WO2004085632A1 - 幹細胞の分化誘導および分化能の制御 - Google Patents
幹細胞の分化誘導および分化能の制御 Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Definitions
- the present invention relates to culturing, maintaining, expanding, and further inducing differentiation of animal cells, particularly pluripotent stem cells, and more specifically, to a technique that can be used in the technical field of regenerative medicine.
- organs such as the heart, lungs, kidneys, blood vessels, digestive tract, and nerves
- ES cells embryonic stem cells
- stem cells including bone marrow cells have pluripotency (or pluripotency) to differentiate into various cells. It has also been reported that such bone marrow-derived cells contribute to regeneration of injured myocardium even in a living mouse body, and that cardiac function is improved.
- cytokin transforming growth factor 1 (a member of the TGF- ⁇ Spa family, ie, TGF-) 8 and It is known that an embryoid body formed by priming with BMP 2) promotes differentiation into a myocardium having a remarkably increased beating region (for example, see Non-Patent Document 1 below). ).
- cytokines a member of the TGF- ⁇ Spa family, ie, TGF- 8 and It is known that an embryoid body formed by priming with BMP 2) promotes differentiation into a myocardium having a remarkably increased beating region (for example, see Non-Patent Document 1 below).
- BMP 2 transforming growth factor 1
- the effects of cytokines on various cells have been investigated as important regulators of such proliferation and differentiation.
- the functions of cytokines are diverse and the functions are duplicated.
- TBR cell line a bone marrow stromal cell line established from transgenic mice of the temperature-sensitive SV-40T antigen gene and showing myogenic, osteogenic and adipogenic differentiation
- OSM oncostatin M
- OSM oncostatin M
- Non-Patent Documents 3 and 4 show that TBR cell lines show phenotypic changes in response to T antigen inactivation and growth conditions, preadipocytes are induced into adipocytes and osteogenic cells, and some preadipocytes It has also been described that cells and endothelial cell lines are induced into muscle cells and adipocytes. These properties suggest that TBR cells are derived from pluripotent mesenchymal stem cells. It is also known that mesenchymal stem cells can induce bone cells, chondrocytes, tendon cells, ligament cells, skeletal muscle cells, fat cells, stromal cells, and the like (see Non-Patent Document 5 below).
- bone marrow-derived cells capable of differentiating into cardiomyocytes can be stochastically transformed into cardiomyocyte, fat cell, and skeletal muscle cell lines by administering a DNA demethylating agent such as 5-azacitidine. Differentiation has been published (for example, see Patent Document 2 below).
- Patent Document 2 discloses that by adding a combination of one of four types of FGF-8, ET1, Midkine, and bone morphogenetic factor 14 (BMP4), and 5-azacitidine in combination. It is also described that myocardium-specific cells can promote myocardium-specific gene expression. Furthermore, Patent Document 2 discloses that when mouse bone marrow cells capable of differentiating into cardiomyocytes are preliminarily treated with 5-azacitidine, and then transplanted into mice, cardiomyocytes and vascular cells are derived from the transplanted cells. It also suggests that cells can be seen.
- Patent Document 1 JP-A-5-292958
- Patent Document 2 pamphlet No. 01-48151, especially page 4, 2-11 line, page 53, page 55
- Non-patent Document 1 The FASEB J. 2002; 16: 1558-1566, Abstract Non-patent Document 2 In Vitro Gel I. Dev. Biol. -Animal 37: 698-704 (2001)
- Non-patent document 3 J. Cellular Physiology 164: 55-64 (1995)
- Non-patent document 4 Experimental Cel I Research 218, 424—429 (1995)
- Non-Patent Document 5 Science 284, 143-147 (1999)
- Patent Document 2 also describes that the direction of differentiation (expression of a new differentiation trait) can be changed by the use of cytokines.
- the present inventors have carefully studied the effects of cytokines on the direction and degree of differentiation of pluripotent stem cells, and found that the diversity of cytokin functions described above is only between different cells. Instead, they were also found in different specific growth phases of the same lineage cells. They also found that the direction of differentiation of animal cells can be controlled by using two or more types of cytokins in combination. The present invention has been completed based on such findings.
- a method for inducing differentiation of a pluripotent stem cell by contacting the cell with an agent in the process of developing the cell,
- Contacting the cell with an agent comprises: i) a first developmental stage, ii) a second developmental stage, iii) in the first three stages of development, iv) late in the third stage of development, V) early in the fourth stage of development, and vi) in up to four of the developmental stages consisting of the late stage of the fourth stage of development; and
- a method for inducing cell differentiation wherein the agent is a substance capable of promoting and / or suppressing the differentiation of the cell in at least two directions.
- a method for evaluating the ability of a candidate agent to promote or suppress cell differentiation using the method for inducing cell differentiation comprises:
- An evaluation method is provided, wherein the pluripotent stem cell is brought into contact with the cell in up to four phases of the development phase.
