Method for preparing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, and composition for prevention or treatment of cardio-cerebrovascular diseases, comprising prepared vascular endothelial cells

Abstract

The present invention relates to a method for preparing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, and a composition for the prevention or treatment of ischemic disease, comprising the prepared vascular endothelial cells, the method for preparing vascular endothelial cells, according to the present invention, being a method for inducing direct differentiation from embryonic stem cell-derived mesenchymal stem cells into vascular endothelial cells by using ER71. In the present invention, the differentiation into vascular endothelial cells has been optimized by identifying the differentiation efficiency according to various culture additives. In addition, the vascular endothelial cells thus obtained have enhanced immune tolerance properties and enable hindlimb salvage through angiogenesis in a mouse model of hindlimb ischemia, and thus the vascular endothelial cells prepared by the method of the present invention are expected to be usefully employable for the treatment of cardio-cerebrovascular diseases without exhibiting immune rejection when injected in vivo.

Description

Method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells and composition for preventing or treating cerebrovascular and cardiovascular diseases comprising the produced vascular endothelial cells

[0001] The present invention relates to a method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells and a composition for preventing or treating cardiovascular and cerebrovascular diseases comprising the produced vascular endothelial cells.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2024-0021324, filed February 14, 2024, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Angiogenesis is a highly regulated process that occurs in response to various angiogenic factors, such as growth factors, cytokines, and other physiological molecules, as well as hypoxia and low pH. The angiogenic mechanism for the development of new blood vessels requires the cooperation of various molecules that regulate the degradation and remodeling of the extracellular matrix (ECM), migration, proliferation, differentiation, and tube formation. After the initiation of angiogenesis, angiogenic factors such as VEGF, bFGF, and PDGF activate endothelial cells through stimulation of cell surface receptors. The activated cells then undergo cell proliferation, increased expression of cell adhesion molecules, increased secretion of proteolytic enzymes, and increased cell migration and invasion. Additionally, numerous molecules, including members of the integrin, selectin, and immunoglobulin gene superfamilies for cell adhesion, as well as proteolytic enzymes such as matrix metalloproteases and serine proteases for degrading the ECM, promote proliferation and invasion, and furthermore, angiogenesis is formed by inducing lumen formation and differentiation into mature blood vessels by signaling mechanisms derived from cell surface receptors that interact with ECM components and soluble factors.

[0005] Recently, there have been active attempts to treat diseases that depend on angiogenesis, such as cancer, rheumatoid arthritis, psoriasis, ulcers, ischemia, arteriosclerosis, myocardial infarction, angina pectoris, and cerebrovascular diseases, by using factors that induce or inhibit angiogenesis.

[0006] In normal adults, the turnover time of endothelial cells forming blood vessels generally varies from 47 to 20,000 days and is under very strict regulation. Normally, angiogenesis inhibitors such as thrombospondin-1, platelet factor-4, and angiostatin, and angiogenesis promoters such as vascular endothelial growth factor and basic fibroblast growth factor maintain a quantitative balance so that angiogenesis does not occur. However, when a wound or cancer occurs, the quantitative balance of angiogenesis inhibitors and promoters is broken and new blood vessels are formed for the regeneration of wounded tissue and the growth of cancer. At this time, overexpression of angiogenesis promoters is involved.

[0007] Therefore, while excessive angiogenesis can be a major cause of disease exacerbation, angiogenesis also contributes to serious diseases. Angiogenesis is essential for wound healing and tissue regeneration. For example, a placenta with underdeveloped angiogenesis is a significant cause of miscarriage. Necrosis, ulceration, and ischemia caused by angiogenesis can lead to tissue or organ dysfunction or even death.

[0008] Therefore, it is very important to reduce tissue damage caused by hypoxia or hyponutrient conditions due to anemia and to induce or promote new blood vessel formation for smooth tissue regeneration.

[0009] In particular, angiogenesis is essential for the wound healing process, which is essential for the regeneration of injured skin tissue. In the initial stages of wound healing, an inflammatory response occurs, resulting in cell necrosis and vascular destruction. This inflammatory response is followed by a series of events, including devascularization of blood components, platelet activation, and blood coagulation, along with the formation of biological mediators such as kallikrein, thrombin, and plasmin.

[0010] Ischemia is a partial blood shortage condition in which normal angiogenesis is insufficient, resulting in a disruption of arterial blood flow to tissues, organs, or limbs, resulting in cell damage. Ischemia causes a lack of oxygen and glucose, which are necessary for cell metabolism, and can lead to tissue damage or death (ischemic damage). Ischemic diseases are a general term for diseases caused by this ischemic condition, including ischemic lower extremity disease, ischemic stroke, ischemic colitis, and cardiovascular diseases. Ischemic stroke and ischemic heart disease, which are representative ischemic diseases, are caused by ischemia in which the cerebral or coronary artery is occluded by a thrombus or arteriosclerosis, reducing blood flow below a critical level. This damages brain and heart cells, ultimately leading to cell death, and this can lead to cerebral infarction and myocardial infarction. In addition, interruption of blood supply due to ischemia can cause various ischemic diseases such as ischemic heart failure, ischemic enteritis, eye disease, and ischemic lower extremity disease.

[0011] Limb ischemia, which has a separate etiology and standard treatment from cardiac ischemia, results from inadequate blood flow to the limb. Approximately 2 million people in the United States suffer from limb ischemia each year. For 60% of these patients, no effective treatment options are available, and no medications exist that can successfully treat ischemic limb damage. While revascularization of tissue and / or the use of various skin substitutes are common treatments, these treatments are not always sufficient to salvage the damaged limb, leading to amputation, the only viable treatment option.

[0012] Treatment options for ischemic diseases include drug therapy, coronary angioplasty using stents to dilate narrowed blood vessels, and, in the case of the heart, arterial bypass grafting. However, these treatments may not be adequate if the blood vessels are too hardened, all available blood vessels for transplantation have been used, or recurrence persists despite repeated angioplasties due to restenosis. Therefore, to overcome the limitations of these surgical treatments, cell therapies have been developed to induce angiogenesis in patients with ischemic cardiovascular disease. However, the technology for mass-producing and injecting endothelial cells has not been available, and instead, multipotent stem cells have been injected in the hope that they will differentiate into endothelial cells. These cell therapies using multipotent stem cells have only been effective in promoting endothelial cell growth around ischemic tissue by supplying growth factors and cytokines, rather than inducing true endothelial cell differentiation in the target organ. Accordingly, there is a need for a technology to mass-produce vascular endothelial cells that can directly promote angiogenesis, and in particular, it is necessary to produce vascular endothelial cells with immune tolerance for transplantation into other people's tissues and organs.

[0013] Meanwhile, previous studies have reported that terminally differentiated fibroblasts can be directly converted into functional endothelial cells in mice and humans by inducing a combination of key transcription factors. This direct conversion technique has the advantage of not requiring pluripotency induction. However, one major limitation of this technique is that cells do not regenerate during the reprogramming process. Directly reprogrammed cells maintain their cell age, suggesting that direct endothelial conversion of fibroblasts from elderly patients can generate aged endothelial cells. Given that most candidates for cell therapies based on direct conversion technology are likely to have age-related diseases, and that aged cells have low proliferative capacity and limited cellular function, this limitation will likely limit the therapeutic applications of direct reprogramming.

[0014] In contrast, induced pluripotent stem cell (iPSC) technology rejuvenates cells during reprogramming. However, current protocols for deriving endothelial cells from iPSCs or embryonic stem cells (ESCs) require complex redifferentiation and redifferentiation processes, with modest yields (10-60%). Furthermore, this process carries the risk of contamination by pluripotent cells. Given the infinite self-renewal capacity of pluripotent cells, even a small fraction of contaminated pluripotent stem cells cannot be ignored.

[0015] Therefore, the ideal endothelial cell progenitor cell should satisfy the following conditions: (1) it should be a progenitor cell that maintains a young cell age, (2) it should not be pluripotent to avoid the risk of tumorigenesis, and (3) it should be easily obtained and ready for use.

[0016] Mesenchymal stem cells (MSCs) are excellent candidates for these conditions. MSCs from various tissues, including bone marrow, adipose tissue, and umbilical cord, have been reported, and possess multilineage potential, capable of differentiating into mesenchymal tissue lineages including bone, cartilage, fat, tendon, and muscle. Furthermore, MSCs possess high proliferative potential, and one of their most interesting advantages is their immune tolerance, enabling transplantation across major histocompatibility barriers.

[0017] Accordingly, the present inventors developed a method for directly differentiating MSCs into vascular endothelial cells using direct differentiation based on the induction of key transcription factors, and aimed to apply the manufactured vascular endothelial cells to a new treatment for regenerative medicine in cardiovascular and cerebrovascular diseases, including ischemic diseases.

[0018]

[0019] The present invention was devised to solve the problems of the prior art as described above, and confirmed that ER71 directly induces differentiation of embryonic stem cell-derived mesenchymal stem cells into endothelial cells, and optimized differentiation into endothelial cells by confirming the differentiation efficiency according to various culture additives. In addition, it was confirmed that the vascular endothelial cells obtained through this have enhanced immune tolerance characteristics and are capable of saving the lower extremities through angiogenesis in a mouse lower extremity ischemia model, and confirmed that they can exhibit therapeutic effects on cardiovascular and cerebrovascular diseases without showing an immune rejection response when injected in vivo, and based on this, the present invention was completed.

[0020] Accordingly, the present invention provides a method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, which comprises a step of introducing an ER71 (E26 transformation-specific related protein 71) gene into embryonic stem cell-derived mesenchymal stem cells.

[0021] In addition, the present invention provides a pharmaceutical composition for preventing or treating cerebrovascular and cardiovascular diseases, which comprises vascular endothelial cells produced by the above method as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0022]

[0023] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0024]

[0025] To achieve the above purpose, the present invention provides a method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, which comprises a step of introducing an ER71 (E26 transformation-specific variant transcription factor 2) gene into embryonic stem cell-derived mesenchymal stem cells.

[0026] As one embodiment of the present invention, the vascular endothelial cells may be directly differentiated from mesenchymal stem cells derived from embryonic stem cells, but are not limited thereto.

[0027] As another embodiment of the present invention, the step of introducing the ER71 gene may be performed in a medium containing at least one selected from the group consisting of SB431542, vascular endothelial growth factor (VEGF), and ascorbic acid, but is not limited thereto.

[0028] As another embodiment of the present invention, the manufactured vascular endothelial cells may have immune tolerance characteristics, but are not limited thereto.

[0029] In another embodiment of the present invention, the immune tolerance property may be mediated by, but is not limited to, the Ikaros protein.

[0030] As another embodiment of the present invention, the manufactured vascular endothelial cells may be, but are not limited to, vascular endothelial cell (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM1) double positive cells.

[0031] As another embodiment of the present invention, the manufactured vascular endothelial cells can induce blood vessel formation, but are not limited thereto.

[0032] In addition, the present invention provides a pharmaceutical composition for preventing or treating cerebrovascular and cardiovascular diseases, which comprises vascular endothelial cells produced by the above method as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0033] As one embodiment of the present invention, the cerebrovascular disease may be at least one selected from the group consisting of lower extremity vascular disease, ischemic cerebral disease, and heart disease, but is not limited thereto.

[0034] As another embodiment of the present invention, the lower extremity vascular disease may be at least one selected from the group consisting of lower extremity arterial occlusive disease, lower extremity arterial stenotic disease, and lower extremity arteriosclerosis, but is not limited thereto.

[0035] As another embodiment of the present invention, the ischemic cerebral disease may be at least one selected from the group consisting of ischemic stroke, vascular dementia, Alzheimer's dementia, Huntington's disease, and Parkinson's disease, but is not limited thereto.

[0036] As another embodiment of the present invention, the heart disease may be at least one selected from the group consisting of coronary artery disease, congestive heart failure, chronic heart failure, cardiomyopathy, and myocardial infarction, but is not limited thereto.

[0037] As another embodiment of the present invention, the cardiomyopathy may be at least one selected from the group consisting of ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and valvular cardiomyopathy, but is not limited thereto.

[0038] In addition, the present invention provides a method for preventing or treating cerebrovascular and cardiovascular diseases, comprising a step of administering a pharmaceutically effective amount of vascular endothelial cells produced by the above method or a composition containing the same as an active ingredient to a subject in need thereof, wherein the vascular endothelial cells are characterized in that they are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0039] In addition, the present invention provides a use for preventing or treating cerebrovascular and vascular diseases using vascular endothelial cells produced by the above method or a composition containing the same as an effective ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0040] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating cerebrovascular and cardiovascular diseases using vascular endothelial cells manufactured through the above method or a composition containing the same as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0041]

[0042] The method for producing vascular endothelial cells according to the present invention is a method for inducing direct differentiation of embryonic stem cell-derived mesenchymal stem cells into vascular endothelial cells using ER71. In the present invention, differentiation into vascular endothelial cells was optimized by confirming the differentiation efficiency according to various culture additives. In addition, the vascular endothelial cells obtained through this method have enhanced immune tolerance characteristics and are capable of saving the lower extremities through angiogenesis in a mouse lower extremity ischemia model. Therefore, the vascular endothelial cells produced by the method of the present invention are expected to be useful in the treatment of cardiovascular and vascular diseases without showing an immune rejection response when injected in vivo.

[0043]

[0044] Figure 1a is a drawing showing the results of surface marker characteristic analysis of embryonic stem cell-derived mesenchymal stem cells (EMSCs) according to one embodiment of the present invention.

[0045] FIG. 1b is a diagram schematically illustrating a process for directly differentiating EMSCs into vascular endothelial cells (ECs) according to one embodiment of the present invention.

[0046] Figure 1c is a drawing showing the results of confirming direct differentiation of MSCs into ECs through confirmation of VE-cadherin / PECAM1 double positive cells according to one embodiment of the present invention.

[0047] FIG. 1d is a drawing confirming the morphological characteristics of classified VE-cadherin / PECAM1 double positive cells according to one embodiment of the present invention (scale bar = 250 μm).

[0048] