WO2010114136A1 - 多能性幹細胞及び心筋細胞以外の分化細胞に対する細胞死誘導方法 - Google Patents
多能性幹細胞及び心筋細胞以外の分化細胞に対する細胞死誘導方法 Download PDFInfo
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Definitions
- the present invention relates to pluripotent stem cells and pluripotent stem cell-derived cardiomyocytes as a means for purification of cardiomyocytes when differentiating cardiomyocytes from pluripotent stem cells such as embryonic stem cells and inducible pluripotent stem cells
- the present invention relates to a method for inducing cell death in other differentiated cells.
- Known methods for obtaining cardiomyocytes include a method in which stem cells (for example, embryonic stem cells and various adult stem cells) are differentiated and used from a fetus.
- stem cells for example, embryonic stem cells and various adult stem cells
- a factor that suppresses differentiation feeder cell, leukemia inhibitory factor (LIF), etc.
- LIF leukemia inhibitory factor
- a factor (feeder) that suppresses differentiation when human pluripotent stem cells are used.
- Cells, basic fibroblast growth factor (bFGF), transforming growth factor (TGF), etc.) are removed from the culture medium, thereby forming a cell mass (embryoid body), thereby actively The ability to induce differentiation is known as a method for differentiating stem cells.
- the differentiation mode from stem cells to cardiomyocytes outside the body follows a part of the physiological developmental stage in vivo, especially with respect to the physiological development occurring in fertilized egg cells and in vitro differentiation with respect to the early developmental events. There is much in common with this style.
- the lineage of differentiation into cardiomyocytes outside the body is the same as the physiological development, after stem cells first differentiate into undifferentiated mesoderm cells, and some of them differentiate into planned cardiomyocytes (pre-cardiac mesoderm) Differentiate into cardiomyocytes.
- pluripotent stem cells are cells that have the ability to differentiate into all the cells that make up the organ, it is technically difficult to differentiate into only cardiomyocytes. On the other hand, since it is very difficult to simultaneously induce all pluripotent stem cells to the differentiation stage, undifferentiated stem cells often remain in the embryoid body.
- Non-patent Document 1 a method in which some marker gene (eg, GFP) is introduced into a stem cell gene (genome) in advance (Non-patent Document 1).
- GFP marker gene
- the present invention induces cell death in differentiated cells other than pluripotent stem cells such as embryonic stem cells and inducible pluripotent stem cells and cardiomyocytes derived from pluripotent stem cells without genetic modification, It is an object to develop a method that does not induce cell death for cardiomyocytes. Another object of the present invention is to develop a method for producing safe and high-purity cardiomyocytes from pluripotent stem cells without the risk of teratoma by such a method.
- the present inventors have added a substance that has not been confirmed to have physical toxicity or cell death-inducing action to the culture conditions of pluripotent stem cells or non-cardiomyocytes.
- the above problem can be solved without genetic modification by constructing a method for inducing cell death to differentiated cells other than cardiomyocytes very efficiently. Since this method does not induce cell death in cardiomyocytes, it is also an efficient method for purifying cardiomyocytes.
- the present invention has a osmotic pressure of 370 mOsm / kg or more for a cell population containing pluripotent stem cells, differentiated cells other than pluripotent stem cells-derived cardiomyocytes, and pluripotent stem cells-derived cardiomyocytes. It has been clarified that the above-mentioned problems can be solved by providing a method of inducing differentiated cells other than pluripotent stem cells and cardiomyocytes derived from pluripotent stem cells by culturing in a hypertonic solution. Specifically, the present invention specifically relates to the following matters.
- the hypertonic solution having an osmotic pressure of 370 mOsm / kg or more contains 0.1 to 1 M carbohydrate.
- the saccharide is a sugar alcohol, a saccharide, or a betaine.
- the sugar alcohol, saccharide, or betaine is selected from the group consisting of mannitol, sorbitol, xylitol, glycerol, sucrose, glucose, and trimethylglycine.
- a cell population comprising a pluripotent stem cell, a differentiated cell other than a pluripotent stem cell-derived cardiomyocyte, and a pluripotent stem cell-derived cardiomyocyte by the method of the present invention, an undifferentiated pluripotency in the cell population Sexual stem cells and non-cardiomyocytes can be removed efficiently, and at the same time, only cardiomyocytes can survive, so that cardiomyocytes can be efficiently concentrated and purified.
- FIG. 1 shows immunostaining of embryonic stem cell (ES cell) -derived cardiomyocytes and remaining undifferentiated embryonic stem cells.
- FIG. 2 shows mannitol treatment in a culture system in which embryonic stem cell-derived cardiomyocytes and remaining embryonic stem cells are mixed.
- FIG. 3 shows immunostaining for Nkx2.5 and Oct-3 / 4 in cells after mannitol treatment.
- FIG. 4 shows the cell death-inducing action of mannitol 0.45M on mouse embryonic stem cells.
- FIG. 5 shows the effect of mannitol on marmoset embryonic stem cells. Life / death was determined using a mitochondrial membrane potential sensitive dye TMRM.
- FIG. 6 shows the effect of mannitol on marmoset embryonic stem cells.
- FIG. 7 shows the effect of mannitol on human embryonic stem cells (FACS analysis).
- FIG. 8 shows the effect (immediately after) of mannitol on human embryonic stem cells.
- FIG. 9 shows the effect of mannitol on human embryonic stem cells (after 5 hours).
- FIG. 10 shows mannitol treatment of human embryonic stem cell embryoid bodies.
