WO2017033415A1 - 細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法 - Google Patents
細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法 Download PDFInfo
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- WO2017033415A1 WO2017033415A1 PCT/JP2016/003626 JP2016003626W WO2017033415A1 WO 2017033415 A1 WO2017033415 A1 WO 2017033415A1 JP 2016003626 W JP2016003626 W JP 2016003626W WO 2017033415 A1 WO2017033415 A1 WO 2017033415A1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/145—Thrombopoietin [TPO]
Definitions
- the present invention relates to an agent for promoting the expression of c-MPL receptor on the surface of mesenchymal cells, a method for producing mesenchymal cells in which the expression of c-MPL receptor on the surface of cells is promoted,
- the present invention relates to a method for promoting the expression of c-MPL receptor on the surface.
- Platelet transfusion is the only treatment for thrombocytopenia caused by bleeding in the event of accidents or the use of anticancer drugs, and the platelet preparations used at that time are currently 100% dependent on bona fide donation. Platelets are very fragile and there is no method to date that allows long-term storage of platelets for therapeutic purposes. Actually, the shelf life of platelets is 4 days at the latest medical institutions, but considering the time required for testing and shipping, the actual shelf life at clinical sites including clinics is about 3 days. Is done. In this way, many blood banks have always had the difficulty of keeping and storing platelets fresh, and the supply of platelet products that depend on blood donation has decreased blood donors and suffered from viral infections. The situation is easily affected by the increase in blood donors.
- Non-patent Document 1 Non-patent Document 1
- hematopoietic stem cells umbilical cord blood stem cells
- ES embryonic stem
- Non-patent Document 2 a technique for producing mature megakaryocytes and platelets from human ES cells has already been reported (Non-patent Document 2).
- the production efficiency of platelets is poor, and tens of thousands of petri dishes are required to produce a single blood transfusion preparation.
- refractory platelet transfusion is a problem.
- HLA human leukocyte antigen
- platelets having human leukocyte antigen (HLA) different from that of the patient can be used, but by repeating the transfusion, a specific antibody against this HLA is produced in the patient, and as a result, the transfused platelets are rapidly Rejected.
- platelets also have a human blood alloantigen (HPA), which is a unique blood type, and transfusion refractory due to this compatible type difference is also observed.
- HPA human blood alloantigen
- iPS human induced pluripotent stem
- Non-Patent Document 4 a method for producing platelets from fibroblasts by a technique called direct reprogramming. According to this technique, the period until the production of platelets can be greatly shortened and the platelet production can be achieved in about 14 days as compared with the method using iPS cells.
- gene transfer is required for direct reprogramming using fibroblasts, and there is concern about the impact on safety caused by the mixture of vectors for gene transfer.
- MKLI medium megakaryocyte lineage induction medium
- MKLI medium megakaryocyte lineage induction medium
- the MKLI medium was prepared by adding Ismoff-modified Dulbecco medium (IMDM) to 2 mM L-glutamine, 100 U / mL penicillin-streptomycin solution, 0.5% bovine serum albumin, 4 ⁇ g / mL LDL cholesterol, 200 ⁇ g / mL pig iron saturated transferrin (iron binding).
- IMDM Ismoff-modified Dulbecco medium
- Type transferrin a medium supplemented with 10 ⁇ g / mL insulin, 50 ⁇ M 2- ⁇ -mercaptoethanol, nucleotides (ATP, UTP, GTP and CTP each 20 ⁇ M), and 50 ng / mL thrombopoietin (TPO) (non-patented) Reference 5).
- the present inventors have been conducting research on techniques for inducing differentiation from cells other than hematopoietic stem cells into megakaryocytes and platelets.
- Patent Document 1 adipose precursor cells derived from human subcutaneous adipose tissue (non-facial cells) It has been found that Patent Documents 5 and 6) and mouse-derived adipose precursor cells (Non-Patent Documents 5 and 7) can be differentiated into megakaryocytes and platelets. The present inventors have further studied and found a better method capable of producing megakaryocytes and / or platelets (Patent Document 1). The production method of Patent Document 1 is characterized in that mesenchymal cells are cultured in a basic medium for mesenchymal cell culture containing iron ions and an iron transporter, and megakaryocytes and / or platelets are collected from the culture.
- megakaryocytes having the ability to produce platelets and / or platelets having the ability to form thrombus can be obtained from mesenchymal cells such as preadipocytes without adding TPO or the like to the medium. It is possible to manufacture in vitro in a simple and large amount, and at a lower cost or more efficiently in vitro.
- the production method of Patent Document 1 is an excellent production method that eliminates the disadvantages of the conventional production method of platelets using hematopoietic stem cells, ES cells, or iPS cells.
- the types of surface antigens of mesenchymal cells such as preadipocytes before differentiation into megakaryocytes and platelets, and the efficiency of differentiation into megakaryocytes when differentiation of the mesenchymal cells into megakaryocytes, etc.
- the case where c-MPL receptor-positive mesenchymal cells were used compared with the case where c-MPL receptor-negative mesenchymal cells were used, It was shown to differentiate into megakaryocytes and platelets with very high efficiency.
- the ratio of c-MPL receptor-positive cells among mesenchymal cells such as preadipocytes was very small, about 0.5 to 1%.
- C-MPL receptor is known as a TPO receptor in vivo. That is, TPO is known as a ligand for c-MPL receptor in vivo.
- c-MPL is a glycoprotein expressed in hematopoietic stem cells and megakaryocyte cells, belongs to the cytokine receptor gene family, and is deeply involved in platelet hematopoiesis as a receptor for new factors involved in platelet hematopoiesis.
- TPO is synthesized as a 353 amino acid precursor protein in the liver, and a 21 amino acid signal peptide is cleaved into a mature molecule.
- Non-patent Document 8 The mature molecule is composed of two domains highly homologous to erythropoietin and a highly glycosylated carboxy terminus important for the stability of the protein. It has been reported that mice lacking the TPO gene or c-MPL receptor gene have a platelet count reduced to about 10-20% of that of wild-type mice (Non-patent Document 9). It has been shown that the body is involved in the regulation of the pathway that produces platelets from hematopoietic stem cells.
- Non-Patent Document 10 describes that the activity of the c-MPL receptor gene promoter is induced by TPO in CMK cells, which are human megakaryoblastic leukemia cell lines.
- CMK cells which are human megakaryoblastic leukemia cell lines.
- the activity of the c-MPL receptor gene promoter is measured by luciferase activity by expression of a luciferase gene linked downstream of the promoter.
- Non-Patent Document 10 does not confirm the effect of TPO on the expression of c-MPL receptor on the cell surface.
- Non-Patent Document 11 shows that TPO reduces the expression of c-MPL receptor on the surface of CMK cells.
