KR20240078988A - Composition for promoting organoid maturation - Google Patents

Abstract

The present invention relates to a composition for maturing cardiovascular organoids and its use. In the present invention, LIMK promotes maturation of cardiomyocytes in immature cardiovascular organoids and promotes vascular network formation to form vascularized mature ventricular type cardiovascular organoids. Since it has been confirmed that it is manufactured, it can be used as a composition for maturing cardiovascular organoids, and the mature cardiovascular organoids manufactured using it can be usefully used in artificial hearts, disease modeling, drug screening, and toxicity evaluation of new drugs.

Description

{Composition for promoting organoid maturation}

The present invention relates to compositions for maturing cardiovascular organoids and uses thereof.

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. 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 a regenerative medicine or cell therapy agent 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 attracted attention because it can 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.

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.

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.

An object of the present invention is to provide a composition for maturation of cardiovascular organoids.

Additionally, an object of the present invention is to provide a support composition for cardiovascular organoid transplantation.

Additionally, an object of the present invention is to provide a method for producing mature cardiovascular organoids in vitro .

Additionally, an object of the present invention is to provide a method for maturing immature cardiovascular organoids in vitro .

In order to solve the above problems, the present invention provides a composition for cardiovascular organoid maturation containing LIMK (LIM Kinase) protein, a vector containing a polynucleotide encoding the LIMK protein, a LIMK protein expression promoter, or a LIMK protein activator as an active ingredient. provides.

Additionally, the present invention provides a support composition for cardiovascular organoid transplantation comprising the composition.

Additionally, the present invention provides a method for producing mature cardiovascular organoids in vitro .

Additionally, the present invention provides a method for maturing immature cardiovascular organoids in vitro .

According to the present invention, it has been confirmed that LIMK promotes the maturation of cardiomyocytes in immature cardiovascular organoids and promotes the formation of a vascular network to produce vascularized mature ventricular type cardiovascular organoids, which can be used as a composition for maturing cardiovascular organoids. It can be used as a mature cardiovascular organoid manufactured using it, and can be useful for artificial hearts, disease modeling, drug screening, and new drug toxicity evaluation.

Figure 1 is a diagram confirming the optimal cell distribution density for forming cell aggregates when producing cardiovascular organoids derived from human pluripotent stem cells.
Figure 2 is a diagram confirming the stem function and differentiation ability of cell aggregates when producing cardiovascular organoids.
Figure 3 is a diagram showing the morphological characteristics of cardiovascular organoids after dividing them by size.
Figure 4 shows the differentiation efficiency of CO (H-CO) differentiated from cell aggregates cultured for 2 days with relatively high stem function and CO (L-CO) differentiated from cell aggregates cultured for 5 days with low stem function and cardiomyocytes This is a diagram that confirms the type.
Figure 5 is a diagram confirming the formation of chambers of H-CO and L-CO.
Figures 6 and 7 are diagrams confirming the structural and metabolic maturity of H-CO and L-CO.
Figure 8 is a diagram confirming the junction between H-CO and L-CO cardiomyocytes.
Figures 9 to 12 are diagrams analyzing the functional maturity of H-CO and L-CO and action potentials by cardiomyocyte type.
Figure 13 is a diagram confirming the formation of vascular networks of H-CO and L-CO.
Figure 14 is a diagram showing the results of immunofluorescence staining and gene expression analysis for the production of fibroblasts.
Figures 15 and 16 are diagrams confirming the vascular maturity of H-CO and L-CO.
Figures 17 and 18 are diagrams analyzing the gene signaling mechanism pathways involved in cardiomyocyte maturation and vascular network formation in cardiovascular organoids.
Figure 19 shows the results of analyzing the ventricular/myocardial maturation and angiogenic differentiation mechanisms of H-CO and L-CO.
Figure 20 is a diagram confirming CM maturity and angiogenesis in CO through inhibition of the LIMK/Cofilin signaling pathway.
Figure 21 is a diagram showing the production of induced pluripotent stem cell-derived CO and analysis of its characteristics.

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.

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.

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.

