WO2020242045A1 - Procédé pour favoriser la différenciation de cellules souches en cardiomyocytes matures par un traitement avec de la tomatidine - Google Patents

Procédé pour favoriser la différenciation de cellules souches en cardiomyocytes matures par un traitement avec de la tomatidine Download PDF

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WO2020242045A1
WO2020242045A1 PCT/KR2020/004841 KR2020004841W WO2020242045A1 WO 2020242045 A1 WO2020242045 A1 WO 2020242045A1 KR 2020004841 W KR2020004841 W KR 2020004841W WO 2020242045 A1 WO2020242045 A1 WO 2020242045A1
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cardiomyocytes
tomatidine
differentiated
stem cells
composition
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Korean (ko)
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김재호
김예슬
윤정원
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부산대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method for promoting differentiation and maturation of stem cells into cardiomyocytes by treatment with tomatidine.
  • Differentiated cardiomyocytes simultaneously contract and relax repeatedly, express a sarcomeric protein, have a heart cell action potential, and show transient increases and decreases in Ca 2+ concentration (calcium transients). It is known to exhibit a function similar to that of cardiomyocytes during development due to contractile relaxation and signal transmission due to nerve hormones.
  • the differentiated cardiomyocytes remain immature and have a poor contractile relaxation function compared to adult cardiomyocytes, and show a protein expression that is not morphologically appropriate. Therefore, the differentiated cardiomyocytes are used to treat diseases or develop new drugs. There is a limit to use. Accordingly, a step of maturing the differentiated cardiomyocytes is required.
  • a representative recent study related to this is that when treating cardiomyocytes derived from human pluripotent stem cells with T3 (tri-iodo-L-thyronine), the optimal growth hormone for heart formation, the size and sarcomere length of the cardiomyocytes are reduced. Increased, and the expression of cardiac structural proteins has been reported to be increased.
  • tomatidine belongs to a class of steroidal alkaloids made through chemical decomposition of ⁇ -tomatine, which is produced from two molecules of glucose (D-glucose). These steroidal alkaloid substances are mostly found in the plant, animal, and fungal systems.
  • SAT Marty Dean mTORC1 by increasing the signal transmission and reduce muscle atrophy, it is possible to increase skeletal muscle strength and exercise capacity, the SKN-1 / Nrf2 pathway in C.elegans It has been reported that it can improve longevity and health through selective mitophage.
  • the effect of improving the structure and function of cardiomyocytes when the tomatidine is treated on stem cells has not yet been reported.
  • an object of the present invention is a composition for inducing maturation of differentiated or differentiated cardiomyocytes from stem cells containing tomatidine, and of differentiated or differentiated cardiomyocytes from stem cells into cardiomyocytes using the composition. It is to provide a method of inducing maturation.
  • the present invention provides a composition for inducing maturation of differentiated cardiomyocytes or differentiated into cardiomyocytes from stem cells containing tomatidine.
  • the present invention provides a medium composition for inducing maturation of differentiated cardiomyocytes or differentiated from stem cells into cardiomyocytes comprising the composition.
  • the present invention provides a method of inducing maturation of differentiated cardiomyocytes or differentiated from stem cells into cardiomyocytes, comprising the step of culturing stem cells or differentiated cardiomyocytes in a medium containing tomatidine.
  • the present invention provides a cell therapy agent for preventing or treating heart disease, including the matured differentiated cardiomyocytes.
  • the present invention increases the differentiation efficiency of cardiomyocytes by treating stem cells with tomatidine during differentiation into cardiomyocytes, and the cardiomyocytes produced through the tomatidine treatment have a structure and function with mature cardiomyocytes of humans. Shows a similar remarkable effect. Accordingly, the present invention is expected to be usefully utilized in an evaluation platform for developing a cell therapy agent for treating myocardial infarction, cardiac toxicity test, or drug for treating heart disease.
  • FIG. 1 is a schematic diagram of an experimental protocol for differentiation or maturation from human embryonic stem cells to cardiomyocytes using tomatidine.
  • Figure 2 shows the effect on the differentiation of human embryonic stem cells to cardiomyocytes according to the concentration of tomatidine (One-way Anova; *p-value ⁇ 0.05, **p-value ⁇ 0.01, *** p-value ⁇ 0.005)
  • FIG. 3 shows the expression level of each cardiomyocyte marker upon treatment with tomatidine 1 ⁇ M
  • A flow cytometry
  • B Western blot analysis to determine the differentiation efficiency from human embryonic stem cells to cardiomyocytes according to the tomatidine treatment. I confirmed it.
  • Figure 4 shows the results of comparing the size of cells when treated with tomatidine 1 ⁇ M through (A) an optical microscope and (B) a confocal laser microscope (student's t-test; **p-value ⁇ 0.01).
  • Figure 5 shows genes encoding musculoskeletal proteins in cardiomyocytes (MYL2 (MLC2v), MYL7 (MLC2a), MYH7 (Beta myosin heavy chain, ⁇ MHC), MYH6 (Alpha myosin heavy chain, ⁇ MHC), TNN) when treated with 1 ⁇ M of tomatidine.
  • I Type 1 and TNN I Type 3 expression changes were recovered from human embryonic stem cells differentiated cardiomyocytes by differentiation days (14, 30 and 60 days) and confirmed by (A) Western blot and (B) quantified. (Unpaired student's t-test; * p-value ⁇ 0.05, **p-value ⁇ 0.01, ***p-value ⁇ 0.005).
  • FIG. 6 is a diagram showing the results of analyzing the expression level of cTnI in mature cardiomyocytes with respect to cardiomyocytes 30 days after differentiation using a flow cytometer.
  • NPPA disease-related gene
  • FIG. 8 shows the expression of BIN1, a component of t-tubule (rhabdominus), in cardiomyocytes differentiated by treatment with tomatidine, stained with BIN1 antibody, and compared and confirmed through a confocal laser microscope.
  • FIG. 9 shows the expression levels of BIN1 and JPH2, proteins constituting t-tubule (rhabdominus), in cardiomyocytes differentiated by treatment with tomatidine (A) confirmed by Western blot and (B) quantified (unpaired student's t-test; *p-value ⁇ 0.05, **p-value ⁇ 0.01, *** p-value ⁇ 0.005).
  • FIG. 10 is a diagram showing the results of confirming the arrangement of t-tubules (rhabdomyotes) through Di-8-ANEPPS staining in cardiomyocytes differentiated by treatment with tomatidine.
  • Figure 11 shows the quantification of the density of t-tubules (rhabdomyotes) in cardiomyocytes differentiated by treatment with tomatidine (unpaired student's t-test; *p-value ⁇ 0.05, **p-value ⁇ 0.01, ***p-value ⁇ 0.005).
  • FIG. 12 is a diagram showing the results of performing a multi-electrode array assay to determine whether the differentiated cardiomyocytes matured by treatment with tomatidine (student's t-test; *p-value ⁇ 0.05, **p-value ⁇ 0.01, ***p-value ⁇ 0.005).
  • the present invention provides a composition for inducing the differentiation of stem cells containing tomatidine into cardiomyocytes and/or maturation of differentiated cardiomyocytes.
  • the "tomatidine” is 5 alpha, 20 beta (F), 22 alpha (F), 25 beta (F), 27- azaspirostan-3 beta- in the art to which the present invention belongs.
  • Tomatidine is a steroidal alkaloid-based substance, and is a compound made by chemically decomposing two molecules of D-glucose and one molecule of -xylose and -galactose in tomato extracted from tomato. Tomatidine is present in green-colored tomatoes, usually immature tomatoes.
  • the tomatidine may be characterized in that it is contained in a concentration of 0.01 to 100 ⁇ M.
  • methanol (CH 3 OH): chloroform (CHCl 3 ) 1:1 prepared by diluting tomatidine dissolved in 10-50 mM in DMSO to 1 mM in a mixed solution, and the final cell treatment concentration of 1 ⁇ M Alternatively, 0.1% in cell culture medium was used in the experiment.
  • stem cell is a cell that is the basis of cells or tissues constituting an individual, and its characteristic is that it can self-renewal by repetitive division, and can perform specific functions according to the environment. It refers to a cell that has the ability to differentiate into a cell with multiple differentiation.
  • the stem cells may be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation ability.
  • Pluripotent stem cells are pluripotency cells with the potential to differentiate into all cells.
  • the stem cells may be pluripotent stem cells, preferably human pluripotent stem cells, but are not limited thereto.
  • One embodiment of the present invention discloses a method of differentiating or maturing cardiomyocytes from stem cells using human embryonic stem cells.
  • cardiomyocyte includes, without limitation, cells of all differentiation stages of cardiomyocyte progenitor cells, fetal cardiomyocytes, and adult cardiomyocytes having the ability to become functional cardiomyocytes in the future. At least one described, preferably refers to a cell that can be identified by at least one, preferably a plurality of markers or criteria by a plurality of methods. Expression of various markers specific to cardiomyocytes can be detected by known biochemical or immunochemical methods, and these methods can be used without limitation. In this method, a polyclonal antibody or a monoclonal antibody specific for a marker that binds to cardiomyocyte progenitor cells or cardiomyocytes can be used.
  • Antibodies targeting individual specific markers may be commercially available or prepared by known methods, without limitation.
  • myocardial progenitor cells or markers specific for cardiomyocytes include ⁇ -sarcomeric actinin (aSA), myosin heavy/light chain (MHC/MLC), cardiac troponin-T (cTnT), myosin light chain-2 atrium (MLC2a), and MLC2v. (myosin light chain-2 ventricle), MYH7 (Beta myosin heavy chain, ⁇ MHC), MYH6 (Alpha myosin heavy chain, ⁇ MHC), TNN I Type 1, TNN I Type 3, ANP, GATA4, Nkx2.5 and MEF-2c And the like.
  • aSA myocardial progenitor cells or markers specific for cardiomyocytes
  • MHC/MLC myosin heavy/light chain
  • cTnT cardiac troponin-T
  • MLC2a myosin light chain-2 atrium
  • the present invention improves the differentiation rate of tomatidine into stem cells, especially human pluripotent stem cells such as human embryonic stem cells or induced pluripotent stem cells, and matures in human embryonic stem cells or induced pluripotent stem cells.
  • stem cells especially human pluripotent stem cells such as human embryonic stem cells or induced pluripotent stem cells
  • matures in human embryonic stem cells or induced pluripotent stem cells There are technical features in promoting the differentiation into cardiomyocytes, and for increasing the expression of the structural protein of the t-tubule (rhabdomyo) and its genes in the musculoskeletal system of differentiated cardiomyocytes.
  • the tomatidine is aSA ( ⁇ -Sarcomeric Actinin), cTnT (cardiac troponin-T), MLC2a (myosin light chain-2 atrium or MYL7), MLC2v (myosin light chain-2 ventricle or MYL2), cTnI (cardiac troponin 1), MYH7 (Beta myosin heavy chain), MYH6 (Alpha myosin heavy chain), JPH2 (Junctophilin-2), and BIN1 (Bridging integrator 1) one or more genes or proteins selected from the group consisting of It is characterized in that it can increase the expression level of.
  • SA ⁇ -Sarcomeric Actinin
  • cTnT cardiac troponin-T
  • MLC2a myosin light chain-2 atrium or MYL7
  • MLC2v myosin light chain-2 ventricle or MYL2
  • cTnI cardiac troponin 1
  • MYH7 Be
  • the tomatidine is characterized in that it reduces the expression level of the NPPA (natriuretic peptide precursor type A) gene or protein in differentiated cardiomyocytes.
  • NPPA neuropeptide precursor type A
  • the tomatidine is characterized in that the arrangement of t-tubules in differentiated cardiomyocytes is aligned in one direction.
  • cardiomyocyte-specific markers is not limited to a specific method, but is a conventional method for amplifying, detecting, and analyzing mRNA encoding an arbitrary marker protein, such as reverse transcriptase-mediated polymerase chain reaction (RT-PCR) or hybridization analysis. It can be confirmed by molecular biological methods commonly used in Nucleic acid sequences encoding marker proteins specific for cardiomyocytes are already known and can be obtained from public databases such as GenBank, and marker-specific sequences required for use as primers or probes can be easily determined.
  • RT-PCR reverse transcriptase-mediated polymerase chain reaction
  • t-tubules rhabdominal tubules
  • electrophysiological criteria may be additionally used.
  • the present invention provides a medium composition for inducing maturation of differentiated cardiomyocytes or differentiated from stem cells into cardiomyocytes comprising the composition.
  • the medium composition is CHIR99021(6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino ]ethyl]amino]-3-pyridinecarbonitrile) and IWP2(N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2 -d]pyrimidin-2-yl)thio]-acetamide) may be further included.
  • the CHIR99021 is used as a glycogen synthase kinase (GSK) inhibitor/Wnt signaling inducing agent.
  • GSK glycogen synthase kinase
  • all GSK inhibitors including 1-AKP (1-azakenpaullon) involved in the GSK signaling process are It will be apparent to those skilled in the art that it may fall within the scope of the present invention without limitation.
  • the IWP2 is used as a Wnt signaling inhibitor, and it will be apparent to those skilled in the art that all Wnt signaling inhibitors other than IWP2 may belong to the scope of the present invention without limitation.
  • Each of the inducers included in the medium composition according to the present invention may be purchased and used or prepared and used on the market, and the effective concentration may be adjusted according to factors well known in the art, such as the type of the culture medium and the culture method.
  • the CHIR99021 and IWP2 may be added to the medium at a concentration of 8 to 16 ⁇ M and 1 to 10 ⁇ M, respectively, and in one embodiment of the present invention, CHIR99021 and IWP2 were added to each of 10 to 14 ⁇ M and 5 ⁇ M. , But is not limited thereto.
  • the medium composition includes all medium culture solutions commonly used for stem cell culture in the technical field to which the present invention belongs.
  • the culture medium used for cultivation generally contains a carbon source, a nitrogen source, and a trace element component.
  • the present invention provides a method of inducing maturation of differentiated cardiomyocytes or differentiated from stem cells into cardiomyocytes, comprising the step of culturing stem cells or differentiated cardiomyocytes in a medium containing tomatidine.
  • the method comprises before culturing stem cells or differentiated cardiomyocytes in a medium containing tomatidine, (a) the stem cells are added to CHIR99021 in a medium containing B27 supplement from which insulin is removed. Incubating for 30 hours to differentiate into cardiomyocytes; (b) culturing the differentiated cardiomyocytes for 1 to 3 days in a medium to which CHIR99021 is not added; And (c) culturing the cultured cardiomyocytes in a medium to which IWP2 is added for 1 to 3 days; may further include.
  • the method is before culturing stem cells or differentiated cardiomyocytes in a medium containing tomatidine, (a) the stem cells are added to CHIR99021 in a medium containing B27 supplement from which insulin is removed for 24 hours. Culturing during the differentiation into cardiomyocytes; (b) culturing the differentiated cardiomyocytes for 2 days in a medium to which CHIR99021 is not added; And (c) culturing the cultured cardiomyocytes in a medium to which IWP2 is added for 2 days.
  • the method is a differentiated cardiomyocyte that is matured by treating the differentiated cardiomyocytes with tomatidine, and then cultured in a medium (Glucose-, Lactate) containing no glucose and adding lactic acid. It may further include the step of purifying.
  • the purification step is characterized in that tomatidine is not included in the medium.
  • the method may further include the step of purifying the differentiated cardiomyocytes, and then culturing by adding tomatidine to RPMI1640 medium (RBin) containing B27 supplement from which insulin has been removed.
  • RPMI1640 medium RBin
  • glucose is not included, and cultured in a medium (Glucose-, Lactate) to which lactic acid is added for 4 days to differentiate into cardiomyocytes
  • a medium Glucose-, Lactate
  • lactic acid is added for 4 days to differentiate into cardiomyocytes
  • tomatidine was added to RPMI1640 medium (RBin) containing insulin-removed B27 supplement, and cultured continuously for 6 days to prepare matured differentiated cardiomyocytes.
  • the present invention provides a cell therapy agent for preventing or treating heart disease, comprising differentiated cardiomyocytes matured by the method according to the present invention.