- FIG. 11 shows the death of Oct-3 / 4 positive cells by mannitol treatment of human embryonic stem cell embryoid bodies.
- FIG. 12 shows the action (FACS analysis) of mannitol on human-induced pluripotent stem cells (iPS cells).
- FIG. 13 shows cell death-inducing action on human embryonic stem cells in various carbohydrates other than mannitol.
- FIG. 13 shows cell death-inducing action on human embryonic stem cells in various carbohydrates other than mannitol.
- FIG. 14 shows the action of glycerol on human embryonic stem cell-derived cardiomyocytes / non-cardiomyocytes.
- FIG. 15 shows the state of cells treated for 48 hours or 72 hours with a culture solution containing 0.2 M mannitol. An enlarged image of each colony is shown in the lower row, and representative cell morphology is illustrated.
- the present inventors used a hypertonic solution in which a high concentration of mannitol was dissolved in a mixed cell system containing embryonic stem cells, cardiomyocytes, and non-cardiomyocytes. We found the fact that non-cardiomyocytes are prone to cell death. With regard to this finding, as a result of examining the relationship between mannitol concentration and exposure time to mannitol and cell death of embryonic stem cells and non-cardiomyocytes in more detail, long-term exposure was effective at low concentrations of mannitol and embryonic stem cells and Cell death was induced in non-cardiomyocytes, and it was found that 1M, which is a nearly saturated concentration, induces cell death of embryonic stem cells in a short time.
- the method of the present invention for culturing under such conditions is not only an efficient method for removing embryonic stem cells and non-cardiomyocytes, but also an efficient method for enriching and purifying cardiomyocytes.
- the present invention permeates a cell population containing undifferentiated pluripotent stem cells, differentiated cells other than pluripotent stem cell-derived cardiomyocytes, and pluripotent stem cell-derived cardiomyocytes at a rate of 370 mOsm / kg or more.
- a method for inducing differentiated cells other than pluripotent stem cells and cardiomyocytes derived from pluripotent stem cells by culturing in a hypertonic solution having pressure is provided.
- embryoid bodies 3 to 6 days after the start of differentiation induction contain mesoderm or planned cardiomyocytes. Cardiomyocytes appear on the 7th day after the start of differentiation (10th day for human embryonic stem cells).
- embryoid bodies include undifferentiated cells, endothelial epithelial cells, nerve cells, and the like. Examining in more detail the cells that make up the embryoid body, 70-80% of the cell population that makes up the embryoid body is differentiated cells other than cardiomyocytes, among which are undifferentiated embryonic stem cells. May be included. These contaminating cells proliferate vigorously, and their proportion increases with time.
- the inventors of the present invention exposed embryonic bodies having such a cell structure to a suitable range of high osmotic pressure in a culture solution, thereby allowing embryos other than embryonic stem cells and cardiomyocytes remaining in the embryoid bodies to be exposed. It was found that differentiated cells cause cell death. The present inventors have also found that, even when cultured under this condition, cardiomyocytes do not exhibit cell death, but are replaced with a physiologically osmotic pressure medium to resume autonomous pulsation. It was.
- the cell source may be a cell population derived from any source as long as it is a cell population including cardiomyocytes.
- pluripotent stem cells embryonic stem cells (ES cells)
- Biological stem cells Biological stem cells
- inducible pluripotent stem cells iPS cells
- cell populations containing cardiomyocytes differentiated using known cardiomyocyte induction conditions fetal tissue-derived cell populations
- the method of the present invention can be carried out.
- pluripotent stem cells and differentiated cells other than cardiomyocytes derived from pluripotent stem cells May be included.
- the method of the present invention is characterized by culturing such a cell population in a hypertonic solution having an osmotic pressure of 370 mOsm / kg or more.
- the osmotic pressure used in the method of the present invention induces cell death in cells other than cardiomyocytes (pluripotent stem cells and differentiated cells other than cardiomyocytes), but does not induce cell death in cardiomyocytes. Pressure.
- Such osmotic pressure is 370 mOsm / kg or more, preferably 370 mOsm / kg to 1600 mOsm / kg, more preferably 370 mOsm / kg to 1000 mOsm / kg, more preferably 480 mOsm / kg to 1000 mOsm / kg, most preferably 700 mOsm / kg. kg to 1000 mOsm / kg.
- these values are very high compared to the in vitro osmotic pressure of about 200 to 300 mOsm / kg (also referred to as normal osmotic pressure or physiological osmotic pressure), When cultured under these conditions, cells other than cardiomyocytes die.
- the method of the present invention preferably comprises culturing in such a hypertonic solution for 2 hours or more. Is 2 to 72 hours, more preferably 2 to 48 hours, still more preferably 2 to 24 hours, and most preferably 4 to 12 hours.
- conditions under which cell death can be induced only in undifferentiated pluripotent stem cells including human ES cells and iPS cells, or differentiated cells other than cardiomyocytes are determined by the degree of osmotic pressure of the hypertonic solution. And the time of exposure of the cells to the hypertonic solution. That is, regardless of the cell type used to induce cardiomyocytes, the higher the osmotic pressure of the hypertonic solution, the shorter the exposure time of the cells to the hypertonic solution, while the lower the osmotic pressure of the hypertonic solution. If set, it will be necessary to increase the exposure time of the cells to the hypertonic solution.
- cells other than cardiomyocytes are led to cell death (ie, cell death is induced or cell death is induced). Give a signal).
- the cells thus led to cell death cause cell death in the hypertonic solution, or cell death after being returned from the hypertonic solution to a normal culture solution.