- evaluation methods include, but are not limited to, screening for substances (peptidic or non-peptidic) similar to or improved by the cytokines described below that can regulate proliferation and Z or differentiation of pluripotent stem cells. Can be used.
- the cytokinin in which two or more species are combined is used as an active ingredient, and the differentiation direction of three or more pluripotent stem cells, for example, bone marrow stromal cells, is determined, and the differentiation of each cell is determined.
- such a set of cytokines can be used in an environment selected from the group consisting of in vitro, exvivo, and invivo.
- Specific examples of such modes of use include a method for screening for cytokines that can also serve as the above-mentioned components and other agents having an equivalent effect, and a direct or indirect regenerative medicine, for example, When transplanting multipotent cells into a living body, the set of cytokines is administered in combination with these cells, or cells collected from the recipient are treated in advance with the set of cytokins.
- a method of expressing a desired differentiation trait, or further, a method of inducing differentiation into a cell or tissue having a specific function and then transplanting it to a living body can be mentioned.
- Preferred embodiments of the screening method include: (A) preparing pluripotent stem cells, particularly pluripotent bone marrow stromal cells derived from a transgenic mouse carrying a temperature-sensitive SV-40 T antigen gene; (B) culturing the cells in a medium capable of growing the cells in the presence of an agent expected to have the ability to differentiate,
- the present invention provides a method for screening a drug capable of differentiating vertebrate cells, which is used as an indicator of the effect of vertebrate cells on the differentiation ability.
- a further preferred embodiment includes a step in which a cytokine selected from the above-mentioned BMP-2, BMP-4, OSM, GDF-5 and TGF- ⁇ 2 is used as a comparative agent.
- Figure 1 (a) is a photograph of a cell showing the result of TBR32-2 differentiation induction, and (b) is a photo of a cell showing the result of TBR10-1 differentiation induction. Is true.
- FIG. 2 is a graph showing the growth curve and development stage of TBR32-2.
- FIG. 3 is a graph showing the growth curve and development stage of TBR 10-1.
- Figure 4 (a) is a photograph replacing the figure showing the results of Western blotting after gel electrophoresis for Examples 1-5 and Comparative Example 1, and (b) for Examples 6-12. .
- FIG. 5 (a) shows the results of Western blotting after gel electrophoresis for Examples 13 to 17 and Comparative Example 2 and (b) for Examples 18 to 24 and Comparative Example 3. This is a photograph replacing the figure.
- FIG. 6 is a photograph showing the result of the WB method in Example 25 (the protein was separated by SDS polyacrylamide gel electrophoresis, and the protein on the separated gel was transferred to a membrane).
- Pluripotent stem cells that can be used in the present invention include ES cells and other agents that are used to generate two or more cells from native cells by any agent, as long as they are in line with the purpose of the present invention.
- Cells that can become functional cells or tissues or organs that express new differentiation traits include cells that can be isolated from bone marrow, brain, liver, and other organs, as well as primary cells of the cells, Any of subcultured cells and immortalized cells may be used.
- stem cells that are mesenchymal cells derived from bone marrow can be used.
- the cell line is an isolated or established cell line and is preliminarily referred to as a bone marrow stromal cell or a mesenchymal stem cell, as long as the object of the present invention is met, the present invention And pluripotent stem cells.
- the cells showing pluripotency described in Non-patent Documents 3 and 4 above are more specific ones. If they have the same characteristics as the cells described in the direction of differentiation and the degree of differentiation, they can be used irrespective of the origin of the cells. For example, mice, rats, guinea pigs, egrets, cats and cats And cells derived from mammals such as goats, pomas, pigs, pests, monkeys, and humans.
- pluripotent stem cells that are convenient for confirming the action and effect of the present invention include temperature-sensitive pluripotent stem cells as described in the aforementioned Non-patent Document 3 or 4.
- SV-40 T antigen gene includes a TBR strain (or strain) established from a transgenic mouse.
- non-patent documents 3 and 4 include specific strains of the above-mentioned TBR strains, for example, TBR 31-2, as including co-author Dr. Masuo Bando, one of the present inventors.
- the 10-1 mag is subdivided from the Tohoku University Institute of Aging Medicine 4-1 without limitation, assuming that it will be used for testing and research purposes.
- the process of developing such pluripotent stem cells can be any of in vitro, exvivo and invivo development processes.
- developmental processes in the former two which include various stages of development (or development phase) of cells in artificial culture of cells. It consists of steps. i) 1st stage of development: Young immature, cells with vigorous elongation of cell processes and not in the growth stage are observed;
- Second developmental stage the stage immediately before the cell number increases, and the cell projections vigorously elongate, immediately before and immediately after contact between adjacent cell projections occurs, and the expansion of the cell volume increases. The largest cells are observed;
- the first stage of the third growth stage From the cells in which logarithmic growth has started and contact between the projections of adjacent cells has occurred more than 900/0, the cell bodies have completely contacted each other. When the state where there is no area for proliferation is set at 100, cells in which the cell density has reached 6006, that is, cells that have reached a confluence of 60% are observed;
- Late fourth stage of development Mature cells that have completed the cell growth stage are observed. See also FIGS. 2 and 3.
- cytokines when cytokines are used, cells are cultured in a medium containing cytodynamics throughout the culture, so that cells are continuously cultured with cytokines from the beginning of culture to the end of culture.
- the cells promote differentiation of the cells in at least two directions in at least four of the development phases i) to vi), and It is important that it be contacted with a suppressive substance (preferably any site force-in). Whether or not such contact is performed depends on whether the medium is changed during the cell growth process, typically during cell culture, for example, every 2 to 4 days. It can be easily done depending on whether or not it is present.
- Up to four phases can consist of, for example, iii) early third developmental stage, iv) late third developmental stage, V) early fourth developmental stage, and vi) late fourth developmental stage, etc.
- i) First stage of development and ii) Second stage of development and iii) Early stage of third stage of development, etc. i) First stage of development and ii) Second stage of development, iv) Late stage of third stage of development And V) the fourth stage of development, etc., and also any one of the six phases.
- any of the cytokins is preferably added to the medium in any of these phases at a concentration capable of inducing differentiation of the growing cells.
- site forces include, but are not limited to, loncostatin M (OSM), bone morphogenetic factor-2 (BMP-2), bone morphogenetic factor-4 (BMP-4), growth differentiation.
- OSM loncostatin M
- BMP-2 bone morphogenetic factor-2
- BMP-4 bone morphogenetic factor-4
- GDF-5 transforming growth factor
- TGF-; S2 transforming growth factor
- Non-Patent Document 2 when the cell line is continuously brought into contact with OSM throughout the whole culture period of TBR 31-2 (the growth period of i) to vi) described above).
- osteogenic differentiation is stimulated (or the induction of differentiation into osteogenic cells is promoted).
- adipogenic differentiation is suppressed (or the induction of differentiation into adipocytes is suppressed).
- the present invention even when OSM is present not only in the development phase of the above-mentioned iv) to vi) but also in only the development phase of the above-mentioned V i), the adipocytes are obtained from Induction of differentiation into cells is suppressed.
- TBR type bone marrow stromal cell
- test substance determines whether the test substance has the ability to induce differentiation of pluripotent stem cells or the action of promoting or suppressing the differentiation of cells
- the test substance was not included, but bone formation factor 1 (BMP-2), bone formation factor 4 (BMP-4), oncostatin M (OSM), growth ⁇
- BMP-2 bone formation factor 1
- BMP-4 bone formation factor 4
- OSM oncostatin M
- GDF-5 'Differentiation' factor
- TGF-j82 transforming growth factor
- cytokines in which two or more kinds of combined cytokins are used as an active ingredient, in the present invention, cytokines can be used simultaneously or individually at different times, places, etc. May be used. In addition, they are used in combination (or as a set), but once such use is made, the use of only one of them is also included in the use of the cytokine set of the present invention.
- the direction of differentiation of 3 or more means that when a pluripotent stem cell is proliferated, three or more new characteristics (differentiation traits) are expressed, or a functional cell or tissue or organ is directed. In short, it means the tendency to reach any functional cell, for example, a smooth muscle cell, a skeletal muscle cell, or the like.
- the degree of each differentiation means, for example, the rate of appearance of each differentiated cell in the presence of serum without the addition of a cytokine, in which the smooth muscle cells or skeletal muscle cells are a control, for example, in an isolated state. It means the appearance rate is slightly higher than that of.
- the direction of such differentiation is, specifically, whether pluripotent stem cells have expressed each differentiation trait such as smooth muscle cells, skeletal muscle cells, cardiomyocytes, endothelial cells or adipocytes, or It means the property leading to each differentiated cell, but is not limited thereto.
- pluripotent stem cells By using a set of two or more cytokins according to the present invention, it is possible to direct pluripotent stem cells to at least three types of each of the above differentiated cells, and to obtain each of the differentiated cells obtained by directing.
- the degree of expression or appearance of the differentiation trait can be controlled. These degrees are observed when multifunctional stem cells are cultured in essentially the same medium, except that the cytoforce is not added, but serum is supplemented.
- each differentiation trait or the appearance of each differentiated cell Compared to the degree of expression of each differentiation trait or the appearance of each differentiated cell, it means that it is promoted (or increased) or suppressed (or decreased) to the extent that it can be detected. This degree is preferably promoted or suppressed by at least 100/0 as compared with the expression of differentiation traits or the appearance of differentiated cells brought about in the presence of the serum. Thus, according to the present invention, the appearance rate or existence ratio of any specific differentiated cells can be selectively increased. Degree and level of expression of the above differentiation traits Examples of detecting the degree of appearance of transformed cells are specific in the Examples described later, but may be according to any technique known in the art.
- the site force-in constituting a combination (or set) of two or more cytokines capable of inducing pluripotent stem cells as described above is in accordance with the purpose of the present invention, for example, as described immediately above, Any cytokine may be used as long as it can selectively increase the appearance rate or the percentage of the specific differentiated cells, and any combination thereof may be used.
- cytokines include, but are not limited to, bone morphogenetic factor-2 (BMP-2), bone morphogenetic factor-4 (BMP-4), oncostin M (OSM), growth ⁇ Differentiation 'factor (GDF-5) and transformin growth factor (TGF-2).
- Examples of such combinations of cytokins include BMP-2 and BMP-4, BMP-2 and OSM, BMP-2 and TGF-) ⁇ 2, ⁇ ⁇ -2 and ⁇ ⁇ -4 and OSM , OSM—BMP 1-4, OSM and TG F—yS 2, OSM and GDF—5, OSM and GDF—5 and BM P—4, OSM and GDF—5 and TGF—) 82 and BMP—4, BMP—2 OSM, 60-5, and 1 ⁇ 1-4 ⁇ 81 ⁇ 1 — 2, 051 ⁇ 1, 60 DO5, and Ding 0-) 82 and BMP-4.
- pluripotent stem cells can be combined with BMP-2 and OSM to differentiate into autonomously beating cardiomyocytes with a high incidence and endothelium with a high incidence.
- BMP-2 or OSM can be applied to such pluripotent stem cells, or BMP-2 and OSM, depending on the purpose. Can be applied.
- culture conditions known per se which can culture animal cells including TBR cell lines, or It can be used after improvement.
- a culture medium is basically supplemented with essential nutrients.
- the essential amino acids L-arginine, L-cystine, L-tyrosine, L-histidine, L-isoleucine, L-mouth isine, L-Lysine, Shi-methionine, L-Fenilalanine, L-Threonine, L-Tributofuan, L-valine, and Shi-glutamine are added to the base, and in some cases, to improve growth, Any medium containing the essential amino acids L-glycine and L-serine can be used.
- Culture temperature and other conditions are equal, such as temperature conditions, that in addition, a buffer agent to be added in order to stabilize the medium p H and jointly with C 0 2 of C 0 2 incubator one in the gas phase as, since the cells require hydrogen carbonate ion, or in general adding bicarbonate Natoriu ⁇ solution of bicarbonate-based, further added such HEPES, from C 0 2 incubator out re taken out
- the pH may be kept stable.
- Antibiotics may be added as appropriate to prevent bacterial contamination. According to the present invention, if necessary, serum and other agents are removed from the medium, and a cytodynamic enzyme is added.
- a form containing cytokins in a medium that does not adversely affect the physiological or cytokine used can be employed. Examples of such media include, but are not limited to, sterile water, saline, and phosphate buffered saline.
- the amount of each cytokine to be used in vitro and ex vivo can be determined by referring to the amount used in Examples described later and, if necessary, conducting further small experiments to determine the optimal amount.
- the dose can usually be from 0.1 ng ZmI to 20 ng ZmI.
- the culture temperature can be between 33 ° C and 37 ° C.
- a method for identifying cells in which differentiation has been induced can be performed by a method known per se, and a specific example may be a method described in Examples described later.
- a regenerative medicine preparation comprising mainly the differentiated cells obtained by the above-described differentiation inducing method
- vascular organs such as the heart, capillaries, arteries and veins
- gastrointestinal organs such as the stomach, esophagus, colon, rectum, small intestine, and large intestine, limb muscles, trachea, and uterus
- Means that can be used for regenerative medicine such as vagina and adipose tissue can be provided.
- a screening system for an agent capable of controlling the degree of each differentiation can be constructed.
- a set of two or more cytokines can be combined with or without the above sterile water, saline, phosphate buffered saline, or in a matrix of substances that do not adversely affect the living body. It may be present and used in a sustained-release form.
- C Materials forming such a matrix include polylactic acid (porous material), collagen, collagen sponge, beta calcium triphosphate, porous hydroxyapatite, polylactic acid Microspheres, polylactic acid-coated gelatin sponge, lactate-ethylene glycol copolymer, agarose, polyvinyl alcohol, alginic acid, agarose heparin, amylopectin, fibrin gel, collagen mini-pellet, ethylene-vinyl acetate copolymer, Lactic acid-glycolic acid copolymer , It may be mentioned chitosan one ethylene glycol copolymer lactic Z glycolic acid, or titanium in Brandt.
- the above-mentioned TBR stromal cell line provided by a part of the present inventors, which can be easily used and manipulated, is used.
- the direction of differentiation of multi-mode stem cells can be determined, and a screening system for an agent capable of controlling the degree of each of the determined directions can be constructed.
- the set of cytokines according to the invention can be used as comparative samples.
- a candidate agent for screening for example, a candidate agent that can be expected to induce differentiation of a TBR strain
- Induce differentiation of polymorphic stem cells in the desired differentiation direction by using the results of culturing in the presence of one or more of the above as standards (or standards) for evaluating the action of the candidate substance
- a useful agent can be efficiently screened.
- the culture conditions that can be used in such a screening system can be based on those described above.
- Monoclonal Ant i -Myosin Light Chain Kinase mouse Ig G 2 b, clone K 36, Product No. M7905
- sm-A ctin sm-A ctin —reacts specifically with the following antibodies (Sima, Monoclonal Anti-or Smooth Muscle Act in, mouse IgG 2a isotype, clone 1 A4, Product No.A 2547) and a secondary antibody labeled with horseradish peroxidase (HRP) UCN to detect it, UCN, Goat, anti-mouse Ig GF (ab ') 2, IgG, catalog No. 55553).
- HRP horseradish peroxidase
- the cells were cultured at 33 ° C. DiI-Ac-LDL, which was not incorporated by washing three times with PBS for 5 minutes, was removed, and a 3% formaldehyde solution was added, and the mixture was allowed to stand at room temperature for 20 minutes to fix. After washing with distilled water, the cells were observed with a fluorescence microscope using a rhodamine filter.
- Adipocytes were observed microscopically in the cultured cell population, and identified by oil red staining. After removing the culture solution from the culture dish and washing the cells attached to the dish bottom three times with PBS for 3 minutes each, fix the cells by leaving them at room temperature for 10 minutes using a 3% formalin solution. It was then replaced with 80% isopropanol for 1 minute and stained with oil red for 10 minutes at 37 ° C. Separate for 1 minute using 60% isopropanol, wash twice for 3 minutes, and finally treat with Maya's hematoxylin solution (Wako) for 10 minutes for nuclear staining and wash with running water for 2 minutes After, microscopically red The stained fat cells were identified. In addition, since these adipocytes develop into cells with distinct fat globules when matured, they could be easily determined as adipocytes without staining.
- Wako Maya's hematoxylin solution
- the centrifuge used was CF7D, manufactured by Hitachi Kiki Co., Ltd.
- RITC medium 2% FBS RITC80-7 medium
- Table 13 the cells were cultured in a 9 Omm plastic dish until a subconfluent state (about 80% confluent) immediately before the cells joined together.
- 1 ml of EDTA 'trypsin solution 0.02 g EDTA' 3Na 100 ml PBS solution, 0.002 g trypsin 100 ml PBS solution
- This washing operation was performed twice quickly before the cells were peeled off.
- Table 2 shows the effect of the addition of cytokines on the differentiation potential of cells.
- RITC medium described in —3, the medium was replaced with a new RITC medium 12 hours after the start of culture. Medium changes were performed every 3 or 4 days. Cytokines were added each time the medium was replaced, and the amount is as shown in Table 14. Table I 4 Amount of cytokine added
- the cell differentiation was determined according to the criteria described below. That is, for smooth muscle cells, micronucleated mononuclear spindle-shaped cells were observed, and myosin light chain kinase protein specifically expressed in smooth muscle cells from a protein mixture extracted from the cultured cell population. Alternatively, the expression of ⁇ -actin protein was determined by the WB method described above. For fat cells, the number of cells (3.8 mm 2 ) forming fat globules was measured, and the average value was determined.
- Examples 1 to 5 We have developed a technology that can be freely changed with 0.7% TC3 1% TC medium and cytokines (Examples 1 to 5). That is, as shown by the expression of actin, a differentiation marker for smooth muscle cells (see Table 16 and FIG. 4 (a)), and the results of adipocyte differentiation (Examples 1 to 5), OSM of Example 1 While it inhibits both smooth muscle and adipocyte differentiation, BMP-2 in Example 2 and BMP-4 in Example 3 both promote differentiation into smooth muscle and adipocytes. On the other hand, GDF-5 in Example 4 is smooth Inhibits muscle differentiation but promotes fat differentiation. Conversely, TG-2 in Example 5 promotes smooth muscle differentiation but suppresses adipocyte differentiation. Table 1 5 ⁇ — ⁇ ⁇ medium
- the direction of differentiation can be freely controlled by the combination of site forces.
- TBR3 1-2 The development stage of TBR3 1-2 will be described below with reference to FIG. First stage of development: young immature cells that have prominently extended cell projections and have not entered the proliferation stage.
