratory results or activins such as commercially available assays or instrumentation. Can be supported by the disappearance of Treatment can be resumed by recurrence of a disease associated with activin or the like (eg, a blood system disease).

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may optionally be attached to such containers, which may be made, used or sold for administration to humans. Represents approval by government agencies.

  Plasma half-life and biodistribution of drugs and metabolites in plasma, tumors and major organs can also be determined to facilitate selection of the most appropriate drug to inhibit the disorder. Such a measurement can be performed. For example, HPLC analysis can be performed on the plasma of a drug-treated animal, and the location of the radiolabeled compound can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that exhibit strong inhibitory activity in screening assays but lack pharmacokinetic characteristics can be optimized by chemical structure changes and retests. In this regard, compounds that exhibit good pharmacokinetic characteristics can be used as models.

  Toxicity studies can also be performed by testing the compositions of the present invention. For example, toxicity studies can be performed in appropriate animal models such as: (1) a compound is administered to mice (untreated control mice should also be used); (2) each Blood samples are periodically obtained from one mouse in the treatment group via the tail vein; and (3) the sample is obtained from the number of red blood cells and white blood cells, the composition and the ratio of lymphocytes to polymorphonuclear cells Analyze about. Comparison of the results for each dosing regimen with controls shows whether there is toxicity.

  Further studies can be performed by sacrificing the animals at the end of each toxicology study (preferably, the American Veterinary Medical Guidelines Report of the American Veterinary Medical Asc. E. A. 93). Vet.Med.Assoc.202: 229-249). A representative animal from each treatment group can then be tested by global examination for direct evidence of metastasis, abnormal illness or toxicity. Overall abnormalities in the tissue are described and the tissue is examined histologically. Compounds that cause weight loss or blood component loss are not preferred, as are compounds that have adverse effects on major organs. In general, the greater the adverse effect, the less preferred the compound.

(Differentiation of hematopoietic cells)
Hematopoietic cells are made in the bone marrow, differentiate into erythrocytes, platelets, leukocytes, etc. and flow through the peripheral blood. Looking at the differentiation of myeloid cells, the largest source is pluripotent stem cells, followed by hematopoietic stem cells specialized in hematopoietic cells, which differentiate into pluripotent progenitor cells, and then myeloid cells Differentiating into progenitor cells and lymphoid progenitor cells In the myeloid system, pluripotent stem cells differentiate into cells called CFU-GEMM. The cells differentiate from CFU-GEM cells to CFU-GM cells, followed by myeloblasts, promyelocytes, and myelocytes. These are cells present in the bone marrow, and when they differentiate, they become neutrophils and flow in the peripheral blood. When going to the next line, it goes from the cell called CFU-GM toward monocytes and differentiates into monocytes, promonocytes, and monocytes. These monocytes appear in the peripheral blood. In the third line, CFU-GEMM cells are differentiated into BFU-E cells, and then differentiated into pre-erythroblasts, erythroblasts, and erythrocytes. There is also a megakaryocyte system, which differentiates into CFU-Meg (abbreviation for megacaryosite), megakaryocyte, megakaryocyte, and platelet.

  In this differentiation process, leukemia results from abnormal pluripotent stem cells. Therefore, the present invention can be applied to the treatment of leukemia in the sense of improving such abnormalities.

  In the lymphocyte lineage, CML differentiates from pluripotent stem cells into lymphoid stem cells, which are divided into B cell lines and T cell lines. Separately, there is a line that separates towards NK cells. The B cell line is differentiated into precursor B cells, precursor precursor B cells (pre-pre-B-cell), early B cells (early-B-cell), etc., and intermediate B cells (intermediate-B cells). -Cell), mature B cells (mature B-cell), plasmacytoid-B-cells, and plasma cells (plasma-cell). As T cell lines, thymic progenitor cells, immature thymocytes, common thymocytes, and mature thymocytes are differentiated. There are other routes to go to helper / inducer T cells and to differentiate from mature thymocytes into suppressor / cytotoxic T cells.

(Method of identifying hematopoietic stem cells)
A typical method for identifying hematopoietic stem cells will be described below.

(A. Spleen colony formation method)
When hematopoietic cells of a syngeneic mouse are intravenously injected into a mouse irradiated with a lethal dose of radiation, a bulge (colony) is observed on the surface of the spleen on the 8th to 14th days. Each colony consists of various blood cells. One colony is derived from one stem cell, and the mother cell forming this spleen colony is called CFU-S (colony forming unit in splene). . Cells forming splenic colonies (Day 8 CFU-S) formed on the 8th day are mainly erythroblasts, whereas spleen colonies formed on the 12th day (Day 12 CFU-S) are red. In addition to blasts, granulocytes, megakaryocytes, and even B lymphocytes are included and have high proliferation ability and are derived from pluripotent stem cells. Day 12 CFU-S is used as an indicator of pluripotent stem cells. In addition, hematopoietic stem cells remaining after administration of 5-fluoro-uracil (5-FU), which is an anticancer agent, exhibit a very high proliferative ability and are therefore referred to as pre-CFU-S, which is a mother cell of CFU-S.

(B. Long-term bone marrow remodeling ability)
This is a method of reconstructing the hematopoietic system of mice irradiated with lethal dose radiation by transplanted cells and observing whether they can be maintained for a long time. Currently, it is the most reliable in terms of pluripotency and self-renewal ability of hematopoietic stem cells. As the marker, expression of neomycin resistance gene, male and female sex chromosomes, congenic mice and the like are used. Although this method was difficult to quantify, recently, a competitive repopulation assay has been used in which recipient hematopoietic cells are transplanted together with donor hematopoietic cells and the rate of reconstitution is examined. In humans, since it is difficult to establish an in vivo transplantation experiment system like mice, human hematopoietic stem cells are transplanted into immunodeficient mice (scid mouse) that do not cause rejection due to lack of lymphocytes. Sci-hu mice are used. In this system, the human hematopoietic mechanism can be maintained for a long time in the mouse.