- the cell death in the method of the present invention is induced by exposing the cells to physiological stress, the surviving cells are not genetically damaged, and other than the target cells. It is much better than inducing cell death by physical conditions (radiation stress, oxidative stress, etc.) or chemical conditions (compound stress) that directly damage the gene in that it can induce cell death only in cells . This is because the cells that survived the physiological stress survived the normal cell function again by returning to the culture conditions that removed the physiological stress to induce the cell death. Function can be demonstrated. This feature is very easy to use in the field of regenerative medicine performed by transplanting a tissue prepared ex vivo or cells constituting the tissue into the body.
- the term “hypertonic solution having an osmotic pressure of 370 mOsm / kg or more” means a hypertonic solution having only osmotic pressure of 370 mOsm / kg or more without affecting cell metabolism.
- An example is one prepared by adding a saccharide (carbohydrate) to a liquid.
- carbohydrates that can be used in the present invention include carbohydrates that can increase the osmotic pressure of a culture solution without affecting cell metabolism, that is, can be added to the culture solution of the present invention.
- carbohydrates include, but are not limited to, saccharides (monosaccharides, oligosaccharides, polysaccharides), glycosaminoglycans, aminoglycosides, sugar alcohols, and betaines. More specifically, the substances listed in Table 1 can be mentioned.
- the effects of the present invention are also recognized when using substances that are physiologically involved in osmotic pressure regulation in adults, such as sorbitol (sugar alcohols) and trimethylglycine (betaines).
- sorbitol sucgar alcohols
- betaines trimethylglycine
- Betaine, taurine, inositol, glycerophosphocholine, etc. can be used as pressure regulators (organic osmolites).
- the hypertonic solution When preparing a hypertonic solution having an osmotic pressure of 370 mOsm / kg or more by adding a carbohydrate to the culture solution, the hypertonic solution contains 0.1 to 1 M (mol / L) of carbohydrate, preferably 0 It is characterized by containing 1 to 0.6 M carbohydrate.
- carbohydrate concentration (mol / L) and osmotic pressure (mOsm / kg) can be approximated as a substantially linear relationship, and a hypertonic solution containing 0.1 M carbohydrate is about 370 mOsm / kg.
- a hypertonic solution having an osmotic pressure of about 1300 to 1600 mOsm / kg corresponds to a hypertonic solution having an osmotic pressure of 1M.
- the hypertonic solution contains 0.1 to 0.6 M glycerol, preferably 0.1 to 0.5 M glycerol, and the hypertonic solution is characterized in that The culture is performed for 10 hours or longer, for example, 10 to 24 hours, preferably 10 to 18 hours.
- the following table shows typical carbohydrates in the relationship between the carbohydrate concentration and the osmotic pressure when a hypertonic solution is prepared using each carbohydrate.
- a cell population is cultured in a hypertonic solution having an osmotic pressure of 370 mOsm / kg or more, cell death is induced in pluripotent stem cells or non-cardiomyocytes, and then normal osmotic pressure (that is, It is preferable to return to the culture solution having an osmotic pressure of 200 to 300 mOsm / kg) and further culture.
- cardiomyocytes do not cause cell death but may temporarily stop pulsation. However, even in such a case, by returning to the normal osmotic pressure of the culture solution, pulsation can be started again to function as a cardiomyocyte.
- the method of the present invention shows similar results in all the cells examined in the examples (that is, mouse-derived embryonic stem cells, marmoset-derived embryonic stem cells, human-derived embryonic stem cells, human-derived iPS cells). I was able to. Since the method of the present invention did not depend on animal species, it was shown that the method of the present invention can be applied to cells derived from all mammals from mice to humans. Furthermore, since the object could be achieved using the method of the present invention in both embryonic stem cells and inducible pluripotent stem cells, stem cells in which pluripotent stem cells have not been genetically manipulated (eg, embryonic stem cells). It was also shown that the present invention can be applied regardless of whether it is a stem cell) or a genetically engineered stem cell (eg, iPS cell).
- embryonic stem cells or iPS cells are differentiated from a culture medium for differentiation (for example, ⁇ -MEM (Minimum Essential Medium) (SIGMA), 10% FBS (EQUITEC BIO), 100 units / ml penicillin, 50 ⁇ g / ml streptomycin (GIBCO).
- a culture medium for differentiation for example, ⁇ -MEM (Minimum Essential Medium) (SIGMA), 10% FBS (EQUITEC BIO), 100 units / ml penicillin, 50 ⁇ g / ml streptomycin (GIBCO).
- a culture medium for differentiation for example, ⁇ -MEM (Minimum Essential Medium) (SIGMA), 10% FBS (EQUITEC BIO), 100 units / ml penicillin, 50 ⁇ g / ml streptomycin (GIBCO).
- suspension culture is performed by the hanging drop method or the like to induce appropriate myocardial differentiation to form an embryoid body containing cardiomyocytes.
- cardiomyocytes After culturing with exposure to hypertonic solution, only the cardiomyocytes selectively survive in the culturing conditions by replacing the treated cells with normal osmotic pressure and further culturing. be able to. If necessary, non-myocardial cells that have undergone cell death are actively removed by washing the cells using any or a combination of methods such as cell dispersal by enzyme treatment, medium exchange, and centrifugation. It can also be removed.
- Cell death can be induced in 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 100% of the cells.