- Second developmental stage The stage immediately before the cell number increases, and the cell projections elongate vigorously, immediately before and immediately after contact between adjacent cell projections occurs, and the expansion of the cell volume is maximized. Cells.
- Late third stage of development 60% confluent cells that are growing exponentially logarithmically growing are arresting 100 cells that are moving toward confluent state and are moving toward mature cells cell.
- Late fourth stage of development Mature cells that have completed their growth phase.
- Example 7 On the other hand, in the case of adipocyte differentiation, when OSM is given for 24 consecutive days, the differentiation into adipocytes is suppressed (Example 6), but this inhibitory effect is excluded in the third and fourth development stages. Suppressed at all stages of development (Examples 7, 8, 9, and 12) c However, when OSM is given during the late stage to the early stage of the fourth stage of development, differentiation into adipocytes is promoted ( Examples 10 and 11).
- the suppression of differentiation of stem cells into smooth muscle cells during the development stage occurs in mature cells that have completed the proliferation stage (fourth development stage), and the suppression of differentiation into adipocytes is in mature cells that have completed proliferation. Occurred (late stage 4).
- the promotion of differentiation into smooth muscle cells occurs in cells in the logarithmic growth phase, which are actively growing from cells that are still immature (first to third growth stages), and the promotion of differentiation into fat cells is promoted. Occurs in cells facing mature cells just before arrest (late 3rd stage to late 4th stage). See Table 1 (or Figure 4 (b)) for the results.
- Example 6 0 * Example 7 ++ + 8 Example 8 + 2 Example 9 + + 12 Example 10 ++ 19 Example 11 + + 30 Example 12 + 5 Comparative example 2 + + 16
- cytokines differ depending on the stage of stem cell development, and the ratio of differentiation into smooth muscle cells and adipocytes can be freely controlled by selecting the timing at which they are given. Can be.
- TBR 10-1 Another cell line, TBR 10-1, will be described as an example.
- the established mesenchymal stem cells TBR10-1 used in Examples 13 to 24 (Examples 13 to 17 are reference examples) and Comparative Example 2 are described in the aforementioned Non-Patent Document 3 or 4. And was shown to differentiate into smooth muscle cells (FEBS Letters 481: 193-196, 2000).
- the present inventors studied the culture conditions in detail using the same strain, a MEM medium shown in Table 15 and a high serum concentration of fetal bovine serum (10% FBS), and found that in addition to smooth muscle cells, They found that they differentiate into endothelial cells, and revealed that they are pluripotent stem cells with the ability to differentiate into three phenotypes (see Figure 1-b and Table 18). In addition, the ratio of these differentiations was reduced to 0.70 / 0 FB We have developed a technology that can be freely changed using S RITC medium and cytokines.
- myosin light chain kinase protein a differentiation marker for smooth muscle cells, and differentiation into ossified cells (Examples 13 to 17; see Table 18 and FIG. 5 (a)).
- the differentiation of this strain into smooth muscle is inhibited by OSM and TGF-2.
- BMP-2, BMP-4 and GDF-5 they promote smooth muscle differentiation.
- differentiation into gliding cells is promoted in the presence of OSM and GDF-5.
- OSM induces the differentiation of smooth muscle and ossified cells in opposite directions, which means that bone differentiation and smooth muscle differentiation are controlled in a close relationship. There may be multiple stages of stem cell differentiation that differ in their ability to differentiate.
- the development process of the stem cells can be roughly divided into four stages as shown in FIG.
- Example 18 As shown in Tables 8 to 24 (see Table 9 below and Fig. 5 (b)), when cytokines were given at each stage of the growth process of the TBR10-1 strain, they were given continuously. It shows a completely different differentiation direction. In other words, if BMP-2 is given continuously, smooth muscle differentiation proceeds predominantly.
- the effect of BM P-2 to promote smooth muscle differentiation is not necessarily required to be continuously exerted, and is predominantly observed from the third growth stage where cell proliferation is active to the fourth growth stage where mature cells are present.
- the effect of BMP-2 on promoting smooth muscle differentiation is not observed (eg, t
- FIG. 5B shows the level of expression of a smooth muscle differentiation marker, sm-Actin, as detected by chemical light emission.
- ⁇ weak but clear expression
- 1 no expression.
- a stem cell culture system was prepared using a microwell or the like.
- new drugs such as differentiation inducers and inhibitors
- cell line TBR52 (see Non-Patent Documents 3 and 4 described above) was used, and an ⁇ -MEM medium having a composition shown in Table 10 below was used as a medium.
- Identification was performed by a combination of identification methods such as stamp lotting (WB), immunostaining, and reverse transcription polymerase chain reaction (RT-PCR) described below.
