KR102482470B1 - Composition for promoting angiogenesis and method for producing thereof - Google Patents

Abstract

In the present specification, a composition for promoting angiogenesis, including endothelial cells differentiated from stem cells and HA-CA hydrogel, and a method for preparing the same are provided. In addition, in the present specification, a cell therapy composition having a preventive or therapeutic effect on cardiovascular diseases, including the composition for promoting angiogenesis provided by the present invention, is provided.

Description

Composition for promoting angiogenesis and method for producing the same {COMPOSITION FOR PROMOTING ANGIOGENESIS AND METHOD FOR PRODUCING THEREOF}

The present invention relates to a novel composition for promoting angiogenesis and a method for preparing the same.

Neovascularization is a process in which new blood vessels are directly generated from angioblasts (vasculogenesis), a process in which new blood vessels are formed from existing blood vessels (angiogenesis), and a process in which vascular muscle cells are added to form new blood vessels (arteriogenesis). ) may be included. Such blood vessel formation may be involved in various physiological and pathological phenomena such as wound repair, embryogenesis, tumor formation, chronic inflammation, and obesity.

Angiogenesis may be a particularly essential phenomenon for wound healing or tissue regeneration. For example, when there is a lack of angiogenesis in the body, necrosis, ulceration, and ischemia may occur, resulting in tissue or organ dysfunction. Furthermore, as the blood supply is not smooth, cardiovascular diseases such as ischemic heart disease, arteriosclerosis, myocardial infarction and angina pectoris may also be caused. Accordingly, there has been a demand for the development of a therapy that induces or promotes angiogenesis in order to reduce tissue damage due to angiogenesis deficiency and treat cardiovascular diseases caused thereby.

On the other hand, in cardiovascular diseases associated with the induction of angiogenesis and its deficiency, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) Factor) and platelet-derived endothelial growth factor (PDEGF) are being studied. However, the above factors have a problem that it is difficult to isolate or purify them, and as the therapeutic effect on cardiovascular diseases due to lack of angiogenesis is insignificant, they have limitations in clinical application.

For this reason, development of a method for promoting angiogenesis, which can induce angiogenesis and can provide a fundamental therapeutic effect on vascular diseases caused by angiogenesis defects, particularly cardiovascular diseases, is continuously required.

The background description of the invention has been prepared to facilitate understanding of the present invention. It should not be construed as an admission that matters described in the background art of the invention exist as prior art.

On the other hand, human embryonic stem cells (hESCs) isolated from embryos and human induced pluripotent stem cells (hiPSCs) made from somatic cells are endothelial cells that play an important role in blood vessel formation. cell), so it can be used for blood vessel regeneration treatment. Accordingly, as a new strategy for regenerating damaged blood vessels and further inducing the formation of blood vessels, a blood vessel regeneration treatment using endothelial cells differentiated from human pluripotent stem cells has been proposed.

More specifically, revascularization therapy using endothelial cells differentiated from stem cells is the process of angiogenesis described above, that is, a process in which new blood vessels are directly generated from angioblasts (vasculogenesis), and new blood vessels are formed from existing blood vessels. It can be a very useful treatment because blood vessels are formed by involving both angiogenesis and arteriogenesis, in which smooth muscle cells are added to form new blood vessels.

On the other hand, the inventors of the present invention recognized the importance of the in vivo survival rate and maintenance rate of endothelial cells differentiated from induced pluripotent stem cells in the effect of revascularization treatment.

Accordingly, the inventors of the present invention maintain angiogenic ability by enhancing the intracellular viability of endothelial cells having angiogenic ability from various cell lines differentiated from human induced pluripotent stem cells, and new angiogenesis contributing to the enhancement of the therapeutic effect The composition for promotion was studied.

The inventors of the present invention, among materials having various biocompatibility, have catechol group-conjugated hyaluronic acid HA, which has sufficient adhesive strength to attach to the surface of various types of materials without causing cytotoxicity and inflammatory reactions in vivo. -CA (catechol modified hyaluronic acid) hydrogel was noted.

The inventors of the present invention paid attention to the characteristics of the HA-CA hydrogel, and encapsulated human induced pluripotent stem cell-derived endothelial cells in HA-CA and directly injected (injected) them into ischemic myocardial tissue or lower extremity ischemia, thereby making existing cell therapy products For the first time, we came to the idea that cells can be delivered more efficiently and therapeutic effects can be expected.

In particular, the inventors of the present invention, after the HA-CA hydrogel has delivered the desired cells into the tissue, the in vivo time-by-biodegradation kinetics in which HA-CA is degraded by the enzyme (hyaluronidase) present in the tissue ) relationship was discovered for the first time, and thus, it was recognized that HA-CA hydrogel has biocompatibility to the extent that it can be directly injected into ischemic tissue. In addition, the inventors of the present invention could directly inject endothelial cells differentiated from HA-CA hydrogel and human induced pluripotent stem cells into ischemic tissue and expect long-term therapeutic effects thereof.

As a result, the inventors of the present invention confirmed that, when the HA-CA hydrogel is applied together with endothelial cells differentiated from human induced pluripotent stem cells, it is possible to maintain a high survival rate and maintenance rate of endothelial cells introduced in vivo.

In particular, the inventors of the present invention found that, when endothelial cells differentiated from human induced pluripotent stem cells were encapsulated by HA-CA hydrogel and injected into the tissue, compared to the case of administration together with previously known and used bioactive substances, the tissue It was confirmed that the internal adhesion rate, the survival rate of the cells administered into the tissue, and the effect of promoting angiogenesis were more excellent.

On the other hand, the inventors of the present invention, as HA-CA in gel form is directly injected into the ischemic site, it was more noteworthy that its concentration may affect angiogenesis.

Furthermore, the inventors of the present invention found that the mRNA and protein expression levels of CDH5 among various endothelial cell markers were remarkably high in differentiated endothelial cells. Endothelial cells could be isolated with high purity.

At this time, the inventors of the present invention found that when the composition for promoting angiogenesis was transplanted into an ischemic tissue, not only the promotion of angiogenesis but also the ability to regenerate blood vessels was excellent. Accordingly, the inventors of the present invention recognized that the composition for promoting angiogenesis according to one embodiment of the present invention can be clinically applied to the prevention or treatment of cardiovascular diseases.

Accordingly, the problem to be solved is to provide a composition for promoting angiogenesis, including high-purity endothelial cells differentiated from human induced pluripotent stem cells and HA-CA, and a method for preparing the same.