- An object of the present invention is to provide a method for easily and efficiently promoting the expression of c-MPL on the surface of mesenchymal cells.
- the inventors of the present invention have made extensive studies to solve the above-mentioned problems.
- mesenchymal cells such as preadipocytes are maintained and cultured in a basic medium
- TPO is added to the basic medium
- the mesenchyme is The present inventors have found that the expression of c-MPL receptor on the surface of the cell line is promoted, thereby completing the present invention.
- Non-Patent Document 11 showed that TPO decreased the expression of c-MPL receptor on the surface of CMK cells, the above findings found by the present inventors were surprising. .
- the present invention (1) a c-MPL receptor expression promoter containing c-MPL receptor agonist on the surface of mesenchymal cells, (2) The above-mentioned substance wherein the c-MPL receptor agonist is one or more substances selected from the group consisting of TPO or a derivative thereof, Romiplostim, and Eltrombopag olamine ( 1) the promoter of c-MPL receptor expression on the surface of mesenchymal cells, (3) The present invention relates to the expression promoter for c-MPL receptor on the surface of mesenchymal cells according to (1) or (2) above, wherein the mesenchymal cells are preadipocytes.
- the present invention also provides: (4) Step A of culturing mesenchymal cells in a basic medium for culturing mesenchymal cells containing a c-MPL receptor agonist; and Obtaining a mesenchymal cell with enhanced expression of c-MPL receptor on the cell surface; B; A method for producing mesenchymal cells in which expression of c-MPL receptor on the cell surface is promoted, (5)
- the aforementioned c-MPL receptor agonist is one or more substances selected from the group consisting of TPO or a derivative thereof, Romiplostim, and Eltrombopag olamine ( 4)
- the method for producing mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted, (6) The method for producing mesenchymal cells in which the expression of c-MPL receptor on the cell surface according to (4) or (5) above, wherein the mesenchymal cells are preadipocytes, (7) Production of mesenchymal cells with
- the present invention relates to a method for producing mesenchymal cells in which the expression of c-MPL receptor on the surface is promoted.
- the present invention provides (9) Promoting the expression of c-MPL receptor on the surface of mesenchymal cells, comprising the step A of culturing mesenchymal cells in a basic medium for culturing mesenchymal cells containing a c-MPL receptor agonist Regarding the method.
- the expression of c-MPL on the surface of mesenchymal cells can be promoted simply and efficiently. That is, according to the present invention, mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted can be produced simply and efficiently.
- mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface have a high rate of differentiation when induced to differentiate into megakaryocytes or platelets (see, for example, Example 2 described later), c on the cell surface -Mesenchymal cells in which the expression of MPL receptor is promoted can be suitably used for the efficient production of megakaryocytes and platelets.
- the number of mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface does not increase so much during differentiation induction into megakaryocytes and platelets, and therefore, from mesenchymal cells to megakaryocytes and platelets. The significance of the present invention for inducing differentiation is great.
- Method for producing mesenchymal cells in which expression of c-MPL receptor is promoted As a method for producing mesenchymal cells in which the expression of the c-MPL receptor of the present invention is promoted (hereinafter simply referred to as “the production method of the present invention”), culturing mesenchymal cells in a basic medium for culturing mesenchymal cells containing a c-MPL receptor agonist; and Step B of obtaining mesenchymal cells in which expression of c-MPL receptor is promoted; There is no particular limitation as long as it is a method including: “medium” herein refers to a state in which water is added to a “medium component” capable of culturing cells.
- mesenchymal cells such as preadipocytes are cultured in a basic medium for culturing mesenchymal cells containing a c-MPL receptor agonist
- a c-MPL receptor expression promoter on the surface of mesenchymal cells of the present invention described later may be used.
- the step A is not particularly limited as long as it is a step of culturing mesenchymal cells in a basic medium for mesenchymal cell culture containing a c-MPL receptor agonist. Such a culture process is an in vitro culture process.
- the mesenchymal cells used in the present invention are mesenchymal cells that can promote the expression of c-MPL receptor on the cell surface by culturing in a basic medium for mesenchymal cell culture containing a c-MPL receptor agonist. There is no particular limitation as long as it is a cell line.
- the mesenchymal cells include (a) preadipocytes or adipose progenitor cells, (b) mesenchymal stem cells, (c) stromal cells, and the like.
- Examples of the mesenchymal stem cells include subcutaneous adipose tissue-derived mesenchymal stem cells and bone marrow mesenchymal stem cells.
- stromal cells examples include adipose tissue-derived stromal cells, bone marrow stromal cells, and prostate-derived cells. Examples include stromal cells and endometrial stromal cells.
- Preferred mesenchymal cells include adipose precursor cells and subcutaneous adipose tissue-derived mesenchymal stem cells.
- the mesenchymal cells used in the present invention may be cultured cell lines or cells collected from tissues (including primary cultured cells and subcultured cells). More specific examples of mesenchymal cells used in the present invention include human primary cultured adipose precursor cells (HPAD cells), primary mouse cultured precursor cells (derived from subcutaneous adipose tissue), and established mouse stromal cells (OP9 cells). ), Established mouse bone marrow mesenchymal stem cells (HS-22 cells), established mouse adipose precursor cells (3T3-L1 cells), and the like.
- mesenchymal cell culture cell lines and mesenchymal cells collected from tissues can be obtained from Lonza, PromoCell, CELL APPLICATIONS, National Institute of Pharmaceutical Sciences (National Research and Development Corporation The name may be changed to “Research Institute”.
- Commercially available products from companies such as the JCRB cell bank may be used, but “obtained while the expression of c-MPL receptor on the cell surface is promoted” obtained by the production method of the present invention.
- a cell line of the prepared mesenchymal cell may be used.
- the method for establishing a mesenchymal cell line is not particularly limited, and a known method or the like can be used.
- a method for stocking a preadipocyte a method for establishing a mature adipocyte after inducing differentiation of a preadipocyte into a mature adipocyte as described in Example 15 of Patent Document 1 in the background art
- a method for obtaining a preadipocyte cell line can be mentioned by applying a known ceiling culture method.
- mesenchymal cell lines are stored frozen, the expression of “c-MPL receptor on the cell surface is promoted.
- the mesenchymal cells can be rapidly produced, and when megakaryocytes and platelets are required, the mesenchymal cells can be used to immediately start production of megakaryocytes and platelets. is there.
- “mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface” is used for producing megakaryocytes and platelets, it is used in the present invention from the viewpoint of producing megakaryocytes and platelets more efficiently.
- “Mesenchymal cells (preferably preadipocytes)” are preferably CD31 negative and CD71 positive, more preferably CD31 negative, CD71 positive and c-MPL receptor positive.