In one aspect, the present invention provides cardiovascular organoids (CO) containing LIMK (LIM Kinase) protein, a vector containing a polynucleotide encoding the LIMK protein, a LIMK protein expression promoter, or a LIMK protein activator as an active ingredient. It relates to a composition for maturation.

In one embodiment, the expression promoter may be a transcription factor that increases the expression of the LIMK gene.

In one embodiment, the composition of the present invention can promote the maturation of cardiomyocytes, structural maturation and functional maturation of cardiomyocytes, structural maturation including advanced/organized intercalated disc and maturation of mitochondria. And, functional maturity may mean having ventricular-type beat characteristics and action potentials.

In one embodiment, the composition of the present invention can promote the formation of a vascular network, and the vascular network can include vascular endothelial cells, perivascular cells, vascular smooth muscle cells, and basement membrane cells.

In one embodiment, the composition of the present invention can increase gene and protein expression of cTnT, MLC2v, MLC2a or JPH2 in cardiovascular organoids compared to immature cardiovascular organoids.

In one embodiment, the compositions of the invention can increase gene and protein expression of PDGFRβ, αSMA or vWF in cardiovascular organoids compared to immature cardiovascular organoids.

In one embodiment, the composition of the present invention can promote in vitro maturation of immature cardiovascular organoids derived from human pluripotent stem cells.

In one embodiment, the human pluripotent stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

In one embodiment, cardiovascular organoids matured by treatment with a composition of the present invention have CD31, vWF, EFNB2, EPHB4, PROX1, PDGFRα, PDGFRβ, CALPONIN1, COL1A, COL3A1, COL4A1, COL4A2, α- compared to immature cardiovascular organoids. Expression of SMA, LAMA5, LAMB1, ITGA3, ITGB1, ITGB3, ITGB4, PITX1, PITX3, FGFR4, LaminA, FN1, ROCK1, LIMK1, pFAK, pPDGFRα, pLIMK, pCofilin, VEGFR2, pVEGFR2 or peNOS may be increased.

In one embodiment, the cardiovascular organoids matured by treatment with the composition of the present invention may be vascularized mature ventricular type cardiovascular organoids.

In one embodiment of the present invention, it was confirmed that the higher the stem function of the human pluripotent stem cell-derived cell aggregate, the better the myocardial maturation and vascular network formation of the cardiovascular organoid. Specifically, cardiovascular organoids (H-CO, COs generated from CAs with high stemness) generated from cell aggregates maintaining high stemness function of human pluripotent stem cells are cardiovascular organoids generated from cell aggregates with low stemness function. Compared to noids (L-CO, COs generated from CAs with low stemness), they contain cardiomyocytes, vascular endothelial cells, perivascular cells, smooth muscle cells, and basement membrane with higher structural, metabolic, and functional maturity and differentiation into the ventricular type. It was confirmed that a mature vascular network was formed. In addition, it was confirmed that LIMK plays an important role in the generation of cardiovascular organoids containing mature cardiomyocytes and blood vessels.

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.

As used herein, “LIMK protein expression promoter” refers to a substance that enhances the expression of LIMK protein. The expression promoter for the LIMK protein includes all substances that enhance the expression of LIMK at the transcription level or protein level.

As used herein, “LIMK protein activator” refers to a substance that interacts directly with the LIMK protein or acts indirectly to positively regulate the activity of the LIMK protein. For example, the activator of the LIMK protein may be a substance that binds to the LIMK protein and enhances the activity of the LIMK protein.

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. 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 dedifferentiation 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 both differentiation pluripotency and self-replication ability. However, preferably, the pluripotent stem cells may be mammalian cells, more preferably human-derived pluripotent stem cells.

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 perfectly mimics organs that actually interact in vivo, and can be an excellent model for observing and developing treatments for diseases.

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.

In one aspect, the present invention relates to a support composition for cardiovascular organoid transplantation comprising the composition for cardiovascular organoid maturation of the present invention.

In one embodiment, immature cardiovascular organoids can be matured in the support composition of the invention.

In one aspect, the present invention includes the steps of a) dispensing and culturing human pluripotent stem cells to form cell aggregates (CA); b) preparing cardiovascular organoids by differentiating cell aggregates; and c) treating the cardiovascular organoid with the composition for maturing cardiovascular organoids of the present invention.