Another problem to be solved by the present invention is to provide a cell therapy composition for preventing or treating cardiovascular diseases, including the composition for promoting angiogenesis according to an embodiment of the present invention.

The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, a composition for promoting angiogenesis comprising endothelial cells differentiated from stem cells and catechol-modified hyaluronic acid (HA-CA) hydrogel is provided.

At this time, according to the features of the present invention, the composition for promoting angiogenesis according to an embodiment of the present invention may further include an oxidizing agent and a pH adjusting agent. For example, the oxidizing agent may include at least one of the group consisting of sodium periodate, hydrogen peroxide, horseradish peroxidase, and tyrosinase, , the pH adjusting agent is sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide hydroxide), strontium hydroxide, and barium hydroxide.

According to another feature of the present invention, HA-CA in the composition for promoting angiogenesis according to an embodiment of the present invention is HA (hyluronic acid) carboxyl group (carboxy group) of dopamine (amine group) (amine group) of dopamine (dopamine) And may be formed by combining with an amide bond. At this time, Formula 1 of HA-CA is as shown below, L in the formula is -(CH2)-K, K is an integer from 1 to 10, X is independently H or OH, and N is It is an integer from 2 to 700, and m is smaller than n and may mean an integer from 1 to 300.

[Formula 1]

According to another feature of the present invention, the stem cells in the composition for promoting angiogenesis according to an embodiment of the present invention may be human pluripotent stem cells or human induced pluripotent stem cells.

According to another feature of the present invention, the endothelial cells in the composition for promoting angiogenesis according to an embodiment of the present invention contain 98% or more of endothelial cells expressing CDH5, and the expression level of CDH5 mRNA at this time is: It can be 10 times higher than that of undifferentiated stem cells or mesodermally-differentiated stem cells. Furthermore, the aforementioned endothelial cells are encapsulated by the HA-CA hydrogel, and the in vivo viability of the endothelial cells is 10 to 15 times higher than that of the endothelial cells not encapsulated by the HA-CA hydrogel. can be twice as high. These endothelial cells may be 75 vol % to 90 vol % of the total volume of the composition for promoting angiogenesis, and the concentration at this time may be 1 X 10 ⁵ CFU/ml to 6 X 10 ⁶ CFU/ml. .

According to another feature of the present invention, the content of the HA-CA hydrogel in the composition for promoting angiogenesis according to an embodiment of the present invention, based on the total volume of the composition for promoting angiogenesis, is 1% to 5% by volume may be %.

The composition for promoting angiogenesis according to an embodiment of the present invention may be administered by at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intratracheal and topical application to the skin.

According to one embodiment of the present invention, culturing to differentiate from stem cells into endothelial cell lineage cells, sorting homogenous endothelial cells having a CDH5 gene expression level of a predetermined level or higher among the differentiated endothelial cell lineage cells There is provided a method for preparing a composition for promoting angiogenesis comprising the step of mixing the HA-CA hydrogel with a homogeneous endothelial cell suspension.

At this time, according to the features of the present invention, the culturing step in the manufacturing method of the composition for promoting angiogenesis according to an embodiment of the present invention described above includes culturing stem cells to differentiate into mesodermal lineage cells and mesodermal lineage cells. It may include culturing to differentiate into endothelial cell lineage cells. Furthermore, the step of culturing to differentiate into endothelial cell lineage cells described above may include the step of differentiating into the endothelial cell lineage cells by culturing the mesodermal lineage cells in a DLL4-treated medium.

According to another feature of the present invention, the step of treating the HA-CA hydrogel in the method for producing a composition for promoting angiogenesis according to an embodiment of the present invention may be performed for one period of 2 to 7 days. . Furthermore, the method for preparing the composition for promoting angiogenesis according to an embodiment of the present invention may further include mixing an oxidizing agent and a pH adjusting agent after mixing the HA-CA hydrogel.

According to one embodiment of the present invention, there is provided a cell therapy composition for preventing or treating cardiovascular diseases, including the above-described composition for promoting angiogenesis.

At this time, the cardiovascular disease is at least one of the group consisting of thrombosis, peripheral arterial disease, ischemic heart disease, angina pectoris, cerebrovascular disease, coronary artery disease, angina pectoris, myocardial infarction, arteriosclerosis, arrhythmia, hypertension, and diabetic vascular disease. can include

Hereinafter, the present invention will be described in more detail through examples. However, since these examples are merely intended to illustrate the present invention by way of example, the scope of the present invention should not be construed as being limited by these examples.

The present invention relates to an angiogenesis-stimulating composition containing high-purity endothelial cells that do not induce an immune response generated by the use of animal-derived serum or feeder cells and HA-CA hydrogel, which is a highly biocompatible material, and a method for preparing the same By providing, there is an effect that can be stably applied to clinical practice.

Specifically, the present invention purifies endothelial cells having a high expression level of CDH5, thereby increasing high-purity endothelial cells and their in vivo survival rate and promoting angiogenesis, including HA-CA hydrogels that facilitate their migration to surrounding tissues. A composition for promoting and a method for preparing the same can be provided.

Furthermore, the present invention promotes angiogenesis and provides an angiogenesis-stimulating composition with excellent blood vessel regeneration and a method for preparing the composition, which can be used as a cell therapeutic agent effective for preventing or treating cardiovascular diseases.

In addition, the present invention can be directly injected into ischemic tissue to increase intracellular viability and provide effective amounts of endothelial cells and HA-CA hydrogel in the composition for effective angiogenesis and blood vessel regeneration.

Effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

Figure 1a shows the procedure of the manufacturing method of the composition for promoting angiogenesis according to an embodiment of the present invention.
Figure 1b illustratively illustrates each step in the method for preparing a composition for promoting angiogenesis according to an embodiment of the present invention.
Figure 2a shows the results of flow cytometric analysis of CDH5 expression in endothelial cells differentiated according to the method for preparing a composition for promoting angiogenesis according to an embodiment of the present invention.
2b to 2c show the results of flow cytometry for the expression of specific markers in endothelial cells selected based on CDH5 expression.
3 shows the results of nitric oxide detection of endothelial cells selected based on CDH5 expression and the tube forming ability of endothelial cells.
Figure 4 shows the synthetic structural formula of HA-CA.
5A-5B show images of endothelial cells encapsulated by the HA-CA hydrogel.
6a to 6b show images of endothelial cell viability observed by the HA-CA hydrogel.
Figures 7a to 7b show the in vivo degradability observation results of the HA-CA hydrogel used in the composition for promoting angiogenesis of the present invention.
8a to 8c show results of observation of blood flow recovery in a mouse model of lower extremity ischemia for the composition for promoting angiogenesis according to an embodiment of the present invention.
9 illustrates an anatomical image of blood flow restoration in a lower limb ischemic mouse model for the composition for promoting angiogenesis according to an embodiment of the present invention.
10a to 11c show results of immunohistochemical analysis of tissues isolated from a mouse model of lower limb ischemia injected intramuscularly with a composition for promoting angiogenesis according to an embodiment of the present invention.
12a to 12c show the results of immunohistochemical analysis of the heart induced with myocardial infarction (MI) injected with a composition for promoting angiogenesis according to an embodiment of the present invention.

Advantages and features of the present invention, and methods of achieving them, will become clear with reference to the detailed description of the following embodiments taken in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various forms different from each other, only these embodiments make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to completely inform the person who has the scope of the invention, and the present invention is only defined by the scope of the claims.

As used herein, the term "stem cell" may refer to pluripotent stem cells having the ability to differentiate into various body tissues. On the other hand, pluripotent stem cells can be used for various cell regeneration treatments including blood vessel regeneration treatments. In the use of revascularization treatment, pluripotent stem cells may preferably be human embryonic stem cells or human induced pluripotent stem cells. Accordingly, the stem cells used within the present specification may be used interchangeably with "human induced pluripotent stem cells".

Furthermore, in blood vessel regeneration treatment, endothelial cells differentiated from human pluripotent stem cells can be transplanted into a living body to regenerate damaged blood vessels and induce blood vessel formation or new blood vessel formation.

As used herein, the term "angiogenesis" or "angiogenesis" may refer to any phenomenon of forming new blood vessels, and thus "angiogenesis" or "angiogenesis" may be used interchangeably.

As used herein, the term "endothelial cell" may refer to a squamous cell constituting a layer covering the inner walls of blood vessels and lymphatic vessels. Thus, endothelial cells may be used in the same sense as "vascular endothelial cells". Furthermore, "endothelial cells" used in various embodiments of the present specification may mean "endothelial cells differentiated from human induced pluripotent stem cells".

As used herein, the term "cardiovascular disease" may mean a disease that occurs in the heart and major arteries. The cause of this may be poor blood supply due to lack of blood vessel formation. In the present disclosure, cardiovascular disease may include thrombosis, peripheral arterial disease, ischemic heart disease, angina pectoris, cerebrovascular disease, coronary artery disease, angina pectoris, myocardial infarction, arteriosclerosis, arrhythmia, and hypertension. The composition for promoting angiogenesis according to another embodiment of the present invention or the cell therapy composition for preventing or treating cardiovascular disease according to another embodiment of the present invention may be particularly effective in ischemic cardiovascular disease among various cardiovascular diseases. there is. However, the effects of these compositions are not limited to ischemic cardiovascular disease.

As used herein, the term "cell therapy" refers to the proliferation, selection, or alteration of biological properties of living autologous, allogenic, or xenogeneic cells in vitro in order to restore cell and tissue functions. It can mean all medicines used for treatment, diagnosis, and prevention through a series of actions, such as ordering. In the present specification, cell therapy may refer to cells themselves that can be transplanted to repair damaged tissues. For example, the cell therapy agent may be endothelial cells differentiated from human induced pluripotent stem cells that are transplanted into an ischemic area and contribute to blood vessel formation. In this case, the cell therapy agent may be a differentiated endothelial cell encapsulated by HA-CA, but is not limited thereto.

As used herein, the term "differentiation" means that cells develop into a specific cell or tissue complex or individual level having a specific function.

As used herein, the term "proliferation" refers to an increase in the number of cells and is used in the same sense as growth.

As used herein, the term "renewal ability" may refer to the ability of a cell to produce an identical copy of itself, and when the regeneration ability is improved, the proliferative ability of the cell may be excellent.

Hereinafter, with reference to FIGS. 1A to 7B, a method for preparing a composition for promoting angiogenesis will be described in detail.

Figure 1a shows the procedure of the manufacturing method of the composition for promoting angiogenesis according to an embodiment of the present invention. Hereinafter, for convenience of description, it will be described with reference to FIGS. 1B to 7B.

Referring to FIG. 1A , the method for preparing a composition for promoting angiogenesis according to an embodiment of the present invention includes culturing stem cells to differentiate into cells of endothelial cell lineage (S110), differentiated cells of endothelial cell lineage. (S120) and mixing the HA-CA hydrogel with the homogeneous endothelial cell suspension (S130). A composition for promoting angiogenesis may be prepared in the order of.

More specifically, referring to Figure 1b, each step in the manufacturing method of the composition for promoting angiogenesis according to an embodiment of the present invention is shown by way of example.

First, the step of differentiating endothelial cells from stem cells (S110) includes culturing stem cells to differentiate into mesodermal lineage cells (S111) and culturing mesodermal lineage cells to differentiate into endothelial lineage cells (S112). can include In this case, the stem cells may be human induced pluripotent stem cells (hi PSC), but are not limited thereto. Furthermore, the temperature of the culture environment conditions is 36 °C to 38 °C, preferably 36.5 °C to 37.5 °C, supply oxygen (O ₂ ) is 1% to 25%, and supply carbon dioxide (CO ₂ ) is 1% to 15%. can

First, in the step of culturing to differentiate stem cells into mesodermal lineage cells (S111), undifferentiated human induced pluripotent stem cells are seeded on a plate, and 3 to 5 ng/ml of FGF2 as a growth factor Differentiation from stem cells to mesodermal lineage cells can be induced by culturing in a medium containing 2 to 4 μM of CHIRR99021, a GSK3β inhibitor, and 10 to 30% serum replacement, replacing the medium every day for 3 days.

Accordingly, it appears that the stem cells suspended in a spherical shape have differentiated into cells of the mesodermal lineage attached to the plate.

At this time, FGF2 (Fibroblast growth factor) is a growth factor related to various biological processes such as cell proliferation, cell differentiation, promotion of proliferation, angiogenesis, bone formation, and nerve growth.

In addition, CHIRR99021 is a substance that inhibits the activity of GSK (Glycogen synthase kinase). More specifically, as GSK is inhibited, ß-catenin, a signal transduction system involved in cell proliferation, is not degraded by GSK, and the expression level of genes involved in cell proliferation is increased, so that cell survival and proliferation can be improved.

Furthermore, the plate is not limited as long as it can be used for cell culture, and various materials such as flasks, tissue culture flasks, dishes, petri dishes, micro plates, micro well plates, micro slides, pamber slides, petri dishes, tubes, trays and culture bags are used. A shaped plate may be used and may include a cell adhesion layer coating film on the upper surface. More specifically, the coating film of the plate may include at least one of collagen, fibronectin, ramidine, ramidine fragment, vitronectin, basement membrane matrix, gelatin, hyaluronic acid, polylysine, and vitronectin, and 1 mg/ml or less , preferably 0.1 mg/ml of collagen. Thus, as cultured on a plate containing a collagen coating of 0.1 mg/ml, cell adhesion and extension are promoted, and differentiation efficiency of mesodermal lineage cells may be increased.

Then, in the step of culturing the mesodermal lineage cells to differentiate into endothelial lineage cells (S112), the differentiated mesodermal lineage cells were treated with growth factors 3 to 5 ng/ml of FGF2, 3 to 7 ng/ml of EGF, and 5 medium daily for one of 5 to 9 days in medium containing VEGF-A at 15 ng/ml, DLL4 at 20 to 30 ng/ml, a Notch signaling ligand, and 10 to 30% serum replacement. By replacing and culturing, it is possible to differentiate into cells of the endothelial cell lineage. Furthermore, the efficiency of differentiation into the endothelial cell lineage can be increased by selectively using 3 to 7 units/ml of heparin.

At this time, EGF (epidermal growth factor) is a growth factor capable of promoting proliferation, growth, and differentiation of cells by binding to its receptor, and may have an activity that promotes the proliferation of endothelial cells.

In addition, VEGF-A (vascular endothelial growth factor) is a signaling substance involved in the formation of embryonic circulation and blood vessel formation by activating VEGF signaling, and can stimulate cell division and cell migration of endothelial cells.

In addition, DLL4 (delta-like ligand 4) reduces the growth and migration of endothelial cells, the determination of arterial/venous differentiation, the determination of tip/stock cells, and the formation of tip cells, thereby inhibiting Notch, which plays a role in suppressing excessive angiogenesis. It is a signaling substance that acts on receptors to properly regulate angiogenic sprouting. In particular, due to the addition of DLL4, the Notch signal, which acts to distinguish and maintain cell characteristics, is regulated, and the expression levels of endothelial cell characteristics, that is, endothelial cell markers CDH5, KDR, PECAM1, TEK, and VWF, are increased. can

Endothelial cell lineage cells derived from human induced pluripotent stem cells can be obtained on days 8 to 12 through the culturing step (S110) to differentiate endothelial cells from the above stem cells.

Next, in the step of selecting homogenous endothelial cells having a CDH5 gene expression level higher than a predetermined level among differentiated endothelial cell lineage cells (S120), specific surface markers of endothelial cells from various cell lines differentiated from stem cells are selected. Endothelial cells of high purity can be obtained by sorting the endothelial cells using the. For example, the expression level of the CDH5 gene in differentiated endothelial cells can be significantly higher than in undifferentiated stem cells or mesodermally differentiated stem cells.

More specifically, referring to FIG. 2A , results of flow cytometry analysis for CDH5 expression in endothelial cells differentiated according to the method for preparing a composition for promoting angiogenesis according to an embodiment of the present invention are shown. At this time, isotype-Allophycocyanin (APC) was used as a negative control to see the non-specific binding of the antibody.

Among the endothelial cells differentiated from human induced pluripotent stem cells, 98.5% of the cells appear as CDH5 expression-positive cells. Accordingly, in the step of selecting endothelial cells (S120), CDH5 is used as an endothelial cell marker, and based on its intracellular expression level, endothelial cells can be separated with high purity among various differentiated cell lines.

Accordingly, referring to FIG. 2B , flow cytometry analysis results for expression of a specific marker in endothelial cells selected based on CDH5 expression are shown. At this time, isotype-APC (isotype-Allophycocyanin, APC), isotype-PE (isotype-Phycoerythrin) and isotype-FITC (isotype-Fluorescein) were used as negative controls to see the non-specific binding of the antibody. was used

In the endothelial cells selected based on CDH5 expression (CDH5+), the number of cells expressing the endothelial cell-specific markers CDH5, KDR, PECAM1, TEK, and VWF was 94.1%, 44.9%, 28.5%, 12.9%, and 88.0%, respectively. appear.

The expression level of these markers appears to be significantly higher than CDH5 expression-negative (CDH5-) endothelial cells or unselected endothelial cells (endothelial lineage cells themselves differentiated from induced pluripotent stem cells).

More specifically, referring to FIG. 2C , a graph comparing flow cytometry results of endothelial cell-specific marker expression with and without selection based on CDH5 expression is shown.

First, CDH5 expression-positive (CDH5+) endothelial cells show 3.3 times higher CDH5 expression than unselected endothelial cells.

Then, CDH5 expressing positive (CDH5+) endothelial cells showed 3.3 times higher KDR expression than CDH5 expressing negative (CDH5−) cells and unselected endothelial cells.

Then, CDH5 expression-positive (CDH5+) endothelial cells showed 2.25 times higher PECAM1 expression than CDH5 expression-negative (CDH5-) cells, and 15 times higher PECAM1 expression than non-selected endothelial cells.

Then, CDH5 expression-positive (CDH5+) endothelial cells showed lower VWF expression than CDH5 expression-negative (CDH5-) cells, but showed 1.6 times higher VWF expression than non-selected endothelial cells.

Then, CDH5 expression-positive (CDH5+) endothelial cells showed 2-fold higher TEK expression than CDH5-expressing negative (CDH5-) cells, and 3-fold higher TEK expression than non-selected endothelial cells.

Then, CDH5 expression-positive (CDH5+) endothelial cells showed 1.5 times higher NOS3 expression than CDH5 expression-negative (CDH5-) cells, and 2.15 times higher NOS3 expression than non-selected endothelial cells.