(C. In vitro colony method)
By culturing hematopoietic cells (bone marrow cells, splenocytes, etc.) in the presence of various cytokines in a semi-solid medium such as methylcellulose and soft agar, and estimating the number and properties of hematopoietic stem cells from the formed cell population (colony) is there. By analyzing this colony, it is possible to observe and measure various hematopoietic progenitor cells and hematopoietic stem cell differentiation and proliferation processes in vitro. Mixed colonies (CFU-Mix, CFU-GEMM) and highly differentiated colonies (HPP-CFC) are from cells that form a single colony (CFU-GM, BFU-E), etc. Undifferentiated and blast colony forming cells (CFU-blast) are considered to be the most undifferentiated. By this method, it is possible to capture the proliferation and differentiation processes of hematopoietic stem cells and progenitor cells in vitro. Furthermore, in recent years, the action of various cytokines involved in hematopoiesis can be estimated by using a serum-free medium or performing single cell culture of hematopoietic stem cells.

(D. Co-culture system with stromal cells)
The hematopoietic microenvironment is closely involved in the differentiation and proliferation of hematopoietic stem cells. In 1977, Dexter et al. Showed that hematopoietic stem cells can be cultured on bone marrow stromal (stromal) cells for a long period of time of several months or longer (Dexter culture method). It was possible to reproduce the hematopoietic microenvironment in vitro. In this culture system, hematopoietic progenitor cells lose colony forming ability early, whereas undifferentiated hematopoietic stem cells can maintain colony forming ability and bone marrow remodeling ability for a long period of time. For this reason, it is also used for measurement of undifferentiated hematopoietic stem cell activity. Especially in humans, since the in vivo system is difficult to use, cells that can maintain colony-forming ability for a long time on stromal cells are used as LTC-IC (Long Term Culture-Initiating Cells) as an indicator of undifferentiated hematopoietic stem cells. Yes.

(Best Mode for Carrying Out the Invention)
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 obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  In one aspect, the present invention provides a method for preparing hematopoietic stem cells. The method comprises activin for a time sufficient to differentiate A) providing undifferentiated cells, B) dissociating the undifferentiated cells, and C) the undifferentiated cells after dissociation into hematopoietic stem cells. Culturing in a medium. A typical example of this method is shown in FIG. By adjusting the concentration of activin, the present invention succeeded in inducing various differentiations. Such a thing is not known conventionally, and this invention shows a remarkable effect with respect to a prior art. For example, if the conditions are 5 animal caps in a single culture and 3 hours of activin treatment, an irregular epidermis is formed when no activin is added, and blood cells are formed if about 1 ng / ml, and 10 ng If it is about / ml, muscles and notochord are formed, and if it is about 100 ng / ml, endoderm cells are formed. FIG. 2 shows an example of culturing under the conditions of 5 animal caps in single culture and 3 hours of activin treatment time. For blood islands specifically intended in the present invention, as a condition for differentiation into blood islands, 10 dissociated animal caps were treated with 1 ng / ml of activin (Human recombinant Activin A) for 3 hours to be reassembled and sandwiched. The condition of differentiation into blood islands when cultured was established.

  Here, in one embodiment, the undifferentiated cells can use cells derived from late blastocysts. Such late blastocysts are preferable because they are relatively large in number and have sufficient undifferentiation ability, but are not limited thereto.

  In one embodiment, the present invention further comprises the step of contacting the cells obtained by step D) C) with undifferentiated cells. In such a case, it was unexpectedly observed that the differentiation efficiency was further improved. FIG. 3 shows an example of culturing under the conditions of 5 animal caps in the case of contact (here, sandwich) and activin treatment time of 3 hours.

  Another preferred embodiment is shown in FIG. Here, the time condition is 5 hours. As an example at 5 hours, FIG. 5 shows an example of single culture and FIG. 6 shows an example of sandwich culture.

  Another preferred embodiment is shown. Here, the time condition is 10 hours. Examples at 10 hours are shown in FIGS.

  In another embodiment, the contact between activin and cells in the present invention is achieved by sandwiching the cells obtained in step C) with a sheet of undifferentiated cells. Without wishing to be bound by theory, one reason may be, but is not limited to, that the culture in the sandwich state was in a suitable form to achieve differentiation.

  In one embodiment, the undifferentiated cells used in the present invention comprise late blastula animal pole cells. Although not wishing to be bound by theory, it is believed that having an animal pole (so-called “animal cap”) improves reactivity to activin, but is not limited thereto.

  In another embodiment, in the present invention, the contact between activin and an undifferentiated cell is performed for a time sufficient to contact the undifferentiated cell from the initial contact to the final differentiation. Such time is usually at least 1 day, more preferably 1 day or more and 3 days or less.

  The activin used in the present invention may be in any form / subtype as long as it has an action as activin, and examples thereof include activin-A, activin-B, inhibin and activin-C. Yes, but not limited to them. Since activins are highly conserved in animals and amphibian activins are also effective against mammalian organisms, it is understood herein that any activin can be used in any organism .

  In one embodiment, the activin used in the present invention comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12.

  In one embodiment, activin used in the present invention is applied at a concentration of 0.5-1 ng / ml. Activin given at this concentration induces blood islands. If it is 5 ng to 10 ng, muscle cells or notochord cells are induced. At 50 ng to 100 ng, heart cells are induced. Studies have reported that activin A, activin AB, and activin B have the same inducing ability (Nakamura et al., Isolation and charactarization of native activin B. J Biol. Chem. 1992, 267, 16385-9). Activin-A is a dimer of inhibin βA (human inhibin βA accession number NM002192; SEQ ID NOs: 1 and 2). Activin-AB is a dimer of inhibin βA and inhibin βB (human inhibin βB accession number NM002193; SEQ ID NOs: 3 and 4). Activin-B is a dimer of inhibin βB. Activin-C is a dimer of inhibin βC (human inhibin βC accession number NM005538; SEQ ID NOs: 5 and 6). Inhibin is a dimer of inhibin α (human inhibin α accession number NM002191; SEQ ID NOs: 7 and 8).