  • the term "cell therapy” refers to a drug used for treatment, diagnosis, and prevention of cells and tissues manufactured through isolation, culture and special manipulation from humans (US FDA regulations), and functions of cells or tissues It refers to medicines used for treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic or heterogeneous cells in vitro to restore, or changing the biological properties of cells in other ways.
  • prevention refers to any action that suppresses or delays progression of heart disease by administration of the cell therapy agent according to the present invention.
  • treatment refers to any action in which heart disease is improved or beneficially changed by administration of the cell therapy agent according to the present invention.
  • the heart disease may be at least one selected from the group consisting of myocardial infarction, angina, ischemic myocardial disease, primary myocardial disease, secondary myocardial disease, and heart failure, but is not limited thereto.
  • the cardiomyocytes are matured to a degree that is morphologically and functionally very similar to human adult cardiomyocytes compared to cardiomyocytes differentiated by conventional differentiation methods according to the treatment with tomatidine.
  • Example 1 Treatment of tomatidine in the process of differentiation of human embryonic stem cells into cardiomyocytes
  • IWP2(N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-) inhibiting the Wnt signaling pathway d]pyrimidin-2-yl)thio]-acetamide) 5 ⁇ M was added to RBin and cultured for 48 hours, and then tomatidine 1 ⁇ M was added to the culture solution. From the 8th day of differentiation treated with the GSK inhibitor, they were cultured in RPMI1640 medium containing B27 supplementation in the presence of 1 ⁇ M of tomatidine.
  • the concentration of tomatidine was 0.1, 0.5, 1, 2 and 5 ⁇ M.
  • the myocardial skeletal protein aSA ⁇ -sarcomeric actinin expressed in cardiomyocytes differentiated from human embryonic stem cells was analyzed by Western blot on the 20th day of differentiation. The results are shown in FIG. 2.
  • the expression ratio of the cardiomyocyte structural protein was significantly higher in the differentiated cardiomyocytes compared to the control group according to the treatment with tomatidine 1 ⁇ M. This expression pattern was also confirmed through flow cytometry. Through the above results, it was confirmed that the expression level of the cardiomyocyte-specific protein was significantly increased through the treatment with tomatidine, and that human embryonic stem cells were successfully differentiated into cardiomyocytes. In addition, it was confirmed that the concentration of tomatidine that maximizes the differentiation rate was 1 ⁇ M.
  • Cardiomyocytes differentiated from human embryonic stem cells were treated with lactic acid, placed on a cover glass coated with 0.1% gelatin, fixed with 4% paraformaldehyde (PFA), and washed 3 times with phosphate buffer solution (PBS). , Blocked with 5% bovine serum albumin (BSA; sigma Aldrich) for 30 minutes. Next, the primary antibodies against aSA, cTnT and MLC2v (abcam) were reacted overnight at 4° C. and fluorescently labeled through a secondary antibody to which alexa-fluro 488 or 647 was linked.
  • PFA paraformaldehyde
  • BSA bovine serum albumin
  • FIG. 4A it was confirmed that maturation of differentiated cardiomyocytes was induced by the tomatidine treatment through the morphological difference in which the cell size was significantly larger in the tomatidine treatment group compared to the control group.
  • FIG. 4B it was observed that the patterns of aSA, cTnT and MLC2v staining in the tomatidine-treated group also showed a more pronounced tendency compared to the control group.
  • cardiomyocytes differentiated from human embryonic stem cells were recovered by differentiation days (14, 30 and 60 days), and the expression level of the cardiomyocyte marker was confirmed by Western blot using an antibody.
  • genes encoding musculoskeletal proteins (MYL2 (MLC2v), MYL7 (MLC2a), MYH7 (Beta myosin heavy chain, ⁇ MHC), MYH6 (Alpha myosin heavy chain, ⁇ MHC)) in cardiomyocytes differentiated by treatment with tomatidine , TNN I Type 1 and TNN I Type 3) was performed with real-time PCR to determine whether the expression was increased. The results are shown in FIG. 5.
  • NPPA pathological disease
  • NPPA neuropeptide precursor type A
  • t-tubule contains a membrane microdomain rich in ion channels and signaling molecules. Immunofluorescence staining was performed on cells to confirm whether tomatidine induces formation of t-tubule, which is important for contraction and relaxation of cardiomyocytes.