That is, only high-purity endothelial cells, that is, homogenous endothelial cells can be obtained through selection based on CDH5 expression. In this case, the term "homogeneous" used herein may refer to a homogeneous cell type having the same morphological shape and marker expression observed under a microscope. Further, a marker is any substance capable of distinguishing a target cell from other surrounding cells, and may be at least one of a group consisting of proteins, glycolipids, nucleic acids, and combinations thereof, but is not limited thereto. More specifically, markers for vascular endothelial cells may be substances specifically expressed in vascular endothelial cells, and may include CDH5, KDR, PECAM1, VWF, TEK, and NOS3.

The use of such high-purity endothelial cells may be associated with angiogenesis or angiogenesis effect. For example, when low purity endothelial cells including undifferentiated stem cells or mesodermal stem cells are transplanted into an ischemic tissue, the effect of angiogenesis or blood vessel regeneration is lower than when high purity endothelial cells are transplanted. can Accordingly, it may be very important to isolate high-purity endothelial cells.

On the other hand, the characteristics of endothelial cells can be identified through intracellular nitric oxide (NO). Nitric oxide is a vascular homeostasis factor, and is produced by a protein called endothelial nitric oxide synthase (eNOS) in endothelial cells, that is, vascular endothelial cells. Thus, functional characteristics of endothelial cells can be identified through nitric oxide.

More specifically, referring to (a) of FIG. 3 , nitric oxide detection results of endothelial cells selected based on CDH5 expression are shown. At this time, nitric oxide was observed using the DAF-FM reagent, and human umbilical vein endothelial cells (HUVEC) were used as a control. Endothelial cells (hiPSC-ECs) selected based on CDH5 expression appear to excrete nitric oxide at similar levels to human umbilical vein endothelial cells (HUVECs). That is, CDH5 expression-positive cells may be endothelial cells that synthesize nitric oxide.

Furthermore, referring to FIG. 3 (b), an image of endothelial cells selected based on CDH5 expression observed on Matrigel is shown. Endothelial cells (hiPSC-EC) selected based on CDH5 expression appear to form tubes, that is, have a reticular structure similar to human umbilical vein endothelial cells (HUVEC).

Thus, endothelial cells selected based on CDH5 expression appear to have functional and morphological characteristics similar to human umbilical vein endothelial cells, ie, vascular endothelial cells in vivo.

High-purity endothelial cells can be obtained through the step of selecting homogeneous endothelial cells having a CDH5 gene expression level higher than a predetermined level among the differentiated endothelial cell lineage cells (S120). Furthermore, the composition for promoting angiogenesis according to an embodiment of the present invention provides high-purity endothelial cells, so that the ability to promote angiogenesis may be superior to endothelial cells differentiated from unselected human induced pluripotent stem cells.

Again, referring to FIG. 1B, in the step of mixing the HA-CA hydrogel with the selected homogeneous endothelial cell suspension (S130), by increasing the cell persistence and viability in the tissue for the endothelial cells selected based on CDH5, HA-CA hydrogels capable of enhancing angiogenesis can be treated.

The content of the treated HA-CA hydrogel may be 1 vol % to 5 vol %, preferably 1.2 vol % to 3.2 vol %, based on the total volume of the total composition. However, it is not limited to its content, and may be variously set according to the type and condition of the target site and the desired effect. Furthermore, the content of endothelial cells may be 75% by volume to 90% by volume based on the total volume of the total composition, but is not limited thereto. Preferably, the final concentration of endothelial cells may be 1 X 10 ⁵ cells/ml to 6 X 10 ⁶ cells/ml. However, the concentration thereof is not limited thereto, and may be variously set according to the type and condition of the target site and the desired effect.

In this case, the term "HA-CA (catechol-modified hyaluronic acid)" used herein may mean a form in which dopamine is conjugated to the backbone of hyaluronic acid (HA).

More specifically, referring to Figure 4, the synthetic structural formula of HA-CA is shown. HA-CA is a form in which the carboxyl group (-COOH) of hyaluronic acid and the amine group (-NH2) of dopamine are bonded through an amide bond. Such HA-CA is added with EDC and NHS in the same molar ratio as hyaluronic acid, stirred for 30 minutes in an environment having pH 5, and after 30 minutes, dopamine in the same molar ratio as hyaluronic acid is added and stirred for more than 18 hours to obtain carbodiimide. It can be obtained by inducing a coupling reaction (carbodiimide coupling reaction).

Furthermore, HA-CA may have the form of a hydrogel. At this time, the term "hydrogel" used herein may refer to a three-dimensional structure of a hydrophilic polymer holding a sufficient amount of moisture, and the hydrogel structure formed by a crosslinking reaction has more improved bonding strength. can have On the other hand, HA-CA used in the composition for promoting angiogenesis and its manufacturing method according to an embodiment of the present invention may mean a hydrogel formed only by its own oxidizing power without the addition of a crosslinking agent through catechol having high oxidizing power, but , but is not limited thereto.

On the other hand, after the step of mixing the HA-CA hydrogel with the selected homogeneous endothelial cell suspension (S130), the step of mixing an oxidizing agent and a pH adjusting agent with the mixed solution of the endothelial cell suspension and the HA-CA hydrogel is further included. can do. More specifically, by adding an oxidizing agent, the HA-CA hydrogel can further improve cross-linking between catechol groups in the hyaluronic acid derivative. Furthermore, by adding a pH adjusting agent, HA-CA hydrogel can further improve cross-linking between benzoquinone groups in hyaluronic acid derivatives. Thus, due to the addition of the oxidizing agent and the pH adjusting agent, the mutual bonding strength between the hyaluronic acid derivatives is further improved, and the HA-CA hydrogel can have more improved adhesive strength. Furthermore, the hydrogel with improved adhesion can more firmly encapsulate the endothelial cells in the hydrogel, and furthermore, when injected into the body, the adhesion rate in the tissue can be increased.

At this time, the concentration of the oxidizing agent may be 3 to 5 mg/ml, and the oxidizing agent of 3 to 5 mg/ml may be mixed with a solution in which the endothelial cell suspension and the HA-CA hydrogel are mixed at a volume ratio of 1:3. . Furthermore, the oxidizing agent may include at least one of the group consisting of sodium periodate, hydrogen peroxide, horseradish peroxidase, and tyrosinase, but preferably may be sodium periodate. However, it is not limited thereto.

In addition, the concentration of the pH adjusting agent may be 0.4 M, and gelation may be further induced by inducing a slightly alkaline environment of pH 8 through the pH adjusting agent. Furthermore, the pH adjusting agent is sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide hydroxide), strontium hydroxide and barium hydroxide, but preferably sodium hydroxide. However, it is not limited thereto.

