WO2016136986A1 - 間葉系幹細胞培養用培地、間葉系幹細胞の培養方法及び間葉系幹細胞 - Google Patents
間葉系幹細胞培養用培地、間葉系幹細胞の培養方法及び間葉系幹細胞 Download PDFInfo
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Definitions
- the present invention relates to a mesenchymal stem cell culture medium, a mesenchymal stem cell culture method, and a mesenchymal stem cell.
- somatic stem cells generally cannot differentiate into all organs and tissues, but differentiate into specific tissues and organs. There are various types of somatic stem cells, and mesenchymal stem cells are one of them.
- the umbilical cord is rich in somatic stem cells that are the source of many types of cells. For example, it contains hematopoietic stem cells derived from umbilical cord blood and has already been used for the treatment of intractable blood diseases such as leukemia and aplastic anemia.
- the umbilical cord includes cells classified as mesenchymal stem cells and pluripotent stem cell-like cells capable of differentiating into various cells such as nerves, muscles, heart, blood vessels, bones, and skin. Contains various stem cells.
- the present invention has been made in view of the above-described prior art, and is a medium that can be cultured in a large amount, efficiently and over a long period of time while maintaining the undifferentiated nature of mesenchymal stem cells, particularly mesenchymal stem cells.
- an object is to provide a mesenchymal stem cell culture medium more suitable for culturing umbilical cord stem cells.
- the gist of the present invention is as follows.
- the mesenchymal stem cell culture medium according to (1) or (2) which comprises a PTEN inhibitor, a p53 inhibitor, a p38 inhibitor, a Wnt signal activator and a ROCK inhibitor.
- the mesenchymal stem cell culture medium according to any one of (1) to (3) further containing at least one component selected from the group consisting of growth factors and steroidal compounds.
- the p38 inhibitor is selected from the group consisting of SB203580, SB202190, BIRB796, LY2288820, VX-702, PH-79804, TAK-715, VX-745, and Skepone-L, (1) to ( The medium for mesenchymal stem cell culture according to any one of 6).
- the Wnt signal activator is LiCl or a complement molecule C1q.
- a method for culturing mesenchymal stem cells comprising culturing mesenchymal stem cells using the medium for culturing mesenchymal stem cells according to any one of (1) to (9).
- the undifferentiated state can be maintained over a long period of time.
- the medium of the present invention can proliferate more efficiently while maintaining a good cell state over a long period of time, as compared with a conventional medium.
- FIG. 2 is a photograph of umbilical cord stem cells and adipose-derived stem cells on the third day of culture in the medium of the present invention. It is a graph which shows the proliferation of the umbilical cord stem cell and the fat origin stem cell by the culture medium of this invention. It is a photograph of umbilical cord stem cells and adipose-derived stem cells on the eighth day of culture in the medium of the present invention. It is a figure which shows the cell surface marker expression of the fat origin stem cell of the culture
- the mesenchymal stem cell culture medium of the present invention comprises at least two components selected from the group consisting of a PTEN inhibitor, a p53 inhibitor, a p38 inhibitor, a Wnt signal activator and a ROCK inhibitor, and animal cell culture. Basal medium for use. By containing these components, the medium of the present invention can be cultured while maintaining the undifferentiation of mesenchymal stem cells over a long period of time.
- “over a long period” is 8 days or more, preferably 15 days or more, more preferably 30 days or more, further preferably 50 days or more, and particularly preferably 60 days or more. is there.
- the medium of the present invention can efficiently proliferate mesenchymal stem cells while maintaining a good cell state for a long period of time. Whether or not the state of the cell is good can be determined based on the common general technical knowledge of those skilled in the art from the cell morphology, growth rate, and the like. Furthermore, as an effect that could not be predicted, oxidative stress resistance of mesenchymal stem cells can be improved by culturing in the medium of the present invention.
- the medium of the present invention preferably further contains at least one component selected from the group consisting of growth factors and steroidal compounds.
- the culture medium of this invention may contain the other component in the range which does not impair the effect of invention. Below, the culture medium of this invention is demonstrated.
- a mesenchymal stem cell means a cell that has the ability to differentiate into cells belonging to the mesenchymal system (bone cells, chondrocytes, fat cells, etc.) and can proliferate while maintaining the ability.
- the umbilical cord stem cells refer to umbilical cords, umbilical cord-related tissues, and stem cells derived from umbilical cord blood. Specifically, it is a mesenchymal stem cell derived from the umbilical cord and a structure surrounding the umbilical cord, and more specifically, the umbilical cord, and placenta, amniotic membrane, chorion, decidua, egg membrane, Walton's jelly (Wharton's jelly; Whatton ' s Jelly) and other structures around the umbilical cord and mesenchymal stem cells derived from umbilical cord blood.
- an adipose stem cell or an adipose-derived stem cell refers to a stem cell derived from an adipose tissue. Specifically, it is a mesenchymal stem cell derived from adipose tissue.
- the PTEN inhibitor refers to a PTEN (Phosphatase and Tensin Homolog Deleted from Chromosome 10) gene or all substances having a function of inhibiting the action of the PTEN protein.
- the PTEN gene is located at 10q23.3 on the chromosome and has been identified as a tumor suppressor.
- PTEN protein is widely expressed in systemic cells and catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-triphosphate (PIP3), an inositol phospholipid. Known as an enzyme.
- PIP3 is synthesized intracellularly by PI3 kinase (PI3K) and causes activation of protein kinase B (PKB) / AKT.
- PTEN is said to be responsible for the dephosphorylation of PIP3 and to convert it into phosphatidylinositol 4,5-bisphosphate (PIP2). Therefore, PTEN controls the PI3K / AKT information transmission system negatively.
- PIP3 When the activity of PTEN is inhibited, PIP3 accumulates in the cell and the PI3K / AKT signal transduction system is activated.
- a compound containing vanadium is preferable.
- pV phenbig
- bpV dipotassium bisperoxo
- bpV dipotassium bisperoxo
- VO-OHPic trihydrate ((OC-6-45)
- Aqua 3-hydroxy-2-piperidinecarboxylato-kapaN1, kapaO2
- VO-OHPic, HOpic, and pV are more preferable. Any of VO-OHPic, HOpic, and pV can provide a sufficient effect, but pV is more preferable. These may be used alone or in combination of two or more.
- the concentration of the PTEN inhibitor in the medium for culturing mesenchymal stem cells of the present invention is preferably 10 nM to 10 ⁇ M, more preferably 50 nM to 1 ⁇ M, further preferably 100 nM to 750 nM, from the viewpoint of the effect of the present invention. 250 nM to 750 nM is particularly preferable.