- Example 1 Immunostaining of embryonic stem cell-derived cardiomyocytes and remaining undifferentiated embryonic stem cells
- cell clusters embryonic stem cell-derived cardiomyocytes
- cell clusters embryonic stem cells
- embryonic stem cell-derived cardiomyocytes embryonic stem cell-derived cardiomyocytes
- Mouse embryonic stem cells (cell line name EB3, Nat Genet 2000; 24: 372-376) were kindly provided by Dr. Hitoshi Niwa of RIKEN.
- the mouse embryonic stem cells were obtained by using an existing method (Bader A, et al., Differentiation 2001, 68, p31-43) (that is, medium [ ⁇ -MEM (Minimum Essential Medium) (SIGMA), 10% FBS (EQUITEC BIO ), 100 units / ml penicillin, 50 ⁇ g / ml streptomycin (GIBCO)], and 75 embryonic stem cells per EB as a cell mass by the hanging drop method for a total of 7 days) After differentiation into lumps, embryoid bodies were adhered to the culture dish and cultured at 37 ° C. under 5% CO 2 for 3-5 days.
- the obtained embryoid body was fixed with 4% paraformaldehyde, and the cell membrane was semi-solubilized with 0.1% Triton X100.
- An antibody against Nkx2.5 (goat anti-Nkx2.5 antibody (Santacruz No. N-19)), which is blocked by 4% BSA solution and expressed in the most premature cardiomyocytes, and mouse embryonic stem cells 1 each of antibodies against the transcription factor Oct-3 / 4 known to play an important role in maintaining undifferentiated potential (mouse anti-Oct-3 / 4 monoclonal antibody, BD Transduction Laboratories No. 0847720)
- the secondary antibody was diluted 100 times with a blocking solution and then allowed to permeate at 4 ° C. for 12 hours.
- a donkey anti-goat antibody (Molecular Probe) labeled with Alexa Flow488 and a rabbit anti-mouse antibody (DAKO) labeled with TRITC were both diluted 1/200.
- DAPI Molecular probe
- Example 2 Carbohydrate (sugar alcohols) treatment in a culture system in which embryonic stem cell-derived cardiomyocytes and remaining embryonic stem cells are mixed
- a cell mass containing cardiomyocytes formed from embryonic stem cells ( The purpose of this study was to confirm the cultured state of cardiomyocytes and the cultured state of other cells after sugar (sugar alcohol) treatment on the embryoid body).
- Mouse-derived embryonic stem cells were formed into embryoid bodies by the method described in Example 1 and differentiated to a stage containing planned cardiomyocytes (anterior heart mesoderm).
- the obtained embryoid body was partly dispersed by carefully digesting enzyme treatment (trypsin, collagenase) so as not to cause cell damage, and re-adherent culture was performed.
- trypsin, collagenase trypsin, collagenase
- FIG. 3 The results of immunostaining for Nkx2.5 and Oct-3 / 4 by the same method as in Example 1 for the culture shown in FIG. 2 are shown in FIG.
- FIG. 3 regarding the cells surrounded by a square in the phase contrast image shown in the upper left, lower left: Nkx2.5 antibody staining (green), upper right: DAPI staining (blue), and lower right: Nkx2.5 antibody A superimposed image of staining (green) and DAPI staining (blue) is shown.
- 98% or more of the obtained cells are Nkx2.5 positive cardiomyocytes (see lower left and lower right in FIG. 3), and it is clear that there are no Oct-3 / 4 positive undifferentiated cells. became.
- Example 3 Cell death-inducing action of carbohydrates (sugar alcohols) on mouse embryonic stem cells
- carbohydrates sucgar alcohols
- the mitochondrial membrane potential is lost in dead cells, it is derived from this reagent when stained with TMRM (Molecular Probes), a mitochondrial membrane potential sensitive reagent that detects the membrane potential as an indicator of survival and emits fluorescence.
- TMRM Molecular Probes
- the fluorescence signal is high in live cells and low in dead cells.
- embryonic stem cells cultured by the method described in Example 1 were treated with 0 h (Pre-) in an ⁇ -MEM culture medium containing 0.45 M mannitol (corresponding to about 720 mOsm / kg) and 1 ⁇ M TMRM. )
- the collected embryonic stem cells were washed and subjected to FACS analysis, and the survival state of the stem cells was confirmed based on the fluorescence intensity of the mitochondrial membrane potential sensitive reagent TMRM. Indicated.
- the cells existing above the boundary line indicated in the dot plot are live cells, and from the boundary line indicated in the dot plot. The lower cells are dead cells.
- Example 4 Cell death-inducing action of carbohydrates (sugar alcohols) on marmoset embryonic stem cells
- carbohydrates sucgar alcohols
- Marmoset embryonic stem cells were obtained from the Institute for Experimental Animal Research (Stem Cells. 2005 Oct; 23 (9): 1304-13). The basic culture method was also in accordance with this document. That is, the terminal differentiation maintenance culture of marmoset embryonic stem cells was performed using mouse embryonic fibroblasts (MEFs) that were proliferated and inactivated by mitomycin C treatment.
- MEFs mouse embryonic fibroblasts
- KO-DMEM As the culture solution, KO-DMEM (GIBCO), 20% KO-SERUM (GIBCO), 1.6 mM L-glutamine, 0.1 mM non-essential amino acid (MEM), 0.2 mM ⁇ -mercaptoethanol (2-ME; Sigma) ), 100 IU / ml penicillin, 100 ⁇ g / ml streptomycin sulfate, and 8 ng / ml recombinant human leukemia inhibitory factor (LIF; Chemicon), recombinant human basic fibroblast growth factor (bFGF; Peprotech).