Furthermore, the method of enhancing the encapsulation method of endothelial cells by the HA-CA hydrogel is not limited to the oxidizing agent and the pH adjusting agent described above. For example, methods for promoting cross-linking between hyaluronic acid derivatives may include chemical cross-linking, physical cross-linking, and biological cross-linking methods. Chemical crosslinking may include photo-crosslinking and crosslinking using a reactive crosslinker, and biological crosslinking may include crosslinking using the binding force of heparin and growth factors and crosslinking using complementary bonds such as DNA. physical cross-linking may include cross-linking by hydrogen bonding, cross-linking by hydrophobic interaction, and cross-linking by electrostatic interaction.

Thus, the HA-CA hydrogel with improved adhesion can stably encapsulate endothelial cells. More specifically, referring to FIG. 5A , an image of human umbilical vein endothelial cells encapsulated by the HA-CA hydrogel is shown. At this time, human umbilical vein endothelial cells were cultured for 24 hours with HA-CA hydrogel for encapsulation. After 24 hours of incubation, human umbilical vein endothelial cell populations are shown to be wrapped in HA-CA hydrogel. That is, human umbilical vein endothelial cells are shown to be encapsulated by HA-CA hydrogel.

Furthermore, referring to FIG. 5B , an image of endothelial cells derived from stem cells encapsulated by the HA-CA hydrogel is shown. At this time, PA-RGDS and PA-YIGS, which are amphiphilic peptide biomaterials, were used as controls. The encapsulation of stem cell-derived endothelial cells (hiPSC-EC) appears to be the most uniform for the HA-CA hydrogel. That is, it may mean that the HA-CA hydrogel is more suitable than conventional biomaterials for encapsulation of endothelial cells derived from stem cells.

Through the step of mixing the HA-CA hydrogel with the selected homogeneous endothelial cell suspension (S130), the endothelial cells are encapsulated to protect the endothelial cells from the microenvironment, so that the survival rate and differentiation rate can be increased.

More specifically, referring to FIG. 6A , an image obtained by observing the viability of endothelial cells by the HA-CA hydrogel is shown. At this time, endothelial cells differentiated from human induced pluripotent stem cells were cultured with HA-CA hydrogel for 7 days, calcein AM was used to identify live cells, and ethidium homodimer-1 was used to identify dead cells. did

After 7 days of culture, the HA-CA hydrogel-treated endothelial cells showed fewer dead cells than the HA-CA hydrogel-untreated control group. That is, endothelial cells appear to have a higher survival rate due to HA-CA treatment.

Furthermore, referring to FIG. 6B , an image showing the viability of endothelial cells by the HA-CA hydrogel under oxidative stress environmental conditions is shown. At this time, the oxidative stress environment conditions were created through H ₂ O ₂ treatment. Endothelial cells treated with the HA-CA hydrogel show that endothelial cells survive at a level similar to that of the untreated control group even under oxidative stress conditions. In contrast, endothelial cells not treated with HA-CA hydrogels appear to have few viable cells.

That is, these results may mean that the HA-CA hydrogel can protect endothelial cells from a harmful microenvironment without having cytotoxicity against endothelial cells differentiated from human induced pluripotent stem cells. Therefore, the composition for promoting angiogenesis according to an embodiment of the present invention provides high purity endothelial cells together with the HA-CA hydrogel, thereby providing high cell viability in vivo, thereby promoting angiogenesis for a longer period of time. can contribute

Furthermore, referring to FIG. 7A, an in vivo degradability observation image of the HA-CA hydrogel used in the composition for promoting angiogenesis of the present invention is shown. At this time, the HA-CA hydrogel labeled with FITC (Fluorescein isothiocyanate) was implanted into mouse hind limb muscle tissue and observed.

The HA-CA hydrogel appears to disappear over time after being injected into the muscle tissue of the hind limb of the mouse, that is, to be degraded in vivo, and is hardly observed after 17 days (day 17).

More specifically, referring to FIG. 7B , the result of the graph of FIG. 7A is shown. The HA-CA hydrogel was rapidly degraded from the first day (week 0) of injection into the muscle tissue of the hind limb of the mouse, showing a residual rate of 25% at week 1 and 18% at week 2 (week 2). It appears that the retention rate is That is, HA-CA injected into the body appears to be rapidly decomposed by about 14 to 17 days (2 weeks).

In addition, this in vivo degradability appears to have similar results in a mouse model of hind limb ischemia.

Therefore, when endothelial cells encapsulated by HA-CA hydrogel are injected in ischemic tissue, endothelial cells continuously induce angiogenesis in ischemic tissue and HA-CA hydrogel is degraded in vivo. . At this time, the HA-CA hydrogel is degraded in vivo on ischemic tissue, but can provide a sufficiently high survival rate for endothelial cells within cells.

The composition for promoting angiogenesis prepared according to the above manufacturing method includes high-purity endothelial cells and HA-CA hydrogel capable of stably protecting them to improve efficiency, and thus can have more improved blood vessel formation and blood vessel regeneration. there is.

Example 1: Angiogenesis-stimulating ability of the composition for promoting angiogenesis of the present invention

Hereinafter, the angiogenic effect of various compositions of the present invention will be described with reference to FIGS. 8A to 11C . In the following experiments, a hind limb ischemia (HLI) mouse model was used.

Referring to FIG. 8A , an image of blood flow recovery observation in a lower limb ischemic mouse model for the composition for promoting angiogenesis according to an embodiment of the present invention is shown. At this time, PBS was used as a control group, endothelial cells encapsulated with HA-CA hydrogel in Examples, HA-CA hydrogels in Comparative Example 1 and endothelial cells in Comparative Example 2 were used, and each composition was injected intramuscularly into a mouse model of lower limb ischemia. has been used Furthermore, blood flow restoration observation images were measured using a laser Doppler perfusion imager (LDPI).

After 4 weeks of injecting each composition, the example shows that the foot blood flow is restored the fastest. In contrast, in the control group, Comparative Examples 1 and 2, blood flow to the feet was not completely restored, resulting in foot loss.

More specifically, referring to 8b, it is a result graph for the experimental animals of FIG. 8a. At this time, referring to (a) of FIG. 8C, red in the graph means limb salvage, purple means limb muscle atrophy, and light blue means foot loss and necrosis. and necrosis), blue color means limb loss, and the results for each are expressed as a percentage of the number of experimental animals.