- the p53 inhibitor refers to all substances having a function of inhibiting the action of the p53 gene or p53 protein.
- the p53 gene is located at 17p13.1 on the chromosome and is also known as a tumor suppressor gene.
- the p53 protein acts as a transcription factor and has various physiological activities.
- Examples of the p53 inhibitor in the present invention include sodium orthovanadate, pifthrin- ⁇ , MDM2 protein and the like. Of these, sodium orthovanadate, pifthrin- ⁇ , and MDM2 protein are preferred. Sufficient effects can be obtained with any of sodium orthovanadate, pifisulin- ⁇ , and MDM2 protein, but pifisulin- ⁇ is more preferable. These may be used alone or in combination of two or more.
- the concentration of the p53 inhibitor in the mesenchymal stem cell culture medium of the present invention is preferably 100 nM to 1 mM, more preferably 500 nM to 500 ⁇ M, and more preferably 1 ⁇ M to 100 ⁇ M from the viewpoint of the effects of the present invention. More preferably.
- the p38 inhibitor refers to all substances having a function of inhibiting the action of the p38 gene or p38 protein.
- p38 is one of MAP kinase (Mitogen-Activated Protein Kinase) which is a serine / threonine kinase.
- MAP kinase has been shown to be involved in signal molecules that transmit external stimuli, cell proliferation, differentiation, gene expression, apoptosis, and the like.
- Examples of the p38 inhibitor in the present invention include SB203580 (Methyl [4- [4- (4-fluorophenyl) -5- (4-pyridinyl) -1H-imidazol-2-yl] phenyl] sulfoxide), SB202190 (4- [4- (4-Fluorophenyl) -5- (4-pyridinyl) -1H-imidazol-2-yl] phenol), BIRB796 (Doramapimod; 1- [5-tert-Butyl-2- (4-methylphenyl) -2H -Pyrazole-3-yl] -3- [4- (2-morpholinoethoxy) -1-naphthyl] urea), LY2288820 (5- [2- (1,1-Dimethylethyl) -5- (4-fluorophenyl) -1H-imidazol-4-yl] -3- (2,2-dimethylpropyl) -3H
- SB203580, SB202190, BIRB796, LY2288820, VX-702, PH-79784, TAK-715, VX-745, and Skepineone-L are preferable.
- SB203580, SB202190, BIRB796, LY2288820, VX-702, PH-79804, TAK-715, VX-745, and Skepineone-L can provide sufficient effects, but SB203580 and SB202190 are more preferred, and SB203580 is preferred. Further preferred. These may be used alone or in combination of two or more.
- the concentration of the p38 inhibitor in the mesenchymal stem cell culture medium of the present invention is preferably 1 nM to 1 ⁇ M, more preferably 10 nM to 500 nM, and more preferably 50 nM to 250 nM from the viewpoint of the effects of the present invention. More preferably it is.
- the Wnt signal activator refers to all substances that activate the Wnt signal.
- Wnt is a secreted intercellular signal transduction protein and is involved in intracellular signal transduction. This signal transduction pathway controls functions such as cell proliferation and differentiation, movement, body axis formation and organ formation during early embryogenesis.
- Wnt acts on cells and involves several separate activated intracellular signaling mechanisms.
- Wnt signaling pathway promotes ⁇ -catenin pathway that regulates gene expression via ⁇ -catenin, PCP (planar cell polarity) that regulates in-plane polarity of cells, and intracellular mobilization of Ca 2+ Ca2 + pathways are known.
- the Wnt signal activator may activate any of the pathways.
- the Wnt signal activator in the present invention includes both a catenin-dependent activator such as Wnt-3a and a catenin-independent activator such as Wnt-5a.
- a catenin-dependent activator such as Wnt-3a
- a catenin-independent activator such as Wnt-5a
- lithium chloride (LiCl) LiCl
- complement molecule C1q and the like can also be used.
- Wnt-3a, Wnt-5a, LiCl, and complement molecule C1q are preferable, and any of them can provide a sufficient effect, but LiCl and complement molecule C1q are more preferable, and LiCl is more preferable. These may be used alone or in combination of two or more.
- the concentration of the Wnt signal activator in the mesenchymal stem cell culture medium of the present invention is preferably 1 ⁇ M to 10 mM, more preferably 10 ⁇ M to 10 mM, more preferably 100 ⁇ M to 1 mM, from the viewpoint of the effect of the present invention. More preferably, it is 100 ⁇ M to 500 ⁇ M.
- the ROCK inhibitor refers to all substances that inhibit the action of Rho kinase (ROCK).
- Rho kinase (ROCK) is a serine threonine protein kinase that has been identified as a target protein for Rho, a low molecular weight GTP binding protein. Rho kinase is involved in physiological functions such as muscle contraction, cell proliferation, cell migration, and other gene expression induction.
- ROCK inhibitor in the present invention examples include Y-27632 [(R)-(+)-trans-N- (4-pyrylyl) -4- (1-aminoethyl) -cyclohexanecarboxamide ⁇ 2HCl ⁇ H 2 O], K -115 (lipasyl hydrochloride hydrate), Fasudil hydrochloride (fasudil hydrochloride; [HA1077 / 1- (5-Isoquinolinesulfonyl) homoperazine Hydrochloride]) and the like.
- K-115, and fasudil hydrochloride are preferable, and any of these can provide a sufficient effect, but Y-27632 is more preferable.
- the concentration of the ROCK inhibitor in the mesenchymal stem cell culture medium of the present invention is preferably 1 nM to 10 ⁇ M, more preferably 10 nM to 1 ⁇ M, and more preferably 50 nM to 500 nM from the viewpoint of the effects of the present invention. More preferably it is.
- the mesenchymal stem cell culture medium of the present invention contains at least two components selected from the group consisting of these PTEN inhibitors, p53 inhibitors, p38 inhibitors, Wnt signal activators and ROCK inhibitors. From the viewpoint of the effect of the present invention, it is preferable to include any three components, more preferably any four components, and even more preferably all five components. As a combination of the above two components, any of the above five components may be combined.
- any of the above five components may be combined.
- a PTEN inhibitor, a p53 inhibitor and a p38 inhibitor a PTEN inhibitor, a p53 inhibitor and a Wnt PTEN inhibitor, p53 inhibitor and ROCK inhibitor; PTEN inhibitor, p38 inhibitor and Wnt signal activator; PTEN inhibitor, p38 inhibitor and ROCK inhibitor; PTEN inhibitor, Wnt signal activity Agent and ROCK inhibitor; p53 inhibitor, p38 inhibitor and Wnt signal activator; p53 inhibitor, p38 inhibitor and ROCK inhibitor; p53 inhibitor, Wnt signal activator and ROCK inhibitor; p38 inhibitor , A combination of a Wnt signal activator and a ROCK inhibitor. As the combination with the above four components, any of the above five components may be combined.
