WO2024128482A1 - Composition for producing mature cardiac organoid - Google Patents

Composition for producing mature cardiac organoid Download PDF

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WO2024128482A1
WO2024128482A1 PCT/KR2023/013900 KR2023013900W WO2024128482A1 WO 2024128482 A1 WO2024128482 A1 WO 2024128482A1 KR 2023013900 W KR2023013900 W KR 2023013900W WO 2024128482 A1 WO2024128482 A1 WO 2024128482A1
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organoids
cardiac
dcn
mature
composition
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임도선
송명화
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고려대학교 산학협력단
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to compositions for producing mature cardiac organoids.
  • Ischemic heart disease has a very high prevalence worldwide and ranks first among all single diseases causing death. In Korea, the number of patients with ischemic heart disease is also rapidly increasing due to socio-economic development and westernized lifestyles. Moreover, for patients with severe end-stage heart failure, there is no cure other than heart transplantation or mechanical left ventricular assist device. However, it has problems such as lack of donor organs, difficulty in securing organs, high mortality rate, and expensive treatment costs.
  • Transplantation methods for reconstructing damaged tissues in the human body include xenotransplantation, allogeneic transplantation, and autotransplantation.
  • Xenotransplantation has the problems of immune incompatibility and transmission of zoonotic pathogens, including retroviruses.
  • allogeneic transplantation has the problem of immune rejection and unavailability of the donor, and autotransplantation has the problem of difficulty in obtaining appropriate tissue in the required amount and increased trauma to the patient.
  • technologies that attempt to transplant artificial substitutes or cultured and organized cells are attracting attention.
  • Human pluripotent stem cells which are undifferentiated cells that can differentiate into various types of cells, can differentiate into cardiomyocytes, and much research is being conducted on human pluripotent stem cell-derived cardiomyocytes generated in a two-dimensional culture system. These human pluripotent stem cell-derived cardiomyocytes can be widely used in cardiac development, drug screening, disease modeling, and cardiac recovery research. However, unlike mature adult cardiomyocytes, human pluripotent stem cell-derived cardiomyocytes generated from existing two-dimensional culture systems exhibit structurally and functionally immature characteristics similar to embryonic or fetal stages, making them suitable for disease modeling, drug screening, and cardiotoxicity. There are limitations to its use.
  • organoids also called artificial organs or mini-organs
  • Organoids are three-dimensional cell structures formed through development and differentiation from stem cells, and are small organ mimics that reproduce the structural and functional characteristics of the organ.
  • Organoids are three-dimensional cell structures that are composed of various cells that make up tissues and provide a microenvironment containing extracellular matrix (ECM), cell types, and soluble factors, making them superior to existing two-dimensional culture systems. It has recently received attention for its ability to form heart tissue. Therefore, it is widely used in various applied research fields such as basic biology research, new drug development, disease modeling, and regenerative therapy. Self-organization refers to the phenomenon of creating self-organization through interactions between cells without external pressure.
  • ECM extracellular matrix
  • Cardiac organoids have the ability to self-organize and are effectively produced by mimicking the heart in vivo. However, they lack the formation of a vascular network that promotes oxygen and nutrient distribution, so they depend on the culture medium, cannot be cultured for long periods of time, and are difficult to implement in vivo. . Therefore, in order to establish organoids containing mature vascular networks, many researchers have established organoids containing blood vessels by co-culturing vascular endothelial cells or treating them with exogenous vascular inducing factors.
  • vascular endothelial cells in order to achieve self-organization in the body, it is necessary to co-culture vascular endothelial cells or induce simultaneous differentiation from human pluripotent stem cells into various cells that constitute the heart without treating exogenous vascular inducing factors, which can contribute to cardiogenesis and cardiac development. It is important for replicating an organization.
  • a fertilized egg undergoes differentiation into three germ layers including ectoderm, endoderm, and mesoderm, forming all cells and organs during organogenesis.
  • the heart and blood vessels belong to the mesoderm. Therefore, efficient induction of differentiation from human pluripotent stem cells into mesoderm is important for the formation of cardiac organoids containing vascular networks.
  • An object of the present invention is to provide a composition for producing mature cardiac organoids.
  • an object of the present invention is to provide a method for producing mature cardiac organoids.
  • an object of the present invention is to provide cardiac organoids.
  • transplantation material comprising cardiac organoids.
  • an object of the present invention is to provide a method for evaluating drug reactivity.
  • an object of the present invention is to provide a method for evaluating drug toxicity.
  • an object of the present invention is to provide a use of the DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator for producing mature cardiac organoids.
  • an object of the present invention is to provide a method of treating cardiovascular disease.
  • the present invention provides a composition for producing mature cardiac organoids containing DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as active ingredients. .
  • the present invention provides a method for producing mature cardiac organoids.
  • the present invention provides cardiac organoids prepared by the above production method.
  • the present invention provides a transplant material comprising the cardiac organoid.
  • the present invention provides a method for evaluating drug responsiveness using the cardiac organoids.
  • the present invention provides a method for evaluating drug toxicity using the cardiac organoids.
  • the present invention also provides the use of mature cardiac organoids produced by the method of the present invention as an artificial heart.
  • the present invention provides a method of treating cardiovascular disease, comprising transplanting a mature cardiac organoid prepared by the method of the present invention into an individual suffering from cardiovascular disease.
  • a mature ventricular type cardiac organoid can be produced by treatment with DCN, and the formation and arrangement of sarcomeres is better than that of human pluripotent stem cell-derived cardiac organoids that are not treated with DCN, and T -
  • the expression of tubules shows structural maturation with increased expression, the pulsating characteristics of cardiomyocytes have more uniform and synchronized characteristics, and they are metabolically mature, so it can replace existing animal experiments, solving ethical issues. It has the effect of being used as a three-dimensional heart tissue simulator that can reduce differences in reactivity to drugs between different species.
  • Figure 1 is a photograph of a cardiac organoid produced by dispensing various numbers of human induced pluripotent stem cells.
  • Figure 2 is a diagram showing the beating rate of cardiac organoids produced by dispensing various numbers of human induced pluripotent stem cells.
  • Figure 3 is a diagram confirming the gene expression of cardiac differentiation markers and cardiac constituent cell markers in cardiac organoids produced under conditions of 5,000 cells/well and 7,500 cells/well.
  • Figure 4 is a schematic diagram showing DCN processing conditions when inducing differentiation from human induced pluripotent stem cells into cardiac organoids.
  • Figure 5 is a diagram confirming gene expression of total cardiomyocyte markers, atrial cardiomyocyte markers, nodal cardiomyocyte markers, collagen markers, and ventricular cardiomyocyte markers:
  • day 5 Heart organoids produced by processing DCN from day 5 to day 30 of differentiation;
  • day 10 Heart organoids produced by processing DCN from day 10 to day 30 of differentiation;
  • day 15 Heart organoids produced by processing DCN from day 15 to day 30 of differentiation.
  • day 25 Heart organoids produced by processing DCN from day 25 to day 30 of differentiation.
  • Figure 6 is a schematic diagram showing the conditions for processing DCN from the 5th day of differentiation induction, which is the optimal processing condition for DCN for inducing maturation and differentiation of cardiac organoids.
  • Figure 7 is a diagram confirming the morphology and beating of heart organoids treated with DCN on the 5th day of differentiation (DCN treated from the 5th day to the 30th day of differentiation) and the control heart organoids.
  • FIG. 8 is a diagram illustrating the differentiation of control and DCN-treated cardiac organoids by heart type using polymerase chain reaction (FIG. 8A), Western blot analysis (FIG. 8B), and fluorescent staining (FIG. 8C).
  • FIG. 9 is a diagram showing cardiac constituent cells in control and DCN-treated cardiac organoids confirmed by polymerase chain reaction (FIG. 9a) and Western blot analysis (FIG. 9b).
  • Figure 10 is a diagram showing the structure of blood vessels in control and DCN-treated cardiac organoids analyzed using fluorescent staining (Figure 10a) and transmission electron microscopy (Figure 10b).
  • Figure 11 is an image showing the structure of blood vessels in control and DCN-treated heart organoids confirmed by fluorescent staining ( Figure 11a), and the width, length, and junction density of blood vessels confirmed through this ( Figure 11b). .
  • Figure 12 is a diagram confirming the maturation and type-specific differentiation of blood vessels in control and DCN-treated cardiac organoids by polymerase chain reaction.
  • Figure 13 is a diagram confirming the beating characteristics (number of beats per minute, beat interval, and beat duration) of control and DCN-treated heart organoids.
  • Figure 15 is a diagram showing intracellular Ca 2+ transient analysis of control and DCN-treated cardiac organoids.
  • Figure 16 is a diagram confirming the expression of potassium channel markers in control and DCN-treated heart organoids by polymerase chain reaction.
  • Figure 17 is a diagram showing analysis of potassium ions in control and DCN-treated heart organoids using a K + indicator.
  • Figure 18 is a diagram showing the ultrastructure and sarcomere length of cardiomyocytes in control and DCN-treated cardiac organoids analyzed by fluorescence staining and transmission electron microscopy.
  • Figure 19 is a diagram showing T-tubules in control and DCN-treated heart organoids confirmed by polymerase chain reaction (Figure 19a), Western blot analysis ( Figure 19b), and fluorescent staining ( Figure 19c).
  • Figure 20 is a diagram confirming the mitochondrial arrangement in control and DCN-treated heart organoids using fluorescence staining.
  • Figure 21 is a diagram confirming the metabolic maturity of control and DCN-treated heart organoids by the number of mitochondria and density of cristae.
  • Figure 22 is a diagram confirming the metabolic maturity of control and DCN-treated heart organoids by mitochondrial activity.
  • Figure 23 shows the metabolic maturity of control and DCN-treated cardiac organoids confirmed by oxygen consumption rate analysis (basal respiration rate, maximum respiration rate, non-ATP-bound oxygen consumption rate, and ATP-bound oxygen consumption rate).
  • Figure 24 is a diagram confirming the amount of ATP in control and DCN-treated cardiac organoids.
  • Figure 25 is a diagram confirming gene expression and protein expression of cardiac metabolism-related markers in control and DCN-treated cardiac organoids.
  • Figure 26 is a diagram analyzing gene expression and protein expression of heart-related extracellular matrix markers in control and DCN-treated heart organoids.
  • Figure 27 is a diagram confirming the expression of integrins in control and DCN-treated cardiac organoids.
  • Figure 28 is a diagram confirming the expression and phosphorylation of proteins related to focal adhesion in control and DCN-treated heart organoids by Western blot analysis.
  • Figure 29 is a diagram confirming gene expression and protein expression related to the LEFTY signaling mechanism in control and DCN-treated cardiac organoids.
  • Figure 30 is a diagram evaluating reactivity in control and DCN-treated cardiac organoids by treatment with a drug that blocks potassium channels (E-4031):
  • Figure 31 is a schematic diagram showing the DCN-related signaling mechanism in cardiac organoids matured by DCN treatment.
  • the present invention provides a mature cardiac organoid (cardiac) containing a DCN (decorin) protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as an active ingredient.
  • a composition for producing organoids (CO) relates to a composition for producing organoids (CO).
  • the expression promoter may be a transcription factor that increases the expression of the DCN gene.
  • compositions of the present invention can increase the width of blood vessels, the length of blood vessels, or the density of junctions of blood vessels in cardiac organoids.
  • the mature cardiac organoid may be a mature ventricular-like cardiac organoid, and may be a functionally, structurally or metabolically mature cardiac organoid.
  • functionally mature cardiac organoids have fewer beats per minute, beat intervals and beat durations are wider and more uniform compared to immature cardiac organoids; Calcium channel markers increase; and potassium ions may increase.
  • structurally mature cardiac organoids have increased well-aligned long sarcomeres compared to immature cardiac organoids, and Z-lines are observed in the sarcomeres; The length of the sarcomere increases; T-tubules are present; And mitochondria can be arranged along the sarcomere arrangement.
  • metabolically mature cardiac organoids have an increased number of mitochondria and density of cristae compared to immature cardiac organoids; Mitochondrial activity increases; Metabolism through aerobic respiration increases; and cardiac metabolism may increase,
  • the composition of the present invention comprises MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1 ⁇ , It can increase the expression of TFAM, CPT1 ⁇ , COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2.
  • compositions of the present invention are capable of increasing co-expression of cTnT and TOM20.
  • composition of the present invention can increase phosphorylation of FAK or phosphorylation of Cofilin.
  • the present invention relates to a composition for cardiac organoid maturation, comprising DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as an active ingredient.
  • the composition of the present invention comprises MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1 ⁇ ,
  • the expression of TFAM, CPT1 ⁇ , COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2 can be increased, and the cardiovascular organoids matured by treatment with the composition of the present invention are immature cardiovascular organoids. Compared to , the expression of the gene or protein may be increased.
  • composition of the present invention can promote in vitro maturation of immature cardiac organoids derived from human pluripotent stem cells.
  • the human pluripotent stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the present invention includes the steps of a) dispensing and culturing human pluripotent stem cells to form cell aggregates; b) Differentiating cell aggregates into cardiac organoids; and c) inducing maturation and differentiation of the cardiac organoid.
  • differentiating cell aggregates into cardiac organoids includes: differentiating in medium containing a GSK-3 inhibitor; And it may include the steps of treating and culturing a Wnt inhibitor.
  • steps b) and c) may be performed for 30 days.
  • step c) may be from the 5th day to the 30th day of differentiation induction.
  • inducing maturation and differentiation of cardiac organoids may include treating the composition for maturation of cardiac organoids of the present invention.
  • the method includes i) culturing human pluripotent stem cells in E8 medium containing a Rho kinase inhibitor; ii) cultured in RPMI1640 medium containing B27-insulin (B-27 Supplement, minus insulin) containing GSK-3 inhibitor; iii) treating and culturing IWP2; iv) Cultivated in RPMI1640 culture medium containing B27-insulin (B-27 Supplement, minus insulin); and v) culturing in RPMI1640 culture medium containing B27-vitamin A (B-27 Supplement, minus vitamin A), and the composition for cardiac organoid maturation of the present invention may be treated in steps iv) or v). It can be processed from the 5th day of eruption to the 30th day of eruption.
  • the present invention includes the steps of a) dispensing and culturing human pluripotent stem cells to form cell aggregates; and b) differentiating the cell aggregates into cardiac organoids, wherein in step b), the composition for producing mature cardiac organoids of the present invention is treated.
  • the differentiation of step b) may be performed for 20 to 40 days.
  • differentiating the cell aggregates into cardiac organoids includes: i) culturing the cell aggregates in a medium containing a GSK-3 inhibitor; ii) Wnt inhibitor treatment and culturing; iii) culturing in a medium containing B27-insulin (B-27 Supplement, minus insulin); and iv) culturing in a medium containing B27-vitamin A (B-27 Supplement, vitamin minus A).
  • the composition for producing mature cardiac organoids of the present invention may be treated from the 5th day to the 15th day of differentiation, and it is more preferable to process from the 5th day to the 30th day of differentiation. do.
  • human pluripotent stem cells may be dispensed at a density of 1 x 10 3 cells/cm 2 to 1 x 10 5 cells/cm 2 , and more preferably 2.34 x 10 4 cells/cm 2 And, the stem cells can be cultured for 0.5 to 2 days in medium containing a Rho kinase inhibitor.
  • the medium containing the GSK-3 inhibitor may be RPMI1640 medium containing B27-insulin, and differentiation is induced from the moment the medium is treated.
  • differentiation may be performed for 1 to 3 days in a medium containing a GSK-3 inhibitor.
  • the cells may be treated with a Wnt inhibitor and cultured for 1 to 3 days.
  • the medium containing B27-insulin or B27-vitamins may be RPMI1640 medium containing B27-insulin or B27-vitamins.
  • step iii) may be on the 3rd to 5th day of differentiation, and may be cultured in a medium containing B27-insulin (B-27 Supplement, minus insulin) for 2 to 4 days.
  • B27-insulin B-27 Supplement, minus insulin
  • step iv) may be on the 6th to 8th day of differentiation, and may be cultured in a medium containing B27-vitamin A (B-27 Supplement, minus vitamin A) until the 20th to 40th day of differentiation.
  • B27-vitamin A B-27 Supplement, minus vitamin A
  • cardiac organoids prepared by treating DCN with the above method may have increased blood vessel width, blood vessel length, or blood vessel junction density compared to heart organoids that have not been treated with DCN.
  • cardiac organoids prepared by treating DCN with the above method may be functionally, structurally, or metabolically mature compared to cardiac organoids that have not been treated with DCN.
  • the cardiac organoids prepared by processing DCN by the above method have fewer beats per minute, and the beat interval and beat duration are wider and more uniform compared to cardiac organoids without DCN treatment; Calcium channel markers increase; and potassium ions may increase.
  • cardiac organoids prepared by treating DCN with the above method have increased well-arranged long sarcomeres compared to cardiac organoids that have not been treated with DCN, and Z-lines are observed in the sarcomeres; The length of the sarcomere increases; T-tubules are present; And mitochondria can be arranged along the sarcomere arrangement.
  • cardiac organoids prepared by treating DCN with the above method have an increased number of mitochondria and density of cristae compared to cardiac organoids not treated with DCN; Mitochondrial activity increases; Metabolism through aerobic respiration increases; and cardiac metabolism may increase,
  • cardiac organoids prepared by treating DCN with the above method have MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, Expression of CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1 ⁇ , TFAM, CPT1 ⁇ , COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2 may be increased. .
  • cardiac organoids prepared by treating DCN with the above method may have increased co-expression of cTnT and TOM20 compared to cardiac organoids that have not been treated with DCN.
  • cardiac organoids prepared by treating DCN with the above method may have increased phosphorylation of FAK or Cofilin compared to cardiac organoids that are not treated with DCN.
  • DCN (Decorin)
  • decorin (DCN) is a protein belonging to the SLRP (small leucine rich proteoglycan) class and is composed of 10-12 leucine rich repeats, and the core region is in the form of an arch.
  • TGF tumor growth factor
  • vector refers to any nucleic acid containing a competent nucleotide sequence that is inserted into a host cell, recombines with and is inserted into the host cell genome, or replicates spontaneously as an episome.
  • vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.
  • DCN protein expression promoter refers to a substance that enhances the expression of DCN protein.
  • the DCN protein expression promoter includes all substances that enhance the expression of DCN at the transcription level or protein level.
  • DCN protein activator refers to a substance that interacts directly with DCN protein or acts indirectly to positively regulate the activity of DCN protein.
  • the activator of the DCN protein may be a substance that binds to the DCN protein and enhances the activity of the DCN protein.