- WB stamp lotting
- RT-PCR reverse transcription polymerase chain reaction
- sm-MLCK myosin light chain kinase
- HRP Peroxidase
- ICN Peroxidase
- ICN Peroxidase
- PI Providium Iodide
- PBS phosphoric acid buffer
- the primary antibody (1.5 ml) was replaced with 1.5 ml of 5% skin milk-PBS used in the previous 30 minutes treatment, and reacted at 4 ° C. for about 14 hours. Thereafter, the plate was washed three times for 5 minutes using PBS.
- Cardiomyocytes are microscopically mononuclear and have a rod-shaped morphology in a cell population cultured in a 35 mm plastic dish, and autonomously cyclically contract (pulsation). )
- connexin 43 protein was confirmed using the WB method and following the same procedure as described above.
- the primary antibody as an anti-connexin 43 (Ghemicon, product No. MAB 3068) using, as the secondary antibody anti-mouse secondary antibody (I CN manufactured, Goat, anti-mouse I g GF
- RNA extraction reagent Isogen (Futan Gene) It was recovered by the phenol-chloroform method. Then 2.5 Ug of recovered! ⁇ One RNA was prepared by the Oligo (d T) method according to the manual attached to the RT-PCR kit to prepare cDNA, and adjusted to 10 times with DEPC water.
- Examples 26 to 30 Control of differentiation by cytokinin
- Cytokines to be tested were added to RITC 80-7 medium having the composition shown in Table 11 below to confirm the effect of each cytokine.
- Table 1 R I TC 80-7 medium Item Addition amount Nomiri Q Remarks
- FBS ⁇ fetal serum 20 ml Gibco 'BRL lot No. A0247611
- TBR52 was cultured in a 9 Omm plastic dish until a subconfluent state (about 80% confluent) immediately before the cells came into contact with each other.
- 1 ml of EDTA 'trypsin solution 0.02 g EDTA. 3 Na 100 ml PBS solution, 0.002 g trypsin 10 OmI PBS solution
- This washing operation was performed twice quickly before the cells were detached.
- the trypsin solution was added, and immediately after the cells spread evenly on the cell surface, the trypsin solution was quickly sucked and removed with a pasteur pit. Then, after confirming that the cells were completely peeled off with a microscope, the 1 MEM medium shown in Table 10 was added, and the cell concentration was adjusted to 310 ⁇ 5 ZmI. 1.5 ml of this cell suspension was added to a 35 mm plastic dish. At this time, a 25 mm Poly-Llysine coat cover glass (4925-040, Asahi Kunograss, Tokyo) was pre-laid on the 35 mm plastic dish. One hour after the addition of one MEM medium, the medium was replaced with a new one MEM medium. The medium was changed every 2 or 3 days for 30 days.
- one MEM medium was used for cell culture, and a new Of-MEM medium was replaced 24 hours after the addition of one MEM medium. Medium changes were performed every 2 or 3 days for 30 days. Cytokines were added each time the medium was replaced, and the amount was as shown in Table 12 below.
- the cell differentiation was determined according to the criteria described below. That is, for the smooth muscle, cells showing a spindle shape (spindle shape) in a mononuclear state were observed microscopically, the area occupied by a single area was visually measured, and the average value was expressed in%. Furthermore, the expression of myosin light chain kinase protein specifically expressed in smooth muscle cells from the protein mixture extracted from the cultured cell population was determined by the WB method described above.
- the number of elongated blended cells pulsating periodically was counted, and the average was determined.
- endothelial cells cells showing a cobblestone-like morphology were observed microscopically, the area occupying a single area was visually measured, and the average value was expressed in%.
- fat cells the number of cells per unit forming fat globules was measured, and the average value was determined. The results are summarized in Tables 13 and 14.
- Example 27 when OSM was added to the medium, differentiation into cardiomyocytes was It was suppressed by 1 Z 2. In addition, differentiation into skeletal muscle and adipocytes was almost completely suppressed, and microscopic morphological observation did not confirm multinucleated myotube-like skeletal muscle and adipocytes containing fat globules. . This inhibitory effect on skeletal muscle cell differentiation could be confirmed by analysis of expression of fast-muscle skeletal muscle myosin heavy chain II protein by the WB method. Adipocyte differentiation was also suppressed by the addition of OSM. However, it had little effect on smooth muscle and endothelial cell differentiation.
- Example 28 the addition of GDF5 to the medium slightly promoted cardiomyocyte and adipocyte differentiation. However, the addition of GDF5 suppressed the expression of fast skeletal muscle myosin heavy chain II protein specific to skeletal muscle cells. The differentiation of smooth muscle and endothelial cells was not affected by the addition of GDF5.
- Example 29 when TGF-2 was added to the medium, differentiation into smooth muscle, myocardium and adipocytes was suppressed, but differentiation into endothelial cells was not affected. Differentiation into skeletal muscle cells could not be observed at all from morphological observation with a microscope, but cells showing the morphology of skeletal muscle cells could be observed. Expression of chain II protein was enhanced by the WB method. However, while a clear explanation is not currently available for the expression of this protein, the key to clarifying the relationship between morphological cell fusion that occurs during the process of differentiation into skeletal muscle cells and the early induction phase of skeletal muscle differentiation. It might be.