In one embodiment, the culture of step a) may be performed for 1 to 6 days.

In one embodiment, the cell aggregates in step b) include: culturing in E8 medium containing a Rho kinase inhibitor; Culturing in a culture medium containing RPMI1640, B27, and insulin containing a GSK-3 inhibitor; and IWP2 processing and culturing.

In one embodiment, the mature cardiovascular organoid can be a vascularized mature ventricular type cardiovascular organoid.

In one aspect, the invention relates to mature cardiovascular organoids prepared by the above method.

In one aspect, the present invention relates to a method for maturing immature cardiovascular organoids in vitro , comprising treating immature cardiovascular organoids derived from human pluripotent stem cells with the composition for maturing cardiovascular organoids of claim 1. .

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.

Example 1. Determination of optimal cell distribution density for generating cardiovascular organoids

To determine the optimal conditions for forming cell aggregates for the production of human pluripotent stem cell-derived cardiovascular organoids, the cell distribution density was confirmed. Specifically, different numbers of human embryonic stem cells (0.25, 0.5, 1, 2, 3, and 4 × 10 ⁶ , respectively) were dispensed into each well of a methacrylate-coated 6-well culture plate. As a result, it was confirmed that aggregates aggregated together at a dispensing density of 2 to 3 × 10 ⁶ , and that aggregates were not formed well at a dispensing density of 0.25 to 0.5 × 10 ⁶ (Figure 1). Therefore, the optimal cell distribution density for forming cell aggregates when manufacturing cardiovascular organoids (CO) was determined to be 1 × 10 ⁵ cells/cm ² .

Example 2. Cardiovascular organoids derived from human pluripotent stem cells manufacturing

2-1. Confirmation of CO production and spontaneous differentiation into stem function and triploblasts

Matrigel and DMEM/F12 were mixed at a volume ratio of 1:100, treated on a cell culture plate, coated at room temperature for at least one hour, and then human embryonic stem cells were placed on the coated cell culture plate. The cells were cultured using Essential 8 ^TM medium at 37°C and 5% CO ₂ conditions, and subcultured when the cells grew to about 60% on the plate. On the first day of subculture, Thiazovivin was added at a concentration of 2 μM to increase cell survival. During subculture, the medium was changed daily. Afterwards, the cultured human embryonic stem cells were separated into single cells using Accutase. These were distributed in ^a methacrylate-coated 6-well culture plate at ^a density ^of 1 Cell aggregates were formed by culturing (thiazovivin was added at a concentration of 2 μM on the first day of culture to increase cell survival rate), and to induce differentiation of cell aggregates into CO, 6 μM CHIR99021, a GSK3β inhibitor, was added in RPMI/B27 minus insulin medium. It was added and treated for 48 hours. On the second day of differentiation induction, 2 μM IWP2 was added to the WNT inhibitor RPMI/B27 minus insulin medium and treated for 48 hours. On days 4 and 5 of differentiation induction, the medium was replaced with RPMI/B27 minus insulin. On the 7th day of differentiation induction, the medium was replaced with RPM/B27 minus vitamin A and then cultured with the same medium every 2 days to prepare cardiovascular organoids. At this time, in order to confirm stem function and spontaneous differentiation into three germ layers during the period of cell aggregate formation, changes in the shape, diameter, and cell number of the cell aggregates were observed (Figure 2A). Additionally, gene expression patterns of cell lineage markers were investigated.

As a result, the average diameters of cell aggregates on days 1 to 5 of culture were 32.6, 72.1, 102.2, 135.2, and 156.2 μm, respectively, confirming that the average diameter increased over time (Figure 2B). Additionally, examining the gene expression patterns of cell lineage markers, we found that pluripotency markers (OCT4 and NANOG) gradually decreased over time, followed by ectodermal markers (OTX and ZIC), mesodermal markers (MESP1 and T), and endodermal markers ( CXCR4 and AFP) were confirmed to increase (Figure 2C). In addition, through immunofluorescence staining, it was confirmed that ectodermal marker (NESTIN), mesodermal marker (T, MIXL1, and VEGFR2), and endodermal marker (FOXA2) were increased (Figure 2D). In addition, the number of cells in the cell aggregates cultured for 2 and 5 days was confirmed to be 166.6 and 418.1, respectively (Figures 2E to F).

2-2. Selection of uniformly sized cardiovascular organoids

On days 7 and 11 of differentiation induction in Example 2-1, cardiovascular organoids were selected into sizes of <100 μm, 100 - 200 μm, and > 200 μm using strainers with mesh sizes of 100 μm and 200 μm. After dividing them into groups, each group was cultured for up to 30 days to confirm its characteristics (Figure 3A). As a result, on the 7th day of culture, the average diameter of CO in each group of <100 μm, 100 - 200 μm, and >200 μm was 80.1 μm, 152.4 μm, and 279.7 μm, respectively, and the average number was 22.1, 42.0, and 279.7 μm, respectively. It was found to be 6.8 (Figures 3A and B). At 30 days of culture, the average percentage of each group (<100 μm, 100 - 200 μm, and >200 μm) of beating human embryonic stem cell-derived cardiovascular organoids was 27.5%, 65.0%, and 7.5%, respectively (Figure 3D). .

2-3. Confirmation of differentiation efficiency, cardiomyocyte type and characteristics of cardiovascular organoids

In Example 2-1, human pluripotent stem cell-derived cardiovascular organoids (100 - 200 μm in size) differentiated through cell aggregates cultured for 2 days with relatively high stem function and cell aggregates cultured for 5 days with low stem function, respectively. The selected cardiovascular organoids were named H-CO (early cell aggregate-derived CO) and L-CO (late cell aggregate-derived CO), respectively, and their differentiation efficiency, myocardial type and structure were confirmed (Figures 4A and B) ). The average beating percentage of H-CO and L-CO was examined from day 10 to day 30 of differentiation, and the ratio was found to gradually increase, with the beating rate of H-CO being higher than that of L-CO on days 10 and 15 of differentiation. It was found to be high, and similar on days 20 and 30 of differentiation (Figure 4C). In addition, as a result of confirming the types of cardiomyocytes of H-CO and L-CO using polymerase chain reaction, Western blot, and immunofluorescence staining, MLC2v, a marker of ventricular type cardiomyocytes, and MLC2a, a marker of atrial-type cardiomyocytes, were found in H-CO. Although the expression increased, the expression of TBX18, a nodal-type cardiomyocyte marker, appeared to increase in L-CO (Figures 4D to F), indicating that H-CO is mainly composed of ventricular-like and atrial-like cardiomyocytes, and L-CO It can be seen that it is composed of nodal type cardiomyocytes. In addition, as a result of confirming the formation of chambers, which are a specific structure of the heart, it was found that H-CO increased the formation of microchambers compared to L-CO (Figures 5A and B).

Through this, it was confirmed that a micro-ventricular type cardiovascular organoid simulating heart development and heart tissue derived from human pluripotent stem cells was manufactured.

Example 3. Confirmation of structural and metabolic maturity of CO

In order to evaluate the structural maturity of the mature ventricular type cardiovascular organoid prepared in Example 2, the fine structure of sarcomeres and mitochondria was examined through transmission electron microscopy on day 30. As a result, more distinct Z-lines, aligned mitochondria, and organized sarcomeres were observed in H-CO compared to L-CO, and the average length of sarcomeres in H-CO (1.6 μm vs. 1.1 μm) and Width (1.4 μm vs. 0.6 μm) appeared to increase compared to L-CO (Figures 6A-C and Figure 7). In addition, T-tubule formation, which is responsible for cardiac contraction and ion channel signaling, was found to be more increased in H-CO compared to L-CO (Figure 6A). Additionally, using immunofluorescence staining, CAV3 ⁺ and JPH2 ⁺ myocardium showed more uniform and higher expression in H-CO than in L-CO. Gene and protein expression of cTnT, cTnI, CAV3, and JPH2 were significantly higher in H-CO than in L-CO (Figure 6D to F). In addition, the structure of mitochondria was confirmed using transmission electron microscopy to evaluate the maturity of mitochondria that control metabolic energy. As a result, the structure of mitochondria was found to be more developed in H-CO than in L-CO (Figure 6G ), the number, size, and crista formation of mitochondria were significantly increased in H-CO compared to L-CO (Figure 6H to J). As a result of analyzing mitotracker activity to confirm mitochondrial activity, it was confirmed that it increased more in H-CO compared to L-CO (Figure 6K and J). Additionally, to quantify mitochondrial content, the ratio of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) was analyzed through polymerase chain reaction. The ratio of mtDNA/nDNA was higher in H-CO compared to L-CO (Figure 6M). As a result of comparative analysis of the expression of metabolic activity markers in H-CO and L-CO through polymerase chain reaction and Western blot, it was confirmed that the expression of metabolic activity markers PGC1α, CPT1β, and TFAM was significantly increased in H-CO. (Figure 6N and O).