- LIF human leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- a sample of cells was prepared by treating the stained cells for 2 hours in an ⁇ -MEM culture medium containing 0.45M mannitol (corresponding to about 720 mOsm / kg), and then treated for 2 hours in an ⁇ -MEM culture medium not containing mannitol.
- a cell sample was prepared as a control. Prior to the FACS analysis, the cells adhered to each other to form a cell mass in the control, so that the TE treatment was performed again to obtain disjoint cells. Thereafter, cells treated with mannitol for 2 hours and control cells were analyzed for the presence or absence of mitochondrial membrane potential by FACS based on the fluorescence intensity of TMRM, and the survival state of the stem cells was confirmed, and shown in FIG. As a result, it was found that almost all cells lost the membrane potential by treatment with mannitol for 2 hours as compared with the control (FIG. 5 (a)) (FIG. 5 (b)).
- Example 5 Cell death-inducing action of carbohydrates (sugar alcohols) on human embryonic stem cells
- carbohydrates sucgar alcohols
- Human embryonic stem cells Obtained from Stem Cell Medical Research Center (ES Cell Center by National BioResource Project) attached to Research Institute for Regenerative Medicine, Kyoto University (reference: Suemori H, et al., Biochem. Biophys. Res. Commun., Vol. 345). 2006, p. 926-932).
- the basic culture method was also in accordance with the above document. That is, undifferentiated maintenance culture of human embryonic stem cells was performed using mouse embryonic fibroblasts (MEFs) that were proliferated and inactivated by mitomycin C treatment.
- MEFs mouse embryonic fibroblasts
- F12 / DMEM (1: 1) SIGMA, product number D6421
- 20% KO-SERUM GAC
- 1.6 mM L-glutamine 0.1 mM non-essential amino acid (MEM)
- 0.1 mM ⁇ -Mercaptoethanol (2-ME; Sigma)
- 100 IU / ml penicillin 100 ⁇ g / ml streptomycin sulfate
- bFGF human basic fibroblast growth factor
- a sample of cells was prepared by treating the stained cells in an ⁇ -MEM culture solution containing 0.45M mannitol (corresponding to about 720 mOsm / kg) for 2, 3 or 4 hours, and an ⁇ -MEM culture solution containing no mannitol was prepared.
- Samples of cells treated for 2 hours in were prepared as controls. Prior to FACS analysis, cells adhered to each other to form a cell mass in the control, and thus the same trypsin and EDTA treatment was performed again on both to disperse the cells.
- Cells treated with mannitol for 2, 3 or 4 hours and control cells were analyzed for the presence or absence of mitochondrial membrane potential by FACS based on the fluorescence intensity of TMRM, and the survival state of stem cells was confirmed. Indicated. As a result, it was found that almost all cells lost the membrane potential by treatment with mannitol for 2 hours as compared with the control (FIG. 7 (a)) (FIG. 7 (b)).
- TMRM-stained photographs of the cells treated with mannitol for 2 hours and control cells were taken (FIG. 8).
- Cells that were not treated with mannitol (FIG. 8 top) formed self-aggregates by cell-cell adhesion, whereas cells treated with mannitol (FIG. 8 bottom) remained dispersed (FIG. 8). .
- Example 6 Cell death-inducing action of carbohydrates (sugar alcohols) on the remaining stem cells in human embryonic stem cell-derived embryoid bodies and enrichment of cardiomyocytes
- carbohydrates sucrose alcohols
- cardiomyocytes formed from human embryonic stem cells
- the purpose of this study was to confirm the cultured state of cardiomyocytes and the cultured state of other cells after the treatment of the cell mass (embryoid body) containing saccharides (sugar alcohols).
- Embryoid bodies that had passed 3 months after the start of differentiation still maintained autonomous pulsatility. These were partially treated with 0.1% collagenase (Wortonton) and 0.1% trypsin (DIFCO) to form fine cell clumps. Divide this into two, add 0.45M mannitol (equivalent to about 720 mOsm / kg) to one side, treat for 2 hours, and use the same amount as the control (control) group when treated with mannitol.
- Physiological Osmotic Solution (116.4 mM NaCl, 5.4 mM KCl, 5.6 mM Dextrose, 10.9 mM NaH 2 PO 4 , 405.7 ⁇ M MgSO 4 , 20 mM Hepes, pH 7.3) was added for 2 hours. Processed. After treatment, both cell groups were adherently cultured in a DMEM solution containing 10% fetal bovine serum for 3 days.
- FIG. 10 shows the results for the control group among the samples divided into two
- FIG. 11 shows the results for the experimental group in which the mannitol treatment was performed for 2 hours.
- the upper part of FIG. 10 shows cardiomyocytes, and the lower part shows embryonic stem cells.
- this treatment method is a method for efficiently inducing cell death to stem cells even in human embryonic stem cells.
- cell death was not induced for cardiomyocytes
- the method can also be used as a method for concentrating cardiomyocytes.
- One week after mannitol treatment the type and number of viable cells were examined using an immunohistochemical technique. As a result, in contrast to control cells (non-treated group), most viable cells were Nkx2.5-positive cardiomyocytes. And Oct-3 / 4 negative cells were hardly found (FIG. 11 (b)).
- Example 7 Cell death-inducing action of carbohydrates (sugar alcohols) on human-induced pluripotent stem cells (iPS cells)
- iPS cells human-induced pluripotent stem cells
- the purpose was to confirm the viability of iPS cells after treatment with alcohols.
- Human iPS cells were obtained from the Center for Stem Cell Medicine, Institute for Regenerative Medicine, Kyoto University (ES Cell Center by National BioResource Project). The basic culture method was the same as that of human embryonic stem cells, and culture was performed by the same method as described in Example 5.