  • pluripotent stem cell refers to a stem cell capable of induced differentiation into any type of cell constituting the body, and pluripotent stem cells include embryonic stem cells.
  • ESCs induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • embryonic stem cells are derived from the inner cell mass of a blastocyst at the pre-implantation stage. The induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation.
  • induced pluripotent stem cells can refer to pluripotent differentiated cells created by dedifferentiating from somatic cells in the body, and somatic cells can be transformed through a process called reprogramming, such as cell fusion, nuclear replacement, and overexpression of pluripotency regulators. It is formed by creating a state very similar to embryonic stem cells.
  • pluripotent stem cells are not limited to embryonic stem cells and induced pluripotent stem cells, and may include both cells with pluripotency and self-replication ability.
  • the pluripotent stem cells may be mammalian cells, more preferably human-derived induced pluripotent stem cells.
  • human induced pluripotent stem cells refers to stem cells generated through a pluripotent differentiation step from human somatic cells and blood, and refers to any stem cell that constitutes the body. It refers to stem cells that can undergo induced differentiation into any type of cell.
  • human induced pluripotent stem cells can be formed by bringing somatic cells into a state very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear replacement, and overexpression of pluripotency regulators.
  • organoid refers to a 'mini-like organ' created using stem cells to perform minimal functions. It is made of a three-dimensional structure and is characterized by creating an environment similar to an actual body organ in the laboratory. am.
  • organoid refers to cells with a 3D three-dimensional structure, and refers to a model similar to organs such as nerves and intestines manufactured through an artificial culture process rather than collected or acquired from animals, etc.
  • the origin of the cells constituting it is not limited.
  • the organoid may have an environment that allows cells to interact with the surrounding environment during cell growth. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, in the present invention, the 3D organoid almost completely mimics organs that actually interact in vivo, and can be an excellent model for observing and developing treatments for diseases.
  • the term “differentiation” refers to a phenomenon in which cells divide and proliferate and become specialized in their structure or function while the entire organism grows. In other words, it refers to the process by which cells, tissues, etc. of an organism change into an appropriate form and function to perform the roles given to each. For example, the process by which pluripotent stem cells change into ectoderm, mesoderm, and endoderm cells, as well as the process by which progenitor cells become specific Any expression of differentiation traits can be included in differentiation.
  • the present invention relates to cardiac organoids produced by the production method of the present invention.
  • the invention relates to transplantation material comprising the cardiac organoids of the invention.
  • the present invention provides a method comprising reacting a drug with a cardiac organoid prepared by the production method of the present invention; and a method of evaluating drug responsiveness using cardiac organoids, including the step of checking in vivo dynamics.
  • the in vivo kinetics may be metabolism, absorption, membrane permeability, drug interaction, induction of drug metabolizing enzymes, or induction of drug transporters.
  • the present invention provides a method comprising reacting a drug with a cardiac organoid prepared by the production method of the present invention; and measuring conduction displacement, beat rate variation, or velocity in the cardiac organoid.
  • the present invention relates to a method of evaluating drug efficacy for cardiovascular disease, comprising the step of evaluating drug responsiveness after treating cardiac organoids prepared by the production method of the present invention with a drug.
  • the invention relates to the use of a DCN protein, a vector comprising a polynucleotide encoding a DCN protein, a DCN protein expression promoter, or a DCN protein activator in producing mature cardiac organoids.
  • the invention relates to the use of mature cardiac organoids produced by the method of the invention as an artificial heart.
  • the invention relates to a method of treating cardiovascular disease, comprising transplanting a mature cardiac organoid prepared by the method of the invention into an individual suffering from cardiovascular disease.
  • human induced pluripotent stem cells were separated into single cells using accutase and then treated with 2 ⁇ M Thiazovivin (Rho kinase inhibitor).
  • the percentage of beating organoids was 10% at 2,500 cells/well, 95.8% at 5,000 cells/well, 97.3% at 7,500 cells/well, 53.3% at 10,000 cells/well, and 95.8% at 20,000 cells/well. It was observed at 41.8% in the well ( Figure 2).
  • Example 1-1 heart differentiation markers and cardiac constituent cell markers were analyzed in organoids of 5,000 cells/well and 7,500 cells/well of organoids with a high beating rate through polymerase chain reaction. Specifically, total RNA was extracted from organoids using TRIzol, and the concentration and purity of RNA were measured using a Nanodrop absorbance device. cDNA was generated by reacting the extracted total RNA in an amount of 20 ⁇ L for 50 minutes at 37°C using M-MLV reverse transcriptase, and polymerase chain reaction was performed using iQTM SYBR Green supermix, followed by MYIQ2 Detection System.
  • cTnT a total cardiomyocyte marker
  • CD31 a vascular endothelial cell marker
  • ⁇ -SMA a smooth muscle cell marker
  • FSP1 a fibroblast marker
  • human induced pluripotent stem cells When constructing cell aggregates from human induced pluripotent stem cells to induce differentiation into cardiac organoids, human induced pluripotent stem cells were incubated with accutase to establish DCN processing conditions for inducing mature differentiation of cardiac organoids. ) were used to separate cells into single cells, and then dispensed into 96-well plates at 7.5 While inducing differentiation into cardiac organoids under the same conditions as in 1, cardiac organoids were prepared by treating DCN (100 ng/mL) from the 5th, 10th, or 15th day of differentiation to the 30th day (30th day of differentiation) ( Figure 4), on day 30 of differentiation, differentiation by heart type was compared with the control group not treated with DCN through polymerase chain reaction.
  • DCN 100 ng/mL
  • cTnT a marker for total cardiomyocytes
  • MLC2a a marker for atrial cardiomyocytes
  • TBX18 a nodal cardiomyocyte marker
  • COL1A1 and COL3A1 among collagen markers did not show significant differences in each group, but MLC2v , a marker for ventricular cardiomyocytes, and COL4A1, a collagen marker.
  • the expression of was found to increase in the group treated with DCN from the 5th day of differentiation (culture) ( Figure 5). Accordingly, the optimal processing conditions for DCN for inducing maturation and differentiation of cardiac organoids were determined to be processing DCN from the 5th day to the 30th day of differentiation induction (FIG. 6).
  • the heart type of cardiac organoids treated with DCN from day 5 to day 30 of differentiation was confirmed by polymerase chain reaction analysis, Western blot analysis, and fluorescent staining. Specifically, on day 30 of differentiation, polymerase chain reaction was performed by the method described in the above example.
  • polymerase chain reaction was performed by the method described in the above example.
  • Western blot analysis each heart organoid was washed in PBS and then lysed with 1X cell lysate containing 1 mM phenylmethylsulfonyl fluoride. Dissolved with buffer. After measuring the protein concentration through Bradford assay, 15 ⁇ g of protein was mixed with 1X loading dye and boiled for 8 minutes.
  • each protein sample was separated by electrophoresis on a 10% SDS acrylamide gel and then transferred to a polyvinylidene fluoride membrane.
  • the membrane was blocked in TBST containing 5% skim milk or BSA for 1 hour at room temperature, and then the membrane was incubated with anti-MLC2a (1:1000), anti-MLC2v (1:1000), and anti-TBX18 (1:1000). ) was reacted with the primary antibody overnight at 4°C. Afterwards, the membrane was washed three times with TBST and reacted with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour.
  • Protein bands were visualized using ECL and ECL plus and obtained using X-ray film and the ChemiDoc imaging system. Additionally, for fluorescent staining, heart organoids were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes. Fixed cells were permeabilized for 30 minutes using 0.25% Triton X-100 and blocked for 1 hour at room temperature using 5% normal goat serum (NGS). Afterwards, the cardiac organoids were reacted with the primary antibodies for each marker, anti-cardiac troponin T (cTnT, 1:400) and anti-Myosin light chain 2v (MLC2v, 1:400), respectively, overnight at 4°C.
  • PFA paraformaldehyde
  • Each cell was washed three times with PBST containing 0.1% Tween 20, and then incubated with secondary antibodies Alexa Fluor 488 chicken anti-rabbit IgG (1:1000) and Alexa Fluor 594 goat anti- for 2 hours at room temperature. They were reacted with mouse IgG (1:1000) and Alexa Fluor 594 goat anti-rabbit IgG (1:1000) antibodies, respectively, and nuclei were stained with 1 ⁇ g/mL DAPI. Fluorescence images were obtained using fluorescence microscopy and confocal fluorescence microscopy.
  • the cardiomyocyte marker CD31 the vascular cell marker CD31, and the fibroblast marker FSP1 were used. Gene and protein expression was confirmed through polymerase chain reaction and Western blot analysis.
  • Sections of 1 ⁇ m thickness were obtained using a Reichert-Jung Ultracut E ultramicrotome, stained with toluidine blue, and images were obtained using a Carl Zeiss Axio microscope. Afterwards, 60 nm thick sections per block were picked from Formvar-coated slot grids, stained with uranyl acetate/lead citrate, and samples were recorded in TEM. TEM images were analyzed using Image J image processing software, and statistics were expressed as means ⁇ standard deviation (SD) using Prism software.
  • SD standard deviation
  • FIGS. 10A and 11 As a result of fluorescence staining analysis, it was confirmed that the blood vessel width, blood vessel length, and blood vessel junction density in DCN-treated heart organoids were significantly increased compared to the control group ( FIGS. 10A and 11 ). In addition, as a result of transmission electron microscopy, it was confirmed that blood vessel lumen was generated in both control and DCN-treated heart organoids, and that large blood vessels were generated in DCN-treated heart organoids (FIG. 10b).
  • Example 2 Videos were taken for functional evaluation of the control and DCN-treated cardiac organoids prepared in Example 2, and the pulsating characteristics of the beating organoids were confirmed. Beating characteristics are determined by setting up spots in five regions with a certain area in the beating heart organoid in the video, and calculating the number of beats per minute and peak to peak duration within that area. ) and contraction-relaxation duration were measured and then graphed and analyzed (FIG. 13). In addition, since heartbeat is related to calcium channels and calcium ions, calcium channel marker expression was analyzed by polymerase chain reaction, and gene expression of calcium channel markers RyR2 and CACNA1C was found to increase in DCN-treated cardiac organoids ( Figure 14).
  • each cardiac organoid was reacted with 4 ⁇ g/mL of Fluo-4 AM for 45 minutes at 37°C to label intracellular calcium, and then analyzed using fluorescence microscopy and confocal fluorescence microscopy. After observing the fluorescence, the intensity was measured using LAS As a result, it was confirmed that DCN-treated heart organoids had fewer beats per minute compared to the control group, and that the beat interval and beat duration were wider and more uniform, and the intensity of Fluo 4-AM in the DCN-treated organoids was higher than that of the control group. It appeared strong and uniform (Figure 15).
  • T-tubules are observed in mature cardiomyocytes
  • the gene and protein expression of T-tubule markers CAV3, JPH2, and BIN1 were confirmed through polymerase chain reaction, Western blot analysis, and fluorescent staining.
  • gene expression of CAV3 , JPH2 , and BIN1 was found to be increased in DCN-treated heart organoids compared to the control group ( Figure 19a).
  • protein expression analysis also showed that CAV3 and JPH2 were increased in DCN-treated heart organoids.
  • Expression was found to be increased compared to the control group ( Figure 19b), and high expression of JPH2 was observed in DCN-treated heart organoids in fluorescence staining analysis (Figure 19c).
  • control organoids and DCN-treated heart organoids were each distributed in a 96-well plate and cultured in medium containing 1 ⁇ M of oligomycin, 2.5 ⁇ M of carbonyl cyanide-4-phenylhydrazone (FCCP), and 1 ⁇ M of antimycin A. Wow, an extracellular O2 probe was added to each well.
  • DCN-related signaling changes were confirmed in DCN-treated cardiac organoids. Specifically, it has been reported that DCN can increase its strength by binding to extracellular matrices such as collagen and fibronectin, and therefore, the heart-related extracellular matrix collagen markers (COL1A1, COL3A1, and COL4A1), fibronectin marker (FN1), and As a result of confirming the expression of the laminin marker (LAMA2) through polymerase chain reaction and Western blot analysis, it was confirmed that the expression of COL3A1, COL4A1, FN1, and LAMA2 was increased in DCN-treated heart organoids compared to the control group (FIG. 26).
  • LAMA2 laminin marker
  • DCN-treated heart organoids or control heart organoids were treated with E-4031, which blocks potassium channels, and the heart rate was measured.
  • E-4031 the group treated with DCN-treated heart organoids with E-4031 (DCN+E-4031) was observed to have a rhythm more similar to arrhythmia (FIG. 30).
  • a mature ventricular-type cardiac organoid was produced by treating cell aggregates with self-organization ability in hiPSC with DCN, and the DCN-treated cardiac organoid produced in this way had better structural and structural properties of cardiomyocytes compared to the control cardiac organoid. Metabolic, functional and molecular maturity was improved, and genes related to cardiac maturation and ventricular type cardiomyocytes were upregulated.
  • DCN-treated cardiac organoids have 1) more organized sarcomere structures, 2) more organized mitochondria, and 3) well-ordered T-tubule structures; Gene expression of 4) ventricular type cardiomyocyte markers, 5) cardiac metabolic markers, 6) T-tubule formation markers, 7) calcium and potassium ion channel markers, and 8) vascular cell markers was increased; 9) faster movement vector velocity, 10) decreased beats per minute rate, 11) increased Peak-to-Peak duration, and 12) prolonged action potential duration. Additionally, the expression of the LEFTY-PITX2 signaling mechanism, which has been reported to be activated in mature cardiac organoids, was increased ( Figure 31).
  • the mature ventricular heart organoid produced through the DCN treatment of the present invention can be usefully used for drug screening and disease modeling in the cardiac field.

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Abstract

The present invention relates to a composition for producing a mature cardiac organoid, and according to the present invention, a mature ventricular type cardiac organoid can be produced by treatment with DCN, formation and arrangement of sarcomeres is improved compared to that of human pluripotent stem cell-derived cardiac organoids not treated with DCN, expression of T-tubules shows increased structural maturation, and beating characteristics of cardiomyocytes have more uniform and synchronized characteristics and are metabolically mature, and accordingly, existing animal testing can be replaced, thereby solving ethical issues, and the effect of being capable of use as a three-dimensional heart tissue mimic that can reduce differences in reactivity to drugs between different species is provided.

Description

성숙된 심장 오가노이드 제조용 조성물Composition for producing mature cardiac organoids
본 발명은 성숙된 심장 오가노이드 제조용 조성물에 관한 것이다.The present invention relates to compositions for producing mature cardiac organoids.
허혈성 심장질환은 전세계적으로 매우 높은 유병률을 보이고 있으며, 단일질환 전체 사망원인 중 1위를 차지하고 있다. 한국 역시 사회·경제적인 발전과 서구화된 생활습관으로 인하여 허혈성 심장질환 환자들이 급증하고 있다. 더욱이 중증의 말기 심부전 환자의 경우, 심장이식술이나 기계적 좌심실 보조장치 이외에는 완치법이 없는 실정이다. 하지만 공여장기의 부족, 장기확보의 어려움, 높은 사망률 및 고가의 치료비용 등의 문제점을 가지고 있다. Ischemic heart disease has a very high prevalence worldwide and ranks first among all single diseases causing death. In Korea, the number of patients with ischemic heart disease is also rapidly increasing due to socio-economic development and westernized lifestyles. Moreover, for patients with severe end-stage heart failure, there is no cure other than heart transplantation or mechanical left ventricular assist device. However, it has problems such as lack of donor organs, difficulty in securing organs, high mortality rate, and expensive treatment costs.
인체의 손상된 조직을 재건하기 위한 이식방법에는 이종이식, 동종이식, 자가이식이 있다. 이종이식은 면역 부적합성 및 레트로바이러스를 포함하는 인수공통 병원체 전달의 문제를 갖는다. 또한, 동종이식은 제공자의 면역거부 및 이용불가능성의 문제를 가지며, 자가이식은 필요한 양만큼의 적절한 조직을 얻기 어려우며 환자에 대한 외상의 증가의 문제를 가진다. 이러한 문제점을 해결하기 위하여, 인공 대체물이나 세포를 배양하여 조직화시킨 것을 그대로 이식하려고 하는 기술이 주목받고 있다. 여러 종류의 세포로 분화할 수 있는 미분화 세포인 인간 다능성 줄기세포는 심근세포로 분화 가능하며, 2차원 배양 시스템에서 생성된 인간 다능성 줄기세포 유래 심근세포에 관한 많은 연구를 진행하고 있다. 이러한 인간 다능성 줄기세포 유래 심근세포는 심장발달, 약물 스크리닝, 질병 모델링 및 심장 회복 연구에 광범위하게 사용될 수 있다. 하지만, 기존 2차원 배양 시스템으로부터 생성된 인간 다능성 줄기세포 유래 심근세포는 배아 또는 태아 단계와 유사한 구조적 및 기능적으로 미성숙한 특성을 나타내기 때문에 성숙한 성인 심근세포와 달리 질병 모델링, 약물 스크리닝 및 심장 독성을 위한 활용에 한계점을 갖고 있다. 2004년 순수한 세포 자체를 이용하여 제작한 셀 시트(cell sheet)를 이용한 망막 재생에 관한 연구가 New England journal of Medicine에 발표된 뒤 조직공학 분야에서 세포만을 이용한 3차원 인공조직에 관한 연구가 활발히 진행되고 있다. 조직공학 분야에서 최근 인공 조직보다 주목받고 있는 것이 인공 장기 또는 미니 장기라고 불리는 오가노이드(organoid)이다. 현재 생체 내 여러 장기의 줄기세포에서 유래하는 다양한 종류의 오가노이드 모델이 구축되어 있으며, 재생의학이나 세포치료제로써 오가노이드에 대한 연구가 활발하게 진행되고 있다. 오가노이드는 줄기세포로부터 발생 및 분화를 통하여 형성된 3차원 세포 구조체로서, 해당 장기의 구조적, 기능적 특성을 재현하는 소형 장기 모사체이다. 오가노이드는 3차원 세포 구조체로서 조직을 구성하고 있는 다양한 세포로 구성되어 있고 세포외 기질(ECM, extracellular matrix), 세포 유형 및 가용성 인자를 포함하는 미세 환경을 제공해 주어 기존의 2차원 배양 시스템보다 우수한 심장 조직을 형성할 수 있다는 점에서 최근에 주목받고 있다. 따라서, 기초 생물학 연구 분야에서부터 신약개발, 질병 모델링, 재생 치료 등 다양한 응용 연구 분야에 이르기까지 폭넓게 활용되고 있다. 자가조직화는 외부의 압력 없이, 세포 간의 상호작용을 통해 스스로 조직화를 만들어내는 현상을 말한다. Transplantation methods for reconstructing damaged tissues in the human body include xenotransplantation, allogeneic transplantation, and autotransplantation. Xenotransplantation has the problems of immune incompatibility and transmission of zoonotic pathogens, including retroviruses. In addition, allogeneic transplantation has the problem of immune rejection and unavailability of the donor, and autotransplantation has the problem of difficulty in obtaining appropriate tissue in the required amount and increased trauma to the patient. In order to solve these problems, technologies that attempt to transplant artificial substitutes or cultured and organized cells are attracting attention. Human pluripotent stem cells, which are undifferentiated cells that can differentiate into various types of cells, can differentiate into cardiomyocytes, and much research is being conducted on human pluripotent stem cell-derived cardiomyocytes generated in a two-dimensional culture system. These human pluripotent stem cell-derived cardiomyocytes can be widely used in cardiac development, drug screening, disease modeling, and cardiac recovery research. However, unlike mature adult cardiomyocytes, human pluripotent stem cell-derived cardiomyocytes generated from existing two-dimensional culture systems exhibit structurally and functionally immature characteristics similar to embryonic or fetal stages, making them suitable for disease modeling, drug screening, and cardiotoxicity. There are limitations to its use. In 2004, a study on retinal regeneration using cell sheets made using pure cells itself was published in the New England Journal of Medicine, and research on three-dimensional artificial tissues using only cells has been actively conducted in the field of tissue engineering. It is becoming. In the field of tissue engineering, organoids, also called artificial organs or mini-organs, are receiving more attention than artificial tissues recently. Currently, various types of organoid models derived from stem cells of various organs in vivo have been constructed, and research on organoids as regenerative medicine or cell therapy is actively underway. Organoids are three-dimensional cell structures formed through development and differentiation from stem cells, and are small organ mimics that reproduce the structural and functional characteristics of the organ. Organoids are three-dimensional cell structures that are composed of various cells that make up tissues and provide a microenvironment containing extracellular matrix (ECM), cell types, and soluble factors, making them superior to existing two-dimensional culture systems. It has recently received attention for its ability to form heart tissue. Therefore, it is widely used in various applied research fields such as basic biology research, new drug development, disease modeling, and regenerative therapy. Self-organization refers to the phenomenon of creating self-organization through interactions between cells without external pressure.