Referring to (b) of FIG. 8c, in the example, 88% of the experimental animals recovered their lower limbs, and in the control group and Comparative Examples 1 and 2, 29%, 43%, and 33% of the experimental animals recovered their lower limbs, respectively. appears as one That is, the composition for promoting angiogenesis according to an embodiment of the present invention including HA-CA hydrogel and endothelial cells appears to most effectively promote angiogenesis in an ischemic area.

Further referring to FIG. 8C , perfusion rates relative to FIG. 8A are shown. In this case, the perfusion ratio means the ratio of the ischemic foot to the non-ischemic foot.

Comparative Example 2 appears to have a high perfusion rate up to the 2nd week (2 wk), but from the 3rd week (3 wk), the Example appears to be the highest. That is, it appears that the composition for promoting angiogenesis according to an embodiment of the present invention including the HA-CA hydrogel and endothelial cells can most continuously maintain the angiogenic effect. This may mean that, as the viability of the endothelial cells encapsulated by the HA-CA hydrogel improves, the vascular regeneration effect in vivo lasts for a long time.

Accordingly, referring to FIG. 9 , an anatomical image of blood flow restoration in a lower limb ischemic mouse model for the composition for promoting angiogenesis according to an embodiment of the present invention is shown. At this time, the composition for promoting angiogenesis was intramuscularly injected into the lower extremity ischemic region, and 8 months later, the intramuscularly injected region was dissected and compared with normal tissue.

The lower extremity ischemic area observed after 8 months appeared to be similar to normal tissue as the lower extremity ischemia was recovered, and no loss or necrosis of the limbs or feet appeared.

Furthermore, referring to FIGS. 10A to 11C , results of immunohistochemical analysis of tissues isolated from a lower limb ischemic mouse model intramuscularly injected with a composition for promoting angiogenesis according to an embodiment of the present invention are shown. Hereinafter, endothelial cells in the composition for promoting angiogenesis according to an embodiment of the present invention are labeled by CM-Dil and appear in red light, and blood vessels having a function to allow blood flow are FITC-stained ILB4 (isolectin B4) By perfusing the solution intravascularly, it appears as green light. Furthermore, nuclei and chromatin were contrasted using DAPI staining. At this time, the control group refers to a group injected with a composition consisting only of endothelial cells, and the embodiment refers to a group injected with the composition for promoting angiogenesis according to an embodiment of the present invention.

First, referring to Figure 10a, after 3 to 4 weeks after intramuscular injection of each composition, the examples appear to have more endothelial cells of red light than the control group. That is, it may mean that the composition for promoting angiogenesis according to an embodiment of the present invention has higher retention and viability in vivo than the control group consisting only of endothelial cells.

Accordingly, referring to FIG. 10B , endothelial cells in the composition for promoting angiogenesis according to an embodiment of the present invention are shown to survive up to 12 months after being injected into the body. That is, it may mean that the viability and engraftment ability of endothelial cells are increased by the HA-CA hydrogel, so that the endothelial cells can last up to 12 months in vivo.

Furthermore, referring to Figure 11a, the endothelial cells in the composition for promoting angiogenesis according to an embodiment of the present invention are injected into the body and then 8 weeks after the red endothelial cells are linearly arranged (see the yellow dotted line) appears as This may mean that endothelial cells serve as guides for linear angiogenesis.

Accordingly, referring to FIG. 11B , along the linearly arranged red endothelial cells, green light blood vessels appear to be newly formed.

Furthermore, referring to FIG. 11C , it appears that red-colored endothelial cells are included in green-colored blood vessels.

That is, the composition for promoting angiogenesis according to an embodiment of the present invention serves as a guide for angiogenesis in ischemic tissue and can induce angiogenesis on its own, and furthermore, restores damaged blood vessels in existing blood vessels. That is, regeneration can be induced. As a result, the composition for promoting angiogenesis according to an embodiment of the present invention can simultaneously induce a process of directly generating new blood vessels from endothelial cells (vasculogenesis) and a process of forming new blood vessels from existing blood vessels (angiogenesis).

As a result of the above Example 1, the composition for promoting angiogenesis according to an embodiment of the present invention has improved adhesion and viability of endothelial cells through the HA-CA hydrogel, so that endothelial cells are applied for a long period of time in the tongue blood tissue Angiogenesis can be effectively induced for a longer period of time.

Example 2: Therapeutic and preventive effects of the composition for promoting angiogenesis of the present invention in cardiovascular diseases

Hereinafter, the therapeutic and preventive effects of the composition for promoting angiogenesis according to an embodiment of the present invention on cardiovascular diseases will be described with reference to FIGS. 12a to 12c.

12a to 12c, the results of immunohistochemical analysis of the heart induced with myocardial infarction (MI) injected with the composition for promoting angiogenesis according to an embodiment of the present invention are shown.

First, referring to FIG. 12A, a 4-week immunohistochemical analysis image of a heart induced with myocardial infarction (MI) injected with a composition for promoting angiogenesis according to an embodiment of the present invention is shown. Endothelial cells in the composition for promoting angiogenesis according to one embodiment of the present invention are shown to survive up to 4 weeks after being injected into the heart in which myocardial infarction was induced. More specifically, endothelial cells of red light survive up to 4 weeks, appear to be present in heart tissue, and appear to overlap inside and outside blood vessels of green light. That is, the composition for promoting angiogenesis according to an embodiment of the present invention can be used to directly generate new blood vessels from endothelial cells (vasculogenesis) and to form new blood vessels from existing blood vessels (angiogenesis), as well as to muscle cells such as the heart. A process of forming new blood vessels (arteriogenesis) by adding vascular myocytes in a tissue in which cells are present may be induced.

Accordingly, referring to FIG. 12B , an enlarged image of a site where myocardial infarction occurred in FIG. 12A is shown. In the composition for promoting angiogenesis according to an embodiment of the present invention, endothelial cells (EC/HA-CA) are lined up linearly, and linear blood vessels (FITC-IB4) appear to be newly formed along the line. That is, it may mean that the composition for promoting angiogenesis according to an embodiment of the present invention serves as a guide for angiogenesis even in heart tissue where myocardial infarction has occurred, and can induce vasculogenesis.

Furthermore, referring to FIG. 12c , it is shown that endothelial cells in the composition for promoting angiogenesis according to an embodiment of the present invention are contained and present in existing blood vessels in heart tissue. That is, it may mean that endothelial cells attach to and restore (angiogenesis) existing damaged blood vessels.