  In another embodiment, the hematopoietic stem cells used in the present invention are characterized by expressing a stem cell leukemia protein (SCL) marker.

In one embodiment, the dissociation performed in the present invention is accomplished by culturing in a medium that does not contain Ca ²⁺ or Mg ²⁺ .

In another embodiment, the dissociation performed in the present invention is accomplished by culturing in a medium without Ca ²⁺ or Mg ^{2+ for} at least 10 minutes.

In another embodiment, the dissociation performed in the present invention is accomplished by culturing in a medium that does not contain Ca ²⁺ or Mg ^{2+ for} 10 to 20 minutes.

  These dissociations are desirable for inducing differentiation in the present invention. It was not clear in the prior art that such a treatment is preferable.

  In a preferred embodiment, in the method of the present invention, in the step of culturing the undifferentiated cells after dissociation in a medium containing activin for a time sufficient to differentiate into hematopoietic stem cells (step C), the cells are dissociated and reassembled. Is formed. Formation of dissociated reassemblies is usually accomplished by culturing in the medium for at least 30 minutes, and may be cultivated longer.

  In another embodiment, the activin treatment in the present invention is performed for at least 3 hours, but is not limited thereto, and may be shorter if the effect is seen, or 3 hours if the effect is insufficient. You may do it above.

  In a preferred embodiment, the undifferentiated cells used are provided by animal caps. The idea of using animal caps did not exist in conventional hematopoietic stem cell preparations. The present invention provides a method for efficiently preparing hematopoietic stem cells using such a novel concept. In the present invention, 5 to 10 animal caps are preferably used, but are not limited thereto.

  In one embodiment, the present invention relates to the step of culturing the undifferentiated cells after dissociation in a medium containing activin for a time sufficient to differentiate into hematopoietic stem cells, wherein dissociated reassemblies are formed, The method further includes the step of culturing the aggregate together with the undifferentiated cells.

  More preferably, in the method of the present invention, in the step of culturing the undifferentiated cells after dissociation in a medium containing activin for a time sufficient to differentiate into hematopoietic stem cells, dissociated reassemblies are formed, The method further includes the step of sandwich-culturing the aggregate with the undifferentiated cells.

  In one embodiment, the cells used are vertebrate cells, preferably targeted to, but not limited to, amphibian cells (eg, frogs such as Xenopus).

  In another aspect, the present invention provides a cell prepared by the method for preparing the hematopoietic stem cell of the present invention. Such cells are very useful as hematopoietic stem cells.

  In still another aspect, the present invention provides a blood island prepared by the method for preparing the hematopoietic stem cell of the present invention. Such a blood island is a novel artifact because it could not be induced by the prior art.

In another aspect, the present invention provides a medium used in the step of dissociating undifferentiated cells in a method for inducing hematopoietic stem cells. This medium is characterized in that it contains neither Ca ²⁺ nor Mg ²⁺ . Since it has not been known so far that such a medium is used in the step of dissociating undifferentiated cells in the method for inducing hematopoietic stem cells, the present invention provides a medium containing neither Ca ²⁺ nor Mg ^2+. Provide new uses. It is understood that the basic components of the medium used here may be any as long as the cells used survive.

In a specific embodiment, the medium of the invention (medium without Ca ²⁺ or Mg ²⁺ ) is 58 mM NaCl, 0.67 mM KCl, 3.0 mM hydroxyethylpiperazinylethanesulfonic acid and 100 mg / L kanamycin sulfate, pH 7 .4 composition.

  In another aspect, the present invention provides a medium used in the step of differentiating undifferentiated cells into hematopoietic stem cells in a method for inducing hematopoietic stem cells. This medium is characterized by containing an effective amount for inducing activin into hematopoietic stem cells (for example, a concentration of 0.5 to 1 ng / ml, preferably about 1 ng / ml). It has not previously been suggested that hematopoietic stem cells can be induced by specific amounts, such as a concentration of 0.5-1 ng / ml, and the present invention provides a significant effect.

In another aspect, the present invention provides a combination of media for use in a method for inducing hematopoietic stem cells. A medium (set or kit) of such a combination is characterized in that it contains no Ca ²⁺ or Mg ^2+, and a medium characterized in that it contains an effective amount for inducing activin into hematopoietic stem cells. including. It was not known that such a combination of media would be used.

  In another aspect, the present invention provides a kit for preparing hematopoietic stem cells. Such a kit comprises a) dissociation means for dissociating the undifferentiated cells, b) differentiation means for differentiating the undifferentiated cells into hematopoietic stem cells, and c) differentiation of the undifferentiated cells after dissociation into hematopoietic stem cells. And instructions for instructing the undifferentiated cells to be differentiated by means of sufficient time differentiation. Here, preferably, the differentiation means is a medium containing activin. Differentiation means can be appropriately designed by those skilled in the art based on the description given above in this specification.

  In another aspect, the present invention provides the use of activin for preparing hematopoietic stem cells. As the activin used here, any of the embodiments described above in this specification can be used.

  In another aspect, the present invention provides a composition for preparing hematopoietic stem cells, the composition containing an effective amount for inducing activin into hematopoietic stem cells. As the constituent elements used here, any of the embodiments described above in this specification can be used. Preferably, activin is present at a concentration of 0.5-1 ng / ml (preferably about 1 ng / ml).

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the embodiments, but only by the claims.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Animals were handled according to the spirit of animal welfare, in compliance with the standards stipulated at the University of Tokyo Animal Experiment Facility.

Example 1
In many of the organ formations using animal caps reported so far, activin treatment was performed on sheet-shaped animal caps, and single culture or sandwich culture was performed. In this method, cells that receive activin signal and cells that do not receive activin are mixed and non-uniform.

  In this example, dissociation and activin treatment were performed, so that each cell uniformly received activin signal, and single culture and sandwich culture were performed (FIG. 7).