심장발달 과정은 줄기세포로부터 심혈관 전구 세포로의 분화를 거쳐 첫 번째 및 두 번째 심장 필드, 심장 루핑(looping heart) 및 챔버 형성(chamber formation)을 거쳐 자가조직화한다. 심장 오가노이드는 자가조직화 능력이 있어 생체 내 심장을 모방하여 효과적으로 생성되나, 산소 및 영양소 분포를 촉진하는 혈관 네트워크 형성이 부족하므로 배양 배지에 의존 및 장기간 배양이 불가능하고, 생체 내 장기를 구현하기 어렵다. 따라서, 성숙화된 혈관 네트워크를 포함하는 오가노이드를 확립하기 위해서 많은 연구자는 혈관내피세포를 공배양하거나, 외인성 혈관 유도 인자를 처리하여 혈관을 포함하는 오가노이드를 확립하였다. 하지만, 신체 내 자가조직화를 구현하기 위해서는 혈관내피세포를 공배양하거나, 외인성 혈관 유도 인자를 처리하지 않고 인간 다능성 줄기세포로부터 심장을 구성하는 다양한 세포로의 동시 분화를 유도하는 것이 심장발생 및 심장조직을 모사하는 것에 중요하다. 배아 발생 과정 동안 수정란으로부터 외배엽(ectoderm), 내배엽(endoderm) 및 중배엽(mesoderm)을 포함하는 삼배엽(three germ layer)으로 분화 과정을 거쳐 기관형성 과정동안 모든 세포와 기관을 형성하게 된다. 이러한 삼배엽 중에서 심장과 혈관은 중배엽에 속한다. 따라서, 혈관 네트워크를 포함하는 심장 오가노이드 형성을 위해서는 인간 다능성 줄기세포로부터 중배엽으로의 효율적인 분화 유도가 중요하다. 종래의 연구에서는 심혈관 오가노이드를 생성할 때 세포 응집체의 크기가 중요한 매개변수라고 보고하였다. 세포 응집체는 심혈관 오가노이드로 유도하기 전에 세포 응집체의 크기 (200 μm - 450 μm)를 최적화하기 위해 일반적으로 4 - 7일 동안 배양하였다. 그러나 배양 기간이 길어질수록 인간 다능성 줄기세포 마커 (OCT4, NANOG 및 SOX2)의 발현은 점진적으로 감소하는 반면, 삼배엽 마커의 발현은 증가하였다. 즉, 세포 응집체의 장기간 배양은 특정 세포 유형에 대한 외부 자극 없이 삼배엽으로의 자발적 분화를 유도할 수 있으며, 이러한 자발적인 삼배엽으로의 분화는 외배엽과 내배엽으로의 분화를 억제시키는 반면, 중배엽 계통으로의 특이적인 분화 유도를 통해 생성되는 심혈관 오가노이드의 고효율 제작을 방해하는 요인이 될 수 있다.The process of cardiac development progresses through differentiation from stem cells into cardiovascular progenitor cells and self-organization through first and second cardiac fields, looping heart, and chamber formation. Cardiac organoids have the ability to self-organize and are effectively produced by mimicking the heart in vivo. However, they lack the formation of a vascular network that promotes oxygen and nutrient distribution, so they depend on the culture medium, cannot be cultured for long periods of time, and are difficult to implement in vivo. . Therefore, in order to establish organoids containing mature vascular networks, many researchers have established organoids containing blood vessels by co-culturing vascular endothelial cells or treating them with exogenous vascular inducing factors. However, in order to achieve self-organization in the body, it is necessary to co-culture vascular endothelial cells or induce simultaneous differentiation from human pluripotent stem cells into various cells that constitute the heart without treating exogenous vascular inducing factors, which can contribute to cardiogenesis and cardiac development. It is important for replicating an organization. During embryonic development, a fertilized egg undergoes differentiation into three germ layers including ectoderm, endoderm, and mesoderm, forming all cells and organs during organogenesis. Among these three germ layers, the heart and blood vessels belong to the mesoderm. Therefore, efficient induction of differentiation from human pluripotent stem cells into mesoderm is important for the formation of cardiac organoids containing vascular networks. Previous studies have reported that the size of cell aggregates is an important parameter when generating cardiovascular organoids. Cell aggregates were typically cultured for 4 - 7 days to optimize the size of the cell aggregates (200 μm - 450 μm) before derivation into cardiovascular organoids. However, as the culture period lengthened, the expression of human pluripotent stem cell markers (OCT4, NANOG, and SOX2) gradually decreased, while the expression of tripartite markers increased. That is, long-term culture of cell aggregates can induce spontaneous differentiation into the tripoderm without external stimulation for specific cell types, and this spontaneous differentiation into the tripoderm inhibits differentiation into the ectoderm and endoderm, whereas it can induce differentiation into the mesoderm lineage. This may be a factor that hinders the highly efficient production of cardiovascular organoids generated through specific differentiation induction.
인간 다능성 줄기세포를 이용하여 심근세포 및 심근 모사조직의 형성을 시도한 많은 연구가 있었으나, 현재까지 구조적, 기능적으로 미성숙 상태에 머물러 있어 성인에서 발병하는 질환 모델링에 사용하기엔 적합하지 않고, 대부분의 심혈관 질환은 성인이 되거나 노화가 진행된 후 발병되기 때문에, 성체 심근세포와 형태적 및 기능적으로 유사한 성숙 심근세포를 포함한 심장 오가노이드를 개발하기 위해서는 고도로 성숙된 심근세포 및 심장 오가노이드가 필요하다. There have been many studies attempting to form cardiomyocytes and myocardial tissue mimics using human pluripotent stem cells, but to date, they remain in a structurally and functionally immature state, making them unsuitable for use in modeling diseases that occur in adults, and are not suitable for use in modeling diseases that occur in adults. Because the disease develops in adulthood or after aging, highly mature cardiomyocytes and cardiac organoids are needed to develop cardiac organoids containing mature cardiomyocytes that are morphologically and functionally similar to adult cardiomyocytes.
본 발명의 목적은 성숙된 심장 오가노이드 제조용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for producing mature cardiac organoids.
또한, 본 발명의 목적은 성숙된 심장 오가노이드 제조방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for producing mature cardiac organoids.
또한, 본 발명의 목적은 심장 오가노이드를 제공하는 것이다. Additionally, an object of the present invention is to provide cardiac organoids.
또한, 본 발명의 목적은 심장 오가노이드를 포함하는 이식 재료를 제공하는 것이다.Additionally, it is an object of the present invention to provide transplantation material comprising cardiac organoids.
또한, 본 발명의 목적은 약물 반응성을 평가하는 방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for evaluating drug reactivity.
또한, 본 발명의 목적은 약물 독성을 평가하는 방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for evaluating drug toxicity.
또한, 본 발명의 목적은 DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제의 성숙된 심장 오가노이드 제조 용도를 제공하는 것이다. Additionally, an object of the present invention is to provide a use of the DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator for producing mature cardiac organoids.
또한, 본 발명의 목적은 본 발명의 방법으로 제조된 성숙된 심장 오가노이드의 인공 심장으로 사용하기 위한 용도를 제공하는 것이다. Furthermore, it is an object of the present invention to provide the use of mature cardiac organoids produced by the method of the present invention as an artificial heart.
아울러, 본 발명의 목적은 심혈관 질환의 치료 방법을 제공하는 것이다. Additionally, an object of the present invention is to provide a method of treating cardiovascular disease.
상기 과제를 해결하기 위하여, 본 발명은 DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제를 유효성분으로 포함하는 성숙된 심장 오가노이드 제조용 조성물을 제공한다.In order to solve the above problems, the present invention provides a composition for producing mature cardiac organoids containing DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as active ingredients. .
또한, 본 발명은 성숙된 심장 오가노이드 제조방법을 제공한다.Additionally, the present invention provides a method for producing mature cardiac organoids.
또한, 본 발명은 상기 제조방법으로 제조된 심장 오가노이드를 제공한다. Additionally, the present invention provides cardiac organoids prepared by the above production method.
또한, 본 발명은 상기 심장 오가노이드를 포함하는 이식 재료를 제공한다. Additionally, the present invention provides a transplant material comprising the cardiac organoid.
또한, 본 발명은 상기 심장 오가노이드를 이용한 약물 반응성을 평가하는 방법을 제공한다.Additionally, the present invention provides a method for evaluating drug responsiveness using the cardiac organoids.
또한, 본 발명은 상기 심장 오가노이드를 이용한 약물 독성을 평가하는 방법을 제공한다.Additionally, the present invention provides a method for evaluating drug toxicity using the cardiac organoids.
또한, 본 발명은 DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제의 성숙된 심장 오가노이드 제조 용도를 제공한다. Additionally, the present invention provides the use of a DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator for producing mature cardiac organoids.
또한, 본 발명은 본 발명의 방법으로 제조된 성숙된 심장 오가노이드의 인공 심장으로 사용하기 위한 용도를 제공한다. The present invention also provides the use of mature cardiac organoids produced by the method of the present invention as an artificial heart.
아울러, 본 발명은 본 발명의 방법으로 제조된 성숙된 심장 오가노이드를 심혈관 질환에 걸린 개체에 이식하는 단계를 포함하는, 심혈관 질환의 치료 방법을 제공한다. In addition, the present invention provides a method of treating cardiovascular disease, comprising transplanting a mature cardiac organoid prepared by the method of the present invention into an individual suffering from cardiovascular disease.
본 발명에 따르면, DCN의 처리에 의해 성숙한 심실 타입의 심장 오가노이드를 제조할 수 있으며, DCN을 처리하지 않은 인간 다능성 줄기세포 유래 심장 오가노이드보다 근절의 형성 및 배열이 잘 형성되어 있으며, T-세관의 발현이 증가된 구조적 성숙화를 보인고, 심근세포들의 박동 특성이 보다 균일한 동기화된 특성을 가지며, 대사적으로 성숙화되어 있으므로, 기존의 동물실험을 대체할 수 있어 윤리적 문제를 해결하고, 이종 간의 약물에 대한 반응성의 차이를 줄일 수 있는 3차원 심장조직 모사체로 이용할 수 있는 효과가 있다.According to the present invention, a mature ventricular type cardiac organoid can be produced by treatment with DCN, and the formation and arrangement of sarcomeres is better than that of human pluripotent stem cell-derived cardiac organoids that are not treated with DCN, and T -The expression of tubules shows structural maturation with increased expression, the pulsating characteristics of cardiomyocytes have more uniform and synchronized characteristics, and they are metabolically mature, so it can replace existing animal experiments, solving ethical issues. It has the effect of being used as a three-dimensional heart tissue simulator that can reduce differences in reactivity to drugs between different species.
도 1은 다양한 수의 인간 유도만능 줄기세포를 분주하여 제작한 심장 오가노이드를 촬영한 도이다.Figure 1 is a photograph of a cardiac organoid produced by dispensing various numbers of human induced pluripotent stem cells.
도 2는 다양한 수의 인간 유도만능 줄기세포를 분주하여 제작한 심장 오가노이드의 박동 비율을 나타낸 도이다.Figure 2 is a diagram showing the beating rate of cardiac organoids produced by dispensing various numbers of human induced pluripotent stem cells.
도 3은 5,000개 세포/웰 및 7,500개 세포/웰의 조건으로 제작된 심장 오가노이드에서의 심장 분화 마커 및 심장 구성 세포 마커의 유전자 발현을 확인한 도이다.Figure 3 is a diagram confirming the gene expression of cardiac differentiation markers and cardiac constituent cell markers in cardiac organoids produced under conditions of 5,000 cells/well and 7,500 cells/well.
도 4는 인간 유도만능 줄기세포로부터 심장 오가노이드로의 분화 유도시 DCN의 처리 조건을 나타낸 모식도이다.Figure 4 is a schematic diagram showing DCN processing conditions when inducing differentiation from human induced pluripotent stem cells into cardiac organoids.
도 5는 전체 심근세포 마커, 심방 심근세포 마커, 노달 심근세포 마커, 콜라겐 마커 및 심실 심근세포 마커의 유전자 발현을 확인한 도이다:Figure 5 is a diagram confirming gene expression of total cardiomyocyte markers, atrial cardiomyocyte markers, nodal cardiomyocyte markers, collagen markers, and ventricular cardiomyocyte markers:
No treat: DCN을 처리하지 않은 대조군 심장 오가노이드;No treat: control heart organoids not treated with DCN;
day 5: 분화 5일차부터 30일까지 DCN을 처리하여 제작한 심장 오가노이드;day 5: Heart organoids produced by processing DCN from day 5 to day 30 of differentiation;
day 10: 분화 10일차부터 30일까지 DCN을 처리하여 제작한 심장 오가노이드;day 10: Heart organoids produced by processing DCN from day 10 to day 30 of differentiation;
day 15: 분화 15일차부터 30일까지 DCN을 처리하여 제작한 심장 오가노이드; 및day 15: Heart organoids produced by processing DCN from day 15 to day 30 of differentiation; and
day 25: 분화 25일차부터 30일까지 DCN을 처리하여 제작한 심장 오가노이드.day 25: Heart organoids produced by processing DCN from day 25 to day 30 of differentiation.
도 6은 심장 오가노이드의 성숙 분화 유도를 위한 DCN의 최적 처리 조건인 분화 유도 5일차부터 DCN을 처리하는 조건을 나타낸 모식도이다.Figure 6 is a schematic diagram showing the conditions for processing DCN from the 5th day of differentiation induction, which is the optimal processing condition for DCN for inducing maturation and differentiation of cardiac organoids.
도 7은 분화 5일차에 DCN을 처리한 (분화 5일차~30일차까지 DCN 처리) 심장 오가노이드와 대조군 심장 오가노이드의 형태 및 박동을 확인한 도이다.Figure 7 is a diagram confirming the morphology and beating of heart organoids treated with DCN on the 5th day of differentiation (DCN treated from the 5th day to the 30th day of differentiation) and the control heart organoids.
도 8은 대조군 및 DCN 처리 심장 오가노이드의 심장 타입별 분화를 중합효소연쇄반응 (도 8a), 웨스턴 블롯 분석 (도 8b) 및 형광염색법 (도 8c)으로 분석한 도이다.Figure 8 is a diagram illustrating the differentiation of control and DCN-treated cardiac organoids by heart type using polymerase chain reaction (FIG. 8A), Western blot analysis (FIG. 8B), and fluorescent staining (FIG. 8C).
도 9는 대조군 및 DCN 처리 심장 오가노이드의의 심장 구성세포를 중합효소연쇄반응 (도 9a) 및 웨스턴블롯 분석 (도 9b)으로 확인한 도이다.Figure 9 is a diagram showing cardiac constituent cells in control and DCN-treated cardiac organoids confirmed by polymerase chain reaction (FIG. 9a) and Western blot analysis (FIG. 9b).
도 10은 대조군 및 DCN 처리 심장 오가노이드의에서 혈관의 구조를 형광염색 (도 10a) 및 투과전자현미경 (도 10b)으로 분석한 도이다.Figure 10 is a diagram showing the structure of blood vessels in control and DCN-treated cardiac organoids analyzed using fluorescent staining (Figure 10a) and transmission electron microscopy (Figure 10b).
도 11은 대조군 및 DCN 처리 심장 오가노이드의에서 혈관의 구조를 형광염색으로 확인한 이미지 (도 11a), 및 이를 통해 확인한 혈관의 너비, 혈관의 길이 및 혈관의 접합 밀도 (도 11b)를 나타낸 도이다.Figure 11 is an image showing the structure of blood vessels in control and DCN-treated heart organoids confirmed by fluorescent staining (Figure 11a), and the width, length, and junction density of blood vessels confirmed through this (Figure 11b). .
도 12는 대조군 및 DCN 처리 심장 오가노이드에서 혈관의 성숙화 및 타입별 분화를 중합효소연쇄반응으로 확인한 도이다.Figure 12 is a diagram confirming the maturation and type-specific differentiation of blood vessels in control and DCN-treated cardiac organoids by polymerase chain reaction.
도 13은 대조군 및 DCN 처리 심장 오가노이드의 박동 특성 (분당 박동 횟수, 박동 간격 및 박동 지속 기간)을 확인한 도이다.Figure 13 is a diagram confirming the beating characteristics (number of beats per minute, beat interval, and beat duration) of control and DCN-treated heart organoids.
도 14는 대조군 및 DCN 처리 심장 오가노이드에서 칼슘채널 마커 발현을 중합효소연쇄반응으로 분석한 도이다.Figure 14 is a diagram showing the analysis of calcium channel marker expression in control and DCN-treated heart organoids by polymerase chain reaction.
도 15는 대조군 및 DCN 처리 심장 오가노이드의 세포 내 Ca2+ transient 분석을 수행한 도이다.Figure 15 is a diagram showing intracellular Ca 2+ transient analysis of control and DCN-treated cardiac organoids.
도 16은 대조군 및 DCN 처리 심장 오가노이드에서 칼륨 채널 마커 발현을 중합효소 연쇄반응으로 확인한 도이다. Figure 16 is a diagram confirming the expression of potassium channel markers in control and DCN-treated heart organoids by polymerase chain reaction.
도 17은 대조군 및 DCN 처리 심장 오가노이드에서 칼륨 이온을 K+ indicator를 이용하여 분석한 도이다.Figure 17 is a diagram showing analysis of potassium ions in control and DCN-treated heart organoids using a K + indicator.
도 18은 대조군 및 DCN 처리 심장 오가노이드에서 심근세포의 미세구조 및 근절의 길이를 형광염색 및 투과전자현미경으로 분석한 도이다.Figure 18 is a diagram showing the ultrastructure and sarcomere length of cardiomyocytes in control and DCN-treated cardiac organoids analyzed by fluorescence staining and transmission electron microscopy.
도 19는 대조군 및 DCN 처리 심장 오가노이드에서 T-세관을 중합효소연쇄반응 (도 19a), 웨스턴 블롯 분석 (도 19b) 및 형광 염색법 (도 19c)으로 확인한 도이다.Figure 19 is a diagram showing T-tubules in control and DCN-treated heart organoids confirmed by polymerase chain reaction (Figure 19a), Western blot analysis (Figure 19b), and fluorescent staining (Figure 19c).
도 20은 대조군 및 DCN 처리 심장 오가노이드에서의 미토콘드리아 배열을 형광염색법으로 확인한 도이다.Figure 20 is a diagram confirming the mitochondrial arrangement in control and DCN-treated heart organoids using fluorescence staining.
도 21은 대조군 및 DCN 처리 심장 오가노이드의 대사적 성숙도를 미토콘드리아 수 및 크리스타의 밀도로 확인한 도이다.Figure 21 is a diagram confirming the metabolic maturity of control and DCN-treated heart organoids by the number of mitochondria and density of cristae.
도 22는 대조군 및 DCN 처리 심장 오가노이드의 대사적 성숙도를 미토콘드리아의 활성으로 확인한 도이다.Figure 22 is a diagram confirming the metabolic maturity of control and DCN-treated heart organoids by mitochondrial activity.
도 23은 대조군 및 DCN 처리 심장 오가노이드의 대사적 성숙도를 산소소비율 분석 (기본 호흡 비율, 최대 호흡 비율, 비 ATP 결합 산소 소비율 및 ATP 결합 산소 소비율)으로 확인한 도이다.Figure 23 shows the metabolic maturity of control and DCN-treated cardiac organoids confirmed by oxygen consumption rate analysis (basal respiration rate, maximum respiration rate, non-ATP-bound oxygen consumption rate, and ATP-bound oxygen consumption rate).
도 24는 대조군 및 DCN 처리 심장 오가노이드에서 ATP의 양을 확인한 도이다.Figure 24 is a diagram confirming the amount of ATP in control and DCN-treated cardiac organoids.
도 25는 대조군 및 DCN 처리 심장 오가노이드에서 심장 대사 관련 마커의 유전자 발현 및 단백질 발현을 확인한 도이다.Figure 25 is a diagram confirming gene expression and protein expression of cardiac metabolism-related markers in control and DCN-treated cardiac organoids.
도 26은 대조군 및 DCN 처리 심장 오가노이드에서 심장과 관련된 세포 외 기질 마커들의 유전자 발현 및 단백질 발현을 분석한 도이다.Figure 26 is a diagram analyzing gene expression and protein expression of heart-related extracellular matrix markers in control and DCN-treated heart organoids.
도 27은 대조군 및 DCN 처리 심장 오가노이드에서 인테그린의 발현을 확인한 도이다.Figure 27 is a diagram confirming the expression of integrins in control and DCN-treated cardiac organoids.
도 28은 대조군 및 DCN 처리 심장 오가노이드에서 국소접착과 관련된 단백질의 발현 및 인산화를 웨스턴 블롯 분석으로 확인한 도이다.Figure 28 is a diagram confirming the expression and phosphorylation of proteins related to focal adhesion in control and DCN-treated heart organoids by Western blot analysis.
도 29는 대조군 및 DCN 처리 심장 오가노이드에서 LEFTY 신호 기전과 관련된 유전자 발현 및 단백질 발현을 확인한 도이다.Figure 29 is a diagram confirming gene expression and protein expression related to the LEFTY signaling mechanism in control and DCN-treated cardiac organoids.
도 30은 칼륨채널을 막는 약물 (E-4031) 처리에 의한 대조군 및 DCN 처리 심장 오가노이드에서의 반응성을 평가한 도이다:Figure 30 is a diagram evaluating reactivity in control and DCN-treated cardiac organoids by treatment with a drug that blocks potassium channels (E-4031):
노란색 화살표: 부정맥과 유사한 박동.Yellow arrow: Rhythm similar to arrhythmia.
도 31은 DCN 처리에 의해 성숙화된 심장 오가노이드에서 DCN 관련 신호전달 기전을 나타낸 모식도이다.Figure 31 is a schematic diagram showing the DCN-related signaling mechanism in cardiac organoids matured by DCN treatment.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.
일 측면에서, 본 발명은 DCN(decorin) 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제를 유효성분으로 포함하는 성숙된(mature) 심장 오가노이드(cardiac organoids, CO) 제조용 조성물에 관한 것이다.In one aspect, the present invention provides a mature cardiac organoid (cardiac) containing a DCN (decorin) protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as an active ingredient. relates to a composition for producing organoids (CO).
일 구현예에서, 발현 촉진제는 DCN 유전자의 발현을 증가시키는 전사인자일 수 있다.In one embodiment, the expression promoter may be a transcription factor that increases the expression of the DCN gene.
일 구현예에서, 본 발명의 조성물은 심장 오가노이드의 혈관의 너비, 혈관의 길이 또는 혈관의 접합 밀도를 증가시킬 수 있다.In one embodiment, the compositions of the present invention can increase the width of blood vessels, the length of blood vessels, or the density of junctions of blood vessels in cardiac organoids.
일 구현예에서, 성숙된 심장 오가노이드는 성숙된 심실 타입(ventricular-like)의 심장 오가노이드일 수 있으며, 기능적, 구조적 또는 대사적으로 성숙된 심장 오가노이드일 수 있다.In one embodiment, the mature cardiac organoid may be a mature ventricular-like cardiac organoid, and may be a functionally, structurally or metabolically mature cardiac organoid.
일 구현예에서, 기능적으로 성숙된 심장 오가노이드는 미성숙된 심장 오가노이드에 비해 분당 박동 횟수는 더 적고, 박동 간격 및 박동 지속 기간은 더 넓고 균일화되며; 칼슘채널 마커가 증가하고; 및 칼륨 이온이 증가할 수 있다.In one embodiment, functionally mature cardiac organoids have fewer beats per minute, beat intervals and beat durations are wider and more uniform compared to immature cardiac organoids; Calcium channel markers increase; and potassium ions may increase.
일 구현예에서, 구조적으로 성숙된 심장 오가노이드는 미성숙된 심장 오가노이드에 비해 잘 배열된 긴 근절이 증가되며, 근절에서는 Z-선(line)이 관찰되고; 근절의 길이가 증가되고; T-세관이 존재하며; 및 근절 배열을 따라 미토콘드리아가 배열될 수 있다.In one embodiment, structurally mature cardiac organoids have increased well-aligned long sarcomeres compared to immature cardiac organoids, and Z-lines are observed in the sarcomeres; The length of the sarcomere increases; T-tubules are present; And mitochondria can be arranged along the sarcomere arrangement.
일 구현예에서, 대사적으로 성숙된 심장 오가노이드는 미성숙된 심장 오가노이드에 비해 미토콘드리아의 수 및 크리스타의 밀도가 증가하고; 미토콘드리아의 활성이 증가하며; 산소호흡에 의한 대사가 증가하고; 및 심장 대사가 증가할 수 있다,In one embodiment, metabolically mature cardiac organoids have an increased number of mitochondria and density of cristae compared to immature cardiac organoids; Mitochondrial activity increases; Metabolism through aerobic respiration increases; and cardiac metabolism may increase,
일 구현예에서, 본 발명의 조성물은 MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 또는 TGFBR2의 발현을 증가시킬 수 있다.In one embodiment, the composition of the present invention comprises MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, It can increase the expression of TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2.
일 구현예에서, 본 발명의 조성물은 cTnT 및 TOM20의 공동발현을 증가시킬 수 있다.In one embodiment, the compositions of the present invention are capable of increasing co-expression of cTnT and TOM20.
일 구현예에서, 본 발명의 조성물은 FAK의 인산화 또는 Cofilin의 인산화를 증가시킬 수 있다.In one embodiment, the composition of the present invention can increase phosphorylation of FAK or phosphorylation of Cofilin.
일 측면에서, 본 발명은 DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제를 유효성분으로 포함하는, 심장 오가노이드 성숙용 조성물에 관한 것이다.In one aspect, the present invention relates to a composition for cardiac organoid maturation, comprising DCN protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as an active ingredient.
일 구현예에서, 본 발명의 조성물은 MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 또는 TGFBR2의 발현을 증가시킬 수 있으며, 본 발명의 조성물 처리에 의해 성숙된 심혈관 오가노이드는 미성숙 심혈관 오가노이드에 비해 상기 유전자 또는 단백질의 발현이 증가될 수 있다.In one embodiment, the composition of the present invention comprises MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, The expression of TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2 can be increased, and the cardiovascular organoids matured by treatment with the composition of the present invention are immature cardiovascular organoids. Compared to , the expression of the gene or protein may be increased.
일 구현예에서, 본 발명의 조성물은 인간 다능성 줄기세포 유래 미성숙(immature) 심장 오가노이드의 in vitro 성숙을 촉진시킬 수 있다.In one embodiment, the composition of the present invention can promote in vitro maturation of immature cardiac organoids derived from human pluripotent stem cells.
일 구현예에서, 인간 다능성 줄기세포는 배아줄기세포(embryonic stem cells, ESCs) 또는 유도 만능 줄기세포(induced pluripotent stem cells, iPSCs, 역분화 줄기세포)일 수 있다.In one embodiment, the human pluripotent stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).
일 측면에서, 본 발명은 a) 인간 다능성 줄기세포를 분주 및 배양하여 세포 응집체를 형성하는 단계; b) 세포 응집체를 심장 오가노이드로 분화시키는 단계; 및 c) 심장 오가노이드의 성숙 분화를 유도하는 단계를 포함하는, 인간 유도만능 줄기세포 유래 심장 오가노이드를 제조하는 방법에 관한 것이다.In one aspect, the present invention includes the steps of a) dispensing and culturing human pluripotent stem cells to form cell aggregates; b) Differentiating cell aggregates into cardiac organoids; and c) inducing maturation and differentiation of the cardiac organoid.
일 구현예에서, 세포 응집체를 심장 오가노이드로 분화시키는 단계는: GSK-3 억제제를 포함하는 배지에서 분화시키는 단계; 및 Wnt 저해제를 처리하고 배양하는 단계를 포함할 수 있다.In one embodiment, differentiating cell aggregates into cardiac organoids includes: differentiating in medium containing a GSK-3 inhibitor; And it may include the steps of treating and culturing a Wnt inhibitor.
일 구현예에서, 상기 단계 b) 및 c)는 30일 동안 수행될 수 있다.In one embodiment, steps b) and c) may be performed for 30 days.
일 구현예에서, 상기 단계 c)는 분화 유도 5일차 부터 30일차까지일 수 있다.In one embodiment, step c) may be from the 5th day to the 30th day of differentiation induction.
일 구현예에서, 심장 오가노이드의 성숙 분화를 유도하는 단계는 본 발명의 심장 오가노이드 성숙용 조성물을 처리하는 단계를 포함할 수 있다.In one embodiment, inducing maturation and differentiation of cardiac organoids may include treating the composition for maturation of cardiac organoids of the present invention.
일 구현예에서, 상기 방법은 i) Rho kinase 억제제가 포함된 E8 배지에서 인간 다능성 줄기세포를 배양하고; ii) GSK-3 억제제가 포함된 B27-인슐린(B-27 Supplement, minus insulin) 함유 RPMI1640 배양액에서 배양하며; iii) IWP2를 처리하고 배양하고; iv) B27-인슐린(B-27 Supplement, minus insulin) 함유 RPMI1640 배양액에서 배양하며; 및 v) B27-비타민 A(B-27 Supplement, minus vitamin A) 함유 RPMI1640 배양액에서 배양하는 단계를 포함할 수 있으며, 본 발명의 심장 오가노이드 성숙용 조성물은 단계 iv) 또는 v)에 처리될 수 있고, 분화 5일차부터 분화 30일차까지 처리할 수 있다.In one embodiment, the method includes i) culturing human pluripotent stem cells in E8 medium containing a Rho kinase inhibitor; ii) cultured in RPMI1640 medium containing B27-insulin (B-27 Supplement, minus insulin) containing GSK-3 inhibitor; iii) treating and culturing IWP2; iv) Cultivated in RPMI1640 culture medium containing B27-insulin (B-27 Supplement, minus insulin); and v) culturing in RPMI1640 culture medium containing B27-vitamin A (B-27 Supplement, minus vitamin A), and the composition for cardiac organoid maturation of the present invention may be treated in steps iv) or v). It can be processed from the 5th day of eruption to the 30th day of eruption.
일 측면에서, 본 발명은 a) 인간 다능성 줄기세포를 분주 및 배양하여 세포 응집체를 형성하는 단계; 및 b) 세포 응집체를 심장 오가노이드로 분화시키는 단계를 포함하며, 상기 단계 b)에 본 발명의 성숙된 심장 오가노이드 제조용 조성물을 처리하는, 성숙된 심장 오가노이드 제조방법에 관한 것이다.In one aspect, the present invention includes the steps of a) dispensing and culturing human pluripotent stem cells to form cell aggregates; and b) differentiating the cell aggregates into cardiac organoids, wherein in step b), the composition for producing mature cardiac organoids of the present invention is treated.
일 구현예에서, 상기 단계 b)의 분화는 20 내지 40일 동안 수행될 수 있다.In one embodiment, the differentiation of step b) may be performed for 20 to 40 days.
일 구현예에서, 상기 세포 응집체를 심장 오가노이드로 분화시키는 단계는: i) 세포 응집체를 GSK-3 억제제를 포함하는 배지에서 배양하는 단계; ii) Wnt 저해제를 처리하고 배양하는 단계; iii) B27-인슐린(B-27 Supplement, minus insulin)을 포함하는 배지에서 배양하는 단계; 및 iv) B27-비타민 A(B-27 Supplement, minus vitamin A)을 포함하는 배지에서 배양하는 단계를 포함할 수 있다.In one embodiment, differentiating the cell aggregates into cardiac organoids includes: i) culturing the cell aggregates in a medium containing a GSK-3 inhibitor; ii) Wnt inhibitor treatment and culturing; iii) culturing in a medium containing B27-insulin (B-27 Supplement, minus insulin); and iv) culturing in a medium containing B27-vitamin A (B-27 Supplement, vitamin minus A).
일 구현예에서, 상기 단계 b)의 분화 기간 중, 본 발명의 성숙된 심장 오가노이드 제조용 조성물을 분화 5일차 내지 분화 15일차부터 처리할 수 있으며, 분화 5일차부터 30일차까지 처리하는 것이 더욱 바람직하다.In one embodiment, during the differentiation period of step b), the composition for producing mature cardiac organoids of the present invention may be treated from the 5th day to the 15th day of differentiation, and it is more preferable to process from the 5th day to the 30th day of differentiation. do.
일 구현예에서, 인간 다능성 줄기세포는 1 x 103개/cm2 내지 1 x 105개/cm2의 밀도로 분주될 수 있으며, 2.34 x 104개/cm2로 분주되는 것이 더욱 바람직하고, 상기 줄기세포는 Rho kinase 억제제가 포함된 배지에서 0.5 내지 2일 동안 배양될 수 있다.In one embodiment, human pluripotent stem cells may be dispensed at a density of 1 x 10 3 cells/cm 2 to 1 x 10 5 cells/cm 2 , and more preferably 2.34 x 10 4 cells/cm 2 And, the stem cells can be cultured for 0.5 to 2 days in medium containing a Rho kinase inhibitor.
일 구현예에서, GSK-3 억제제를 포함하는 배지는 B27-인슐린을 포함하는 RPMI1640 배지일 수 있으며, 상기 배지를 처리하는 순간부터 분화가 유도된다.In one embodiment, the medium containing the GSK-3 inhibitor may be RPMI1640 medium containing B27-insulin, and differentiation is induced from the moment the medium is treated.
일 구현예에서, GSK-3 억제제를 포함하는 배지에서 1 내지 3일 동안 분화시킬 수 있다.In one embodiment, differentiation may be performed for 1 to 3 days in a medium containing a GSK-3 inhibitor.
일 구현예에서, Wnt 저해제를 처리하고 1 내지 3일 동안 배양될 수 있다.In one embodiment, the cells may be treated with a Wnt inhibitor and cultured for 1 to 3 days.
\일 구현예에서, B27-인슐린 또는 B27-비타민을 포함하는 배지는 B27-인슐린 또는 B27-비타민을 포함하는 RPMI1640 배지일 수 있다.\In one embodiment, the medium containing B27-insulin or B27-vitamins may be RPMI1640 medium containing B27-insulin or B27-vitamins.
일 구현예에서, 상기 단계 iii)는 분화 3 내지 5일차일 수 있으며, B27-인슐린(B-27 Supplement, minus insulin)을 포함하는 배지에서 2 내지 4일 동안 배양될 수 있다.In one embodiment, step iii) may be on the 3rd to 5th day of differentiation, and may be cultured in a medium containing B27-insulin (B-27 Supplement, minus insulin) for 2 to 4 days.