Through this, it was confirmed that the cardiovascular organoids constructed through the in vivo heart development simulation strategy of the present invention had structurally and functionally mature mitochondria and formed organized and arranged muscle tissue.

Example 4. CO Functional maturity, action potential analysis by cardiomyocyte cell type, and confirmation of junctions between cardiomyocytes

For functional evaluation of the cardiovascular organoid prepared in Example 2, cardiac pulsatility and electrophysiological reactivity were evaluated, and imaging analysis and graph analysis were performed. As a result, H-CO had a larger pulsation pattern compared to L-CO. While slow, L-CO showed a smaller and faster pulsation pattern (Figures 9A and B, Figure 10, and Figure 11). In addition, it was confirmed that the peak to peak duration and contraction relaxation duration increased in H-CO (FIG. 9E and F). On the other hand, cardiomyocyte beats per minute were confirmed to increase in L-CO (Figure 9D). In addition, while H-COs were all differentiated into ventricular-type cardiomyocytes, L-COs were found to contain all ventricular-type, atrial-type, and nodal-type cardiomyocytes (Figure 9I). In addition, it was confirmed that H-CO had a lower frequency and higher premature heart rate than L-CO ( FIGS. 9F to H and FIG. 12 ). Additionally, to assess the functional maturity of CM, we measured Ca ²⁺ in H-CO and L-CO using the Ca ²⁺ indicator Fluo-4 AM, and found that Flo-4 AM fluorescence intensity was higher in H-CO than in L-CO. It was found to be significantly higher in CO (Figure 9J), indicating that H-CO is functionally more mature than L-CO (Figure 9K). In addition, unlike L-CO, highly specialized/organized intercalated discs containing adherence junctions, gap junctions, and desmosomes were identified in H-CO (Figure 8A ). As a result of confirming the expression of Cx43, a gap junction marker, through immunofluorescence staining analysis, it was confirmed that Cx43 was evenly expressed at the cardiomyocyte junction in H-CO, whereas it was partially expressed in L-CO (Figure 9L), and the expression of Cx43 Expression of genes and proteins also appeared to be increased in H-CO compared to L-CO (Figure 9M and N).

Through this, it was confirmed that the cardiovascular organoids constructed through the in vivo heart development simulation method of the present invention were differentiated into functionally mature ventricular types and formed organized and arranged muscle tissue.

Example 5. Evaluation of vascular network formation in CO

To evaluate the vascular network formation of the cardiovascular organoid prepared in Example 2, structural analysis and vascular-related gene expression were confirmed. As a result, TIE2 and CD117 increased more in L-CO, while CD31, vWF, and vascular type markers EFNB2, EPHB4, and PROX1 were significantly increased in H-CO compared to L-CO ( Figures 13A-C). In addition, through immunofluorescence staining, it was confirmed that the expression of cTnT and CD31 in H-CO increased compared to L-CO, and through quantitative evaluation, it was confirmed that blood vessel area, junction density, and average blood vessel length were also significantly increased. (Figures 13D to G). In addition, an increase in the co-expressed portion of CD31 ⁺ and vWF ⁺ was observed in H-CO, confirming the formation of a complex vascular network including tip cells of vascular endothelial cells and sprouting angiogenesis. (Figures 13H and I). In addition, it was confirmed through immunofluorescence staining and transmission electron microscopy that vascular lumen was created in H-CO (Figures 13J and K). In addition, as a result of confirming the production of fibroblasts, one of the cells constituting the heart, excluding cardiomyocytes and vascular endothelial cells, through immunofluorescence staining and gene expression analysis, it was found that there was no difference between H-CO and L-CO (Figure 14).