- the literature (Watabebe K, et al., Nat. Biotechnol., 2007, 25: 681-686, in which cells were dispersed separately using TE (0.25% trypsin (GIBCO) and 1 mM EDTA). According to Epub 2007 May 27), 10 ⁇ M ROCK inhibitor (Y27632) was added to suppress cell death. At the same time, the mitochondria of living cells were stained using 50 nM TMRM.
- a sample of cells was prepared by treating the stained cells for 2 hours in an ⁇ -MEM culture medium containing mannitol 0.45M (corresponding to about 720 mOsm / kg), and the sample was prepared for 2 hours in an ⁇ -MEM culture medium not containing mannitol. Samples of treated cells were made as controls. Prior to the FACS analysis, the cells adhered to each other in the control sample and formed a cell mass. After treating the cells again with the same trypsin and EDTA to disperse the cells, based on the fluorescence intensity of TMRM The presence or absence of mitochondrial membrane potential was analyzed by FACS, and the survival state of the stem cells was confirmed and shown in FIG.
- Example 8 Cell death-inducing action of various carbohydrates (sugar alcohols, saccharides, betaines) on human embryonic stem cells
- human embryonic stem cells were converted into saccharides other than mannitol (sugar alcohols, saccharides). The purpose of this study was to confirm the survival state of stem cells after treatment with betaines).
- Example 9 Cell death inducing action of various carbohydrates (sugar alcohols, saccharides, betaines) on stem cells remaining in human embryonic stem cell-derived embryoid bodies and enrichment of cardiomyocytes
- carbohydrate sucrose alcohols, saccharides, betaines
- Embryoid bodies that had passed 3 months after the start of differentiation still maintained autonomous pulsatility. These were partially treated with 0.1% collagenase (Wortonton) and 0.1% trypsin (DIFCO) to form fine cell clumps.
- Example 10 Histological analysis of cell death-inducing action of carbohydrates (sugar alcohols) on stem cells remaining in human embryonic stem cell-derived embryoid bodies
- carbohydrates sucrose alcohols
- cardiomyocytes formed from human embryonic stem cells
- the purpose of this study was to confirm the morphology of cells under culture conditions of cardiomyocytes after treatment with a carbohydrate (sugar alcohol) on a cell mass (embryoid body) containing
- glycerol effectively induces cell death in differentiated cells other than pluripotent stem cells (embryonic stem cells / iPS cells) and cardiomyocytes when treated for a long time.
- Example 11 Cell death-inducing action of low concentration carbohydrate (mannitol) on mouse embryonic stem cells
- mannitol carbohydrate
- Embryonic stem cells cultured by the method described in Example 1 were cultured in ⁇ -MEM culture medium containing mannitol 0.2M (corresponding to about 480 mOsm / kg) for 48 hours and 72 hours, and then cell adhesion.
- the survival state of the stem cells was confirmed by non-adhesion and morphological observation, and is shown in FIG.
- a culture solution [ ⁇ -MEM (Minimum Essential Medium) (SIGMA), 10% FBS (EQUITEC BIO), 100 units / ml penicillin, 50 ⁇ g / ml streptomycin (GIBCO)] was added.
- SIGMA Minimum Essential Medium
- FBS FBS
- GEBCO streptomycin
- pluripotent stem cells By treating a cell population containing pluripotent stem cells, cells other than pluripotent stem cells-derived cardiomyocytes, and pluripotent stem cells-derived cardiomyocytes by the method of the present invention, embryonic stem cells and non-cardiomyocytes in the cell population Since the cells can be efficiently removed and at the same time only the cardiomyocytes can survive, the cardiomyocytes can be efficiently concentrated and purified.