(Removal of undifferentiated cells)
Xenopus undifferentiated cells were removed by the method described below. Undifferentiated and multipotent sheet-like cells at the animal pole of Xenopus embryos that reached late blastocysts were cultured in a culture solution (Steinberg's Solution (SS); 58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca). (NO ₃ ) ₂ , 0.83 mM MgSO ₄ , 3.0 mM hydroxyethylperazinyl etheric acid acid, and 100 mg / L kanamicin sulfate, pH 7.4) was transferred to a petri dish with a 3% agarose filled with 3% agarose. Was taken out to 0.5 mm square.

(Dissociation of cells)
First, as a pretreatment for performing the activin treatment, the sheet-shaped undifferentiated cells taken out from the late Xenopus late blastula were dissociated by the following method. SS (no ^Ca 2+ without calcium and magnesium ions, ^{Mg 2+} Steinberg solution ^{(Ca 2+, Mg 2+ Free Steinberg} 's Solution); 58mM NaCl, 0.67mM KCl, 3.0mM hydroxyethyl piperazinyl ethanesulfonic acid , And 100 mg / L kanamycin sulfate, pH 7.4) were taken out into a culture container (for example, a 96-well culture dish) filled with 100 μL. Approximately 10 sheet-shaped undifferentiated cells (450 when the animal cap of stage 8.5 was cut into 0.5 mm square) were transferred and allowed to stand for 20 minutes. This treatment loosened the cell-cell interaction of the sheet-like undifferentiated cells, and each cell dissociated instead of the sheet. Xenopus cleaves when fertilized. This cleavage is said to occur almost synchronously until the 12th division. A fertilized egg (embryo) in the early cleavage stage is called a morula. An embryo at a time when the cleavage is advanced, the cell size is reduced, and a cavity called a blastocoel can be seen inside the embryo is called a blastocyst. The blastocyst is roughly classified into an early blastocyst (stage 7), an intermediate blastocyst (stage 8), and a late blastocyst (stage 9). In Xenopus laevis, it is said that mRNA transcription begins in the metaphase blastula. In later blastocysts, cell division synchrony is lost. Following the late blastocyst, a morphogenic movement with gastrulation movement begins. Along with gastrulation movement, ectoderm, mesoderm, and endoderm are formed. Embryos in this developmental stage are called gastrulation embryos. Stage 8.5 corresponds to the middle of the middle and late blastocysts (conceptual definition). Xenopus eggs are packed with nutrients (egg yolk). Because it is heavy, it is usually biased in the direction of gravity. The egg yolk side is called the plant pole, and the other side is called the animal pole. The surface layer of the Xenopus egg on the side of the animal pole has a black pigment, usually with the black pigment side (animal pole side) facing up. The plant pole side with many uncolored egg yolks always faces down according to the direction of gravity. The blastocoel is formed on the side of the animal pole, and the upper part from the upper end of the blastocoel is called the animal pole, the lower part to the lower part of the blastocoel is called the plant pole, and the middle is called the band. The animal pole side cell above the blastocoel is called an animal cap. Animal cap cells are known to be undifferentiated and multipotent. So far, it has been used as a reaction system in the search for mesoderm-inducing factors, as a starting material for tissue formation in vitro, and for gene function analysis using an overexpression system. It has been empirically known that animal caps of early blastocysts, intermediate blastocysts, and late blastocysts have different sensitivities to activin. The early blastula is not very sensitive. It is said that the sensitivity to activin is the highest in the mid-blastocyst. Late blastocysts are said to be less sensitive to activin than intermediate blastocysts. In gastrulation, the sensitivity is further reduced (Abe et al., 2004).

(Induction of blood islands (hematopoietic stem cells))
After dissociating the cells, remove SS without calcium and magnesium ions, add 1 ng / mL activin (prepared with SS containing 0.1% BSA), and use a pipette (eg Pasteur pipette) Stir and let stand for 3 hours. This treatment caused the cells to undergo activin treatment while reassembling. The dissociated reassemblies thus treated with activin were transferred to a culture vessel (for example, a 12-well culture dish) filled with SS containing 0.1% BSA. Subsequently, the dissociated and reassembled body was transferred to a petri dish with 3% agarose filled with SS and divided into 5 equal parts. This dissociated and reassembled body divided into 5 equal parts is sandwiched between two sheet-like undifferentiated cells (extracted from Xenopus late blastocysts at 0.7 mm square) and completely adhered. Until 10 minutes. When this explant is transferred to a culture vessel filled with SS containing 0.1% BSA (for example, a 96-well culture dish) and cultured for 3 days, blood islands (hematopoiesis) surrounded by irregularly shaped epidermis Stem cells) were induced at a high rate (FIG. 8a). In addition, since blood islands (hematopoietic stem cells) are formed inside the explant that swells transparently, it was easy to identify and observe blood islands (hematopoietic stem cells) (FIG. 8b). In this culture process, blood islands (hematopoietic stem cells) are not induced when dissociated reassemblies are cultured without being sandwiched between undifferentiated cells in sheet form or when the activin treatment concentration or treatment time is changed. It was.

(Identification of blood islands (hematopoietic stem cells))
As described above, the induced blood island (hematopoietic stem cell) has a tissue structure peculiar to the blood island (FIG. 8a), and a protein (marker protein) peculiar to the blood island (hematopoietic stem cell) by the RT-PCR method. It was confirmed that SCL (stem cell leukemia protein) was expressed (FIG. 9).

(Example 2: Dissociation reassembly method (1): Activin treatment (3 hours))
Animal caps of late blastocysts (st. 8.5) were cut into 0.5 mm squares and 10 pieces were collected. This was treated with a Steinberg solution not containing Ca ²⁺ and Mg ²⁺ for 20 minutes, and then the Steinberg solution was ^removed , an activin solution containing Ca ²⁺ and Mg ²⁺ was added, and the cells were dissociated by pipetting.
Cells were reassembled during the 3 hours of activin treatment. The dissociated reassemblies were washed with Steinberg's solution containing 0.1% BSA, and then cultured alone or sandwiched with two animal caps excised from late blastocysts. The external shape of the explant and the differentiated tissue were observed to examine gene expression (FIG. 10).