As a result of the above Example 2, the composition for promoting angiogenesis according to an embodiment of the present invention can prevent or treat cardiovascular diseases not only in extremities but also in various tissues in which blood vessels are present. More specifically, the composition for promoting angiogenesis according to an embodiment of the present invention is a cell therapeutic agent, and can treat and prevent diseases caused by the absence and disorder of blood vessels by restoring damaged blood vessels or inducing new blood vessels. .

As a result of Examples 1 and 2 above, endothelial cells used in the composition for promoting angiogenesis according to one embodiment of the present invention and the cell therapy composition for preventing or treating cardiovascular diseases according to another embodiment of the present invention and The effects of the HA-CA hydrogel on recovery of ischemic tissue and formation of functional blood vessels were confirmed.

More specifically, the composition for promoting angiogenesis according to an embodiment of the present invention contains homogeneous endothelial cells of high purity, and thus, regenerative treatment effects can be enhanced. Moreover, as the composition for promoting angiogenesis according to an embodiment of the present invention includes the HA-CA hydrogel, it protects the high-purity endothelial cells from the microenvironment to increase the survival rate and increase the in vivo adhesion for a long period of time. Thus, the regenerative treatment effect can be further improved.

Thus, the compositions according to various embodiments of the present invention can be used as a cell therapy having a high transplantation rate in the treatment of cardiovascular diseases.

Each feature of the various embodiments of the present invention can be partially or entirely combined or combined with each other, and as a person skilled in the art can fully understand, various interlocking and driving operations are possible, and each embodiment can be implemented independently of each other. and may be implemented together in a related relationship.

Although the embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and may be variously modified and implemented without departing from the technical spirit of the present invention. . Therefore, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of the present invention should be construed according to the claims below, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

Claims (21)

Including endothelial cells differentiated from stem cells and catechol-modified hyaluronic acid (HA-CA) hydrogel,
The stem cells,
Human pluripotent stem cells or human induced pluripotent stem cells,
The endothelial cells,
It contains more than 98% of endothelial cells expressing CDH5 (Cadherin 5),
further comprising an oxidizing agent and a pH adjusting agent;
The oxidizing agent,
At least one of the group consisting of sodium periodate, hydrogen peroxide, horseradish peroxidase, and tyrosinase,
The concentration of the oxidizing agent is,
3 to 5 mg/ml;
The pH adjusting agent,
Sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide At least one of the group consisting of strontium hydroxide and barium hydroxide,
The HA-CA,
A composition for promoting angiogenesis, which is a compound represented by Formula 1 below.
[Formula 1]

In the above formula, n is an integer from 2 to 700, and m is an integer from 1 to 300 smaller than n. delete delete delete According to claim 1,
The HA-CA,
A composition for promoting angiogenesis formed by binding a carboxyl group of HA (hyluronic acid) to an amine group of dopamine through an amide bond. delete delete delete According to claim 1,
The expression level of CDH5 mRNA in the endothelial cells,
A composition for promoting angiogenesis that is 10 times higher than undifferentiated stem cells or mesodermally-differentiated stem cells. According to claim 1,
The endothelial cells,
Encapsulated by the HA-CA hydrogel,
The in vivo survival rate of the endothelial cells,
A composition for promoting angiogenesis, which is 10 to 15 times higher than endothelial cells not encapsulated by the HA-CA hydrogel. According to claim 1,
The content of the endothelial cells,
75% by volume to 90% by volume relative to the total volume of the composition, the composition for promoting angiogenesis. According to claim 1,
The concentration of the endothelial cells,
1 X 10 ⁵ CFU / ml to 6 X 10 ⁶ CFU / ml, the composition for promoting angiogenesis. According to claim 1,
The content of the HA-CA hydrogel,
Based on the total volume of the composition, 1% by volume to 5% by volume, the composition for promoting angiogenesis. According to claim 1,
The composition,
A composition for promoting angiogenesis in a form administered by at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intratracheal and skin topical application. Cultivating stem cells to differentiate into endothelial cell lineage cells;
Selecting homogenous endothelial cells having a CDH5 (Cadherin 5) gene expression level of a predetermined level or higher among the differentiated endothelial cell lineage cells;
Mixing the HA-CA hydrogel with the homogeneous endothelial cell suspension,
Further comprising the step of mixing the oxidizing agent and the pH adjusting agent,
The predetermined level is,
is 98%,
The oxidizing agent,
At least one of the group consisting of sodium periodate, hydrogen peroxide, horseradish peroxidase, and tyrosinase,
The concentration of the oxidizing agent is,
3 to 5 mg/ml;
The pH adjusting agent,
Sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide At least one of the group consisting of strontium hydroxide and barium hydroxide,
The HA-CA,
A method for producing a composition for promoting angiogenesis, which is a compound represented by Formula 1 below.
[Formula 1]

In the above formula, n is an integer from 2 to 700, and m is an integer from 1 to 300 smaller than n. According to claim 15,
The culturing step is
culturing the stem cells to differentiate into cells of the mesodermal lineage; and
A method for producing a composition for promoting angiogenesis comprising culturing the mesodermal lineage cells to differentiate into endothelial lineage cells. According to claim 16,
The step of culturing to differentiate into the endothelial cell lineage cells,
A method for producing a composition for promoting angiogenesis, comprising the step of differentiating into the endothelial cell lineage cells by culturing the mesodermal lineage cells in a medium treated with DLL4 (Delta Like Canonical Notch Ligand 4). According to claim 15,
The step of processing the HA-CA hydrogel,
A method for producing a composition for promoting angiogenesis, which is performed for one period of 2 to 7 days. According to claim 15,
After the step of mixing the HA-CA hydrogel, further comprising the step of mixing an oxidizing agent and a pH adjusting agent, a method for producing a composition for promoting angiogenesis. A cell therapy composition for preventing or treating cardiovascular diseases, comprising the composition for promoting angiogenesis according to any one of claims 1, 5, and 9 to 14. 21. The method of claim 20,
The cardiovascular disease,
of cardiovascular disease, including at least one of the group consisting of thrombosis, peripheral arterial disease, ischemic heart disease, angina pectoris, cerebrovascular disease, coronary artery disease, angina pectoris, myocardial infarction, arteriosclerosis, arrhythmic hypertension, and diabetic vascular disease Cell therapy composition for prophylaxis or treatment.

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