- a PTEN inhibitor, a p53 inhibitor, a p38 inhibitor and a Wnt signal activator a PTEN inhibitor P53 inhibitor, p38 inhibitor and ROCK inhibitor; PTEN inhibitor, p53 inhibitor, Wnt signal activator and ROCK inhibitor; PTEN inhibitor, p38 inhibitor, Wnt signal activator and ROCK inhibitor; p53 A combination of an inhibitor, a p38 inhibitor, a Wnt signal activator and a ROCK inhibitor.
- TGF transforming growth factor
- EGF epidermal growth factor
- IGF Insulin-like growth factor
- nerve growth factor nerve growth factor
- BDNF brain-derived neurotrophic factor
- VEGF vascular endothelial growth factor
- G-CSF granulocyte colony stimulating factor
- GM-CSF granulocyte macrophage colony stimulating factor
- PDGF platelet derived growth factor
- EPO erythropoietin
- TPO thrombopoietin
- HGF hepatocyte growth factor
- Further examples include albumin, transferrin, lactoferrin, fetuin and the like. These may be used alone or in combination of two or more.
- concentration of the growth factor in the mesenchymal stem cell culture medium of the present invention an appropriate concentration is appropriately used depending on the type of the growth factor. In general, from the viewpoint of the effect of the present invention, it is preferably 1 nM to 100 mM, more preferably 10 nM to 10 mM.
- steroidal compounds As the steroidal compound in the culture medium for stem cell culture of the present invention, any steroidal compound known to those skilled in the art can be used. Typically, steroid hormones such as estradiol, progesterone, testosterone, cortisone, cortisol, hydrocortisone can be used, but are not limited thereto. These may be used alone or in combination of two or more.
- concentration of the steroidal compound in the mesenchymal stem cell culture medium of the present invention an appropriate concentration is appropriately selected depending on the type of the steroidal compound. In general, from the viewpoint of the effects of the present invention, it is preferably 0.1 nM to 1 mM, more preferably 1 nM to 100 ⁇ M, and even more preferably 10 nM to 1 ⁇ M.
- the basal medium for animal cell culture in the present invention refers to a medium containing a carbon source, a nitrogen source, an inorganic salt and the like essential for culturing animal cells.
- animal cells refer to mammalian cells, particularly human cells.
- the basal medium for animal cell culture in the present invention contains as little biological material as possible in consideration of the possibility of using cells obtained by culturing and the culture supernatant thereof for the treatment of diseases of animals (including humans).
- a medium for example, a serum-free medium
- the basic culture medium for animal cell culture may be mixed with trace effective substances such as trace nutrient promoting substances and precursors as necessary.
- animal cell culture medium animal cell culture media known to those skilled in the art can be used.
- minimum essential medium such as Eagle medium, Dulbecco's modified Eagle medium (DMEM), minimum essential medium ⁇ (MEM- ⁇ ), mesenchymal cell basal medium (MSCBM), Ham's F- 12 and F-10 medium, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified Dulbecco medium (IMDM), McCoy modified medium, etc. It is done.
- DMEM / F12 medium is particularly preferably used, but is not limited thereto.
- Additives such as amino acids, inorganic salts, vitamins, carbon sources and antibiotics can be added to the basal medium for animal cell culture.
- concentration of these additives is not particularly limited, and can be used at a concentration used in a normal animal cell culture medium.
- amino acids examples include glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, Examples include L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like.
- inorganic salts include calcium chloride, copper sulfate, iron (III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc. Is mentioned.
- vitamins examples include choline, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, vitamin B7, vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin Bh, vitamin Bt Vitamin Bx, vitamin C (ascorbic acid), vitamin D, vitamin E, vitamin F, vitamin K, vitamin M, vitamin P and the like.
- antibiotics such as penicillin, streptomycin, gentamicin, kanamycin
- carbon sources such as glucose, galactose, fructose, sucrose
- Stem cell differentiation inducers such as ⁇ -glycerophosphate, dexamethasone, rosiglitazone, isobutylmethylxanthine, 5-azacytidine
- antioxidants such as 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine Adenosine 5'-monophosphate, corticosterone, ethanolamine, insulin, reduced glutathione, lipoic acid, melatonin, hypoxanthine, phenol red, progesterone, putre Buffer, pyruvic acid, thymidine, triiodothy
- Examples of the mesenchymal stem cell culture medium of the present invention include, for example, DMEM / F-12 medium, L-glutamine, ascorbic acid, human recombinant albumin, bovine serum-derived fetuin, sodium hydrogen carbonate, HEPES, Lipid mixed solution, ITSE mixed solution, transferrin, bFGF, progesterone, hydrocortisone, VO-OHPic, Pifithrin- ⁇ (pifithrin- ⁇ ), SB203580, lithium chloride and Y-27632 added medium;
- DMEM / F-12 medium L-glutamine, ascorbic acid, human recombinant albumin, fetin from bovine serum, sodium bicarbonate, HEPES, Lipid mixture, ITSE mixture, transferrin, bFGF, progesterone, hydrocortisone, VO- Medium supplemented with OHPic, Pifthrin- ⁇ (Pifthrin- ⁇ ), SB203580;
- the method for preparing the medium of the present invention is not particularly limited, and can be prepared according to a conventionally known conventional method. For example, it can be obtained by adding and mixing each of the above-described components to a basal medium for animal cell culture at room temperature or as necessary.
- the medium of the present invention is preferably a liquid, but may be a gel or a solid medium such as an agar medium if necessary.
- mesenchymal stem cells can be seeded and incubated in a culture vessel or a culture carrier whose surface is not surface-treated.
- the present invention also includes a method for culturing mesenchymal stem cells, wherein the mesenchymal stem cells are cultured using the medium for culturing mesenchymal stem cells of the present invention.
- the culture method of the present invention is not particularly limited as long as it is a culture method using the medium for culturing mesenchymal stem cells of the present invention, and the same method as the conventional one is used. Usually, it is performed at a temperature of 30 ° C. to 37 ° C., in a 2% to 7% CO 2 environment, and in a 5% to 21% O 2 environment.
- the passage time and method of mesenchymal stem cells are not particularly limited as long as they are suitable for each mesenchymal stem cell, and can be performed in the same manner as before while observing the morphology of mesenchymal stem cells.
- the present invention also includes mesenchymal stem cells that are CD29, CD73, CD90, CD105, and CD166 positive, obtained by culturing in the medium for culturing mesenchymal stem cells of the present invention.
- the mesenchymal stem cells obtained by culturing in the medium for culturing mesenchymal stem cells of the present invention are maintained undifferentiated and strongly express CD29, CD73, CD90, CD105 and CD166.
- the mesenchymal stem cells obtained by culturing in the mesenchymal stem cell culture medium of the present invention have high viability and a good state.
- the mesenchymal stem cells obtained by culturing in the medium for culturing mesenchymal stem cells of the present invention have high resistance to oxidative stress. Therefore, the mesenchymal stem cells of the present invention can be effectively used as a pharmaceutical for the treatment of various diseases.
- Examples of diseases in which the mesenchymal stem cells of the present invention can be used as pharmaceuticals include cartilage degradation, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout, psoriasis, multiple sclerosis, and muscle atrophy.
- Lateral sclerosis Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic stenosis, renal failure, lupus, pancreatitis, allergy, fibrosis, anemia, atherosclerosis, restenosis, chemotherapy / radiation-related Complications, type I diabetes, type II diabetes, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease, nonspecific colitis, allergic conjunctivitis, Diabetic retinopathy, Sjogren's syndrome, uveitis allergic rhinitis, asthma, asbestosis, silicosis, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, sarcoidosis , Glomerulonephritis, vasculitis, dermatitis, HIV-related cachexia, cerebral malaria, ankylosing
- the administration method when using the mesenchymal stem cells of the present invention as a pharmaceutical is not particularly limited, but is preferably intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, subcutaneous administration, etc. Intravascular administration is more preferred.
- the dosage when the mesenchymal stem cells of the present invention are used as pharmaceuticals may vary depending on the type of disease, the degree of symptoms, the dosage form, the body weight of the administration target, etc., but 1 ⁇ 10 mesenchymal stem cells per day. It can be administered in the range of 5 to 1 ⁇ 10 9 .
- the administration may be performed once to several times a day.
- the administration may be a single administration or may be performed continuously. In the case of continuous administration, for example, administration can be continued twice or more at a frequency of once or more every 3 days.
- Mammals to be administered when the mesenchymal stem cells of the present invention are used as pharmaceuticals are not particularly limited, but humans, monkeys, mice, rats, hamsters, guinea pigs, cattle, pigs, horses, rabbits, sheep, goats. , Cats, dogs, and the like are preferred, with human beings being more preferred.
- the present invention also includes a method for producing a pharmaceutical comprising the above-described mesenchymal stem cells of the present invention.
- the method for producing a medicinal product containing mesenchymal stem cells of the present invention comprises a step of preparing mesenchymal stem cells according to the above-described method for culturing mesenchymal stem cells, and a pharmaceutically acceptable preservation solution for the obtained mesenchymal stem cells. Including the step of storing in.
- the manufacturing method of the pharmaceutical containing the mesenchymal stem cell of this invention may further include the process of suspending the mesenchymal stem cell of this invention in the buffer solution for administration etc. as needed.
- Each medium (Comparative Example 1 and Examples 1 to 8) was prepared by adding the components shown in Table 2 below to the prepared 14-2-1 medium so as to have the concentrations shown in Table 2. This medium was used for culturing umbilical cord stem cells and adipose-derived mesenchymal stem cells.
- Umbilical cord-derived mesenchymal stem cells (UC-MSC; Umbilical Cord Derived Mesenchymal Stem Cells Wharton's Jelly (HMSC-WJ), FC-0020, LifeLine), and adipose-derived mesenchymal stem cells (AD-MSC; Stem Cells, FC-0034, LifeLine) were conditioned in LifeLine recommended medium at 37 ° C. and 5% CO 2 and then seeded on CellBind Flask at a density of 5,000 cells / cm 2 .
- UC-MSC Umbilical Cord Derived Mesenchymal Stem Cells Wharton's Jelly
- AD-MSC adipose-derived mesenchymal stem cells
- FIG. 2 shows the growth rate up to the 8th day (the number of times the seeded cells had divided before the next passage).
- cell photographs after 3 days and 8 days are shown in FIGS. 1 and 3, respectively.
- AD-MSC showed better cell growth when cultured with the medium of Example 1 than with the recommended medium (FIG. 2).
- the cell morphology is not so different until the third day of culture (FIG. 1), but on the eighth day (FIG. 3), the culture medium of Example 1 is slightly smaller and rounder.
- the condition was good, but when the recommended medium was used, it became a long and narrow shape, and the condition was judged to be slightly worse.
- AD-MSC cultured in the recommended medium had only one expression peak of CD105 and CD73, whereas when cultured in the medium of Example 1, CD105 was partially Its expression decreased in the cells, resulting in two expression peaks.
- the expression intensity of CD73 decreased as a whole.
- the recommended medium seemed more suitable for maintaining the undifferentiated nature of AD-MSC.
- UC-MSC grew better in the medium of Example 1 on the sixth day of culture.
- the medium of Example 1 was better in a state where the cells were small and rounded.
- the expression of the surface marker it was determined that those cultured in the medium of Example 1 had higher expression peaks of CD105 and CD90 and were able to maintain undifferentiation more.
- Test 2 Umbilical cord-derived mesenchymal stem cells (UCMS-WJ), FC-0020, LifeLine) recommended by LifeLine under conditions of 37 ° C. and 5% CO 2 After acclimation in the medium, the medium was replaced with LifeLine recommended medium or the medium of Example 1 (1 ⁇ 10 5 cells / well; 6 well plate). The cells were passaged every 2 to 3 days, and the expression of cell surface markers (CD29, CD73, CD90, 105 and CD166) was analyzed by FACS for the cells on the 11th day after the medium change. The results are shown in FIG.
- the expression of CD73, CD90, 105 and CD166 is higher when cultured in the medium of Example 1 than when cultured in the recommended medium.
- the CD29 peak became more uniform. It was judged that those cultured in the medium of Example 1 were able to maintain the undifferentiated nature of UC-MSC.
- the medium of Example 1 was able to maintain better cell morphology for both AD-MSC and UC-MSC than the medium of Comparative Example 1.
- the surface marker of UC-MSC it can be seen that the medium of Example 1 had higher expression of CD105, sharp peaks, and maintained an undifferentiated and uniform population.
- the media of Examples 2 and 3 significantly promoted UC-MSC growth compared to AD-MSC.
- the cell morphology was good for both AD-MSC and UC-MSC.
- the medium of Example 4 further promoted the growth of UC-MSC compared to AD-MSC.
- the cell morphology was good for both AD-MSC and UC-MSC.
- the peak width was slightly wider and the uniformity of the cell population was slightly reduced.
- the media of Examples 5 and 6 promoted UC-MSC growth more than AD-MSC.
- the cell morphology was slightly elongated with AD-MSC, but UC-MSC was good.
- the surface marker (CD105) of UC-MSC the peak is sharp, indicating that an undifferentiated and uniform population could be maintained.
- the medium of Example 7 significantly promoted the growth of UC-MSC compared to AD-MSC.
- the cell morphology was good for both AD-MSC and UC-MSC.
- the peak width tended to be slightly wider.
- the medium of Example 8 significantly promoted the growth of UC-MSC compared to AD-MSC.
- the cell morphology was good for both AD-MSC and UC-MSC.
- the surface marker (CD105) of UC-MSC the peak is sharp, indicating that an undifferentiated and uniform population could be maintained.
- the medium (Examples 4 to 8) containing any 4 types of PTEN inhibitor, p53 inhibitor, p38 inhibitor, Wnt signal activator and ROCK inhibitor is composed of AD-MSC and UC.
- -It was possible to cultivate while maintaining the morphology of both cells of MSC in good condition.
- UC-MSC could be maintained in a good state almost as well as the medium of Example 1 containing all of the above five factors, and cultured while maintaining undifferentiation.
- the medium of Example 2 containing a PTEN inhibitor, p53 inhibitor and p38 inhibitor, and the medium of Example 3 containing a Wnt signal activator and a ROCK inhibitor are also in good form for both AD-MSC and UC-MSC.
- the effect of promoting the proliferation of UC-MSC was maintained.
- Test 4 Induction of oxidative stress tolerance (UC-MSC; Umbilical Cord Derived Messenical Stem Cells, Wharton's Jelly (HMSC-WJ), FC-0020, Lifeline sslC; And Umbilical Cord-delivered Messenical Stem Cells ATCC) were conditioned in 37 ° C. and 5% CO 2 with each company's recommended medium, and the medium was replaced with each company's recommended medium or the medium of Example 1 (0.3 to 1 ⁇ 10 ⁇ 10). 5 cells / well; 6 well plate). Cells that were passaged every 2-3 days were treated with Rotenone at various concentrations (0 nM, 100 nM, 200 nM, 500 nM, 1 ⁇ M). After 48 hours, staining was performed with Hoechst 33358, and the number of nuclei was counted with ImageXpress. The results are shown in FIG.
- UC-MSC suffers damage depending on the concentration of Rotenone treatment, and the number of cells decreases.
- the UC-MSC can be resistant to the damage caused by Rotenone treatment and oxidative stress is reduced. It became difficult to receive, suggesting the possibility of suppressing the decrease in the number of cells.
- Test 7 Umbilical cord-derived mesenchymal stem cells (UCMS-WJ), FC-0020, LifeLine) recommended by LifeLine under conditions of 37 ° C. and 5% CO 2 After acclimation in the medium, the cells were cultured in the medium of Examples 1 and 4 to 8 in Table 2 for 8 days, and the effects on cell proliferation were compared. Comparison of the effect on cell proliferation was performed by staining the cells with Hoechst 33342, taking images with ImageXpress, and counting the number of cells. The result is shown in FIG.
- a medium (Examples 9 to 18) containing any two of the five factors of a PTEN inhibitor, a p53 inhibitor, a p38 inhibitor, a Wnt signal activator and a ROCK inhibitor (Examples 9 to 18), As compared with the medium of Example 1 containing the above, it was found that the growth effect on UC-MSC for 5 days was almost the same with any medium. Therefore, when a medium containing any two of the five factors of PTEN inhibitor, p53 inhibitor, p38 inhibitor, Wnt signal activator and ROCK inhibitor is used, culture for 5 days against UC-MSC It was found that a sufficiently excellent growth promoting effect was obtained in the system.
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Abstract
Description
(2)PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤からなる群より選択される少なくとも3種の成分と、動物細胞培養用基礎培地とを含有する、(1)記載の間葉系幹細胞培養用培地。
(3)PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤を含有する、(1)又は(2)記載の間葉系幹細胞培養用培地。
(4)増殖因子及びステロイド性化合物からなる群より選択される少なくとも1種の成分をさらに含有する、(1)から(3)のいずれか記載の間葉系幹細胞培養用培地。
(5)前記PTEN阻害剤が、VO-OHPic、HOPic及びpVからなる群より選択される、(1)から(4)のいずれか記載の間葉系幹細胞培養用培地。
(6)前記p53阻害剤が、オルトバナジン酸ナトリウム、ピフィスリン-α及びMDM2タンパク質からなる群より選択される、(1)から(5)のいずれか記載の間葉系幹細胞培養用培地。
(7)前記p38阻害剤が、SB203580、SB202190、BIRB796、LY2228820、VX-702、PH-797804、TAK―715、VX-745、及びSkepinone-Lからなる群より選択される、(1)から(6)のいずれか記載の間葉系幹細胞培養用培地。
(8)前記Wntシグナル活性化剤が、LiCl又は補体分子C1qである、(1)から(7)のいずれか記載の間葉系幹細胞培養用培地。
(9)前記ROCK阻害剤が、Y-27632、K-115及び塩酸ファスジルからなる群より選択される、(1)から(8)のいずれか記載の間葉系幹細胞培養用培地。
(10)(1)から(9)のいずれか記載の間葉系幹細胞培養用培地を用いて、間葉系幹細胞を培養する、間葉系幹細胞の培養方法。
(11)(1)から(9)のいずれか記載の間葉系幹細胞培養用培地で培養された、CD29、CD73、CD90、CD105及びCD166陽性である間葉系幹細胞。
本発明の間葉系幹細胞培養用培地は、PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤からなる群より選択される少なくとも2種の成分と、動物細胞培養用基礎培地とを含有する。本発明の培地は、これらの成分を含有することで、間葉系幹細胞の未分化性を長期に渡って維持しながら培養することができる。ここで「長期に渡って」とは、8日間以上であり、好ましくは15日間以上であり、より好ましくは30日間以上であり、さらに好ましくは50日間以上であり、特に好ましくは60日間以上である。また、本発明の培地は、間葉系幹細胞を長期に渡って良好な細胞状態を維持しながら、効率的に増殖させることができる。なお、細胞の状態が良好であるか否かは、細胞の形態、増殖速度等から当業者の技術常識に基づいて判断することができる。さらに予想もできなかった効果として、本発明の培地で培養することで、間葉系幹細胞の酸化ストレス耐性機能を向上させることができる。本発明の培地は、増殖因子及びステロイド性化合物からなる群より選択される少なくとも1種の成分をさらに含有することが好ましい。なお、本発明の培地は発明の効果を損なわない範囲でその他の成分を含有していてもよい。以下に、本発明の培地について説明する。
本発明においてPTEN阻害剤とは、PTEN(Phosphatase and Tensin Homolog Deleted from Chromosome 10)遺伝子、又はPTENタンパク質の作用を阻害する機能を有するすべての物質をいう。PTEN遺伝子は、染色体上の10q23.3に位置し、腫瘍抑制因子として同定されている。PTENタンパク質は広く全身の細胞に発現しており、イノシトールリン脂質であるフォスファチジルイノシトール 3,4,5-トリフォスフェイト(phosphatidylinositol 3,4,5-trisphosphate;PIP3)の脱リン酸化反応を触媒する酵素として知られている。PIP3は、PI3キナーゼ(PI3K)により細胞内で合成され、プロテインキナーゼB(PKB)/ AKTの活性化を引き起こす。PTENは、このPIP3の脱リン酸化反応を担い、フォスファチジルイノシトール 4,5-ビスフォスフェイト(phosphatidylinositol 4,5-bisphosphate;PIP2)に変換する作用があるとされている。従って、PTENは、PI3K/AKT情報伝達系を負に制御する。PTENの活性が阻害されると、細胞内にPIP3が蓄積し、PI3K/AKT情報伝達系が活性化する。
本発明においてp53阻害剤とは、p53遺伝子又はp53タンパク質の作用を阻害する機能を有するすべての物質をいう。p53遺伝子は、染色体上の17p13.1に位置し、腫瘍抑制遺伝子としても知られている。p53タンパク質は、転写因子として作用し、多様な生理活性を有する。
本発明においてp38阻害剤とは、p38遺伝子又はp38タンパク質の作用を阻害する機能を有するすべての物質をいう。p38は、セリン/スレオニンキナーゼであるMAPキナーゼ (Mitogen-Activated Protein Kinase)の1つである。MAPキナーゼは、外界刺激を伝達するシグナル分子、細胞増殖、分化、遺伝子発現、アポトーシス等への関与が明らかにされている。
本発明においてWntシグナル活性化剤とは、Wntシグナルを活性化させるすべての物質をいう。Wntは、分泌性の細胞間シグナル伝達タンパク質で、細胞内シグナル伝達に関与している。このシグナル伝達経路は細胞の増殖や分化、運動、初期胚発生時の体軸形成や器官形成等の機能を制御している。Wntシグナル経路では、Wntが細胞に作用することにより、いくつかの別々の活性化される細胞内シグナル伝達機構が含まれる。Wntシグナル経路にはβ-カテニンを介して遺伝子発現を制御するβ-カテニン経路、細胞の平面内極性を制御するPCP(planar cell polarity, 平面内細胞極性)経路、Ca2+の細胞内動員を促進するCa2+経路等が知られている。本明細書において、Wntシグナル活性化剤とは、そのいずれの経路を活性化するものであってもよい。
本発明においてROCK阻害剤とは、Rhoキナーゼ(ROCK)の作用を阻害するすべての物質をいう。Rhoキナーゼ(ROCK)は、低分子量GTP結合タンパク質であるRhoの標的タンパク質として同定されたセリン・スレオニンタンパク質リン酸化酵素である。Rhoキナーゼは、筋肉等の収縮、細胞増殖、細胞遊走及び他の遺伝子発現誘導等の生理機能に関与している。
本発明の幹細胞培養用培地における増殖因子としては、当業者に公知のいずれの増殖因子でも用いることができる。代表的には、トランスフォーミング成長因子(TGF)、上皮成長因子(EGF)等が挙げられるがこれに限定されず、インスリン様成長因子(IGF)、神経成長因子(NGF)、脳由来神経栄養因子(BDNF)、血管内皮細胞増殖因子(VEGF)、顆粒球コロニー刺激因子(G-CSF)、顆粒球マクロファージコロニー刺激因子(GM-CSF)、血小板由来成長因子(PDGF)、エリスロポエチン(EPO)、トロンボポエチン(TPO)、塩基性線維芽細胞増殖因子(bFGF又はFGF2)、肝細胞増殖因子(HGF)等が挙げられる。さらにアルブミン、トランスフェリン、ラクトフェリン、フェツイン等も例示される。これらは単独で用いてもよいし、2種以上を組み合わせて用いることもできる。
本発明の幹細胞培養用培地におけるステロイド性化合物としては、当業者に公知のいずれのステロイド性化合物でも用いることができる。代表的には、エストラジオール、プロゲステロン、テストステロン、コルチゾン、コルチゾール、ハイドロコルチゾン等のステロイドホルモンを使用することができるが、これらに限定されない。これらは単独で用いてもよいし、2種以上を組み合わせて用いることもできる。
本発明における動物細胞培養用基礎培地とは、動物細胞の培養に必須の炭素源、窒素源及び無機塩等を含有させた培地をいう。ここで、動物細胞とは、哺乳類細胞、特にはヒト細胞を指す。本発明における動物細胞培養用基礎培地は、培養して得られる細胞やその培養上清を動物(ヒトを含む)の疾患の治療のために用いる可能性を考慮すると、できるだけ生物由来原料を含まない培地(例えば、無血清培地)であることが好ましい。動物細胞培養用基礎培地には、必要に応じて、微量栄養促進物質、前駆物質等の微量有効物質を配合してもよい。このような動物細胞培養用基礎培地としては、当業者に公知の動物細胞培養用培地を使用することができる。具体的には、イーグル培地のような最小必須培地(MEM)、ダルベッコ改変イーグル培地(DMEM)、最小必須培地α(MEM-α)、間葉系細胞基礎培地(MSCBM)、Ham’s F-12及びF-10培地、DMEM/F12培地、Williams培地E、RPMI-1640培地、MCDB培地、199培地、Fisher培地、Iscove改変ダルベッコ培地(IMDM)、McCoy改変培地等、これらの混合培地等が挙げられる。動物細胞培養培地として用いる場合には、特にはDMEM/F12培地が好ましく用いられるがこれに限定されない。