Example 6. Evaluation of vascular maturity of CO

In order to evaluate the vascular maturity of the cardiovascular organoid prepared in Example 2, the formation of various cells constituting the blood vessels was evaluated. As a result, the periphery cell marker PDGFRβ and the vascular smooth muscle cell marker CD31 were expressed in the peripheral area. It was confirmed that αSMA was expressed (Figures 15A and B, and Figure 16). In addition, the formation of the lumen of blood vessels, tight junctions, and a layer formed by the basement membrane were confirmed through transmission electron microscopy (Figure 15C). In addition, as a result of comparative analysis of the expression of vascularization-related genes and proteins in H-CO and L-CO, the expression of PDGFRβ, CALPONIN1, COL4A1, COL4A2, and α-SMA was significantly higher in H-CO compared to L-CO. An increase was confirmed (Figures 15D and E).

Through this, it was confirmed that vascularized cardiac organoids can be produced through the self-assembly heart simulation method of the present invention, which simulates heart development without co-culturing vascular endothelial cells or treating exogenous vascular inducing factors.

Example 7. Analysis of ventricular maturation and angiogenic differentiation mechanisms of micro cardiovascular organoids

In order to identify the gene signaling mechanism pathways involved in cardiomyocyte maturation and vascular network formation in the cardiovascular organoid of the present invention, related transcripts were analyzed at 5, 15, and 25 days after differentiation induction of H-CO and L-CO. analysis (FIGS. 17A and B), and specific differentiation into the heart was confirmed through heatmap analysis (FIG. 18). GO analysis results showed that the expression of genes involved in ventricular cell maturation and genes involved in vascular network formation increased (Figure 17C). In addition, heatmap analysis results showed that structural maturation, metabolic maturation, cardiac cell junction, cardiac conduction and calcium channel activity, vascular development, vascular morphogenesis, and vascular stiffening of the ventricle, myocardium, and heart were significantly increased at 15 and 25 days. appeared to increase in H-CO over L-CO (Figure 17D and E). In addition, as a result of analyzing the main signaling mechanism pathways of myocardial maturation and angiogenesis through GO (gene ontology) analysis, the gene signaling mechanism pathways for extracellular matrix, integrin signaling pathway, focal adhesion, response to TGFβ, and angiogenesis were H. While -CO was found to be increased compared to L-CO (Figure 17F), gene signaling pathways for nervous system development, cell cycle and DNA replication were shown to be decreased (Figure 17G). In addition, the expression of related genes in the major gene signaling pathways analyzed through heatmap was found to increase in H-CO compared to L-CO on days 15 and 25 (Figure 17H). In addition, through gene network analysis, it was confirmed that the molecular networks of extracellular matrix, integrins, focal adhesion, TGFβ signaling, and angiogenesis were shared between ventricular cell maturation and vascular network formation (Figure 17I). Accordingly, as a result of further verifying the signaling pathway through RNA-Seq and polymerase chain reaction analysis, the RNA-Seq results showed the expression of extracellular matrix genes, integrin genes, TGFβ signaling, and angiogenesis genes. On days 15 and 25, H-CO increased compared to L-CO (Figure 17A), and among the genes selected through RNA-Seq analysis, extracellular matrix genes (COL3A1, LAMA5, LAMB1) and integrin genes (ITGA3) , it was confirmed through polymerase chain reaction analysis that TGFβ signaling pathway genes (PITX1 and PITX3) and angiogenic genes (FGFR4) increased in H-CO compared to L-CO (Figure 19B).