(result)
The explants obtained by culturing the untreated dissociated reassemblies alone became irregularly shaped. Activin treatment 1 ng / ml treatment swelled transparently, and activin 10 ng / ml treatment explants. In the activin 100 ng / ml treatment, a white lump was formed (FIG. 11).

  A tissue section of a single culture of dissociated reassortant is shown in FIG.

  When the expression of various markers was observed, blood cells were observed at 1 n / g ml of activin, muscle at 10 ng / ml, and endoderm cells at 100 ng / ml (FIG. 13).

  The outline of the sandwich culture of dissociated reassemblies was observed (FIG. 14).

The explants obtained by sandwich culture of untreated dissociated reassemblies became irregularly shaped.
Even with the activin treatment 1 ng / ml treatment, the explants became irregularly shaped. In the activin 10 ng / ml treatment, a wrinkle-like structure was observed. The activin 100 ng / ml treatment resulted in a transparent bulging profile with cement glands.

  Tissue sections of sandwich cultures of dissociated reassemblies were observed (FIG. 15).

  It was observed that blood islands were differentiated at 1 ng / ml of activin (FIG. 16).

(Example 3: Dissociation reassembly method (2): Activin treatment (5 hours))
Animal caps of late blastocysts (st. 8.5) were cut into 0.5 mm squares and 10 pieces were collected. This was treated with a Steinberg solution not containing Ca ²⁺ and Mg ²⁺ for 20 minutes, and then the Steinberg solution was ^removed , an activin solution containing Ca ²⁺ and Mg ²⁺ was added, and the cells were dissociated by pipetting.

  Cells were reassembled during 5 hours of activin treatment. The dissociated reassemblies were washed with Steinberg's solution containing 0.1% BSA, and then cultured alone or sandwiched with two animal caps excised from late blastocysts. The external shape of the explant and the differentiated tissue were observed to examine gene expression (FIG. 17).

(result)
The explants obtained by culturing the untreated dissociated reassemblies alone became irregularly shaped. Activin treatment 1 ng / ml treatment swelled transparently, and activin 10 ng / ml treatment explants. In the activin 100 ng / ml treatment, a white lump was formed (FIG. 18).

  Endodermal cells were observed at 100 ng / ml of activin (FIGS. 19 and 20).

The explants obtained by sandwich culture of untreated dissociated reassemblies became irregularly shaped.
Activin treatment 1 ng / ml treatment swelled transparently, and activin treatment 10 ng / ml treatment showed a cocoon-like structure. In the activin 100 ng / ml treatment, both pulsating and those with cement glands were seen (FIG. 21).

  A torso structure was seen at 10 ng / ml of activin. At 100 ng / ml, 50% of anterior structures including cement glands and nerve masses and those differentiated into the heart were observed (FIGS. 22 and 23).

(Discussion)
When disaggregated animal caps were treated with activin to form reassemblies and cultured alone, medium concentration of activin treatment produced muscle, and high concentration activin treatment produced endoderm cells regardless of the length of treatment time. Differentiated.

  When the dissociated reassemblies treated with activin for 3 hours were sandwich-cultured with an untreated animal cap, blood islands were differentiated by low concentration activin treatment (1 ng / ml).

  When the dissociated reassemblies treated with activin for 5 hours were sandwich-cultured with an untreated animal cap, the anterior nerve and heart were differentiated by high-concentration activin treatment.

(Example 4: Dissociation reassembly method (3): Activin treatment (5 sheets, 3 hours))
In Example 2, instead of using 10 sheets, an experiment was performed in this example in which 5 sheets were treated with activin exposure for 3 hours. Other procedures were the same as in Example 2.

  As a result, as shown in FIGS. 2 and 3, the same result as when 10 sheets were used could be obtained.

  Therefore, it can be seen that the number of cell sheets used has a certain density range.

(Example 5: Dissociation reassembly method (4): Activin treatment (5 sheets, 5 hours))
In Example 3, instead of using 10 sheets, an experiment was performed in this example in which 5 sheets were treated with activin exposure for 5 hours. Other procedures were the same as in Example 3.

  As a result, as shown in FIGS. 5 and 6, the same results as when 10 sheets were used could be obtained.

  Therefore, it can be seen that the number of cell sheets used has a certain density range.

(Example 6: Implementation with other solutions)
Use other solutions instead of the Steinberg solution used in the procedures shown in Examples 1 and 2 to see if similar effects are obtained.

MMR (pH 7.5, 0.1 M NaCl, 2 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , and 5 mM HEPES) is used as another solution.

  When experimented, differentiation effects similar to Steinberg's solution can be observed.

(Example 7: Demonstration with other activins)
Whether or not activin-B, activin-C and inhibin can be obtained in place of activin-A used in Example 1 is confirmed. The following is used as the drug. These can be performed by producing recombinant protein from genetic information, or activin A (Human Activin A, R & D, catalog number # 338-AC-005), activin AB (Human ActivinAB, R & D, catalog number) # 1066-AB-005), Activin B (Human Activin B, R & D, catalog number # 659-AB-005) can be purchased and used.

Activin-A: dimer of inhibin βA (human inhibin βA accession number NM002192; SEQ ID NOs: 1 and 2 (nucleic acids and amino acids))
Activin-AB: dimer of inhibin βA and inhibin βB (human inhibin βB accession number NM002193; SEQ ID NOs: 3 and 4 (nucleic acids and amino acids))
Activin-B: dimer of inhibin βB.

Activin-C: dimer of inhibin βC (human inhibin βC accession number NM005538; SEQ ID NOs: 5 and 6 (nucleic acids and amino acids))
Inhibin: dimer of inhibin α (accession number NM002191 of human inhibin α; SEQ ID NOs: 7 and 8 (nucleic acids and amino acids))
Xenopus activin A (X68250; SEQ ID NOs: 9 and 10)
Xenopus inhibin βB: (S61773; SEQ ID NOS: 11 and 12).