- “Mesenchymal cells (preferably adipose precursor cells) whose c-MPL receptor expression is promoted on the cell surface” are preferably CD31 negative and CD71 positive.
- CD31 is known as a vascular endothelial cell marker
- CD71 is known as a transferrin receptor.
- CD31 negative, CD71 positive, and c-MPL receptor positive are preferable in mesenchymal cells. ing.
- CD31 negative and CD71 positive adipose precursor cells preferably CD31 negative and CD71 positive and c-MPL receptor positive adipose precursor cells are selected in advance
- the above-mentioned cell line method may be applied to the cells.
- the above-mentioned preadipocytes and subcutaneous adipose tissue-derived mesenchymal stem cells can be collected from adipose tissue such as subcutaneous adipose tissue and visceral tissue, and bone marrow mesenchymal stem cells can be collected from bone marrow tissue. Cells can be collected from adipose tissue, bone marrow tissue, prostate, endometrium, etc.
- adipose precursor cells and subcutaneous adipose tissue-derived mesenchymal stem cells are less invasive and are more numerous.
- a mesenchymal cell derived from an adipose tissue can be preferably mentioned because it can more easily collect mesenchymal cells. Conventional methods can be used to collect mesenchymal cells from tissues.
- Mesenchymal cells having a specific cell surface marker profile such as CD31 negative and CD71 positive mesenchymal cells, or CD31 negative, CD71 positive and c-MPL positive mesenchymal cells are those specific cells Selection can be made from a cell population containing mesenchymal cells using the presence or absence of a surface marker as an index.
- selecting mesenchymal cells having a specific cell surface marker profile only mesenchymal cells having a specific cell surface marker profile are isolated from a cell population containing mesenchymal cells.
- the method for selecting mesenchymal cells having the specific cell surface marker profile described above is not particularly limited, but from the viewpoint of selecting the target mesenchymal cells more easily and quickly, each of the above cell surface markers is selected.
- a preferred example is a method for selecting mesenchymal cells having a specific cell surface marker profile using antibodies (preferably labeled antibodies, more preferably fluorescently labeled antibodies) and using the presence or absence of specific binding of each of these antibodies as an indicator. can do.
- antibodies preferably labeled antibodies, more preferably fluorescently labeled antibodies
- the above-mentioned “selecting with the presence / absence of specific binding of antibody as an index” means that for positive cell surface markers in the profile, cells that exhibit specific binding to the antibody against the marker are selected and negative in the profile.
- a cell surface marker For a cell surface marker, this means that cells to which the antibody against the marker does not show specific binding are selected. For example, when selecting CD31 negative and CD71 positive mesenchymal cells from a mesenchymal cell population, It means that cells that do not show specific binding to the CD31 antibody and show specific binding to the anti-CD71 antibody are selected.
- the method for selecting mesenchymal cells having a specific cell surface marker profile using the presence or absence of specific antibody binding as an index is not particularly limited, and examples include a method using a cell sorter, magnetic beads, a cell adsorption column, or the like. A method using a cell sorter can be preferably mentioned because it can be performed more easily and quickly.
- the method using a cell sorter is based on the flow cytometry method and is well known to those skilled in the art.
- a specific method is described in, for example, Japanese Translation of PCT International Publication No. 2009-513161 as well as an instruction manual of the cell sorter.
- a method using magnetic beads is well known to those skilled in the art as a magnetic separation method.
- a magnetic bead carrying a specific antibody is brought into contact with a cell, and then the magnetic bead is used with a magnet.
- a method of separating cells that specifically bind to the specific antibody by collecting can be mentioned.
- methods using a cell adsorption column are well known to those skilled in the art.
- a specific method a cell group is brought into contact with a cell adsorption column carrying a specific antibody, and a cell other than the target cell is used. Examples thereof include a method for adsorbing cells on a column.
- CD31 negative and CD71 positive mesenchymal cells preferably adipose precursor cells
- CD31 negative and CD71 positive and c-MPL positive mesenchymal cells preferably adipose precursor cells
- CD45 a marker for hematopoietic cells other than erythrocytes and platelets
- Ter119 a marker for erythrocytes and their progenitor cells
- blood contained in adipose tissue It is preferable because cells related to the can be excluded.
- CD45 and Ter119 are not expressed on the surface of mesenchymal cells, it is not necessary to confirm that CD45 and Ter119 are negative if the cells are mesenchymal cells. Further, although not a cell surface marker, it is preferable to use 7-amino-actinomycin D (7-AAD) as an index, since dead cells contained in adipose tissue can be excluded. 7-AAD intercalates into the dead cell DNA strand and emits red fluorescence upon excitation at 488 nm.
- 7-AAD 7-amino-actinomycin D
- the fluorescently labeled anti-CD31 antibody the fluorescently labeled anti-CD71 antibody, the fluorescently labeled anti-c-MPL antibody, the fluorescently labeled anti-CD45 antibody, and the fluorescently labeled anti-Ter119 antibody, those commercially available from Immunobiological Research Institute, BD Biosciences, etc. are used. be able to.
- 7AAD those commercially available from BD Biosciences or the like can be used.
- mesenchymal cells that are CD31-negative and CD71-positive, preferably c-MPL-positive mesenchymal cells when used, cells containing these mesenchymal cells having a cell surface marker profile
- the population may be used for the culture in the above step A without selecting the mesenchymal cells having the profile, but the cell population in which the mesenchymal cells having the profile are selected is used for the culture in the above step A. Is preferred.
- adipose tissue-derived cell population as a cell population containing mesenchymal cells, in addition to the cell surface marker profile of CD31 negative, CD71 positive, and preferably c-MPL positive, CD45 negative and Ter119 negative cells
- a surface marker profile as an index is preferable in that cells related to blood other than mesenchymal cells can be efficiently eliminated. It is also preferable to confirm that 7-AAD is negative because dead cells contained in adipose tissue can be efficiently eliminated.
- the biological species from which the mesenchymal cells are derived is not particularly limited as long as it is a vertebrate, and examples of the vertebrate include mammals, birds, reptiles, amphibians, fish, Preferred examples include mammals such as humans, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, monkeys, sheep, goats, and pigs, with humans being particularly preferred.
- the medium used for the culture of the present invention is a medium in which a basic medium for mesenchymal cell culture contains a c-MPL receptor agonist.
- the c-MPL receptor agonist is not particularly limited as long as it is a substance capable of operating c-MPL receptor, that is, a substance having c-MPL receptor agonist activity, and may be a protein, It may be a low molecular compound having a molecular weight of 10,000 or less.
- proteins include TPO or a derivative thereof; Romiplostim; an agonist antibody to c-MPL receptor (Japanese Patent Laid-Open No. 2009-017881), and the like.
- Low molecular compounds having a molecular weight of 10,000 or less include Eltrombopag olamine; and the like.
- TPO a derivative thereof, and romiplostim
- TPO can be more preferably mentioned.
- Lomiprostim examples include Romiplate (registered trademark) (manufactured by Kyowa Hakko Kirin Co., Ltd.), and examples of the above-mentioned Eltrombopag olamine include Revolade (manufactured by GlaxoSmithKline Co., Ltd.). It is done.
- the TPO or derivative thereof is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 92.5% or more, more preferably 95% with the amino acid sequence shown in SEQ ID NO: 1 or 3. %, More preferably 97% or more, more preferably 98% or more, and still more preferably 99% or more, and a protein having c-MPL receptor agonist activity.
- the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of full-length human TPO
- the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of amino acid numbers 1-163 corresponding to the biologically active domain of natural human TPO. Is an array.
- the nucleotide sequence shown in SEQ ID NO: 2 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1
- the nucleotide sequence shown in SEQ ID NO: 4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3. is there.
- the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3 can be prepared by any method known to those skilled in the art, such as chemical synthesis and genetic engineering techniques.
- DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 DNA having the nucleotide sequence shown in SEQ ID NO: 2
- DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 SEQ ID NO: The DNA having the nucleotide sequence shown in FIG.
- a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 3 can be prepared by any method known to those skilled in the art, such as chemical synthesis and genetic engineering techniques. it can.
- a DNA encoding a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 3 The DNA encoding the protein is incorporated into an appropriate expression vector, and the transformant obtained by introducing the DNA into an appropriate host cell is recovered and an extract is obtained, followed by ion exchange, reverse phase, gel filtration, etc.
- affinity chromatography in which an antibody against a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 3 above is immobilized on a column, or further It is possible to purify and prepare by combining a plurality of columns.
- a DNA encoding a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 3 above has a certain amount (for example, 80% or more) with the nucleotide sequence shown in SEQ ID NO: 2 or 4, respectively. Or more, preferably 85% or more, more preferably 90% or more, further preferably 92.5% or more, more preferably 95% or more, still more preferably 97% or more, more preferably 98% or more, and further preferably 99%. It can be prepared as DNA having the above identity.
- these DNAs can be obtained by using a method of bringing a DNA having the nucleotide sequence shown in SEQ ID NO: 2 or 4 into contact with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering method, or the like.
- Mutant DNA can be obtained by introducing a mutation into.
- Site-directed mutagenesis which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position.
- the c-MPL receptor agonist such as TPO or a derivative thereof may not be chemically or biologically modified. However, as long as it has c-MPL receptor agonist activity, It may be biologically modified. Such modifications include addition of a peptide, protein or polymer to the end of the protein of the above TPO or its derivative in addition to glycosylation of amino acids. Such polymers include polyethylene glycol. Examples of the peptide to be added (fused) to the protein of TPO or a derivative thereof include, for example, FLAG (Hopp, T. P.
- 6 His 6 ⁇ His consisting of (histidine) residues, 10 ⁇ His, influenza agglutinin (HA) fragment, human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E
- HA influenza agglutinin
- HA human c-myc fragment
- VSV-GP fragment p18HIV fragment
- T7-tag HSV-tag
- E E
- peptides such as -tag, SV40T antigen fragment, lck tag, ⁇ -tubulin fragment, B-tag, Protein C fragment and the like can be used.
- proteins to be added (fused) to the above TPO or its derivatives include, for example, GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, ⁇ -galactosidase, MBP (maltose binding protein) etc. are mentioned.
- the above glycosylation can be performed using a glycosylation enzyme.
- the addition (fusion) of the peptide or protein to the end of the protein of the above TPO or its derivative is, for example, DNA encoding the protein of the TPO or its derivative, and DNA encoding the peptide or protein to be added.
- DNA encoding the protein of the TPO or its derivative In the form of ligation, it is incorporated into an appropriate expression vector, and this is introduced into an appropriate host cell.
- the transformant obtained is recovered and an extract is obtained, followed by chromatography such as ion exchange, reverse phase, and gel filtration. Alternatively, it can be purified and prepared by subjecting the antibody against the fusion protein to affinity chromatography fixed on a column, or by further combining a plurality of these columns.
- addition of the polymer to the terminal of the protein of the above TPO or a derivative thereof can be performed, for example, by mixing a polymer having a reactive group and the protein of the above TPO or a derivative
- modified TPO examples include recombinant human TPO (also expressed as rHuTPO or rHTPO), which is a glycosylated molecule having the same full-length amino acid sequence as that of natural TPO, and the biologically active domain of natural TPO.
- Preferred examples include polyethylene glycol (PEG) -linked recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), which is a non-glycosylated molecule containing 1-163 amino acids corresponding to (Blood 2002; 100: 3457-3469, Semin Hematol 2000; 37: 41-49).
- a substance having c-MPL receptor agonist activity refers to a substance that causes intracellular signal transduction related to c-MPL receptor in a cell expressing c-MPL receptor (ie, a substance that activates the c-MPL receptor), (a) STAT phosphorylation in the Jak-STAT pathway, (b) MAPK phosphorylation in the Ras-MAPK pathway, and (c) PI3K-Akt pathway Any one or two (preferably three) phosphorylation substances selected from Akt phosphorylation are preferably included.
- Whether or not a certain substance has c-MPL receptor agonist activity can be confirmed by a known method.
- a method of analyzing downstream signal transduction due to activation of c-MPL receptor include a method of analyzing downstream signal transduction due to activation of c-MPL receptor. Specifically, (a) STAT in the Jak-STAT pathway caused by activation of c-MPL receptor. And (b) MAPK phosphorylation in the Ras-MAPK pathway and (c) Akt phosphorylation in the PI3K-Akt pathway. Such phosphorylation can be analyzed by Western blotting using antibodies specific for phosphorylated STAT, phosphorylated MAPK, and phosphorylated Akt. A commercially available antibody can be used as such a specific antibody. Since the human c-MPL receptor has a known amino acid sequence (see Genbank: NP_005364), it can be obtained by any method known to those skilled in the art, such as chemical synthesis or genetic engineering techniques. Can be made
- the concentration of the c-MPL receptor agonist in the medium used for the culture of the present invention is such that the mesenchymal cells are cultured in a basic medium for culturing mesenchymal cells containing such a concentration of the c-MPL receptor agonist.
- the medium is not particularly limited as long as it is a medium capable of producing “mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted”. For example, it is preferably in the range of 1 ng / mL to 10 ⁇ g / mL.
- concentration range can be particularly preferably mentioned when the c-MPL receptor agonist is TPO or a derivative thereof.