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Abstract
Description
(1)多能性幹細胞、多能性幹細胞由来の心筋細胞以外の細胞、及び多能性幹細胞由来心筋細胞を含む細胞集団を、370mOsm/kg以上の浸透圧を有する高張液で培養することにより、多能性幹細胞及び多能性幹細胞由来の心筋細胞以外の細胞を細胞死に導く方法。
(2)高張液での培養を2時間以上行う、上記(1)に記載の方法。
(3)370mOsm/kg以上の浸透圧を有する高張液が、培養液に対して糖質(炭水化物)を添加することにより調製されたものである、上記(1)又は(2)に記載の方法。
(4)370mOsm/kg以上の浸透圧を有する高張液が、0.1~1Mの糖質を含有するものである、上記(3)に記載の方法。
(5)糖質が、糖アルコール類、糖類、又はベタイン類である、上記(3)又は(4)に記載の方法。
(6)糖アルコール類、糖類、又はベタイン類が、マンニトール、ソルビトール、キシリトール、グリセロール、スクロース、グルコース及びトリメチルグリシンからなる群から選択される、上記(5)に記載の方法。
(7)370mOsm/kg以上の浸透圧を有する高張液が、0.1~0.6Mのグリセロールを含有するものである、上記(4)に記載の方法。
(8)培養を10時間以上行う、上記(7)に記載の方法。
(9)細胞集団を高張液で培養した後、200~300mOsm/kgの通常浸透圧を有する培養液に戻して更に培養する、上記(1)~(8)のいずれか1項に記載の方法。
図2は、胚性幹細胞由来心筋細胞と、残存する胚性幹細胞が混入した培養系におけるマンニトール処理を示す。
図3は、マンニトール処理後の細胞におけるNkx2.5及びOct−3/4に対する免疫染色を示す。
図4は、マウス胚性幹細胞に対する、マンニトール0.45Mの細胞死誘導作用を示す。
図5は、マーモセット胚性幹細胞に対するマンニトールの作用を示す。ミトコンドリア膜電位感受性色素TMRMを用いて生死判定を行った。
図6は、マーモセット胚性幹細胞に対するマンニトールの効果を示す。
図7は、ヒト胚性幹細胞に対するマンニトールの作用(FACS解析)を示す。
図8は、ヒト胚性幹細胞に対するマンニトールの効果(直後)を示す。
図9は、ヒト胚性幹細胞に対するマンニトールの効果(5時間後)を示す。
図10は、は、ヒト胚性幹細胞胚様体のマンニトール処理を示す。
図11は、ヒト胚性幹細胞細胞胚様体のマンニトール処理による、Oct−3/4陽性細胞の死滅を示す。
図12は、ヒト誘導性多能性幹細胞(iPS細胞)に対するマンニトールの作用(FACS解析)を示す。
図13は、マンニトール以外の種々の糖質における、ヒト胚性幹細胞に対する細胞死誘導作用を示す。
図14は、ヒト胚性幹細胞由来心筋・非心筋細胞に対するグリセロールの作用を示す。
図15は、0.2Mマンニトールを含有する培養液で、48時間または72時間処理した細胞の状態を示す。下段にそれぞれのコロニーの拡大像を示し、代表的な細胞形態を図示した。
本実施例においては、幹細胞から心筋細胞を含む細胞塊(胚様体)を形成した場合の、細胞塊(胚様体)中の心筋細胞と未分化細胞との混在状態を確認することを目的とした。
本実施例においては、胚性幹細胞から形成された心筋細胞を含む細胞塊(胚様体)に対して糖質(糖アルコール類)処理を行った後の、心筋細胞の培養状態とそれ以外の細胞の培養状態とを確認することを目的とした。
本実施例においては、マウス胚性幹細胞を糖質(糖アルコール類)にて処理した後の幹細胞の生存状態を確認することを目的とした。
本実施例においては、マーモセット胚性幹細胞を糖質(糖アルコール類)にて処理した後の幹細胞の生存状態を確認することを目的とした。
本実施例においては、ヒト胚性幹細胞を糖質(糖アルコール類)にて処理した後の幹細胞の生存状態を確認することを目的とした。
本実施例においては、ヒト胚性幹細胞から形成された心筋細胞を含む細胞塊(胚様体)に対して糖質(糖アルコール類)処理を行った後の、心筋細胞の培養状態とそれ以外の細胞の培養状態とを確認することを目的とした。
本実施例においては、ヒト誘導性多能性幹細胞(iPS細胞)を糖質(糖アルコール類)にて処理した後のiPS細胞の生存状態を確認することを目的とした。
本実施例においては、ヒト胚性幹細胞をマンニトール以外の糖質(糖アルコール類、糖類、ベタイン類)にて処理した後の幹細胞の生存状態を確認することを目的とした。
本実施例においては、ヒト胚性幹細胞から形成された心筋細胞を含む細胞塊(胚様体)に対してマンニトール以外の糖質(糖アルコール類、糖類、ベタイン類)にて処理を行った後の、心筋細胞の培養状態とそれ以外の細胞の培養状態とを確認することを目的とした。
本実施例においては、ヒト胚性幹細胞から形成された心筋細胞を含む細胞塊(胚様体)に対して糖質(糖アルコール類)にて処理を行った後の、心筋細胞の培養条件下での細胞の形態を確認することを目的とした。
本実施例においては、マウス胚性幹細胞を低濃度のマンニトールにて処理した後の幹細胞の生存状態を確認することを目的とした。
Claims (9)
- 多能性幹細胞、多能性幹細胞由来の心筋細胞以外の細胞、及び多能性幹細胞由来心筋細胞を含む細胞集団を、370mOsm/kg以上の浸透圧を有する高張液で培養することにより、多能性幹細胞及び多能性幹細胞由来の心筋細胞以外の細胞を細胞死に導く方法。
- 高張液での培養を2時間以上行う、請求項1に記載の方法。
- 370mOsm/kg以上の浸透圧を有する高張液が、培養液に対して糖質(炭水化物)を添加することにより調製されたものである、請求項1又は2に記載の方法。
- 370mOsm/kg以上の浸透圧を有する高張液が、0.1~1Mの糖質を含有するものである、請求項3に記載の方法。
- 糖質が、糖アルコール類、糖類又はベタイン類である、請求項3又は4に記載の方法。
- 糖アルコール類、糖類又はベタイン類が、マンニトール、ソルビトール、キシリトール、グリセロール、スクロース、グルコース及びトリメチルグリシンからなる群から選択される、請求項5に記載の方法。
- 370mOsm/kg以上の浸透圧を有する高張液が、0.1~0.6Mのグリセロールを含有するものである、請求項4に記載の方法。
- 培養を10時間以上行う、請求項7に記載の方法。
- 細胞集団を高張液で培養した後、200~300mOsm/kgの通常浸透圧を有する培養液に戻して更に培養する、請求項1~8のいずれか1項に記載の方法。
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WO2018074457A1 (ja) * | 2016-10-17 | 2018-04-26 | 学校法人慶應義塾 | 未分化幹細胞除去剤、及び未分化幹細胞除去方法 |
WO2018110654A1 (ja) | 2016-12-15 | 2018-06-21 | Heartseed株式会社 | 未分化幹細胞除去剤及び未分化幹細胞除去方法 |
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Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9202219D0 (en) * | 1992-02-03 | 1992-03-18 | Connaught Lab | A synthetic heamophilus influenzae conjugate vaccine |
US5543316A (en) * | 1994-04-20 | 1996-08-06 | Diacrin, Inc. | Injectable culture medium for maintaining viability of myoblast cells |
EP1003847A4 (en) * | 1997-09-04 | 2005-04-06 | Science Res Lab Inc | SEPARATION OF CELLS BY USE OF ELECTRIC FIELDS |