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、VO-OHPic、Pifithrin-α(ピフィスリン-α)、SB203580を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、塩化リチウム及びY-27632を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、VO-OHPic、Pifithrin-α(ピフィスリン-α)、SB203580及びY-27632を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、VO-OHPic、Pifithrin-α(ピフィスリン-α)、SB203580及び塩化リチウムを加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、VO-OHPic、SB203580、塩化リチウム及びY-27632を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、Pifithrin-α(ピフィスリン-α)、SB203580、塩化リチウム及びY-27632を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾン、VO-OHPic、Pifithrin-α(ピフィスリン-α)、塩化リチウム及びY-27632を加えた培地;
DMEM/F-12培地に、L-グルタミン、アスコルビン酸、ヒト組み換え型アルブミン、ウシ血清由来Fetuin、炭酸水素ナトリウム、HEPES、Lipid混合液、ITSE混合液、トランスフェリン、bFGF、プロゲステロン、ハイドロコルチゾンを含む基礎培地に、VO-OHPic及びPifithrin-α(ピフィスリン-α);又はVO-OHPic及びSB203580;又はVO-OHPic及び塩化リチウム;又はVO-OHPic及びY-27632;又はPifithrin-α(ピフィスリン-α)及びSB203580;又はPifithrin-α(ピフィスリン-α)及び塩化リチウム;又はPifithrin-α(ピフィスリン-α)及びY-27632;又はSB203580及び塩化リチウム;又はSB203580及びY-27632;又は塩化リチウム及びY-27632を加えた培地等が挙げられる。
本発明は、本発明の間葉系幹細胞培養用培地を用いて、間葉系幹細胞を培養する、間葉系幹細胞の培養方法も含む。本発明の培養方法は、本発明の間葉系幹細胞培養用培地を用いた培養方法であれば特に限定されず、従来と同様の方法が用いられる。通常、30℃~37℃の温度、2%~7%CO2環境下、5%~21%O2環境下で行われる。また、間葉系幹細胞の継代の時期及び方法もそれぞれの間葉系幹細胞に適していれば特に限定されず、間葉系幹細胞の形態を観察しながら、従来と同様に行うことができる。
本発明は、本発明の間葉系幹細胞培養用培地で培養して得られる、CD29、CD73、CD90、CD105及びCD166陽性である間葉系幹細胞も含む。本発明の間葉系幹細胞培養用培地で培養して得られる間葉系幹細胞は、未分化性が維持されており、CD29、CD73、CD90、CD105及びCD166を強く発現している。また、本発明の間葉系幹細胞培養用培地で培養して得られる間葉系幹細胞は、バイアビリティが高く、状態が良好である。さらに、予想もできなかった効果として、本発明の間葉系幹細胞培養用培地で培養して得られる間葉系幹細胞は、酸化ストレスに対する耐性が高いことが挙げられる。そのため、本発明の間葉系幹細胞を医薬品として種々の疾患の治療に有効に用いることができる。
下記表1に示す基本の処方14-2-1培地(比較例1)を調製した。具体的には、DMEM/F12培地に下記表1に記載の成分を記載の濃度となるように添加した。
(試験1)
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)、及び脂肪由来間葉系幹細胞(AD-MSC;Adipose derived Mesenchymal Stem Cells、FC-0034、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化した後、5,000cells/cm2の密度でCellBind Flaskに播種した。翌日、LifeLine社推奨培地又は実施例1の培地に培地交換した(1X105cells/well;6well plate)。3日後、それぞれ同様に播種し直し、さらに3日後(合計では6日後)、細胞数を計数した。同様にさらに2日間培養した(合計では8日間)細胞については形態と表面マーカーを解析した。8日目までの増殖率(播種した細胞が次の継代までに分裂した回数)を図2に示す。また、3日後、8日後の細胞写真をそれぞれ図1、図3に示す。さらに、8日後の細胞について、FACSにて細胞表面マーカー(脂肪由来間葉系幹細胞については、CD105及びCD73、臍帯由来間葉系幹細胞についてはCD105及びCD90)の発現を解析した結果は、図4及び5に示す。
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化後、LifeLine社推奨培地又は実施例1の培地に培地交換した(1X105cells/well;6well plate)。2~3日おきに継代を行い、培地交換から11日目の細胞について、FACSにて細胞表面マーカー(CD29、CD73、CD90、105及びCD166)の発現を解析した。結果を図6に示す。
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)、及び脂肪由来間葉系幹細胞(AD-MSC;Adipose derived Mesenchymal Stem Cells、FC-0034、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化した後、表2に記載の各培地中でそれぞれ5日間培養し(播種時70,000cells/well)、細胞数を計数し、結果を図7にPDLで示した。細胞写真は図8及び9に示す。さらに継代しながら培養を継続し、合計3週間、それぞれの培地中で培養した細胞の細胞表面マーカー(CD105)発現をFACSにて解析した。その結果を図10に示す。
実施例2及び3の培地は、AD-MSCに比べてUC-MSCの増殖を顕著に促進した。細胞の形態は、AD-MSC、UC-MSC共に良好であった。
実施例4の培地は、AD-MSCに比べてUC-MSCの増殖をより促進した。細胞の形態は、AD-MSC、UC-MSC共に良好であった。しかし、UC-MSCの表面マーカー(CD105)については、ピークの幅がやや広く細胞集団の均一性がやや低下した。
実施例5及び6の培地は、AD-MSCに比べてUC-MSCの増殖をより促進した。細胞の形態は、AD-MSCでやや細長くなったが、UC-MSCは良好であった。UC-MSCの表面マーカー(CD105)については、ピークもシャープであり、未分化で均一な集団を維持できたことがわかる。
実施例7の培地は、AD-MSCに比べてUC-MSCの増殖を顕著に促進した。細胞の形態は、AD-MSC、UC-MSC共に良好であった。UC-MSCの表面マーカー(CD105)については、ピークの幅がやや広くなる傾向が見られた。
実施例8の培地は、AD-MSCに比べてUC-MSCの増殖を顕著に促進した。細胞の形態は、AD-MSC、UC-MSC共に良好であった。UC-MSCの表面マーカー(CD105)については、ピークもシャープであり、未分化で均一な集団を維持できたことがわかる。
以上のように、PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤のうちのいずれか4種類を含む培地(実施例4~8)は、AD-MSC及びUC-MSCの両方の細胞の形態を良好な状態に維持しながら培養することができた。また、AD-MSCに比べてUC-MSCの増殖をより促進する傾向があった。さらに、上記5因子の全てを含む実施例1の培地とほぼ同程度にUC-MSCを良好な状態で維持できると共に、未分化性を維持したまま培養することができた。また、PTEN阻害剤、p53阻害剤及びp38阻害剤を含む実施例2の培地、Wntシグナル活性化剤及びROCK阻害剤を含む実施例3の培地も、AD-MSC、UC-MSCともに良好な形態を保ちながら、特にUC-MSCの増殖を促進する効果があった。