일 구현예에서, 상기 단계 iv)는 분화 6 내지 8일차일 수 있으며, B27-비타민 A(B-27 Supplement, minus vitamin A)을 포함하는 배지에서 분화 20 내지 40일차까지 배양될 수 있다.In one embodiment, step iv) may be on the 6th to 8th day of differentiation, and may be cultured in a medium containing B27-vitamin A (B-27 Supplement, minus vitamin A) until the 20th to 40th day of differentiation.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 혈관의 너비, 혈관의 길이 또는 혈관의 접합 밀도가 증가될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method may have increased blood vessel width, blood vessel length, or blood vessel junction density compared to heart organoids that have not been treated with DCN.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 기능적, 구조적 또는 대사적으로 성숙될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method may be functionally, structurally, or metabolically mature compared to cardiac organoids that have not been treated with DCN.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 분당 박동 횟수는 더 적고, 박동 간격 및 박동 지속 기간은 더 넓고 균일화되며; 칼슘채널 마커가 증가하고; 및 칼륨 이온이 증가할 수 있다.In one embodiment, the cardiac organoids prepared by processing DCN by the above method have fewer beats per minute, and the beat interval and beat duration are wider and more uniform compared to cardiac organoids without DCN treatment; Calcium channel markers increase; and potassium ions may increase.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 잘 배열된 긴 근절이 증가되며, 근절에서는 Z-선(line)이 관찰되고; 근절의 길이가 증가되고; T-세관이 존재하며; 및 근절 배열을 따라 미토콘드리아가 배열될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method have increased well-arranged long sarcomeres compared to cardiac organoids that have not been treated with DCN, and Z-lines are observed in the sarcomeres; The length of the sarcomere increases; T-tubules are present; And mitochondria can be arranged along the sarcomere arrangement.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 미토콘드리아의 수 및 크리스타의 밀도가 증가하고; 미토콘드리아의 활성이 증가하며; 산소호흡에 의한 대사가 증가하고; 및 심장 대사가 증가할 수 있다,In one embodiment, cardiac organoids prepared by treating DCN with the above method have an increased number of mitochondria and density of cristae compared to cardiac organoids not treated with DCN; Mitochondrial activity increases; Metabolism through aerobic respiration increases; and cardiac metabolism may increase,
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 또는 TGFBR2의 발현이 증가될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method have MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, Expression of CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2 may be increased. .
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 cTnT 및 TOM20의 공동발현이 증가될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method may have increased co-expression of cTnT and TOM20 compared to cardiac organoids that have not been treated with DCN.
일 구현예에서, 상기 방법으로 DCN을 처리하여 제조된 심장 오가노이드는 DCN을 처리하지 않은 심장 오가노이드에 비해 FAK의 인산화 또는 Cofilin의 인산화가 증가될 수 있다.In one embodiment, cardiac organoids prepared by treating DCN with the above method may have increased phosphorylation of FAK or Cofilin compared to cardiac organoids that are not treated with DCN.
본 발명에서 용어, "DCN(Decorin)"은 실시예에 예시된 데코린뿐만 아니라, 본 발명에서 목표로 하는 성숙된 심장 오가노이드 제작 효율 또는 심장 오가노이드 성숙 효율을 증가시킬 수 있는 데코린의 모든 유사체(homologues)를 포함한다. 본 발명에서 데코린(decorin: DCN)은 SLRP (small leucine rich proteoglycan) 부류에 속하는 단백질로서 10-12개의 루이신 리치 리피트 (leucine rich repeat)로 구성되어 있으며, 코어 부위는 아치 (arch) 형태로 되어 있어 세포외기질에 존재하는 여러 종류의 성장인자 또는 데코린 수용체와 결합이 용이한 구조로 형성되어 있다 (Krusius T, Ruoslahti E., Proc Natl Acad Sci US A, 1986, 83(20):7683-7687; Day AA, et al., Biochem J, 1987, 248(3):801-805; Fisher LW, Termine JD, Young MF., J Biol Chem, 1989, 264(8):4571-4576). 데코린은 인간에서 DCN 유전자에 의해 암호화되며, 콜라겐 및 피브로넥틴과 같은 세포 외 기질의 섬유형성에 관여하며, 종양 성장 인자 (TGF)-β의 활성을 억제시킴으로써 콜라겐의 섬유화를 막고 세포외기질의 구성(matrix assembly)에 관여하고, 종양 세포 성장을 억제하여 종양의 형성과 성장에 천연 길항제 (antagonist)로 작용한다고 알려져 있다 (Iozzo RV., Crit Rev Biochem Mol Biol, 1997, 32(2):141-174; Isaka Y, et al., Nat Med, 1996, 2(4):418-423). In the present invention, the term "DCN (Decorin)" refers not only to the decorin exemplified in the examples, but also to all decorins that can increase the efficiency of producing mature cardiac organoids or the efficiency of cardiac organoid maturation targeted in the present invention. Contains homologues. In the present invention, decorin (DCN) is a protein belonging to the SLRP (small leucine rich proteoglycan) class and is composed of 10-12 leucine rich repeats, and the core region is in the form of an arch. It is formed in a structure that facilitates binding to various types of growth factors or decorin receptors present in the extracellular matrix (Krusius T, Ruoslahti E., Proc Natl Acad Sci US A, 1986, 83(20):7683 -7687; Day AA, et al., Biochem J, 1987, 248(3):801-805; Fisher LW, Termine JD, Young MF., J Biol Chem, 1989, 264(8):4571-4576). Decorin is encoded by the DCN gene in humans and is involved in the fibrogenesis of extracellular matrices such as collagen and fibronectin. It prevents fibrosis of collagen by inhibiting the activity of tumor growth factor (TGF)-β and organizes the extracellular matrix ( It is known to be involved in matrix assembly and to act as a natural antagonist for tumor formation and growth by inhibiting tumor cell growth (Iozzo RV., Crit Rev Biochem Mol Biol, 1997, 32(2):141-174 ; Isaka Y, et al., Nat Med, 1996, 2(4):418-423).
본 발명에서 용어, "벡터"는 숙주 세포에 삽입되어 숙주 세포 게놈과 재조합되고 이에 삽입되거나, 또는 에피좀으로서 자발적으로 복제하는 컴피턴트 뉴클레오티드 서열을 포함하는 임의의 핵산을 의미한다. 이러한 벡터로는 선형 핵산, 플라스미드, 파지미드, 코스미드, RNA 벡터, 바이러스 벡터 등이 있다. As used herein, the term “vector” refers to any nucleic acid containing a competent nucleotide sequence that is inserted into a host cell, recombines with and is inserted into the host cell genome, or replicates spontaneously as an episome. These vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.
본 명세서에 기재된 "DCN 단백질 발현 촉진제"는 DCN 단백질의 발현을 증진시키는 물질을 의미한다. 상기 DCN 단백질의 발현 촉진제는 DCN의 발현을 전사(transcription) 수준 또는 단백질 수준에서 증진시키는 물질을 모두 포함한다.“DCN protein expression promoter” as used herein refers to a substance that enhances the expression of DCN protein. The DCN protein expression promoter includes all substances that enhance the expression of DCN at the transcription level or protein level.
본 명세서에 기재된 "DCN 단백질 활성화제"는 DCN 단백질과 직접 상호작용하거나 간접적으로 작용하여 DCN 단백질의 활성을 양성적으로 조절하는 물질을 의미한다. 예를 들면, 상기 DCN 단백질의 활성화제는 DCN 단백질과 결합하여 DCN 단백질의 활성을 강화하는 물질일 수 있다.“DCN protein activator” as used herein refers to a substance that interacts directly with DCN protein or acts indirectly to positively regulate the activity of DCN protein. For example, the activator of the DCN protein may be a substance that binds to the DCN protein and enhances the activity of the DCN protein.
본 발명에서 용어, "만능 줄기세포(pluripotent stem cell, PSC)"는 몸을 구성하는 어떠한 형태의 세포로도 유도 분화가 가능한 줄기세포를 의미하며, 만능 줄기세포에는 배아줄기세포(embryonic stem cells, ESCs)와 유도 만능 줄기세포 (induced pluripotent stem cells, iPSCs, 역분화 줄기세포)가 포함된다. 구체적으로, 배아 줄기세포는 착상 이전 단계에 배반포(blastocyst)의 내부 세포 덩어리(inner cell mass)로부터 유도된다. 유도된 세포는 특정한 환경에서 유지되며, 무제한적인 배양 및 다능성 분화가 가능하다. 나아가, 유도 만능 줄기세포는 신체 체세포로부터 역분화되어 만들어지는 다능성 분화 세포를 의미할 수 있으며, 세포 융합, 핵 치환, 다분화성 조절 인자의 과발현과 같은 재프로그래밍(reprograming)이라는 과정을 통해 체세포를 배아 줄기세포와 매우 유사한 상태로 만들어줌으로써 형성된다. 나아가, 만능 줄기세포는 배아 줄기세포 및 유도 만능 줄기세포에 제한되는 것은 아니며, 분화 다능성 및 자기 복제능을 겸비한 세포를 모두 포함할 수 있다. 그러나, 바람직하게 만능 줄기세포는 포유동물의 세포, 더욱 바람직하게는 인간 유래의 유도만능 줄기세포일 수 있다.In the present invention, the term "pluripotent stem cell (PSC)" refers to a stem cell capable of induced differentiation into any type of cell constituting the body, and pluripotent stem cells include embryonic stem cells. ESCs) and induced pluripotent stem cells (iPSCs) are included. Specifically, embryonic stem cells are derived from the inner cell mass of a blastocyst at the pre-implantation stage. The induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation. Furthermore, induced pluripotent stem cells can refer to pluripotent differentiated cells created by dedifferentiating from somatic cells in the body, and somatic cells can be transformed through a process called reprogramming, such as cell fusion, nuclear replacement, and overexpression of pluripotency regulators. It is formed by creating a state very similar to embryonic stem cells. Furthermore, pluripotent stem cells are not limited to embryonic stem cells and induced pluripotent stem cells, and may include both cells with pluripotency and self-replication ability. However, preferably, the pluripotent stem cells may be mammalian cells, more preferably human-derived induced pluripotent stem cells.
본 발명에서 용어, "인간 유도만능 줄기세포(induced pluripotent stem cells, iPSCs, 역분화 줄기세포)"는 인간의 체세포 및 혈액 등에서 역분화 단계를 통하여 생성된 줄기세포를 의미하며, 몸을 구성하는 어떠한 형태의 세포로도 유도 분화가 가능한 줄기세포를 의미한다. 구체적으로, 인간 유도만능 줄기세포는 세포 융합, 핵 치환, 다분화성 조절 인자의 과발현과 같은 재프로그래밍(reprogramming)이라는 과정을 통해 체세포를 배아줄기세포와 매우 유사한 상태로 만들어줌으로써 형성될 수 있다.In the present invention, the term "human induced pluripotent stem cells (iPSCs)" refers to stem cells generated through a pluripotent differentiation step from human somatic cells and blood, and refers to any stem cell that constitutes the body. It refers to stem cells that can undergo induced differentiation into any type of cell. Specifically, human induced pluripotent stem cells can be formed by bringing somatic cells into a state very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear replacement, and overexpression of pluripotency regulators.
본 발명에서 용어, "오가노이드 (organoid)"는 줄기세포를 이용해 최소 기능을 할 수 있도록 만든 '미니 유사 장기’로서, 3차원 구조로 만들어져 실험실에서도 실제 신체 기관과 비슷한 환경을 만들 수 있는 것이 특징이다. 즉, "오가노이드(organoid)"는 3D 입체구조를 가지는 세포를 의미하며, 동물 등에서 수집, 취득하지 않은 인공적인 배양 과정을 통하여 제조한 신경, 장 등의 장기와 유사한 모델을 의미한다. 이를 구성하는 세포의 유래는 제한되지 않는다. 상기 오가노이드 (organoid)는 세포의 성장 과정에서 주변 환경과 상호 작용하도록 허용되는 환경을 가질 수 있다. 2D 배양과는 달리, 3D 세포 배양은 체외에서 세포가 모든 방향으로 성장할 수 있다. 이에 따라 본 발명에서 3D 오가노이드는 실제로 생체 내에서 상호 작용을 하고 있는 장기를 거의 완벽히 모사하여, 질병의 치료제 개발 및 등을 관찰할 수 있는 훌륭한 모델이 될 수 있다.In the present invention, the term "organoid" refers to a 'mini-like organ' created using stem cells to perform minimal functions. It is made of a three-dimensional structure and is characterized by creating an environment similar to an actual body organ in the laboratory. am. In other words, “organoid” refers to cells with a 3D three-dimensional structure, and refers to a model similar to organs such as nerves and intestines manufactured through an artificial culture process rather than collected or acquired from animals, etc. The origin of the cells constituting it is not limited. The organoid may have an environment that allows cells to interact with the surrounding environment during cell growth. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, in the present invention, the 3D organoid almost completely mimics organs that actually interact in vivo, and can be an excellent model for observing and developing treatments for diseases.
본 발명에서 용어, "분화"는 세포가 분열하여 증식하며 전체 개체가 성장하는 동안에 세포의 구조나 기능이 특수화되는 현상을 의미한다. 즉, 생물의 세포, 조직 등이 각각에게 주어지는 역할을 수행하기 위해 적합한 형태 및 기능으로 변하는 과정을 말하며, 예를 들어, 만능 줄기세포가 외배엽, 중배엽 및 내배엽 세포로 변하는 과정뿐 아니라 전구세포가 특정 분화형질을 발현하게 되는 것도 모두 분화에 포함될 수 있다.In the present invention, the term “differentiation” refers to a phenomenon in which cells divide and proliferate and become specialized in their structure or function while the entire organism grows. In other words, it refers to the process by which cells, tissues, etc. of an organism change into an appropriate form and function to perform the roles given to each. For example, the process by which pluripotent stem cells change into ectoderm, mesoderm, and endoderm cells, as well as the process by which progenitor cells become specific Any expression of differentiation traits can be included in differentiation.
일 측면에서, 본 발명은 본 발명의 제조방법으로 제조된 심장 오가노이드에 관한 것이다.In one aspect, the present invention relates to cardiac organoids produced by the production method of the present invention.
일 측면에서, 본 발명은 본 발명의 심장 오가노이드를 포함하는 이식(移植) 재료에 관한 것이다.In one aspect, the invention relates to transplantation material comprising the cardiac organoids of the invention.
일 측면에서, 본 발명은 본 발명의 제조방법으로 제조된 심장 오가노이드와 약물을 반응시키는 단계; 및 체내 동태를 확인하는 단계를 포함하는, 심장 오가노이드를 이용한 약물 반응성을 평가하는 방법에 관한 것이다.In one aspect, the present invention provides a method comprising reacting a drug with a cardiac organoid prepared by the production method of the present invention; and a method of evaluating drug responsiveness using cardiac organoids, including the step of checking in vivo dynamics.
일 구현예에서, 체내 동태가 대사, 흡수성, 막투과성, 약물 상호 작용, 약물 대사 효소의 유도 또는 약물 트랜스포터의 유도일 수 있다.In one embodiment, the in vivo kinetics may be metabolism, absorption, membrane permeability, drug interaction, induction of drug metabolizing enzymes, or induction of drug transporters.
일 측면에서, 본 발명은 본 발명의 제조방법으로 제조된 심장 오가노이드와 약물을 반응시키는 단계; 및 심장 오가노이드에서 전도 변위(conduction displacement), 심박 변이(beat rate variation) 또는 박동 속도(velocity)를 측정하는 단계를 포함하는, 심장 오가노이드를 이용한 약물 독성을 평가하는 방법에 관한 것이다.In one aspect, the present invention provides a method comprising reacting a drug with a cardiac organoid prepared by the production method of the present invention; and measuring conduction displacement, beat rate variation, or velocity in the cardiac organoid.
일 측면에서, 본 발명은 본 발명의 제조방법으로 제조된 심장 오가노이드에 약물을 처리한 후 약물 반응성을 평가하는 단계를 포함하는, 심혈관질환의 약물 효능을 평가하는 방법에 관한 것이다.In one aspect, the present invention relates to a method of evaluating drug efficacy for cardiovascular disease, comprising the step of evaluating drug responsiveness after treating cardiac organoids prepared by the production method of the present invention with a drug.
일 측면에서, 본 발명은 DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제의 성숙된 심장 오가노이드 제조 용도에 관한 것이다.In one aspect, the invention relates to the use of a DCN protein, a vector comprising a polynucleotide encoding a DCN protein, a DCN protein expression promoter, or a DCN protein activator in producing mature cardiac organoids.
일 측면에서, 본 발명은 본 발명의 방법으로 제조된 성숙된 심장 오가노이드의 인공 심장으로 사용하기 위한 용도에 관한 것이다.In one aspect, the invention relates to the use of mature cardiac organoids produced by the method of the invention as an artificial heart.
일 측면에서, 본 발명은 본 발명은 본 발명의 방법으로 제조된 성숙된 심장 오가노이드를 심혈관 질환에 걸린 개체에 이식하는 단계를 포함하는, 심혈관 질환의 치료 방법에 관한 것이다.In one aspect, the invention relates to a method of treating cardiovascular disease, comprising transplanting a mature cardiac organoid prepared by the method of the invention into an individual suffering from cardiovascular disease.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.
실시예 1. 심장 오가노이드 분화 조건 수립Example 1. Establishment of cardiac organoid differentiation conditions
1-1. 세포 수에 따른 박동 비율 확인1-1. Check the beating rate according to the number of cells
인간 유도만능 줄기세포로부터 심장 오가노이드로 분화시키기 위한 최적 세포의 수를 확립하기 위해, 인간 유도만능 줄기세포를 아큐테이즈(accutase)를 사용해 단일 세포로 분리한 뒤 2 μM의 Thiazovivin (Rho kinase 억제제)이 포함된 E8 배지 (STEMCELL Technologies)를 이용하여 96웰 플레이트에 0.25 내지 1.0 X 104 세포/웰로 분주하였으며, 원심분리기를 이용하여 세포를 가라앉혔다. 그 후, 1일 동안 배양하여 세포 응집체가 생성되면 6 μM의 농도의 GSK-3 억제제인 CHIR99021 (Sigma-Aldrich)을 첨가한 RPMI1640+B27-인슐린 배지로 48시간 동안 배양하고, 분화(배양) 2일차에 2 μM IWP2를 처리한 뒤 2일 동안 배양하였다. 분화(배양) 4일차에 RPMI1640+B27-인슐린 배지로 교체하여 배양하고, 분화(배양) 7일째에 RPMI1640+B27-비타민 A 배지로 교환해주면서 분화 30일차까지 배양한 뒤 (도 1), 박동하는 오가노이드의 비율을 측정하였다. To establish the optimal number of cells for differentiating human induced pluripotent stem cells into cardiac organoids, human induced pluripotent stem cells were separated into single cells using accutase and then treated with 2 μM Thiazovivin (Rho kinase inhibitor). ) were distributed in a 96-well plate using E8 medium (STEMCELL Technologies) containing 0.25 to 1.0 After culturing for 1 day, when cell aggregates were formed, they were cultured for 48 hours in RPMI1640+B27-insulin medium supplemented with CHIR99021 (Sigma-Aldrich), a GSK-3 inhibitor at a concentration of 6 μM, and differentiated (culture) 2 On day 1, cells were treated with 2 μM IWP2 and cultured for 2 days. On the 4th day of differentiation (culture), the medium was replaced with RPMI1640+B27-insulin medium and cultured, and on the 7th day of differentiation (culture), the medium was replaced with RPMI1640+B27-vitamin A medium and cultured until the 30th day of differentiation (Figure 1). The proportion of organoids was measured.