- Basic medium for mesenchymal cell culture As a basic medium for culturing mesenchymal cells used in the present invention, when a c-MPL receptor agonist is added, mesenchymal cells are cultured in the medium to obtain “c-MPL receptor on the cell surface”.
- the medium is not particularly limited as long as it can produce “mesenchymal cells whose expression is promoted”, but is preferably a chemically synthesized medium because it is easy to prepare and prevents variation from lot to lot.
- One or more sugars Class
- saccharide examples include monosaccharides such as glucose, lactose, mannose, fructose, and galactose, and disaccharides such as sucrose, maltose, and lactose, among which glucose is particularly preferable.
- monosaccharides such as glucose, lactose, mannose, fructose, and galactose
- disaccharides such as sucrose, maltose, and lactose, among which glucose is particularly preferable.
- glucose is particularly preferable.
- One or a combination of two or more can also be added.
- the inorganic salts include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron (III) nitrate nonahydrate, iron (II) sulfate heptahydrate, magnesium chloride hexahydrate. , Magnesium sulfate, potassium chloride, sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate monohydrate, dihydrogen phosphate
- One or more inorganic salts (s) selected from sodium dihydrate, sodium selenite pentahydrate, and zinc sulfate heptahydrate can be mentioned. Any inorganic salt or a combination thereof can be used as long as it is a component that advantageously acts on the production of “mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface”.
- amino acids include alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glycine, histidine, glutamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine.
- amino acid (s) selected from tryptophan, tyrosine, valine, etc., preferably L-form amino acids and their derivatives and salts thereof, and derivatives such as hydrates thereof. it can.
- the arginine include arginine derivatives such as L-arginine hydrochloride and L-arginine monohydrochloride.
- aspartic acid examples include L-aspartic acid sodium salt monohydrate, L-asparagine.
- Derivatives of aspartic acid such as acid monohydrate, potassium L-aspartate, magnesium L-aspartate, and the like
- examples of the cysteine include L-cysteine dihydrochloride and L-cysteine hydrochloride monohydrate
- Derivatives of cysteine such as L-lysine hydrochloride and derivatives of lysine such as L-lysine hydrochloride
- the glutamic acid can include derivatives of glutamine such as L-glutamic acid monosodium salt
- Asparagine can include derivatives of asparagine, such as L-asparagine monohydrate.
- tyrosine examples include derivatives of tyrosine such as L-tyrosine disodium dihydrate
- histidine examples include derivatives of histidine such as histidine hydrochloride and histidine hydrochloride monohydrate.
- the lysine examples include lysine derivatives such as L-lysine hydrochloride.
- vitamins include one or two selected from biotin, choline, folic acid, inositol, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, paraaminobenzoic acid (PABA), and ascorbic acid. Mention may be made of more than one type of vitamin (s) and derivatives of each of these components and their salts and their hydrates.
- examples of the choline include choline derivatives such as choline chloride
- examples of niacin include niacin derivatives such as nicotinic acid, nicotinic acid amide, and nicotinic alcohol, and pantothenic acid.
- Can include derivatives of pantothenic acid such as calcium pantothenate, sodium pantothenate, and panthenol.
- pyridoxine include derivatives of pyridoxine such as pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate, and pyridoxamine.
- thiamine examples include thiamine hydrochloride, thiamine nitrate, bis-thiamine nitrate, thiamine dicetyl sulfate, thulamine derivatives such as fursultiamine hydrochloride, octothiamine, benfotiamine and the like, and ascorbic acid Ascorbic acid 2-phosphate ester, ascorbic acid magnesium phosphate, sodium ascorbate sulfate, sodium ascorbyl sulfate, ascorbyl aminopropyl phosphate, sodium ascorbate phosphate, etc. be able to.
- Derivatives such as serum such as fetal bovine serum (FBS), buffering agents such as HEPES, antibiotics such as penicillin and streptomycin, pyruvate, and derivatives and salts thereof, and hydrates thereof Phenol red, and the like.
- Preferred examples of the derivative of the antibiotic include penicillin G sodium, streptomycin sulfate, and a penicillin-streptomycin solution
- the derivative of pyruvic acid includes sodium pyruvate.
- the serum concentration when serum is added to the basic medium for mesenchymal cell culture as other components is in the range of 1 to 20% by weight, preferably in the range of 5 to 15% by weight, A range of 13% by weight is more preferable.
- DMEM Dulbecco's modified Eagle medium
- IMDM Iskov modified Dulbecco medium
- RPMIIM1640 medium
- MEM minimum essential medium
- BME Eagle basic medium
- a known chemical synthesis medium such as F12 medium, a medium in which any two or more of these mediums such as DMEM / F12 medium (medium in which DMEM and F12 medium are mixed at 1: 1) are mixed in an appropriate ratio
- F12 medium a medium in which any two or more of these mediums
- DMEM / F12 medium medium in which DMEM and F12 medium are mixed at 1: 1
- additional amino acids preferably non-essential amino acids
- the added culture medium can be mentioned preferably, especially serum such as FBS in DMEM, IMDM or RPMI 1640 medium, More preferably, a medium further supplemented with an antibiotic (preferably penicillin G sodium, streptomycin sulfate, or penicillin-streptomycin solution) and an additional non-essential amino acid can be mentioned.
- serum preferably FBS
- antibiotics are added to DMEM.
- a medium to which a substance (preferably penicillin G sodium, streptomycin sulfate or penicillin-streptomycin solution) and an additional non-essential amino acid are further added can be particularly preferably mentioned.
- a basal medium for culturing mesenchymal cells particularly suitable in the present invention 10% (final concentration) serum (preferably FBS), 1% (final concentration) non-essential amino acid with respect to DMEM having the composition described later
- the product a medium supplemented with a 100 U / mL (final concentration) penicillin-streptomycin solution (hereinafter referred to as “a particularly preferable basic medium” in the present invention), and each component in a particularly preferable basic medium in the present invention
- a medium containing each component at a concentration in the range of 70% to 130% independently for each component may be mentioned.
- DMEM composition 200 mg / L anhydrous calcium chloride, 0.1mg / L Fe (NO 3 ) 3 ⁇ 9H 2 O, 200mg / L potassium chloride, 97.67mg / L anhydrous magnesium sulfate, 6400mg / L of sodium chloride, 3700mg / L sodium bicarbonate 125 mg / L sodium dihydrogen phosphate monohydrate, 4500 mg / L D-glucose, 15 mg / L phenol red, 110 mg / L sodium pyruvate, 84 mg / L L-arginine hydrochloride, 63 mg / L L-cystine dihydrochloride Salt, 584 mg / L L-glutamine, 30 mg / L glycine, 42 mg / L L-histidine hydrochloride monohydrate, 105 mg / L L-isoleucine, 105 mg / L L-leucine, 146 mg / L L-lysine hydrochloride,
- Examples of the particularly preferable medium used for the culture of the present invention include a medium containing a c-MPL receptor agonist in the above-described particularly preferable basic medium in the present invention. More preferable examples include a medium containing a c-MPL receptor agonist as a single active ingredient in a suitable basic medium.