Example 8. Analysis of signaling mechanisms related to myocardial maturation and vascular network formation in CO

Protein expression of genes selected through transcriptome profiling in Example 7 was verified through Western blot. As a result, extracellular matrix-related proteins (COL1A, LaminA, and FN1) and integrin-related proteins (ITGB1, ITGB3, and ITGB4) were significantly increased in H-CO compared to L-CO on days 15 and 25 (Figure 19C). , expression of Rho-related protein kinases ROCK1, pFAK, and LIMK1 was increased on days 15 and 25 in H-CO compared to L-CO. On the other hand, pLIMK appeared to increase only on day 25. In addition, pCofilin increased on both 15th and 25th days in H-CO compared to L-CO, but there was no difference in pRAC and pMLC (Figure 19D). These results suggest that activation of the ROCK1-pFAK-pLIMK-pCofilin pathway through extracellular matrix-integrin may play an important role in the maturation of cardiomyocytes. Additionally, PITX2, which belongs to the TGFβ superfamily and is downstream of the left-right signaling pathway of LEFTY and NODAL signaling, which is known to regulate left-right asymmetry in mammalian cardiac development, showed no difference in expression in H-CO and L-CO. It was found that pSMAD1/5 was not detected (Figure 19E). In addition, as a result of Western blot analysis of signaling pathway genes involved in vascular network formation selected through transcript profiling analysis, PDGFRα, PDGFRβ, VEGFR2, pPDGFRα, pVEGFR2, and peNOS were found in H-CO on days 15 and 25. Expression increased compared to L-CO. Additionally, HIF2A expression increased on day 25 (Figure 19F).

Example 9. Evaluation of CM maturity and angiogenesis in CO through inhibition of LIMK/Cofilin signaling pathway

Since the LIMK/Cofilin signaling pathway was significantly activated in Example 8, to evaluate the role of the LIMK/Cofilin signaling pathway in CM maturation and angiogenesis, H-CO was added to the control group DMSO or LIMK (DMSO) on day 11 of differentiation. Each was treated with LIMKi3, a LIM Kinase) inhibitor, and qRT-PCR and Western blot analysis were performed on day 15. As a result, pLIMK, LIMK, and pCofilin expression were found to be decreased in the LIMKi3-treated group (Figure 20A), and gene and protein expression levels of CM maturation-related genes cTnT, MLC2v, MLC2a, and JPH2 were decreased in LIMKi3-treated H-CO. (Figures 20B and C). Additionally, the expression levels of angiogenesis-related genes PDGFRβαSMA and vWF were decreased (Figure 20D). These results indicate that LIMK/Cofilin signaling is involved in CM maturation and angiogenesis in H-CO. Additionally, we determined whether ECM-integrin interactions, LEFTY-NODAL, pPDGFRα, pPDGFRβpVEGFR, and peNOS signaling pathways were affected in H-CO treated with LIMKi3, and genes of COL4A2, LAMA2, LAMC1, LAMC3, ITGB3, and ITGA7 were identified. Expression levels were significantly decreased in LIMKi3-treated H-CO, and protein expression of ECM genes (COL1A1, Laminin, and FN1) was decreased in LIMKi-treated H-CO (Figure 20G). NODAL decreased in the LIMKi3-treated group, while the protein level of LEFTY increased in the LIMKi3-treated group compared to the DMSO control group, and phosphorylation of SMAD2 and SMAD3 was found to increase in the LIMKi3-treated group (Figure 20H). Additionally, the protein expression levels of PDGFRα, pPDGFRα and pPDGFRβ were decreased in LIMKi3-treated H-CO, peNOS was downregulated in LIMKi3-treated group compared to DMSO control group, while VEGFR2, pVEGFR2 and HIF2A were downregulated between LIMKi3-treated group and DMSO control group. There appeared to be no difference (Figure 20I).

In conclusion, LIMK/Cofilin signaling is activated together with extracellular matrix-integrin interaction, TGFβ signaling pathway (NODAL, pSMAD2, and pSMAD3) and angiogenic pathway (PDGFRα, pPDGFRα, pPDGFRβ, and peNOS), and in particular, LIMK promotes vascularization. It was confirmed that it plays a role in promoting the maturation of cardiomyocytes and the formation of vascular networks in heart organoids.

Example 9. Production and analysis of CO derived from induced pluripotent stem cells

In order to confirm whether the method for constructing cardiovascular organoids of the present invention generates the same cardiovascular organoids not only from human embryonic stem cells but also from human induced pluripotent stem cells, cardiovascular organoids were performed using human induced pluripotent stem cells by the method of Example 2 above. The nooids were prepared and the differentiation and maturity of cardiac cells such as cardiomyocytes, vascular endothelial cells, and fibroblasts were analyzed, and MLC2v+, CD31+, and FSP-1+ cells were analyzed using H-CO and L-CO fluorescent immunostaining. The ratio was investigated.