- Example 30 when BMP 4 was added to the medium, the differentiation to adipocytes was promoted at a ratio of 10 times or more, as is clear from the comparative example in which BMP 4 was not added, but to smooth muscle and skeletal muscle cells. Was found to be suppressed by the WB analysis. In addition, the number of autonomous beating cells, which is a characteristic of cardiomyocytes, was small, and differentiation into cardiomyocytes was suppressed.
- the addition of BMP4 has little effect on endothelial cell differentiation and these results indicate that pluripotent stem cells can be given various cytokines to predominantly amplify the direction of differentiation into specific cells. Can be reduced, and by adding additional cytokines Thus, it is shown that unnecessary cells can be suppressed, and only the target cells can be amplified in a dominant manner.
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EP04722990A EP1619242A4 (en) | 2003-03-25 | 2004-03-24 | CONTROL OF INDUCTION OF DIFFERENTIATION OF STEM CELLS AND DIFFERENTIATION CAPACITY |
CA002520445A CA2520445A1 (en) | 2003-03-25 | 2004-03-24 | Induction of differentiation of stem cells, and control of differentiation potency of stem cells |
US10/550,436 US20070104679A1 (en) | 2003-03-25 | 2004-03-24 | Induction of differentiation of stem cells, and control of differentiation potency of stem cells |
CN2004800143245A CN1795265B (zh) | 2003-03-25 | 2004-03-24 | 干细胞的分化诱导及分化能力的控制 |
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JP2003095242A JP2004298087A (ja) | 2003-03-31 | 2003-03-31 | 幹細胞の分化誘導 |
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Cited By (4)
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WO2009035612A2 (en) * | 2007-09-11 | 2009-03-19 | The University Of Miami | Multilineage-inducible cells and uses thereof |
CN101835890B (zh) * | 2008-06-27 | 2013-07-24 | 国立大学法人京都大学 | 有效建立诱导的多能干细胞的方法 |
CN101864392B (zh) * | 2005-12-13 | 2016-03-23 | 国立大学法人京都大学 | 核重新编程因子 |
CN110938669A (zh) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | 无机磷酸盐刺激瓣膜***成骨分化的验证方法 |
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WO2007140590A1 (en) * | 2006-06-02 | 2007-12-13 | Mcmaster University | Methods and compositions for inducing bone synthesis |
JP6111510B1 (ja) | 2016-05-02 | 2017-04-12 | インテグリカルチャー株式会社 | 成長誘導システム、成長誘導制御装置、成長誘導制御方法、および、成長誘導制御プログラム |
US11484879B2 (en) | 2016-05-02 | 2022-11-01 | Integriculture Inc. | System for aging induction, control device for aging induction, method for controlling aging induction, and program for controlling aging induction |
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US5733541A (en) * | 1995-04-21 | 1998-03-31 | The Regent Of The University Of Michigan | Hematopoietic cells: compositions and methods |
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US20030027330A1 (en) * | 2001-04-02 | 2003-02-06 | Robert Lanza | Method for facilitating the production of differentiated cell types and tissues from embryonic and adult pluripotent and multipotent cells |
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WO2000030635A1 (en) * | 1998-11-19 | 2000-06-02 | The University Of Western Ontario | Composition and use of rar antagonists for promoting chondrogenesis |
WO2003066856A1 (en) * | 2002-02-06 | 2003-08-14 | Sanbio, Inc. | METHOD OF DIFFERENTIATING/INDUCING BONE MARROW INTERSTITIAL CELLS INTO NERVE CELLS AND SKELETON MUSCLE CELLS BY TRANSFERRING Notch GENE |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101864392B (zh) * | 2005-12-13 | 2016-03-23 | 国立大学法人京都大学 | 核重新编程因子 |
WO2009035612A2 (en) * | 2007-09-11 | 2009-03-19 | The University Of Miami | Multilineage-inducible cells and uses thereof |
WO2009035612A3 (en) * | 2007-09-11 | 2009-05-22 | Univ Miami | Multilineage-inducible cells and uses thereof |
CN101835890B (zh) * | 2008-06-27 | 2013-07-24 | 国立大学法人京都大学 | 有效建立诱导的多能干细胞的方法 |
CN110938669A (zh) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | 无机磷酸盐刺激瓣膜***成骨分化的验证方法 |
CN110938669B (zh) * | 2019-12-20 | 2023-03-28 | 南昌大学第二附属医院 | 无机磷酸盐刺激瓣膜***成骨分化的验证方法 |
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US20070104679A1 (en) | 2007-05-10 |
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