In the above experiment, the following documents can be referred to.
Nakamura etal., Isolation and characterization of native activin B. J Biol. Chem. 1992,267, 16385-9
Uchiyama and Asashima, Specific erythroid differentiation of mouse erythroleukemia cells by activins and its enhancement by retinoic acids. Biochem Biophys. Res. Commun. 1992, 187, 347-52
Fukui et al., Isolation and characterization of Xenopus follistatin and activins. Dev. Biol. 1993, 159, 131-9
Fukui et al., Identification of activins A, AB, and B and follistatin proteins in Xenopusembryos.Dev. Biol. 1994, 163, 279-81
Nakano et al., Comparison of mesoderm-inducing activity with monomeric and dimeric inhibin alpha and beta-A subunits on Xenopus ectoderm. Horm. Res. 1995, 44, Suppl. 2,15-22.

(Example 8: Verification of the optimal example about the timing which takes out an animal cap)
Next, the optimal example is verified about the timing which takes out an animal cap. In Example 1, the following is verified for the animal cap to be taken out.
Stage 7
Stage 8
Stage 8.5
Stage 9
Stage 10
The timing for removing the animal cap was verified by removing the animal cap at each developmental stage described above to dissociate the cells, treating the cells with activin 1 ng / ml, reassembling the cells, and performing sandwich culture. We decided to adopt developmental stage 8.5 where blood islands were formed at the highest rate.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  This technique was the first to realize the formation of blood islands (hematopoietic stem cells) from undifferentiated cells, which had not been realized so far. These blood islands (hematopoietic stem cells) are specifically diagnosed for diseases such as humans that specifically lack or abnormally express proteins involved in hematopoietic stem cell formation, and defects or defects in hematopoietic stem cells such as leukemia There is a possibility of providing a material useful for the purpose of treating abnormalities and diseases such as humans caused by the disease. It is also expected to provide good materials for research on such abnormalities and diseases, and to provide materials for assay systems useful in the development of drugs for treating those abnormalities and diseases.