  • the cells at the 60th day of differentiation were placed on a cover glass coated with 0.1% gelatin, cultured for 2-3 days in a medium mixed with RPMI1640 and 20% fetal bovine serum, and then RPMI1640 medium (RB+) containing B27 supplement, a cardiomyocyte growth medium.
  • the t-tubule membrane and the sarcoplasmic reticulum membrane are physically connected in muscle cells, and the plasmalemmal L-type calcium channel and myoplasmic reticulum for calcium-induced calcium release.
  • Western blot was performed using JPH2 and BIN1 antibodies to confirm the expression levels of JPH2 (Junctophilin-2) and BIN1 (Bridging integrator 1) related to the sarcoplasmic reticulum ryanodine receptor. The results are shown in FIG. 9.
  • the myocardial cells differentiated with Di-8-ANEPPS which becomes a fluorescent label according to ion migration in the t-tubule, were stained to confirm the arrangement and density.
  • Di-8-ANEPPS Di-8-ANEPPS (Thermo) was mixed with RB+, a cardiomyocyte growth medium, and reacted (20 minutes, 37° C.).
  • the cardiomyocytes were washed twice with a medium containing no reagent, and then photographed at 450, 510, and 570 nm using a laser scanning confocal microscope. The photographing results were analyzed using the Image J program. The results are shown in FIGS. 10 and 11.
  • cardiomyocytes that have undergone maturation have an even arrangement of t-tubules, and that as the density of t-tubules increases, contraction and relaxation of cardiomyocytes occur simultaneously in a large area.
  • FIG. 10 it was confirmed that the t-tubule arrangement of the tomatidine-treated group was remarkably even compared to the control group.
  • FIG. 11 compared to the control group, it was confirmed that the tomatidine-treated group had a denser density than the area occupied by t-tubules in the cardiomyocytes.
  • a multi-electrode array assay was performed to measure conductivity, which is an electrophysiological characteristic exhibited through contraction and relaxation of cardiomyocytes.
  • Cardiomyocytes at day 60 of differentiation were washed twice with HBSS, reacted with 0.05% trypsin-EDTA (3 min, 37°C), inactivated with RPMI1640+20% FBS(R20) medium, and then removed by pipetting the cells. .
  • the cells were placed in a fibronectin-coated multi-electrode array plate with R20 and cultured (48 hours, 37°C). Thereafter, the medium was replaced with RB+ and the conductivity was measured. The results are shown in FIG. 12.
  • the tomatidine according to the present invention is treated during the differentiation of human pluripotent stem cells into cardiomyocytes, the differentiation efficiency of cardiomyocytes is increased, and the resulting cardiomyocytes have a structure and structure with mature cardiomyocytes of humans. It was confirmed that the function showed similar remarkable effects. Therefore, if the matured cardiomyocytes prepared using the present invention are used for new drug discovery and toxicity evaluation, as matured human cardiomyocytes that are very similar in morphology and function to adult cardiomyocytes are used, due to differences between species In addition to solving possible differences, it is expected that the efficacy and toxicity of new drugs can be predicted and evaluated before clinical trials, thereby reducing enormous costs required for new drug development and shortening the validation period.

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Abstract

La présente invention concerne un procédé de traitement d'une cellule souche avec de la tomatidine pour favoriser sa différenciation et sa maturation en cardiomyocyte. La présente invention augmente l'efficacité de la différenciation des cardiomyocytes en traitant une cellule souche avec de la tomatidine pendant sa différenciation en cardiomyocyte, et présente un effet remarquable en ce que le cardiomyocyte produit par le traitement avec de la tomatidine est similaire quant à sa structure et sa fonction à un cardiomyocyte humain mature. Ainsi, la présente invention est prévue pour être utilement appliquée à une plateforme d'évaluation pour développer un agent de thérapie cellulaire pour traiter un infarctus du myocarde, ou un médicament pour tester la toxicité cardiaque ou traiter une maladie cardiaque.
PCT/KR2020/004841 2019-05-28 2020-04-09 Procédé pour favoriser la différenciation de cellules souches en cardiomyocytes matures par un traitement avec de la tomatidine WO2020242045A1 (fr)

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