- LDL cholesterol, insulin, 2- ⁇ -mercaptoethanol, iron ions, iron transporters, etc. may be added to the medium used for the culture of the present invention, but differentiation induction into megakaryocytes and platelets will proceed. It is preferable not to add.
- the expression of c-MPL receptor on the cell surface is promoted by culturing mesenchymal cells in a basic medium for mesenchymal cell culture containing a c-MPL receptor agonist.
- a method of adhesion culture in a culture vessel coated with an extracellular matrix can be mentioned, and the culture temperature is usually within the range of 12 to 45 ° C.
- the temperature can be in the range of 15 to 37 ° C.
- the culture period is in the range of 1 to 15 days, in the range of 2 to 10 days, in the range of 2 to 4 days, or in 3 days. be able to.
- mesenchymal cells may be passaged. When subcultured, it is preferable to select and pass the mesenchymal cells whose c-MPL receptor expression is promoted on the cell surface.
- the manufacturing method of the present invention includes the step B after the step A.
- the step B is not particularly limited as long as it is a step of obtaining mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted.
- mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface means that a c-MPL receptor expression promoter is not added to the basic medium for mesenchymal cell culture. Shows the number of c-MPL receptors expressed on the surface of mesenchymal cells obtained by culturing mesenchymal cells in the same manner (hereinafter referred to as “control mesenchymal cells”).
- mesenchymal cell having a large number of c-MPL receptors expressed on the surface of the cell, and when the mesenchymal cell is a cell population, the entire cell population of the control mesenchymal cell Or the average number of c-MPL receptors (average number per cell) expressed on the surface of some cells (eg, cells in any 10-100 range included in the cell population) Compared to all or part of a cell population (eg, contained in the cell population Means a cell population of mesenchymal cells with a high average number of c-MPL receptors (average number per cell) expressed on the surface of cells (within a range of 10 to 100 cells) .
- mesenchymal cells in which expression of c-MPL receptor on the cell surface is promoted is compared with the proportion of c-MPL receptor positive cells in the cell population of control mesenchymal cells.
- a cell population of mesenchymal cells with a high proportion of c-MPL receptor positive cells is preferably included.
- the mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted has a ratio of c-MPL receptor positive cells of 1% or more, preferably 2% or more, A cell population of mesenchymal cells that is preferably 3% or more is suitably included.
- the proportion of c-MPL receptor positive cells is usually less than 0.8% (in Example 1 described later). (Refer to the ratio of c-MPL receptor positive cells in the cell population of preadipocytes (TPO-)).
- Whether the expression of c-MPL receptor on the cell surface is promoted is determined by using an antibody against c-MPL receptor (preferably a labeled antibody, more preferably a fluorescently labeled antibody). As an index, it can be confirmed by selecting mesenchymal cells expressing c-MPL receptor (for example, mesenchymal cells positive for c-MPL receptor).
- a method for performing such selection a method using a cell sorter, a magnetic bead, a cell adsorption column, or the like can be used, and a method using a cell sorter can be preferably mentioned because it is simpler and quicker.
- the method using a cell sorter is based on the flow cytometry method and is well known to those skilled in the art.
- a mesenchymal cell in which the number of c-MPL receptors expressed on the surface of a cell is larger than the number of c-MPL receptors expressed on the surface of a control mesenchymal cell The number of c-MPL receptors expressed on the cell surface is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more, more preferably 4 times or more of control mesenchymal cells.
- mesenchymal cells that are:
- the cell population is compared with the above-mentioned “average number of c-MPL receptors (average number per cell) expressed on the surface of all or part of the cell population of control mesenchymal cells”
- the cell population of mesenchymal cells having a large average number of c-MPL receptors (average number per cell) expressed on the surface of all or some of the cells is all or part of the cell population
- the average number of c-MPL receptors expressed on the cell surface (average number per cell) is 1.5 times or more, preferably 2 times or more, more preferably that of the control mesenchymal cell population.
- Is preferably a cell population of mesenchymal cells that is 3 times or more, more preferably 4 times or more.
- the cell population obtained by the culture in the above-mentioned step A is described as follows: “Expression of c-MPL receptor on cell surface” May be a method of directly obtaining a cell population containing “mesenchymal cells promoted”, or “c-MPL receptor expression on the cell surface is promoted from the cell population obtained by the culture in the above step A”.
- the method may be obtained by selecting “made mesenchymal cells”.
- selecting specific mesenchymal cells includes (p) isolating only specific mesenchymal cells from the cell population, and (q) included in the cell population after selection. It is also included for convenience that the ratio of specific mesenchymal cells (ratio of the number of cells) is higher than the ratio of specific mesenchymal cells (ratio of the number of cells) included in the cell population before selection. .
- the method for selecting “mesenchymal cells in which the expression of c-MPL receptor is promoted on the cell surface” is not particularly limited. However, from the viewpoint of selecting target mesenchymal cells more easily and quickly, c -The use of antibodies against MPL receptors (preferably labeled antibodies, more preferably fluorescently labeled antibodies), while the expression of c-MPL receptors on the cell surface was promoted using the presence or absence of specific binding of each of these antibodies as an index A method for sorting leaf cells can be mentioned. Examples of the method for selecting mesenchymal cells include a method using a cell sorter, a magnetic bead or a cell adsorption column as described above, and a method using a cell sorter in that it is simpler and quicker. Can be preferably mentioned.
- ⁇ Method of promoting expression of c-MPL receptor on the surface of mesenchymal cells As a method for promoting the expression of c-MPL receptor on the surface of the mesenchymal cell of the present invention (hereinafter simply referred to as “promotion method of the present invention”), a mesenchymal cell culture containing a c-MPL receptor agonist is used. As long as it contains the process A which culture
- the c-MPL receptor expression promoter on the surface of the mesenchymal cell of the present invention (hereinafter simply referred to as “expression promoter of the present invention”) is not particularly limited as long as it contains a c-MPL receptor agonist. However, it is preferable to contain a c-MPL receptor agonist as an active ingredient, and it is more preferable to contain a c-MPL receptor agonist as a single active ingredient.
- the expression promoter of the present invention may be composed only of a c-MPL receptor agonist, or a c-MPL receptor agonist may be formulated by a conventional method.
- the dosage form of the expression promoter of the present invention may be a solid preparation such as a powder or granule, or may be a liquid such as a solution, emulsion or suspension.