As a result, the proportion of MLC2v ⁺ cardiomyocytes and CD31 ⁺ vascular endothelial cells increased in H-CO compared to L-CO, while the proportion of FSP-1 + fibroblasts was similar in H-CO and L-CO (Figure 21A ). Additionally, expression of MLC2v and MLC2a increased in H-CO compared to L-CO, and TBX18 expression increased in H-CO compared to L-CO (Figure 21B). In addition, the expression of cardiac differentiation, maturation, and metabolism-related genes such as cTnT, cTnI, CAV3, JPH2, CPT1β, and Cx43 was confirmed to be increased in H-CO compared to L-CO (FIG. 21C to E). In addition, it was confirmed that the expression of genes for vascular network formation, such as CD31, vWF, PDGFRβ, αSMA, and COL4A1, was increased in H-CO compared to L-CO (Figure 21F). Expression of FSP-1 did not differ between H-CO and L-CO (Figure 21G). Therefore, it was confirmed that the mature cardiovascular organoid differentiation protocol established in the present invention is universally applicable not only to human embryonic stem cells but also to human induced pluripotent stem cells.

Through the above examples, in the present invention, the ROCK-pFAK-pLIMK-pCofilin, LEFTY-NODAL, pPDGFRα, pPDGFRβ, pVEGFR and peNOS signaling pathways are activated by extracellular matrix-integrin interaction to activate human pluripotent stem cell-derived blood vessels. It was confirmed that LIMK induces the maturation of cardiomyocytes and the formation of a vascular network in the mature cardiac organoids, and that LIMK promotes the generation of mature cardiovascular organoids through the extracellular matrix-integrin pathway (FIG. 20J).

Claims (13)

A composition for maturing cardiovascular organoids (CO) comprising a LIMK (LIM Kinase) protein, a vector containing a polynucleotide encoding the LIMK protein, a LIMK protein expression promoter, or a LIMK protein activator as an active ingredient. The composition for maturation of cardiovascular organoids according to claim 1, which promotes maturation of cardiomyocytes. The composition for maturation of cardiovascular organoids according to claim 1, which promotes vascular network formation. The composition for cardiovascular organoid maturation according to claim 1, which increases gene and protein expression of cTnT, MLC2v, MLC2a or JPH2. The composition for cardiovascular organoid maturation according to claim 1, which increases gene and protein expression of PDGFRβ, αSMA or vWF. The composition for cardiovascular organoid maturation according to claim 1, which promotes in vitro maturation of immature cardiovascular organoids derived from human pluripotent stem cells. The composition for maturation of cardiovascular organoids according to claim 6, wherein the human pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). A support composition for cardiovascular organoid transplantation comprising the composition for cardiovascular organoid maturation of claim 1. 9. The support composition for cardiovascular organoid transplantation according to claim 8, wherein immature cardiovascular organoids are matured in the support composition. a) dispensing and culturing human pluripotent stem cells to form cell aggregates;
b) preparing cardiovascular organoids by differentiating cell aggregates; and
c) A method of producing in vitro matured cardiovascular organoids, comprising the step of treating the cardiovascular organoids with the composition for maturing cardiovascular organoids of claim 1. 11. The method of claim 10, wherein in step b) the cell aggregates are:
i) culturing in E8 culture medium containing Rho kinase inhibitor;
ii) culturing in a culture medium containing RPMI1640, B27, and insulin containing a GSK-3 inhibitor; and
iii) A method of producing in vitro mature cardiovascular organoids that are differentiated into cardiovascular organoids by a method comprising the steps of IWP2 treatment and culturing. 11. The method of claim 10, wherein the mature cardiovascular organoid is a vascularized mature ventricular type cardiovascular organoid. A method for maturing immature cardiovascular organoids in vitro , comprising treating immature cardiovascular organoids derived from human pluripotent stem cells with the composition for maturing cardiovascular organoids of claim 1.