FIG. 1 shows a typical method scheme of the present invention. FIG. 1 shows a schematic diagram of the induction of blood islands (hematopoietic stem cells) from undifferentiated cells. Animal caps were excised from Xenopus late blastocysts, cells were dissociated in Steinberg's solution excluding calcium and magnesium ions, and treated with activin prepared in Steinberg's solution containing calcium and magnesium ions for 3 and 5 hours. . Tissue differentiation was examined when this activin-treated dissociated reassortant was cultured alone and when it was sandwich-cultured with an untreated animal cap. As a result, dissociated reassemblies that were cultured alone after 3 hours of activin treatment were irregularly shaped epidermis at an activin concentration of 0 ng / ml, blood cells and mesenchyme at 1 ng / ml, and muscle at 10 ng / ml. At ng / ml, they differentiated into endoderm cells. On the other hand, when activin-treated dissociated reassemblies were sandwich-cultured, an activin-treated concentration of 0 ng / ml gave an irregular epidermis, 1 ng / ml a blood island, 10 ng / ml a prorenal tube, 100 ng / ml In ml, differentiation into cement glands was observed. According to this experiment, explants obtained by sandwich culture of dissociated reassemblies treated with activin showed differentiation depending on the concentration and treatment time of activin, and sandwiched dissociated reassemblies treated with 1 ng / ml of activin for 3 hours. It was found that blood islands were induced at a high rate by culturing. FIG. 2 shows an example of culture in a single culture under the conditions of 5 animal caps and an activin treatment time of 3 hours. In this figure, a tissue section photograph is shown. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. Untreated epidermis was observed, blood island formation was observed with 1 ng / ml treatment, muscle notochord was seen with 10 ng / ml treatment, and endoderm cells were seen with 100 ng / ml treatment. FIG. 3 shows a photograph seen in a culture example of sandwich culture under conditions of 5 animal caps and an activin treatment time of 3 hours. A shows the outer shape of the sandwich culture, and B shows a tissue section photograph. In both A and B, the upper left shows 0 ng / ml treatment, the upper right shows 1 ng / ml treatment, the lower left shows 10 ng / ml treatment, and the lower right shows 100 ng / ml treatment. Untreated epidermis was observed, blood island formation was observed with 1 ng / ml treatment, prorenal ducts were seen with 10 ng / ml treatment, and cement glands were seen with 100 ng / ml treatment. FIG. 4 shows another example of a representative method scheme of the present invention. FIG. 4 shows an example of 5 animal caps and 5 hours of activin treatment in a schematic diagram of the induction of blood islands (hematopoietic stem cells) from undifferentiated cells. Other explanations are the same as those in FIG. FIG. 5 shows a photograph seen in a culture example in a single culture under conditions of 5 animal caps and an activin treatment time of 5 hours. A shows a tissue external shape, and B shows a tissue section photograph. In both A and B, the upper left shows 0 ng / ml treatment, the upper right shows 1 ng / ml treatment, the lower left shows 10 ng / ml treatment, and the lower right shows 100 ng / ml treatment. Untreated epidermis was observed, muscle formation was observed with the 1 ng / ml treatment, notochord was seen with the 10 ng / ml treatment, and heart was seen with the 100 ng / ml treatment. FIG. 6 shows a photograph seen in a culture example in sandwich culture under conditions of 5 animal caps and an activin treatment time of 5 hours. A shows a tissue external shape, and B shows a tissue section photograph. In both A and B, the upper left shows 0 ng / ml treatment, the upper right shows 1 ng / ml treatment, the lower left shows 10 ng / ml treatment, and the lower right shows 100 ng / ml treatment. Untreated epidermal epidermis is observed, but 5 animal caps, activin 1 ng / ml, 5 hours treatment, sandwich culture can produce blood cells but not blood islands. At 10 ng / ml treatment, nerves and cement glands were seen. FIG. 7 is an image diagram of dissociation in the method of the present invention. FIG. 8 shows an optical micrograph (a) of a tissue section of a blood island (hematopoietic stem cell) derived from undifferentiated cells and an optical micrograph (b) of an explant containing the blood island. FIG. 9 shows the results of PCR for verifying gene marker expression (SCL, ODCR + and ODCR− from the top) by activin. Expression of SCL, a blood island (hematopoietic stem cell) marker, was observed in blood islands (hematopoietic stem cells, lane 2) derived from undifferentiated cells. 1 to 4 lanes: Activin treatment at each concentration and then sandwiched between undifferentiated cell sheets and cultured for 1 day. The WE (Whole Embryo) in Lane 5 is an untreated whole embryo that has reached the developmental stage 20, SCL is Stem cell leukemia protein, and ODC (Orthineline decarboxylase) is stably expressed at all developmental stages. So it is used as a reference material. FIG. 10 shows another example of a representative method scheme of the present invention. FIG. 10 shows an example of 5 animal caps and 3 hours of activin treatment in a schematic diagram of the induction of blood islands (hematopoietic stem cells) from undifferentiated cells. Other explanations are the same as those in FIG. FIG. 11 shows the external shape of explants obtained by culturing 10 animal caps and dissociated reassemblies after 3 hours of activin treatment alone. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. FIG. 12 shows tissue sections of single cultures of dissociated reassemblies with 10 animal caps and 3 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. Untreated epidermis was observed, blood island formation and body cavity epithelialization were observed with 1 ng / ml treatment, and muscle was seen with 10 ng / ml treatment. FIG. 13 shows gene marker expression by activin with 10 animal caps and 3 hours of activin treatment (from the top edd (endodermal cell marker), Hex (liver marker), ms-actin (muscle marker), epidermal keratin (epidermis) Results of PCR for verifying markers), ODC R + and ODCR-). Expression of SCL, a blood island (hematopoietic stem cell) marker, was observed in blood islands (hematopoietic stem cells, lane 2) derived from undifferentiated cells. Blood cell formation was observed with the activin 1 ng / ml treatment, muscle was seen with the 10 ng / ml treatment, and epidermis was seen with the 100 ng / ml treatment. FIG. 14 shows the external shape of explants obtained by sandwich culture of 10 animal caps and dissociated reassemblies after 3 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. FIG. 15 shows a tissue section of a sandwich culture of 10 animal caps and dissociated reassemblies after 3 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. Untreated epidermis was observed, blood island formation was observed with 1 ng / ml treatment, prorenal ducts were seen with 10 ng / ml treatment, and cement glands were seen with 100 ng / ml treatment. FIG. 16 shows gene marker expression by activin in 10 animal caps and 3 hours of activin treatment (from the top otx-2 (forebrain marker), nrp-1 (neurological marker), SCL (blood island marker), aml-1 (Blood island marker), ODC R + and ODCR-) PCR results for verification. Expression of SCL, a blood island (hematopoietic stem cell) marker, was observed in blood islands (hematopoietic stem cells, lane 2) derived from undifferentiated cells. Blood cell formation was observed with the activin 1 ng / ml treatment, muscle was seen with the 10 ng / ml treatment, and epidermis was seen with the 100 ng / ml treatment. FIG. 17 shows another example of a representative method scheme of the present invention. FIG. 10 shows an example of 10 animal caps and 5 hours of activin treatment in a schematic diagram of induction of blood islands (hematopoietic stem cells) from undifferentiated cells. Other explanations are the same as those in FIG. FIG. 18 shows the external shape of an explant in which 10 animal caps and dissociated reassemblies after 5 hours of activin treatment were cultured alone. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. FIG. 19 shows tissue sections of single cultures of dissociated reassemblies with 10 animal caps and 5 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. Untreated epidermis was observed, blood island formation was observed with 1 ng / ml treatment, notochord with 10 ng / ml treatment, and endoderm cells with 100 ng / ml treatment. FIG. 20 shows gene marker expression in single culture with 10 animal caps and activin treated with activin for 5 hours (from the top edd (endoderm cell marker), Hex (liver marker), ms-actin (muscle marker), It is the result of PCR for verifying epidermal keratin (epidermal marker), ODC R + and ODCR-. Expression of SCL, a blood island (hematopoietic stem cell) marker, was observed in blood islands (hematopoietic stem cells, lane 2) derived from undifferentiated cells. Blood cell formation was observed with the activin 1 ng / ml treatment, muscle was seen with the 10 ng / ml treatment, and endoderm cells were seen with the 100 ng / ml treatment. FIG. 21 shows the external shape of explants obtained by sandwich culture of 10 animal caps and dissociated reassemblies after 5 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. FIG. 22 shows a tissue section of a sandwich culture of 10 animal caps and dissociated reassemblies at 5 hours of activin treatment. Upper left shows 0 ng / ml treatment, upper right shows 1 ng / ml treatment, lower left shows 10 ng / ml treatment, and lower right shows 100 ng / ml treatment. Untreated epidermis is observed, and 10 animal caps, activin 1 ng / ml, 5 hours treatment, sandwich culture cannot form blood islands. At 10 ng / ml treatment, spinal cord was seen, and at 100 ng / ml treatment, cement gland, body cavity epithelium and heart were seen. FIG. 23 shows gene marker expression in sandwich culture with 10 animal caps and activin treated with activin for 5 hours (from top to bottom otx-2 (forebrain marker), nrp-1 (nerve marker), SCL (blood island marker) , Aml-1 (blood island marker), ODC R + and ODCR-). Expression of SCL, a blood island (hematopoietic stem cell) marker, was observed in blood islands (hematopoietic stem cells, lane 2) derived from undifferentiated cells. Blood cell formation was observed with the activin 1 ng / ml treatment, muscle was seen with the 10 ng / ml treatment, and endoderm cells were seen with the 100 ng / ml treatment.