그 결과, 박동하는 오가노이드의 비율이 2,500개 세포/웰에서 10%, 5,000개 세포/웰에서 95.8%, 7,500개 세포/웰에서 97.3%, 10,000개 세포/웰에서 53.3% 및 20,000개 세포/웰에서 41.8%로 관찰되었다 (도 2). As a result, the percentage of beating organoids was 10% at 2,500 cells/well, 95.8% at 5,000 cells/well, 97.3% at 7,500 cells/well, 53.3% at 10,000 cells/well, and 95.8% at 20,000 cells/well. It was observed at 41.8% in the well (Figure 2).
1-2. 심장 마커 확인1-2. check heart markers
상기 실시예 1-1에서 높은 박동 비율을 가지는 5,000개 세포/웰의 오가노이드 및 7,500개 세포/웰의 오가노이드에서 심장으로의 분화 마커 및 심장 구성세포 마커를 중합효소연쇄반응을 통하여 분석하였다. 구체적으로, TRIzol을 이용하여 오가노이드에서 전체 RNA를 추출하고, RNA의 농도와 순도를 Nanodrop 흡광기기로 측정하였다. 추출한 전체 RNA를 M-MLV 역전사 효소를 이용하여 37 ℃에서 50분 동안 20 μL의 양으로 반응시켜 cDNA를 생성하고, iQTM SYBR Green supermix를 이용하여 중합효소연쇄반응을 수행한 뒤, MYIQ2 Detection System을 이용하여 분석 결과를 기록하였다. Ct 값을 기반으로 정량화하여 유전자 발현 수준을 비교하였으며, 참조 유전자로는 GAPDH를 이용하였고, 미토콘드리아의 DNA의 비율을 확인할 때는 핵 DNA를 이용하여 표준화하였다. 지놈 DNA의 증폭을 피하기 위하여, intron-spanning 프라이머를 ProbeFinder software을 이용하여 디자인하였다.In Example 1-1, heart differentiation markers and cardiac constituent cell markers were analyzed in organoids of 5,000 cells/well and 7,500 cells/well of organoids with a high beating rate through polymerase chain reaction. Specifically, total RNA was extracted from organoids using TRIzol, and the concentration and purity of RNA were measured using a Nanodrop absorbance device. cDNA was generated by reacting the extracted total RNA in an amount of 20 μL for 50 minutes at 37°C using M-MLV reverse transcriptase, and polymerase chain reaction was performed using iQTM SYBR Green supermix, followed by MYIQ2 Detection System. The analysis results were recorded using Gene expression levels were compared by quantification based on Ct values, GAPDH was used as a reference gene, and the ratio of mitochondrial DNA was standardized using nuclear DNA. To avoid amplification of genomic DNA, intron-spanning primers were designed using ProbeFinder software.
그 결과, 전체 심근세포 마커인 cTnT, 혈관내피세포 마커인 CD31, 평활근 세포 마커인 α-SMA 및 섬유아세포 마커인 FSP1의 발현 모두 5000 세포/웰 및 7500 세포/웰에서 유의한 차이를 보이지 않았으나 심실 심근세포 마커인 MLC2v의 발현이 7500 세포/웰의 오가노이드에서 증가하는 패턴을 나타냈다 (도 3). 이에, 가장 적절한 세포의 수는 96웰 플레이트를 기준으로 7.5 X 103 세포/웰인 것으로 결정하였다.As a result, the expression of cTnT , a total cardiomyocyte marker, CD31 , a vascular endothelial cell marker, α-SMA , a smooth muscle cell marker, and FSP1 , a fibroblast marker, all showed no significant difference at 5000 cells/well and 7500 cells/well, but the expression of the ventricular cell marker was The expression of MLC2v , a cardiomyocyte marker, showed an increasing pattern in organoids of 7500 cells/well (Figure 3). Accordingly, the most appropriate number of cells was determined to be 7.5 X 10 3 cells/well based on a 96-well plate.
실시예 2. DCN(decorin) 처리 심장 오가노이드 제작Example 2. Fabrication of DCN (decorin) treated cardiac organoids
2-1. DCN 처리 조건 수립2-1. Establishing DCN processing conditions
인간 유도만능 줄기세포로부터 세포 응집체를 구성하여 심장 오가노이드로 분화를 유도할 때 심장 오가노이드의 성숙 분화를 유도하기 위한 DCN의 처리 조건을 확립하기 위해, 인간 유도만능 줄기세포를 아큐테이즈(accutase)를 사용해 단일 세포로 분리한 뒤 2 μM의 Thiazovivin (Rho kinase 억제제)이 포함된 E8 배지 (STEMCELL Technologies)를 이용하여 96웰 플레이트에 7.5 X 103 세포/웰로 분주한 뒤, 상기 실시예 1-1에서와 동일한 조건으로 심장 오가노이드로의 분화를 유도하면서, DCN (100ng/mL)을 분화 5일차, 10일차 또는 15일차부터 30일까지 (분화 30일차) 처리하여 심장 오가노이드를 제작하고 (도 4), 분화 30일차에 심장 타입별 분화를 중합효소연쇄반응을 통해 DCN을 처리하지 않은 대조군과 비교하였다. When constructing cell aggregates from human induced pluripotent stem cells to induce differentiation into cardiac organoids, human induced pluripotent stem cells were incubated with accutase to establish DCN processing conditions for inducing mature differentiation of cardiac organoids. ) were used to separate cells into single cells, and then dispensed into 96-well plates at 7.5 While inducing differentiation into cardiac organoids under the same conditions as in 1, cardiac organoids were prepared by treating DCN (100 ng/mL) from the 5th, 10th, or 15th day of differentiation to the 30th day (30th day of differentiation) ( Figure 4), on day 30 of differentiation, differentiation by heart type was compared with the control group not treated with DCN through polymerase chain reaction.
그 결과, 전체 심근세포 마커인 cTnT, 심방 심근세포 마커인 MLC2a, 노달 심근세포 마커인 TBX18, 콜라겐 마커들 중 COL1A1 및 COL3A1은 각 그룹에서 큰 차이를 보이지 않았으나 심실 심근세포 마커인 MLC2v 및 콜라겐 마커 COL4A1의 발현이 분화(배양) 5일차부터 DCN을 처리한 그룹에서 증가하는 것으로 나타났다 (도 5). 이에, 심장 오가노이드의 성숙 분화 유도를 위한 DCN의 최적 처리 조건을 분화 유도 5일차부터 30일차까지 DCN을 처리하는 것으로 결정하였다 (도 6).As a result, cTnT , a marker for total cardiomyocytes, MLC2a , a marker for atrial cardiomyocytes, TBX18 , a nodal cardiomyocyte marker, and COL1A1 and COL3A1 among collagen markers did not show significant differences in each group, but MLC2v , a marker for ventricular cardiomyocytes, and COL4A1, a collagen marker. The expression of was found to increase in the group treated with DCN from the 5th day of differentiation (culture) (Figure 5). Accordingly, the optimal processing conditions for DCN for inducing maturation and differentiation of cardiac organoids were determined to be processing DCN from the 5th day to the 30th day of differentiation induction (FIG. 6).
2-2. 형태 및 박동 분석2-2. Morphology and beat analysis
분화 5일차부터 분화 30일차까지 DCN을 처리하여 배양한 심장 오가노이드 (도 6)의 형태와 박동을 분석한 결과, 대조군 및 DCN 처리 심장 오가노이드의 형태는 큰 차이가 관찰되지 않았으며, 크기는 약 2,000-2,500 μm를 차지하며, 박동하는 오가노이드는 약 100%를 차지하였다 (도 7).As a result of analyzing the shape and beating of cardiac organoids cultured with DCN from the 5th day of differentiation to the 30th day of differentiation (Figure 6), no significant difference was observed in the morphology of the control and DCN-treated heart organoids, and the size was It occupied approximately 2,000-2,500 μm, and pulsating organoids accounted for approximately 100% (Figure 7).
2-3. 심장 타입별 분화 분석2-3. Differentiation analysis by heart type
분화 5일차부터 분화 30일차까지 DCN을 처리한 심장 오가노이드의 심장 타입을 중합효소연쇄반응 분석, 웨스턴 블롯 분석 및 형광염색법으로 확인하였다. 구체적으로, 분화 30일차에 중합효소연쇄반응을 상기 실시예에 기재된 방법으로 수행하였으며, 웨스턴 블롯 분석을 위해, 심장 오가노이드를 각각 PBS에서 세척한 후, 1 mM의 phenylmethylsulfonyl fluoride가 함유된 1X 세포 용해 버퍼로 용해하였다. Bradford 분석을 통해 단백질의 농도를 측정한 뒤, 15 μg의 단백질을 1X 로딩 염색제와 섞은 후 8분간 끓였다. 이 후, 각 단백질 시료를 10%의 SDS 아크릴아마이드 젤에서 전기영동으로 분리한 뒤 polyvinylidene fluoride 멤브레인으로 트랜스퍼하였다. 상온에서 1시간 동안 멤브레인을 5%의 탈지분유 또는 BSA를 포함하는 TBST에서 블록킹한 뒤, 멤브레인을 항-MLC2a (1:1000), 항-MLC2v (1:1000) 및 항-TBX18 (1:1000)의 1차 항체와 4 ℃에서 밤새 반응시켰다. 그 후, 멤브레인을 TBST로 3번 세척하고 horseradish peroxidase가 컨쥬게이트된 2차 항체와 상온에서 1시간 동안 반응시켰다. 단백질 밴드는 ECL 및 ECL plus를 이용하여 시각화하였으며, X선 필름과 ChemiDoc 이미징 시스템을 이용하여 얻었다. 또한, 형광염색을 위해, 심장 오가노이드를 PBS를 이용하여 2번 세척한 후 4% 파라포름알데하이드(PFA)를 이용하여 20분 동안 고정하였다. 고정된 세포를 0.25%의 Triton X-100을 이용하여 30분 동안 투과시켰으며, 5% 일반 염소 혈청(NGS)를 이용하여 상온에서 1시간 동안 블록킹하였다. 그 후 심장 오가노이드를 각 마커에 대한 1차 항체인 항-cardiac troponin T (cTnT, 1:400) 및 항-Myosin light chain 2v (MLC2v, 1:400) 항체와 각각 4 ℃에서 밤새 반응시켰다. 각 세포를 PBS에 0.1%의 Tween 20이 함유된 PBST로 3번 세척한 후, 상온에서 2시간 동안 2차 항체인 Alexa Fluor 488 chicken 항-rabbit IgG (1:1000), Alexa Fluor 594 goat 항-mouse IgG (1:1000) 및 Alexa Fluor 594 goat 항-rabbit IgG (1:1000) 항체와 각각 반응시켰으며, 핵은 1 μg/mL의 DAPI로 염색하였다. 형광 이미지는 형광현미경과 공초점 형광현미경을 이용하여 얻었다. The heart type of cardiac organoids treated with DCN from day 5 to day 30 of differentiation was confirmed by polymerase chain reaction analysis, Western blot analysis, and fluorescent staining. Specifically, on day 30 of differentiation, polymerase chain reaction was performed by the method described in the above example. For Western blot analysis, each heart organoid was washed in PBS and then lysed with 1X cell lysate containing 1 mM phenylmethylsulfonyl fluoride. Dissolved with buffer. After measuring the protein concentration through Bradford assay, 15 μg of protein was mixed with 1X loading dye and boiled for 8 minutes. Afterwards, each protein sample was separated by electrophoresis on a 10% SDS acrylamide gel and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TBST containing 5% skim milk or BSA for 1 hour at room temperature, and then the membrane was incubated with anti-MLC2a (1:1000), anti-MLC2v (1:1000), and anti-TBX18 (1:1000). ) was reacted with the primary antibody overnight at 4°C. Afterwards, the membrane was washed three times with TBST and reacted with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Protein bands were visualized using ECL and ECL plus and obtained using X-ray film and the ChemiDoc imaging system. Additionally, for fluorescent staining, heart organoids were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes. Fixed cells were permeabilized for 30 minutes using 0.25% Triton X-100 and blocked for 1 hour at room temperature using 5% normal goat serum (NGS). Afterwards, the cardiac organoids were reacted with the primary antibodies for each marker, anti-cardiac troponin T (cTnT, 1:400) and anti-Myosin light chain 2v (MLC2v, 1:400), respectively, overnight at 4°C. Each cell was washed three times with PBST containing 0.1% Tween 20, and then incubated with secondary antibodies Alexa Fluor 488 chicken anti-rabbit IgG (1:1000) and Alexa Fluor 594 goat anti- for 2 hours at room temperature. They were reacted with mouse IgG (1:1000) and Alexa Fluor 594 goat anti-rabbit IgG (1:1000) antibodies, respectively, and nuclei were stained with 1 μg/mL DAPI. Fluorescence images were obtained using fluorescence microscopy and confocal fluorescence microscopy.
중합효소연쇄반응 분석 결과, 심실 심근세포 마커인 MLC2v의 발현이 DCN 처리 심장 오가노이드에서 증가한 것으로 나타났으며 (도 8a), 웨스턴 블롯 분석 결과에서도 중합효소연쇄반응과 유사하게 MLC2v의 발현이 DCN 처리 심장 오가노이드에서 증가한 반면, MLC2a 및 TBX18의 발현은 대조군과 DCN 처리 심장 오가노이드 사이에서 큰 차이를 보이지 않았다 (도 8b). 또한, 형광염색 분석 결과, 대조군에 비해 DCN 처리 심장 오가노이드에서 MLC2v의 발현이 강하게 관찰되었다 (도 8c).Polymerase chain reaction analysis showed that the expression of MLC2v, a marker of ventricular cardiomyocytes, increased in DCN-treated heart organoids (Figure 8a), and Western blot analysis also showed that, similar to polymerase chain reaction, the expression of MLC2v increased in DCN-treated heart organoids. While increased in cardiac organoids, the expression of MLC2a and TBX18 did not show significant differences between control and DCN-treated cardiac organoids (Figure 8b). Additionally, as a result of fluorescence staining analysis, strong expression of MLC2v was observed in DCN-treated heart organoids compared to the control group (Figure 8c).
실시예 3. DCN 처리 심장 오가노이드의 특성 분석Example 3. Characterization of DCN-treated cardiac organoids
3-1. 구성 세포 분석3-1. Constitutive cell analysis
심장에는 심근세포 뿐만 아니라 혈관 세포 및 섬유아세포 등이 존재하므로, DCN 처리 심장 오가노이드와 대조군 심장 오가노이드의 심장 구성 세포를 확인하기 위해, 심근세포 마커 CD31, 혈관 세포 마커 CD31 및 섬유아세포 마커 FSP1의 유전자 및 단백질 발현을 중합효소연쇄반응 및 웨스턴블롯 분석을 통해 확인하였다.Since the heart contains not only cardiomyocytes but also vascular cells and fibroblasts, in order to identify cardiac constituent cells in DCN-treated cardiac organoids and control cardiac organoids, the cardiomyocyte marker CD31, the vascular cell marker CD31, and the fibroblast marker FSP1 were used. Gene and protein expression was confirmed through polymerase chain reaction and Western blot analysis.
중합효소연쇄반응 결과, FSP1의 발현은 두 그룹에서 큰 차이를 보이지 않았으나, cTnT 및 CD31의 유전자 발현은 DCN 처리 심장 오가노이드에서 유의하게 증가된 것으로 나타났다 (도 9a). 또한, 항-cTnT (1:1000), 항-CD31 (1:1000) 및 항-FSP1 (1:1000)의 1차 항체를 이용한 웨스턴 블롯 분석 결과, cTnT 및 CD31의 발현은 DCN 처리 심장 오가노이드에서 증가하였으며, FSP1은 대조군 및 DCN 처리 심장 오가노이드에서 큰 차이를 보이지 않았다 (도 9b).As a result of polymerase chain reaction, the expression of FSP1 did not show a significant difference in the two groups, but the gene expression of cTnT and CD31 was significantly increased in DCN-treated heart organoids (Figure 9a). In addition, Western blot analysis using primary antibodies of anti-cTnT (1:1000), anti-CD31 (1:1000), and anti-FSP1 (1:1000) showed that the expression of cTnT and CD31 was significantly increased in DCN-treated cardiac organoids. and FSP1 showed no significant difference in control and DCN-treated heart organoids (Figure 9b).
3-2. 혈관 구조 분석3-2. Vascular structure analysis
DCN 처리 심장 오가노이드와 대조군 심장 오가노이드의 에서 혈관의 구조를 관찰하기 위하여, 형광염색 및 투과전자현미경을 이용하여 분석을 수행하였다. 구체적으로, 항-cTnT (1:400) 및 항-CD31 (1:400)의 1차 항체를 이용하여 형광염색을 수행하였으며, 투과전자현미경 관찰을 위해, 심장 오가노이드를 각각 2% PFA/2.5% 글루타르알데하이드(glutaraldehyde)를 이용하여 4 ℃에서 밤새 고정시켰다. 그 후 1% 오스뮴 테트록사이드(osmium tetroxide)로 후고정하고, 탈수한 뒤, Eponate-12 레진(resin)으로 감쌌다. Reichert-Jung Ultracut E ultramicrotome을 이용하여 1 μm 두께의 섹션을 얻어, 톨루이딘 블루(toluidine blue)로 염색하고 Carl Zeiss Axio 현미경으로 이미지를 얻었다. 그 후, 블록당 60 nm 두께의 섹션을 Formvar 코팅된 슬롯 그리드(slot grids)에서 골라 우라닐 아세테이트/시트르산 납으로 염색하고 샘플을 TEM에 기록하였다. TEM 이미지는 Image J 이미지 가공 소프트웨어를 이용하여 분석하였으며, 통계는 Prism 소프트웨어를 이용하여 means ± standard deviation(SD)로 나타내었다. To observe the structure of blood vessels in DCN-treated heart organoids and control heart organoids, analysis was performed using fluorescence staining and transmission electron microscopy. Specifically, fluorescent staining was performed using primary antibodies of anti-cTnT (1:400) and anti-CD31 (1:400). For transmission electron microscopy, cardiac organoids were treated with 2% PFA/2.5% PFA/2.5% PFA, respectively. It was fixed overnight at 4°C using % glutaraldehyde. Afterwards, it was post-fixed with 1% osmium tetroxide, dehydrated, and wrapped with Eponate-12 resin. Sections of 1 μm thickness were obtained using a Reichert-Jung Ultracut E ultramicrotome, stained with toluidine blue, and images were obtained using a Carl Zeiss Axio microscope. Afterwards, 60 nm thick sections per block were picked from Formvar-coated slot grids, stained with uranyl acetate/lead citrate, and samples were recorded in TEM. TEM images were analyzed using Image J image processing software, and statistics were expressed as means ± standard deviation (SD) using Prism software.