(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社;UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells ScienCell社;及びUmbilical Cord derived Mesenchymal Stem Cells ATCC社)を、37℃、5%CO2の条件下、各社推奨培地にて馴化後、各社推奨培地又は実施例1の培地に培地交換した(0.3~1X105cells/well;6well plate)。2~3日おきに継代を行った細胞に対してRotenoneを各濃度で処理した(0nM、100nM、200nM、500nM、1μM)。48時間後にHoechest33358で染色し、ImageXpressで核数を計測した。結果を図11に示す。
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jellyc-12971、Promo cell社)を、37℃、5%CO2の条件下、Promo cell社推奨培地にて馴化した後、Promo Media(c-39810 or c-28019、Promo cell社、表2の実施例1の培地で長期に渡って継代培養を続け、合計の細胞数を計数した結果を図12に示す。
(試験6)長期間培養2
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化した後、表2の実施例1の培地又はLifeLine社推奨培地で長期に渡って継代培養を続けた。合計の細胞数を計数した結果を図13に示す。
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化した後、表2の実施例1、4~8の培地で8日間培養し、細胞増殖に対する効果を比較した。細胞増殖に対する効果の比較は、Hoechest33342で細胞を染色後、ImageXpressで撮影して細胞数を計数することにより行った。その結果を図15に示す。
臍帯由来間葉系幹細胞(UC-MSC;Umbilical Cord derived Mesenchymal Stem Cells Wharton’s Jelly(HMSC-WJ)、FC-0020、LifeLine社)を、37℃、5%CO2の条件下、LifeLine社推奨培地にて馴化した後、下記表3の実施例9~18の培地及び、上記表2の実施例1の培地で5日間培養し、細胞増殖に対する効果を比較した。細胞増殖に対する効果の比較は、Hoechest33342で細胞を染色後、ImageXpressで撮影して細胞数を計数することにより行った(細胞数/0.36mm2)。その結果を図16に示す。
Claims (11)
- PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤からなる群より選択される少なくとも2種の成分と、動物細胞培養用基礎培地とを含有する、間葉系幹細胞培養用培地。
- PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤からなる群より選択される少なくとも3種の成分と、動物細胞培養用基礎培地とを含有する、請求項1記載の間葉系幹細胞培養用培地。
- 前記PTEN阻害剤、p53阻害剤、p38阻害剤、Wntシグナル活性化剤及びROCK阻害剤を含有する、請求項1又は2記載の間葉系幹細胞培養用培地。
- 増殖因子及びステロイド性化合物からなる群より選択される少なくとも1種の成分をさらに含有する、請求項1から3のいずれか1項記載の間葉系幹細胞培養用培地。
- PTEN阻害剤が、VO-OHPic、HOPic及びpVからなる群より選択される、請求項1から4のいずれか1項記載の間葉系幹細胞培養用培地。
- 前記p53阻害剤が、オルトバナジン酸ナトリウム、ピフィスリン-α及びMDM2タンパク質からなる群より選択される、請求項1から5のいずれか1項記載の間葉系幹細胞培養用培地。
- 前記p38阻害剤が、SB203580、SB202190、BIRB796、LY2228820、VX-702、PH-797804、TAK―715、VX-745、及びSkepinone-Lからなる群より選択される、請求項1から6のいずれか1項記載の間葉系幹細胞培養用培地。
- 前記Wntシグナル活性化剤が、LiCl又は補体分子C1qである、請求項1から7のいずれか1項記載の間葉系幹細胞培養用培地。
- 前記ROCK阻害剤が、Y-27632、K-115及び塩酸ファスジルからなる群より選択される、請求項1から8のいずれか1項記載の間葉系幹細胞培養用培地。
- 請求項1から9のいずれか1項記載の間葉系幹細胞培養用培地を用いて、間葉系幹細胞を培養する、間葉系幹細胞の培養方法。
- 請求項1から9のいずれか1項記載の間葉系幹細胞培養用培地で培養された、CD29、CD73、CD90、CD105及びCD166陽性である間葉系幹細胞。
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- 2016-02-26 WO PCT/JP2016/055930 patent/WO2016136986A1/ja active Application Filing
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- 2016-02-26 US US15/553,868 patent/US20180066231A1/en not_active Abandoned
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US11707488B2 (en) | 2015-08-28 | 2023-07-25 | Rohto Pharmaceutical Co., Ltd. | ROR1-positive mesenchymal stem cells and method for preparing same, pharmaceutical composition containing ROR1-positive mesenchymal stem cells and method for preparing same, and method for preventing or treating disease using ROR1-positive mesenchymal stem cells |
WO2018159431A1 (ja) * | 2017-03-03 | 2018-09-07 | ロート製薬株式会社 | 間葉系幹細胞及び肝疾患治療剤 |
US11253550B2 (en) | 2017-03-03 | 2022-02-22 | Rohto Pharmaceutical Co., Ltd. | Method for treating fibrotic liver disease |
WO2018164228A1 (ja) * | 2017-03-08 | 2018-09-13 | ロート製薬株式会社 | Ror1陽性の間葉系幹細胞を含有する、線維症を伴う疾患の予防又は処置のための医薬組成物、及びその調製方法、並びにror1陽性の間葉系幹細胞を用いる線維症を伴う疾患の予防又は処置方法 |
CN110418645A (zh) * | 2017-03-08 | 2019-11-05 | 日本乐敦制药株式会社 | 含有ror1阳性的间充质干细胞的、用于预防或处置伴随纤维化的疾病的药物组合物、及其制备方法、以及使用ror1阳性的间充质干细胞的伴随纤维化的疾病的预防或处置方法 |
JPWO2018164228A1 (ja) * | 2017-03-08 | 2020-01-09 | ロート製薬株式会社 | Ror1陽性の間葉系幹細胞を含有する、線維症を伴う疾患の予防又は処置のための医薬組成物、及びその調製方法、並びにror1陽性の間葉系幹細胞を用いる線維症を伴う疾患の予防又は処置方法 |
WO2019022222A1 (ja) | 2017-07-28 | 2019-01-31 | 日産化学株式会社 | 培地添加用組成物及び培地添加用化合物並びにそれらを用いた細胞又は組織の培養方法 |
WO2020158841A1 (ja) | 2019-01-30 | 2020-08-06 | 日産化学株式会社 | ヒドラジド化合物及びキナーゼ阻害剤 |
CN110478368A (zh) * | 2019-08-21 | 2019-11-22 | 中国科学院动物研究所 | 脐带间充质干细胞条件培养基的用途 |
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JP6962814B2 (ja) | 2021-11-05 |
JPWO2016136986A1 (ja) | 2017-12-07 |
EP3263697A1 (en) | 2018-01-03 |
EP3263697A4 (en) | 2018-08-15 |
EP3263697B1 (en) | 2021-03-31 |
US20180066231A1 (en) | 2018-03-08 |
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