- an appropriate pharmaceutically acceptable carrier for example, an excipient, a binder, a solvent, a solubilizing agent, a suspension, etc.
- Agents emulsifiers, isotonic agents, buffers, stabilizers, soothing agents, preservatives, antioxidants, coloring agents, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, diluents, etc. These optional components can be blended.
- the expression promoter of the present invention can be used by adding it to a basic medium for culturing mesenchymal cells and culturing mesenchymal cells in vitro in that medium.
- the concentration added to the basal medium is 1 ng / mL to 2.5 ⁇ g / mL, preferably 5 ng / mL to 500 ng / mL in terms of dry c-MPL receptor agonist concentration. be able to.
- Such a concentration range can be particularly preferably mentioned when the c-MPL receptor agonist is TPO.
- Human primary cultured adipose precursor cells (HumanHPreadipocytes: HPAD) manufactured by CELL APPLICATIONS were prepared. A culture dish was coated with collagen, and a culture medium was added thereto.
- the culture medium includes DMEM medium (Dulbecco's Modified Eagle's Medium, Life Technology), 10% fetal bovine serum (Sigma), 1% non-essential amino acid (Life Technology), 100 U / mL penicillin-streptomycin solution (Life Technology) and a medium supplemented with 50 ng / mL recombinant human TPO (R & DPOSystems) were used.
- the preadipocytes were cultured for 3 days at 37 ° C. in the culture medium. The preadipocytes after culture were collected.
- adipose precursor cell population (hereinafter referred to as “adipose precursor cell population (TPO +)”) are labeled with a fluorescent-labeled anti-c-MPL antibody (BD Bioscience), and fluorescence is obtained using a flow cytometry method. By measuring the ratio of c-MPL receptor positive cells, it was 3.68 ⁇ 0.39%.
- TPO + The result of immunostaining the preadipocyte cell population (TPO +) with “fluorescence-labeled anti-c-MPL antibody” and “DAPI (4 ′, 6-diamidino-2-phenylindole)” is shown in the right panel of FIG. Show.
- fat precursor cells obtained by culturing in the same manner except that recombinant human TPO was not added to the culture medium hereinafter referred to as “cell population of fat precursor cells (TPO ⁇ )”.
- TPO ⁇ cell population of fat precursor cells
- the percentage of c-MPL receptor-positive cells in was measured by the flow cytometry method described above, and was 0.67 ⁇ 0.12%.
- the result of immunostaining the preadipocyte cell population (TPO-) with "fluorescent labeled anti-c-MPL antibody" and "DAPI” is shown in the left panel of FIG. The left panel shows that there are few cells showing fluorescence due to the fluorescently labeled anti-c-MPL antibody around the circular fluorescence of the DAPI-stained nucleus, that is, c-MPL receptor positive cells. .
- MKLI medium megakaryocyte lineage induction medium
- the MKLI medium is IMDM medium (Iscove'soveModified Dulbecco's Medium, Life Technology), 2mM L-glutamine (Life Technology), 100U / mL penicillin-streptomycin solution (Life Technology), 0.5% BSA.
- the “cell population of preadipocytes (TPO +)” obtained in Example 1 above was cultured in the above-described MKLI medium at 37 ° C. under a CO 2 concentration of 5% for 7 days. After the cell population after culture was collected, the percentage (%) of CD41 (specific markers for megakaryocytes and platelets) positive cells in the cell population was measured. Such measurement was performed by directly labeling with FITC (fluorescein isothiocyanate) labeled anti-CD41 antibody and using flow cytometry. The proportion of CD41 positive cells in this cell population was 28.5 ⁇ 2.1%.
- adipose precursor cell population (TPO +) obtained by culturing in a medium to which recombinant TPO has been added
- a medium to which recombinant TPO has not been added It is shown that approximately 1.7 times as many megakaryocytes and platelets can be produced as compared with the case of using adipose precursor cells obtained by culturing in (1) (a population of precursor fat cells (TPO-)). It was done.
- the expression of c-MPL on the surface of mesenchymal cells can be promoted simply and efficiently. That is, according to the present invention, mesenchymal cells in which the expression of c-MPL receptor on the cell surface is promoted can be produced simply and efficiently.
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Abstract
Description
(1)c-MPL受容体作動物質を含有する、間葉系細胞の表面におけるc-MPL受容体の発現促進剤や、
(2)c-MPL受容体作動物質が、TPO又はその誘導体、ロミプロスチム(Romiplostim)、及びエルトロンボパグ オラミン(Eltrombopag olamine)からなる群から選択される1種又は2種以上の物質である上記(1)に記載の間葉系細胞の表面におけるc-MPL受容体の発現促進剤や、
(3)間葉系細胞が脂肪前駆細胞である上記(1)又は(2)に記載の間葉系細胞の表面におけるc-MPL受容体の発現促進剤に関する。
(4)c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程A;及び、
細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞を得る工程B;