US6576464B2 (en) * | 2000-11-27 | 2003-06-10 | Geron Corporation | Methods for providing differentiated stem cells |
US20030194798A1 (en) * | 2001-05-24 | 2003-10-16 | Surber Mark W. | Minicell compositions and methods |
US7790039B2 (en) * | 2003-11-24 | 2010-09-07 | Northwest Biotherapeutics, Inc. | Tangential flow filtration devices and methods for stem cell enrichment |
US20050233446A1 (en) * | 2003-12-31 | 2005-10-20 | Parsons Xuejun H | Defined media for stem cell culture |
NZ567082A (en) * | 2005-10-14 | 2012-08-31 | Univ Minnesota | Differentiation of non-embryonic stem cells to cells having a pancreatic phenotype |
CA2640644C (en) * | 2006-01-31 | 2013-11-26 | Asubio Pharma Co., Ltd. | A method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses |
-
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Non-Patent Citations (14)
Title |
---|
BADER, A. ET AL., DIFFERENTIATION, vol. 68, 2001, pages 31 - 43 |
BIEBERICH, E. ET AL., J. CELL BIOL., vol. 167, 2004, pages 723 - 734 |
CHOO, A.B. ET AL., STEM CELLS, vol. 26, 2008, pages 1454 - 1463 |
HIRATA, H ET AL.: "Coexpression of platelet-derived growth factor receptor alpha and fetal liver kinase 1 enhances cardiogenic potential in embryonic stem cell differentiation in vitro.", J BIOSCI BIOENG., vol. 103, no. 5, May 2007 (2007-05-01), pages 412 - 419, XP022136680 * |
HUBER, I ET AL.: "Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.", FASEB J., vol. 21, no. 10, August 2007 (2007-08-01), pages 2551 - 2563, XP008132356 * |
MULLER, M. ET AL., FASEB J., vol. 14, 2000, pages 2540 - 2548 |
NAT GENET, vol. 24, 2000, pages 372 - 376 |
PAREKKADAN, B ET AL.: "Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood.", TISSUE ENG., vol. 13, no. 10, October 2007 (2007-10-01), pages 2465 - 2473, XP008154242 * |
SCHR6DER, A.R. ET AL., CELL, vol. 110, 2002, pages 521 - 529 |
SCHULDINER, M. ET AL., STEM CELLS, vol. 21, 2003, pages 257 - 265 |
See also references of EP2415862A4 |
STEM CELLS, vol. 23, no. 9, October 2005 (2005-10-01), pages 1304 - 13 |
WATANABE, K. ET AL., NAT. BIOTECHNOL., vol. 25, 27 May 2007 (2007-05-27), pages 681 - 686 |
WU, X ET AL.: "Small molecules that induce cardiomyogenesis in embryonic stem cells.", J AM CHEM SOC., vol. 126, no. 6, 18 February 2004 (2004-02-18), pages 1590 - 1591, XP002324807 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170031152A (ko) | 2014-07-16 | 2017-03-20 | 하트 시드 가부시키가이샤 | 신규 미분화 줄기세포 제거 및 심근 순화 정제 배지 |
WO2018074457A1 (ja) * | 2016-10-17 | 2018-04-26 | 学校法人慶應義塾 | 未分化幹細胞除去剤、及び未分化幹細胞除去方法 |
KR20190052096A (ko) | 2016-10-17 | 2019-05-15 | 각고호우징 게이오기주크 | 미분화 줄기세포 제거제, 및 미분화 줄기세포 제거 방법 |
JPWO2018074457A1 (ja) * | 2016-10-17 | 2019-07-18 | 学校法人慶應義塾 | 未分化幹細胞除去剤、及び未分化幹細胞除去方法 |
WO2018110654A1 (ja) | 2016-12-15 | 2018-06-21 | Heartseed株式会社 | 未分化幹細胞除去剤及び未分化幹細胞除去方法 |
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KR20110134417A (ko) | 2011-12-14 |
EP2415862A4 (en) | 2013-02-20 |
RU2551778C2 (ru) | 2015-05-27 |
EP2415862B1 (en) | 2015-05-06 |
US8735152B2 (en) | 2014-05-27 |
IL215328A0 (en) | 2011-12-29 |
CA2755887A1 (en) | 2010-10-07 |
CN102449139B (zh) | 2016-08-24 |
AU2010232148B2 (en) | 2014-10-09 |
RU2011143857A (ru) | 2013-05-10 |
SG174980A1 (en) | 2011-11-28 |
US20120094383A1 (en) | 2012-04-19 |
AU2010232148A1 (en) | 2011-10-27 |
CA2755887C (en) | 2014-02-04 |
KR101656761B1 (ko) | 2016-09-12 |
EP2415862A1 (en) | 2012-02-08 |
CN102449139A (zh) | 2012-05-09 |
JP5620905B2 (ja) | 2014-11-05 |
BRPI1012715A2 (pt) | 2015-09-15 |
JPWO2010114136A1 (ja) | 2012-10-11 |
IL215328A (en) | 2014-06-30 |
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