(Explanation of sequence listing)
SEQ ID NO: 1 is the nucleic acid sequence of human inhibin βA accession number NM002192.
SEQ ID NO: 2 is the amino acid sequence of accession number NM002192 of human inhibin βA.
SEQ ID NO: 3 is the nucleic acid sequence of human inhibin βB accession number NM002193.
SEQ ID NO: 4 is the amino acid sequence of accession number NM002193 of human inhibin βB.
SEQ ID NO: 5 is the nucleic acid sequence of human inhibin βC accession number NM005538.
SEQ ID NO: 6 is the amino acid sequence of accession number NM005538 of human inhibin βC.
SEQ ID NO: 7 is the nucleic acid sequence of accession number NM002191 for human inhibin α.
SEQ ID NO: 8 is the amino acid sequence of accession number NM002191 for human inhibin α.
SEQ ID NO: 9 is the nucleic acid sequence of accession number: X68250 (Xenopus activin A).
SEQ ID NO: 10 is the amino acid sequence of accession number: X68250 (Xenopus activin A).
SEQ ID NO: 11 is the nucleic acid sequence of inhibin βB (accession number: S61773) for Xenopus laevis.
SEQ ID NO: 12 is the amino acid sequence of inhibin βB (accession number: S61773) for Xenopus laevis.

Claims (41)

A method for preparing hematopoietic stem cells comprising:
A) providing undifferentiated cells;
B) dissociating the undifferentiated cells;
C) culturing the undifferentiated cells after dissociation in a medium containing activin for a time sufficient to differentiate into hematopoietic stem cells;
Including the method.   The method of claim 1, wherein the undifferentiated cells comprise cells derived from late blastocysts. The method according to claim 1, further comprising the step of contacting the cells obtained in step D) C) with undifferentiated cells. The method according to claim 3, wherein the contact is achieved by sandwiching the cells obtained in step C) with a sheet of undifferentiated cells. The method according to claim 3, wherein the undifferentiated cell comprises an animal pole cell of a late blastula. 4. The method of claim 3, wherein the contact with the undifferentiated cell is a time sufficient to contact the undifferentiated cell from initial contact to final differentiation. The method according to claim 3, wherein the contact with the undifferentiated cells is performed for at least one day. The method according to claim 3, wherein the contact with the undifferentiated cells is performed within 1 day to 3 days. The method of claim 1, wherein the activin is an activin selected from the group consisting of activin-A, activin-B, inhibin and activin-C. The method of claim 1, wherein the activin comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The method of claim 1, wherein the activin is applied at a concentration of 0.5-1 ng / ml per animal cap. 2. The method of claim 1, wherein the hematopoietic stem cells express a stem cell leukemia protein (SCL) marker. The method according to claim 1, wherein the dissociation is achieved by culturing in a medium containing neither Ca ²⁺ nor Mg ²⁺ . The method according to claim 1, wherein the dissociation is achieved by culturing in a medium without Ca2 ⁺ or Mg2 ^{+ for} at least 10 minutes. The method according to claim 1, wherein the dissociation is achieved by culturing in a medium containing neither Ca2 ⁺ nor Mg2 ^{+ for} 10 to 20 minutes. The method according to claim 1, wherein a dissociated and reassembled body of the cells is formed in the step C). The method according to claim 16, wherein the formation of dissociated reassemblies in the step C) is achieved by culturing in the medium for at least 30 minutes. The method according to claim 1, wherein the activin treatment in the step C) is performed for at least 3 hours. The method of claim 1, wherein the undifferentiated cell is provided by an animal cap. The method according to claim 19, wherein 5 to 10 animal caps are used. The method according to claim 1, further comprising the step of culturing the dissociated reassemblies together with the undifferentiated cells in step C, wherein dissociated reassemblies are formed. The method according to claim 1, further comprising the step of sandwich-culturing the dissociated reassemblies in the undifferentiated cells in the step C, wherein dissociated reassemblies are formed. 2. The method of claim 1, wherein the cell is a vertebrate cell. 2. The method of claim 1, wherein the cell is an amphibian animal cell. 2. The method of claim 1, wherein the cell is a frog cell. 2. The method of claim 1, wherein the cell is a Xenopus cell. A cell prepared by the method according to any one of claims 1 to 26. A blood island prepared by the method according to any one of claims 1 to 28. A medium used in the step of dissociating undifferentiated cells in a method for inducing hematopoietic stem cells,
A medium characterized in that it contains neither Ca ²⁺ nor Mg ²⁺ . 30. The medium of claim 29, wherein the medium comprises a composition of 58 mM NaCl, 0.67 mM KCl, 3.0 mM hydroxyethylpiperazinylethanesulfonic acid and 100 mg / L kanamycin sulfate, pH 7.4. A medium used in the step of differentiating undifferentiated cells into hematopoietic stem cells in the method for inducing hematopoietic stem cells,
A medium comprising an effective amount for inducing activin into hematopoietic stem cells. 32. The medium of claim 31, wherein the activin is present at a concentration of 0.5-1 ng / ml. 32. The medium of claim 31, wherein the activin is present at a concentration of about 1 ng / ml. A combination of media used in a method for inducing hematopoietic stem cells, characterized in that the combination does not contain Ca ²⁺ or Mg ²⁺ ;
A medium combination comprising a medium comprising an effective amount for inducing activin into hematopoietic stem cells. A kit for preparing hematopoietic stem cells comprising:
a) dissociation means for dissociating the undifferentiated cells;
b) differentiation means for differentiating the undifferentiated cells into hematopoietic stem cells;
c) instructions for instructing to differentiate the undifferentiated cells by means of time differentiation sufficient to differentiate the undifferentiated cells after dissociation into hematopoietic stem cells;
A kit comprising: The kit according to claim 35, wherein the differentiation means is a medium containing activin. Use of activin to prepare hematopoietic stem cells. A composition for preparing hematopoietic stem cells, the composition comprising an effective amount for inducing activin into hematopoietic stem cells. 39. The composition of claim 38, wherein the activin is present at a concentration of 0.5-1 ng / ml. 40. The composition of claim 38, wherein the activin is present at a concentration of about 1 ng / ml. A method for preparing a desired differentiated cell comprising:
A) providing undifferentiated cells;
B) dissociating the undifferentiated cells;
C) culturing the undifferentiated cells after dissociation in a medium containing activin under conditions sufficient to differentiate into desired differentiated cells;
Including the method.