を含む、細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法や、
(5)c-MPL受容体作動物質が、TPO又はその誘導体、ロミプロスチム(Romiplostim)、及びエルトロンボパグ オラミン(Eltrombopag olamine)からなる群から選択される1種又は2種以上の物質である上記(4)に記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法や、
(6)間葉系細胞が脂肪前駆細胞である上記(4)又は(5)に記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法や、
(7)間葉系細胞培養用基本培地がさらに血清を含む上記(4)~(6)のいずれかに記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法や、
(8)工程Bで得られる間葉系細胞の細胞集団におけるc-MPL受容体陽性の間葉系細胞の割合が1%以上である上記(4)~(7)のいずれかに記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法に関する。
(9)c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程Aを含む、間葉系細胞の表面におけるc-MPL受容体の発現を促進する方法に関する。
本発明のc-MPL受容体の発現が促進された間葉系細胞の製造方法(以下、単に「本発明の製造方法」と表示する。)としては、
c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程A;及び、
c-MPL受容体の発現が促進された間葉系細胞を得る工程B;
を含む方法である限り特に制限されず、ここで「培地」とは、細胞を培養できる「培地成分」に水を添加した状態のものをいう。脂肪前駆細胞等の間葉系細胞を、c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で培養すると、作用機序の詳細は不明であるが、間葉系細胞の表面におけるc-MPL受容体の発現が促進される。なお、本発明の製造方法における「c-MPL受容体作動物質」として、後述の本発明の「間葉系細胞の表面におけるc-MPL受容体の発現促進剤」を用いてもよい。
上記工程Aとしては、c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程である限り特に制限されない。かかる培養工程は、生体外での培養工程である。
本発明に用いる間葉系細胞としては、c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で培養することによって、細胞表面におけるc-MPL受容体の発現が促進され得る間葉系細胞である限り特に制限されない。該間葉系細胞としては(a)脂肪前駆細胞(preadipocytesあるいはadipose progenitor cells)、(b)間葉系幹細胞(mesenchymal stem cell)、(c)間質細胞(stromal cell)等を挙げることができ、上記間葉系幹細胞としては、皮下脂肪組織由来間葉系幹細胞、骨髄間葉系幹細胞を挙げることができ、上記間質細胞としては、脂肪組織由来間質細胞、骨髄間質細胞、前立腺由来間質細胞、子宮内膜由来間質細胞を挙げることができる。好ましい間葉系細胞として、脂肪前駆細胞、皮下脂肪組織由来間葉系幹細胞を挙げることができる。本発明に用いる間葉系細胞は、培養細胞株であってもよいし、組織から採取した細胞(初代培養細胞や継代培養細胞を含む)であってもよい。本発明に用いる間葉系細胞のより具体的な例として、ヒト初代培養脂肪前駆細胞(HPAd細胞)、マウス初代培養前駆細胞(皮下脂肪組織由来)、株化されたマウス間質細胞(OP9細胞)、株化されたマウス骨髄間葉系幹細胞(HS-22細胞)、株化されたマウス脂肪前駆細胞(3T3-L1細胞)などを挙げることができる。
本発明の培養に用いる培地は、間葉系細胞培養用基本培地にc-MPL受容体作動物質を含有させた培地である。
本発明に用いる間葉系細胞培養用基本培地としては、c-MPL受容体作動物質を添加した場合に、その培地で間葉系細胞を培養することにより「細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞」を製造し得る培地であれば特に制限されないが、調製が容易であり、ロットごとのばらつきを防ぐ点から化学合成培地が好ましく、1又は2種類以上の糖(類)、1又は2種類以上の無機塩(類)、1又は2種類以上のアミノ酸(類)、及び1又は2種類以上のビタミン(類)、及び、任意で1又は2種類以上のその他成分を含むことが好ましい。
200mg/L 無水塩化カルシウム、0.1mg/L Fe(NO3)3・9H2O、200mg/L 塩化カリウム、97.67mg/L 無水硫酸マグネシウム、6400mg/L 塩化ナトリウム、3700mg/L 炭酸水素ナトリウム、125mg/L リン酸二水素ナトリウム一水和物、4500mg/L D-グルコース、15mg/L フェノールレッド、110mg/L ピルビン酸ナトリウム、84mg/L L-塩酸アルギニン、63mg/L L-シスチン二塩酸塩、584mg/L L-グルタミン、30mg/Lグリシン、42mg/L L-ヒスチジン塩酸塩一水和物、105mg/L L-イソロイシン、105mg/L L-ロイシン、146mg/L L-リジン塩酸塩、30mg/L L-メチオニン、66mg/L L-フェニルアラニン、42mg/L L-セリン、95mg/L L-スレオニン、16mg/L L-トリプトファン、104mg/L L-チロシン二ナトリウム二水和物、94mg/L L-バリン、4mg/L D-パントテン酸カルシウム、4mg/L 塩化コリン、4mg/L 葉酸、7.2mg/L i-イノシトール、4mg/L ニコチン酸アミド、4mg/L ピリドキシン塩酸塩、0.4mg/L リボフラビン、4mg/L 塩酸チアミン。
本発明における培養の方法としては、c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養することにより、「細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞」を製造し得る限り特に制限されないが、例えば細胞外マトリックスでコーティングされた培養容器内で接着培養する方法を挙げることができ、培養温度として通常12~45℃の範囲内、好ましくは15~37℃の範囲内を挙げることができ、培養期間として1~15日間の範囲内や、2~10日間の範囲内や、2~4日間の範囲内や、3日間を挙げることができる。また、かかる培養において、間葉系細胞を継代してもよい。継代する際は、細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞を選別して継代することが好ましい。
本発明の製造方法は、上記工程Aの後に、上記工程Bを含んでいる。上記工程Bとしては、細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞を得る工程である限り特に制限されない。
本発明の間葉系細胞の表面におけるc-MPL受容体の発現を促進する方法(以下、単に「本発明の促進方法」)としては、c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程Aを含んでいる限り特に制限されない。かかる工程Aは、上記の本発明の製造方法における工程Aと同じである。
本発明の間葉系細胞の表面におけるc-MPL受容体の発現促進剤(以下、単に「本発明の発現促進剤」)としては、c-MPL受容体作動物質を含有している限り特に制限されないが、c-MPL受容体作動物質を有効成分として含有することが好ましく、c-MPL受容体作動物質を単独の有効成分として含有することがより好ましい。
c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で培養することによって、間葉系細胞の細胞表面におけるc-MPL受容体の発現が促進するかどうかを調べるために、以下の培養実験を行った。なお、この培養実験では、間葉系細胞として脂肪前駆細胞を用いた。
上記の実施例1で得られた「脂肪前駆細胞の細胞集団(TPO+)」が、巨核球・血小板(巨核球及び/又は血小板)の効率的な製造に実際に用い得るかを確認するために、特許文献1に記載の方法にしたがって、脂肪前駆細胞から巨核球・血小板への分化誘導を行った。
Claims (9)
- c-MPL受容体作動物質を含有する、間葉系細胞の表面におけるc-MPL受容体の発現促進剤。
- c-MPL受容体作動物質が、TPO又はその誘導体、ロミプロスチム(Romiplostim)、及びエルトロンボパグ オラミン(Eltrombopag olamine)からなる群から選択される1種又は2種以上の物質である請求項1に記載の間葉系細胞の表面におけるc-MPL受容体の発現促進剤。
- 間葉系細胞が脂肪前駆細胞である請求項1又は2に記載の間葉系細胞の表面におけるc-MPL受容体の発現促進剤。
- c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程A;及び、
細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞を得る工程B;
を含む、細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法。 - c-MPL受容体作動物質が、TPO又はその誘導体、ロミプロスチム(Romiplostim)、及びエルトロンボパグ オラミン(Eltrombopag olamine)からなる群から選択される1種又は2種以上の物質である請求項4に記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法。
- 間葉系細胞が脂肪前駆細胞である請求項4又は5に記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法。
- 間葉系細胞培養用基本培地がさらに血清を含む請求項4~6のいずれかに記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法。
- 工程Bで得られる間葉系細胞の細胞集団におけるc-MPL受容体陽性の間葉系細胞の割合が1%以上である請求項4~7のいずれかに記載の細胞表面におけるc-MPL受容体の発現が促進された間葉系細胞の製造方法。
- c-MPL受容体作動物質を含む間葉系細胞培養用基本培地で間葉系細胞を培養する工程Aを含む、間葉系細胞の表面におけるc-MPL受容体の発現を促進する方法。
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