형광염색 분석 결과, DCN 처리 심장 오가노이드에서 혈관의 너비, 혈관의 길이 및 혈관의 접합 밀도가 대조군에 비해 유의적으로 증가하는 것을 확인하였다 (도 10a 및 도 11). 또한, 투과전자현미경 관찰 결과, 대조군 및 DCN 처리 심장 오가노이드 모두에서 혈관 내강(lumen)이 생성되었으며, 그 중 DCN 처리 심장 오가노이드에서 큰 혈관이 생성되었음을 확인하였다 (도 10b).As a result of fluorescence staining analysis, it was confirmed that the blood vessel width, blood vessel length, and blood vessel junction density in DCN-treated heart organoids were significantly increased compared to the control group ( FIGS. 10A and 11 ). In addition, as a result of transmission electron microscopy, it was confirmed that blood vessel lumen was generated in both control and DCN-treated heart organoids, and that large blood vessels were generated in DCN-treated heart organoids (FIG. 10b).
3-3. 혈관의 성숙화 및 타입별 분화 분석3-3. Analysis of blood vessel maturation and differentiation by type
혈관의 성숙화 및 타입별 분화를 분석하기 위하여, 중합효소연쇄반응을 실시한 결과, 성숙 혈관내피세포 마커인 vWF의 발현이 증가하였으며, 동맥혈관 세포 마커인 EFNB2 및 NRP1, 정맥혈관 세포 마커인 NRP2 및 EPHB4, 및 림프 혈관 세포 마커인 PROX1 및 LYVE1의 발현이 DCN 처리 심장 오가노이드에서 대조군에 비해 유의하게 증가하였으며, 동맥혈관 세포 마커 중 CXCR4의 발현은 큰 차이를 보이지 않았다 (도 12). To analyze vascular maturation and differentiation by type, polymerase chain reaction was performed, and as a result, the expression of vWF , a mature vascular endothelial cell marker, increased, arterial vascular cell markers EFNB2 and NRP1 , and venous vascular cell markers NRP2 and EPHB4 . , and the expression of lymphatic vascular cell markers PROX1 and LYVE1 were significantly increased in DCN-treated heart organoids compared to the control group, and the expression of CXCR4 among arterial vascular cell markers did not show a significant difference (FIG. 12).
실시예 4. DCN 처리 심장 오가노이드의 기능적 성숙도 분석Example 4. Functional maturity analysis of DCN treated cardiac organoids
상기 실시예 2에서 제조한 대조군 및 DCN 처리 심장 오가노이드의 기능적 평가를 위해 동영상을 촬영하였으며, 박동하는 오가노이드의 박동 특성을 확인하였다. 박동 특성은 동영상에서 박동하는 심장 오가노이드에서 일정한 크기의 면적을 갖는 5개 지역의 스팟(spot)을 설정하여 그 면적 내에서의 분 당 박동 횟수(Beat per minute), 박동 간격(peak to peak duration) 및 박동 지속 기간(contraction-relaxation duration)을 측정한 후 그래프로 나타내어 분석하였다 (도 13). 또한, 심장 박동은 칼슘채널 및 칼슘 이온과 관련되어 있으므로, 칼슘채널 마커 발현을 중합효소연쇄반응으로 분석한 결과, 칼슘채널 마커인 RyR2 및 CACNA1C의 유전자 발현이 DCN 처리 심장 오가노이드에서 증가하는 것으로 나타났다 (도 14). 또한, 세포 내 Ca2+ transient 분석을 위해, 각각의 심장 오가노이드를 4 μg/mL의 Fluo-4 AM와 45분간 37 ℃에서 반응시켜 세포 내의 칼슘을 표지하고, 형광현미경 및 공초점 형광현미경으로 형광을 관찰한 뒤, LAS X 소프트웨어로 강도를 측정하여 그래프로 정량화하였다. 그 결과, DCN 처리 심장 오가노이드는 대조군에 비해 분당 박동 횟수는 더 적으며, 박동 간격 및 박동 지속 기간은 더 넓고 균일화된 것을 확인하였으며, DCN 처리 오가노이드에서 Fluo 4-AM의 강도가 대조군에 비해 강하고 균일한 것으로 나타났다 (도 15). 아울러, 칼륨 이온 및 채널은 심장의 활동전위의 기간을 결정한다고 보고되어 있으므로, 중합효소 연쇄반응 및 K+ indicator를 이용하여 분석을 진행하였다. 중합효소연쇄반응은 상기 실시예에서와 같이 진행하였으며, 세포 내 K+을 분석하기 위해, 각각의 심장 오가노이드를 4 μg/mL의 Potassium indicator로 45분간 37 ℃에서 반응시켜 세포 내의 칼륨을 표지하고, 형광현미경으로 형광을 관찰하였다. 그 결과, 대조군에 비해 DCN 처리 심장 오가노이드에서 칼륨채널 마커인 KCNA4, KCNH2 및 KCNJ2의 발현이 증가하였으며, 칼륨 이온 또한 DCN 처리 심장 오가노이드에서 증가된 것으로 나타났다 (도 16 및 도 17).Videos were taken for functional evaluation of the control and DCN-treated cardiac organoids prepared in Example 2, and the pulsating characteristics of the beating organoids were confirmed. Beating characteristics are determined by setting up spots in five regions with a certain area in the beating heart organoid in the video, and calculating the number of beats per minute and peak to peak duration within that area. ) and contraction-relaxation duration were measured and then graphed and analyzed (FIG. 13). In addition, since heartbeat is related to calcium channels and calcium ions, calcium channel marker expression was analyzed by polymerase chain reaction, and gene expression of calcium channel markers RyR2 and CACNA1C was found to increase in DCN-treated cardiac organoids ( Figure 14). Additionally, for intracellular Ca 2+ transient analysis, each cardiac organoid was reacted with 4 μg/mL of Fluo-4 AM for 45 minutes at 37°C to label intracellular calcium, and then analyzed using fluorescence microscopy and confocal fluorescence microscopy. After observing the fluorescence, the intensity was measured using LAS As a result, it was confirmed that DCN-treated heart organoids had fewer beats per minute compared to the control group, and that the beat interval and beat duration were wider and more uniform, and the intensity of Fluo 4-AM in the DCN-treated organoids was higher than that of the control group. It appeared strong and uniform (Figure 15). In addition, since potassium ions and channels are reported to determine the duration of cardiac action potential, analysis was conducted using polymerase chain reaction and K + indicator. The polymerase chain reaction was performed as in the above example, and to analyze intracellular K + , each heart organoid was reacted with a 4 μg/mL Potassium indicator at 37°C for 45 minutes to label intracellular potassium. , fluorescence was observed using a fluorescence microscope. As a result, the expression of potassium channel markers KCNA4 , KCNH2 , and KCNJ2 increased in DCN-treated heart organoids compared to the control group, and potassium ions also appeared to increase in DCN-treated heart organoids (FIGS. 16 and 17).
이를 통해, 본 발명의 DCN 처리를 통해 체내 심장 발생 모사 방법으로 구축된 심장 오가노이드가 기능적으로 성숙한 심장 오가노이드로 분화되었음을 확인하였다. Through this, it was confirmed that the cardiac organoids constructed by simulating in vivo heart development through the DCN treatment of the present invention were differentiated into functionally mature cardiac organoids.
실시예 5. DCN 처리 심장 오가노이드의 구조적 성숙도 분석Example 5. Structural maturity analysis of DCN treated cardiac organoids
5-1. 심근세포의 미세구조 및 근절 길이 분석5-1. Ultrastructure and sarcomere length analysis of cardiomyocytes
대조군 및 DCN 처리 심장 오가노이드에서 심근세포의 초미세 구조(ultrastructure)을 분석하기 위하여 형광염색 및 투과전자현미경 사진을 이용하여 심근세포의 미세구조를 확인하였다. 그 결과, DCN 처리 심장 오가노이드에서는 잘 배열되고 긴 근절이 관찰되었으며 근절에서는 Z-선(line)이 관찰된 반면, 대조군에서는 불규칙적으로 배열된 짧은 근절 혹은 끊어진 근절 등이 관찰되었다 (도 18a 및 b). 근절의 길이는 심근세포의 성숙도의 지표가 될 수 있으므로, 근절의 길이를 정량화하여 그래프로 나타낸 결과, 근절의 길이는 DCN 처리 심장 오가노이드에서 약 2.1 μm로 나타났으며, 대조군에서는 약 1.8 μm로 나타났다 (도 18c).To analyze the ultrastructure of cardiomyocytes in control and DCN-treated heart organoids, fluorescent staining and transmission electron microscopy were used to confirm the ultrastructure of cardiomyocytes. As a result, well-arranged and long sarcomeres were observed in DCN-treated heart organoids, and Z-lines were observed in the sarcomeres, while irregularly arranged short sarcomeres or broken sarcomeres were observed in the control group (FIGS. 18a and b) ). Since the length of the sarcomere can be an indicator of the maturity of cardiomyocytes, the sarcomere length was quantified and graphed. As a result, the sarcomere length was found to be about 2.1 μm in the DCN-treated cardiac organoids, and about 1.8 μm in the control group. appeared (Figure 18c).
5-2. T-세관(T-tubule) 확인5-2. Identification of T-tubules
성숙한 심근세포에서는 T-세관(T-tubule)이 관찰되므로 중합효소연쇄반응, 웨스턴 블롯 분석 및 형광 염색법을 통해 T-세관 마커인 CAV3, JPH2 및 BIN1의 유전자 발현 및 단백질의 발현을 확인하였다. 그 결과, DCN 처리 심장 오가노이드에서 CAV3, JPH2 및 BIN1의 유전자 발현이 대조군에 비해 증가되는 것으로 나타났으며 (도 19a), 이러한 결과와 유사하게 단백질 발현 분석에서도 CAV3 및 JPH2가 DCN 처리 심장 오가노이드에서 대조군에 비해 발현이 증가되는 것으로 나타났고 (도 19b), 형광염색 분석에서도 DCN 처리 심장 오가노이드에서 JPH2의 높은 발현이 관찰되었다 (도 19c).Since T-tubules are observed in mature cardiomyocytes, the gene and protein expression of T-tubule markers CAV3, JPH2, and BIN1 were confirmed through polymerase chain reaction, Western blot analysis, and fluorescent staining. As a result, gene expression of CAV3 , JPH2 , and BIN1 was found to be increased in DCN-treated heart organoids compared to the control group (Figure 19a). Similar to these results, protein expression analysis also showed that CAV3 and JPH2 were increased in DCN-treated heart organoids. Expression was found to be increased compared to the control group (Figure 19b), and high expression of JPH2 was observed in DCN-treated heart organoids in fluorescence staining analysis (Figure 19c).
5-3. 근절 및 미토콘드리아 배열 확인5-3. Verification of sarcomere and mitochondrial array
성숙한 심근세포에서는 근절의 배열을 따라 미토콘드리아가 배열되므로, 항-cTnT (1:400) 및 미토콘드리아 마커인 항-TOM20 (1:400)의 1차 항체를 이용하여 형광염색 분석으로 이를 확인하였다. 그 결과, cTnT 및 TOM20의 공동발현이 대조군에 비해 DCN 처리 심장 오가노이드에서 증가되는 것으로 나타났다 (도 20).Since mitochondria are arranged along the arrangement of sarcomeres in mature cardiomyocytes, this was confirmed by fluorescence staining analysis using primary antibodies of anti-cTnT (1:400) and anti-TOM20 (1:400), a mitochondrial marker. As a result, co-expression of cTnT and TOM20 was found to be increased in DCN-treated heart organoids compared to the control group (FIG. 20).
이를 통해, 본 발명의 DCN 처리를 통해 체내 심장 발생 모사 방법으로 구축된 심장 오가노이드가 구조적으로 성숙한 심장 오가노이드로 분화되었음을 확인하였다. Through this, it was confirmed that the cardiac organoids constructed by simulating in vivo cardiac development through the DCN treatment of the present invention were differentiated into structurally mature cardiac organoids.
실시예 6. DCN 처리 심장 오가노이드의 대사적 성숙도 분석Example 6. Metabolic Maturity Analysis of DCN-Treated Cardiac Organoids
6-1. 미토콘드리아의 성숙 확인6-1. Confirmation of mitochondrial maturation
미토콘드리아가 성숙될수록 미토콘드리아의 수가 많아지며, 크리스타의 밀도가 높아져 복잡한 구조를 가진다고 보고된 바 있으므로, 대조군 및 DCN 처리 심장 오가노이드의 대사적 성숙도를 분석하기 위해, 투과전자현미경 사진을 통하여 대조군 및 DCN 처리 심장 오가노이드의 미토콘드리아를 비교하여 그래프화 함으로써 미토콘드리아의 성숙을 비교하였다. 그 결과, 미토콘드리아의 수가 DCN 처리 심장 오가노이드에서 대조군에 비해 증가하였으며, 크리스타(cristae) 구조의 밀도가 대조군에 대비하여 DCN 처리 심장 오가노이드에서 조밀하게 형성됨을 관찰할 수 있었다 (도 21). 미토콘드리아의 수와 크리스타의 밀도가 증가되었으므로, 미토콘드리아의 활성을 조사하기 위하여, 전체 살아있는 미토콘드리아를 탐지할 수 있는 MitoTracker와 ROS를 생성하는 미토콘드리아를 탐지할 수 있는 MitoSox를 염색한 결과, MitoTracker 및 MitoSox를 공동발현하는 미토콘드리아의 수가 DCN 처리 심장 오가노이드에서 증가된 것으로 나타났다 (도 22).It has been reported that as mitochondria mature, the number of mitochondria increases and the density of cristae increases, resulting in a complex structure. Therefore, in order to analyze the metabolic maturity of control and DCN-treated cardiac organoids, transmission electron micrographs were used to analyze the control and DCN-treated cardiac organoids. Mitochondrial maturation was compared by comparing and graphing the mitochondria of heart organoids. As a result, it was observed that the number of mitochondria increased in DCN-treated heart organoids compared to the control group, and that the density of cristae structures was densely formed in DCN-treated heart organoids compared to the control group (FIG. 21). As the number of mitochondria and the density of cristae increased, in order to investigate the activity of mitochondria, MitoTracker, which can detect entire living mitochondria, and MitoSox, which can detect mitochondria that produce ROS, were stained. As a result, MitoTracker and MitoSox were used together. The number of expressing mitochondria appeared to be increased in DCN treated cardiac organoids (Figure 22).
6-2. 심장 대사 활성 분석6-2. Cardiometabolic activity assay
미토콘드리아는 심장 대사에 관여하므로, 심장 오가노이드의 대사적 성숙도를 비교하기 위해, 산소호흡에 의한 대사를 Mitochondrial Stress Test Complete Assay Kit를 이용한 산소소비율 분석을 통해 확인하였다. 구체적으로, 대조군 오가노이드 및 DCN 처리 심장 오가노이드를 각각 96웰 플레이트에 분주하고, 1 μM의 올리고마이신, 2.5 μM의 FCCP(carbonyl cyanide-4-phenylhydrazone) 및 1 μM의 안티마이신 A를 포함하는 배지와, extracellular O2 probe를 각 웰에 첨가하였다. 그 후, 심장 오가노이드를 예열된 HS 미네랄오일로 감싸고 SpectraMax　i3x microplate reader을 이용하여 여기(excitation) 및 방출(emission)을 340 nm와 655 nm 파장에서 확인함으로써 형광을 측정하였다. 또한, ATP의 양을 분석하기 위하여 ATP assay를 진행하였다. 구체적으로, 심장 오가노이드를 PBS로 세척하고, 단백질을 제거하였다. 심장 오가노이드를 4 ℃에서 5분간 13,000 x g으로 원심분리하여 불용성 물질을 제거하였으며, 상층액을 96 웰 플레이트의 웰에 50 μL의 ATP assay 버퍼와 함께 배양하였으며, 50 μL의 Reaction Mix를 추가하고, 50 μL의 Background Reaction Mix를 Background control에 넣었다. 그 후 96 웰 플레이트의 빛을 차단하여 실온에서 30분간 배양하고, SpectraMax® i3x microplate reader에서 파장 λ=570 nm에서 측정하였다. 아울러, 중합효소연쇄반응 및 웨스턴 블롯 분석을 통해 심장 대사에 관련된 마커인 SIRT1, PGC1α, TFAM 및 CPT1β의 유전자 및 단백질 발현을 확인하였다.Since mitochondria are involved in cardiac metabolism, in order to compare the metabolic maturity of cardiac organoids, metabolism by aerobic respiration was confirmed through oxygen consumption rate analysis using the Mitochondrial Stress Test Complete Assay Kit. Specifically, control organoids and DCN-treated heart organoids were each distributed in a 96-well plate and cultured in medium containing 1 μM of oligomycin, 2.5 μM of carbonyl cyanide-4-phenylhydrazone (FCCP), and 1 μM of antimycin A. Wow, an extracellular O2 probe was added to each well. Afterwards, the heart organoids were wrapped in preheated HS mineral oil and fluorescence was measured by checking excitation and emission at 340 nm and 655 nm wavelengths using a SpectraMax i3x microplate reader. Additionally, an ATP assay was performed to analyze the amount of ATP. Specifically, heart organoids were washed with PBS and proteins were removed. The heart organoids were centrifuged at 13,000 50 μL of Background Reaction Mix was added to the Background control. Afterwards, the 96-well plate was blocked from light, incubated at room temperature for 30 minutes, and measured at a wavelength of λ=570 nm using a SpectraMax ® i3x microplate reader. In addition, gene and protein expression of SIRT1, PGC1α, TFAM, and CPT1β, markers related to cardiac metabolism, were confirmed through polymerase chain reaction and Western blot analysis.
산소호흡에 의한 대사를 확인한 결과, DCN 처리 심장 오가노이드에서 대조군에 비하여 기본 호흡 비율, 최대 호흡 비율 및 ATP 결합 산소 소비율이 유의적으로 증가하는 것을 확인하였다 (도 23). 또한, ATP의 양이 DCN 처리 심장 오가노이드에서 증가하는 것으로 나타났다 (도 24). 아울러, 심장 대사 관련 마커인 SIRT1, PGC1α, TFAM 및 CPT1β의 유전자 발현이 DCN 처리 심장 오가노이드에서 대조군에 비해 유의적으로 증가하였으며, PGC1α, TFAM 및 CPT1β의 단백질 발현이 DCN 처리 심장 오가노이드에서 증가된 것으로 나타났다 (도 25).As a result of confirming metabolism by aerobic respiration, it was confirmed that the basal respiration rate, maximum respiration rate, and ATP-bound oxygen consumption rate were significantly increased in DCN-treated heart organoids compared to the control group (FIG. 23). Additionally, the amount of ATP was shown to increase in DCN treated cardiac organoids (Figure 24). In addition, gene expression of cardiac metabolism-related markers SIRT1 , PGC1α , TFAM , and CPT1β was significantly increased in DCN-treated heart organoids compared to the control group, and protein expression of PGC1α, TFAM, and CPT1β was increased in DCN-treated heart organoids. It was shown (Figure 25).
실시예 7. DCN 처리 심장 오가노이드의 DCN 관련 신호전달 분석Example 7. Analysis of DCN-related signaling in DCN-treated cardiac organoids
7-1. 세포 외 기질 및 인테그린의 발현 분석7-1. Expression analysis of extracellular matrix and integrins
DCN 처리 심장 오가노이드에서의 DCN 관련 신호전달 변화를 확인하였다. 구체적으로, DCN은 콜라겐 및 피브로넥틴과 같은 세포 외 기질과 결합하여 강도를 증가시킬 수 있다고 보고되어 있으므로, 심장과 관련된 세포 외 기질인 콜라겐 마커들 (COL1A1, COL3A1 및 COL4A1), 피브로넥틴 마커 (FN1) 및 라미닌 마커 (LAMA2)의 발현을 중합효소연쇄반응 및 웨스턴 블롯 분석을 통하여 확인한 결과, COL3A1, COL4A1, FN1 및 LAMA2의 발현이 DCN 처리 심장 오가노이드에서 대조군에 비해 증가된 것을 확인하였다 (도 26). 또한, 세포 외 기질의 하위 신호기전으로 보고된 인테그린을 조사하기 위하여 웨스턴 블롯분석을 진행한 결과, ITGA5, ITGB3 및 ITGB4의 발현이 DCN 처리 심장 오가노이드에서 대조군에 비해 증가된 것으로 나타났다 (도 27).DCN-related signaling changes were confirmed in DCN-treated cardiac organoids. Specifically, it has been reported that DCN can increase its strength by binding to extracellular matrices such as collagen and fibronectin, and therefore, the heart-related extracellular matrix collagen markers (COL1A1, COL3A1, and COL4A1), fibronectin marker (FN1), and As a result of confirming the expression of the laminin marker (LAMA2) through polymerase chain reaction and Western blot analysis, it was confirmed that the expression of COL3A1, COL4A1, FN1, and LAMA2 was increased in DCN-treated heart organoids compared to the control group (FIG. 26). In addition, Western blot analysis was performed to investigate integrins, which have been reported as downstream signaling mechanisms of the extracellular matrix, and the results showed that the expression of ITGA5, ITGB3, and ITGB4 was increased in DCN-treated heart organoids compared to the control group (Figure 27) .
7-3. 국소접착의 발현 분석7-3. Expression analysis of focal adhesion
세포외기질-인테그린의 하류 신호 기전이라 알려진 국소접착과 관련된 단백질의 발현 및 인산화를 웨스턴 블롯 분석으로 확인한 결과, FAK의 인산화가 DCN 처리 심장 오가노이드에서 대조군에 비해 증가되었으며, LIMK의 발현 및 Cofilin의 인산화 또한 DCN 처리 심장 오가노이드에서 대조군에 비해 증가한 것을 확인하였다 (도 28).As a result of confirming the expression and phosphorylation of proteins related to focal adhesion, known as the downstream signaling mechanism of extracellular matrix-integrin, by Western blot analysis, phosphorylation of FAK was increased in DCN-treated heart organoids compared to the control group, and expression of LIMK and Cofilin were increased in DCN-treated heart organoids. Phosphorylation was also confirmed to be increased in DCN-treated heart organoids compared to the control group (FIG. 28).
7-4. LEFTY-PITX2 신호전달 분석7-4. LEFTY-PITX2 signaling analysis
LEFTY 신호 기전과 관련된 유전자 발현 및 단백질 발현을 웨스턴 블롯 분석으로 확인한 결과, LEFTY2, PITX2 및 TGFBR2의 유전자 발현이 DCN 처리 심장 오가노이드에서 대조군에 비해 유의하게 증가되었으며, NODAL의 유전자 발현은 대조군 및 DCN 처리 심장 오가노이드에서 큰 차이가 없는 것으로 나타났다 (도 29). 이와 유사하게 LEFTY 및 PITX2의 단백질 발현이 DCN 처리 심장 오가노이드에서 증가하였으며, NODAL은 큰 차이가 없었다 (도면 29).Gene expression and protein expression related to the LEFTY signaling mechanism were confirmed by Western blot analysis, and the gene expression of LEFTY2, PITX2, and TGFBR2 was significantly increased in DCN-treated cardiac organoids compared to the control group, and the gene expression of NODAL was significantly increased in the control and DCN-treated heart organoids. There appeared to be no significant differences in cardiac organoids (Figure 29). Similarly, protein expression of LEFTY and PITX2 increased in DCN-treated cardiac organoids, while NODAL did not differ significantly (Figure 29).
실시예 8. DCN 처리 심장 오가노이드의 약물 반응성 평가Example 8. Evaluation of drug responsiveness of DCN treated cardiac organoids
심장은 칼슘, 칼륨 및 나트륨의 이온 및 이온채널에 의해 활동전위가 조절되므로, 채널을 막는 약물들을 이용하여 약물에 대한 심장의 반응성을 평가할 수 있다. 이에, 칼륨채널을 막는 E-4031을 DCN 처리 심장 오가노이드 또는 대조군 심장 오가노이드 처리하여 박동을 측정해 본 결과, 대조군, DCN 처리 심장 오가노이드 그룹뿐만 아니라 대조군 오가노이드에 E-4031을 처리한 그룹 (대조군+E-4031)에 비해서도 DCN 처리 심장 오가노이드에 E-4031을 처리한 그룹 (DCN+E-4031)에서 부정맥에 더 유사한 박동이 나타나는 것을 관찰하였다 (도 30).Since the heart's action potential is controlled by ions and ion channels of calcium, potassium, and sodium, the heart's responsiveness to drugs can be evaluated using drugs that block the channels. Accordingly, DCN-treated heart organoids or control heart organoids were treated with E-4031, which blocks potassium channels, and the heart rate was measured. As a result, not only the control and DCN-treated heart organoid groups, but also the control organoids treated with E-4031 were found. Compared to the control group+E-4031, the group treated with DCN-treated heart organoids with E-4031 (DCN+E-4031) was observed to have a rhythm more similar to arrhythmia (FIG. 30).
상기 결과들을 통해, hiPSC에서 자가 조직화 능력을 갖는 세포 응집체에 DCN을 처리하여 성숙한 심실 타입의 심장 오가노이드를 제작하였으며, 이렇게 제작한 DCN 처리 심장 오가노이드는 대조군 심장 오가노이드에 비해 심근세포의 구조적, 대사적, 기능적 및 분자적 성숙도가 향상되었고, 심장 성숙 및 심실 타입 심근세포과 관련된 유전자가 상향 조절되었다. 구체적으로, DCN 처리 심장 오가노이드는 1) 보다 조직화된 근절 구조, 2) 더 조직화된 미토콘드리아 및 3) 잘 배열된 T-세관 구조를 가지며; 4) 심실 타입 심근세포 마커, 5) 심장 대사 마커, 6) T-세관 형성 마커, 7) 칼슘 및 칼륨 이온 채널 마커, 및 8) 혈관세포 마커의 유전자 발현이 증가되었고; 9) 더 빠른 움직임 벡터 속도, 10) 감소된 분당 박동 속도, 11) 증가된 Peak-to-Peak 지속기간, 및 12) 연장된 활동전위 기간과 같은 기능적 성숙을 나타냈다. 또한, 성숙 심장 오가노이드에서 활성화된다고 보고된 LEFTY-PITX2 신호 기전의 발현이 증가되었다 (도 31).Based on the above results, a mature ventricular-type cardiac organoid was produced by treating cell aggregates with self-organization ability in hiPSC with DCN, and the DCN-treated cardiac organoid produced in this way had better structural and structural properties of cardiomyocytes compared to the control cardiac organoid. Metabolic, functional and molecular maturity was improved, and genes related to cardiac maturation and ventricular type cardiomyocytes were upregulated. Specifically, DCN-treated cardiac organoids have 1) more organized sarcomere structures, 2) more organized mitochondria, and 3) well-ordered T-tubule structures; Gene expression of 4) ventricular type cardiomyocyte markers, 5) cardiac metabolic markers, 6) T-tubule formation markers, 7) calcium and potassium ion channel markers, and 8) vascular cell markers was increased; 9) faster movement vector velocity, 10) decreased beats per minute rate, 11) increased Peak-to-Peak duration, and 12) prolonged action potential duration. Additionally, the expression of the LEFTY-PITX2 signaling mechanism, which has been reported to be activated in mature cardiac organoids, was increased (Figure 31).
따라서, 본 발명의 DCN 처리를 통하여 제작된 성숙화된 심실 심장 오가노이드는 심장 분야에서 약물 스크리닝 및 질병 모델링에 유용하게 활용될 수 있다.Therefore, the mature ventricular heart organoid produced through the DCN treatment of the present invention can be usefully used for drug screening and disease modeling in the cardiac field.

Claims (24)

  1. DCN(decorin) 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제를 유효성분으로 포함하는 성숙된(mature) 심장 오가노이드(cardiac organoids, CO) 제조용 조성물.A composition for producing mature cardiac organoids (CO) comprising a DCN (decorin) protein, a vector containing a polynucleotide encoding the DCN protein, a DCN protein expression promoter, or a DCN protein activator as an active ingredient.
  2. 제 1항에 있어서, 혈관의 너비, 혈관의 길이 또는 혈관의 접합 밀도를 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The composition for producing mature cardiac organoids according to claim 1, which increases the width of blood vessels, the length of blood vessels, or the density of junctions of blood vessels.
  3. 제 1항에 있어서, 성숙된 심장 오가노이드는 기능적, 구조적 또는 대사적으로 성숙된 심장 오가노이드인, 성숙된 심장 오가노이드 제조용 조성물.The composition for producing a mature cardiac organoid according to claim 1, wherein the mature cardiac organoid is a functionally, structurally or metabolically mature cardiac organoid.
  4. 제 1항에 있어서, 성숙된 심장 오가노이드는 심장 오가노이드는 성숙된 심실 타입 심장 오가노이드인, 성숙된 심장 오가노이드 제조용 조성물.The composition for producing a mature cardiac organoid according to claim 1, wherein the mature cardiac organoid is a mature ventricular type cardiac organoid.
  5. 제 1항에 있어서, MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 또는 TGFBR2의 발현을 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The method of claim 1, wherein MLC2v, cTnT, CD31, vWF, EFNB2, NRP1, NRP2, EPHB4, PROX1, LYVE1, RyR2, CACNA1C, KCNA4, KCNH2, KCNJ2, CAV3, JPH2, BIN1, SIRT1, PGC1α, TFAM, CPT1β, A composition for producing mature cardiac organoids, which increases the expression of COL3A1, COL4A1, FN1, LAMA2, ITGA5, ITGB3, ITGB4, LIMK, LEFTY2, PITX2 or TGFBR2.
  6. 제 1항에 있어서, cTnT 및 TOM20의 공동발현을 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The composition of claim 1, which increases co-expression of cTnT and TOM20.
  7. 제 1항에 있어서, FAK의 인산화 또는 Cofilin의 인산화를 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The composition for producing mature cardiac organoids according to claim 1, which increases phosphorylation of FAK or phosphorylation of Cofilin.
  8. 제 1항에 있어서, 칼륨 이온을 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The composition for producing mature cardiac organoids according to claim 1, wherein the composition increases potassium ions.
  9. 제 1항에 있어서, ATP의 양을 증가시키는, 성숙된 심장 오가노이드 제조용 조성물.The composition according to claim 1, which increases the amount of ATP.
  10. a) 인간 다능성 줄기세포를 분주 및 배양하여 세포 응집체를 형성하는 단계; 및a) dispensing and culturing human pluripotent stem cells to form cell aggregates; and
    b) 세포 응집체를 심장 오가노이드로 분화시키는 단계를 포함하며, 상기 단계 b)에 제 1항의 성숙된 심장 오가노이드 제조용 조성물을 처리하는, 성숙된 심장 오가노이드 제조방법.b) Differentiating cell aggregates into cardiac organoids, wherein in step b), the composition for producing mature cardiac organoids of claim 1 is treated.
  11. 제 10항에 있어서, 인간 다능성 줄기세포는 1 x 103개/cm2 내지 1 x 105개/cm2의 밀도로 분주되는 성숙된 심장 오가노이드 제조방법.The method of claim 10, wherein the human pluripotent stem cells are dispensed at a density of 1 x 10 3 cells/cm 2 to 1 x 10 5 cells/cm 2 .
  12. 제 10항에 있어서, 인간 다능성 줄기세포를 Rho kinase 억제제가 포함된 배지에서 배양하는, 성숙된 심장 오가노이드 제조방법.The method of claim 10, wherein human pluripotent stem cells are cultured in a medium containing a Rho kinase inhibitor.
  13. 제 10항에 있어서, 인간 다능성 줄기세포를 0.5 내지 2일 동안 배양하는, 성숙된 심장 오가노이드 제조방법.The method of claim 10, wherein human pluripotent stem cells are cultured for 0.5 to 2 days.
  14. 제 10항에 있어서, 세포 응집체를 심장 오가노이드로 분화시키는 단계는:11. The method of claim 10, wherein differentiating cell aggregates into cardiac organoids comprises:
    i) 세포 응집체를 GSK-3 억제제를 포함하는 배지에서 배양하는 단계;i) culturing the cell aggregates in a medium containing a GSK-3 inhibitor;
    ii) Wnt 저해제를 처리하고 배양하는 단계; ii) Wnt inhibitor treatment and culturing;
    iii) B27-인슐린(B-27 Supplement, minus insulin)을 포함하는 배지에서 배양하는 단계; 및 iii) culturing in a medium containing B27-insulin (B-27 Supplement, minus insulin); and
    iv) B27-비타민 A(B-27 Supplement, minus vitamin A)을 포함하는 배지에서 배양하는 단계를 포함하는, 성숙된 심장 오가노이드 제조방법.iv) A method of producing mature heart organoids, comprising culturing in a medium containing B27-vitamin A (B-27 Supplement, minus vitamin A).
  15. 제 10항에 있어서, 단계 b)의 분화는 20 내지 40일 동안 수행되는, 성숙된 심장 오가노이드 제조방법.11. The method of claim 10, wherein the differentiation in step b) is performed for 20 to 40 days.
  16. 제 10항에 있어서, 제 1항의 성숙된 심장 오가노이드 제조용 조성물을 분화 5일차 내지 분화 15일차부터 처리하는, 성숙된 심장 오가노이드 제조방법.The method of claim 10, wherein the composition for producing mature heart organoids of claim 1 is treated from the 5th day to the 15th day of differentiation.
  17. 제 10항의 제조방법으로 제조된 심장 오가노이드.A cardiac organoid prepared by the method of claim 10.
  18. 제 17항의 심장 오가노이드를 포함하는 이식(移植) 재료.A transplant material comprising the cardiac organoid of claim 17.
  19. 제 10항의 제조방법으로 제조된 심장 오가노이드와 약물을 반응시키는 단계; 및 체내 동태를 확인하는 단계를 포함하는, 심장 오가노이드를 이용한 약물 반응성을 평가하는 방법.Reacting the heart organoid prepared by the method of claim 10 with a drug; And a method for evaluating drug responsiveness using cardiac organoids, including the step of checking in vivo dynamics.
  20. 제 19항에 있어서, 체내 동태가 대사, 흡수성, 막투과성, 약물 상호 작용, 약물 대사 효소의 유도 또는 약물 트랜스포터의 유도인, 심장 오가노이드를 이용한 약물 반응성을 평가하는 방법.The method of claim 19, wherein the in vivo kinetics are metabolism, absorption, membrane permeability, drug interaction, induction of drug metabolizing enzymes, or induction of drug transporters.
  21. 제 10항의 제조방법으로 제조된 심장 오가노이드와 약물을 반응시키는 단계; 및 심장 오가노이드에서 전도 변위(conduction displacement), 심박 변이(beat rate variation) 또는 박동 속도(velocity)를 측정하는 단계를 포함하는, 심장 오가노이드를 이용한 약물 독성을 평가하는 방법.Reacting the heart organoid prepared by the method of claim 10 with a drug; and measuring conduction displacement, beat rate variation, or velocity in the cardiac organoid.
  22. DCN 단백질, DCN 단백질을 암호화하는 폴리뉴클레오티드를 포함하는 벡터, DCN 단백질 발현 촉진제 또는 DCN 단백질 활성화제의 성숙된 심장 오가노이드 제조 용도.Use in the production of mature cardiac organoids of a DCN protein, a vector comprising a polynucleotide encoding a DCN protein, a DCN protein expression promoter, or a DCN protein activator.
  23. 인공 심장으로 사용하기 위한, 제 10항의 방법으로 제조된 성숙된 심장 오가노이드의 용도.Use of a mature cardiac organoid prepared by the method of claim 10 for use as an artificial heart.
  24. 제 10항의 방법으로 제조된 성숙된 심장 오가노이드를 심혈관 질환에 걸린 개체에 이식하는 단계를 포함하는, 심혈관 질환의 치료 방법.A method of treating cardiovascular disease, comprising transplanting a mature cardiac organoid prepared by the method of claim 10 into a subject suffering from cardiovascular disease.
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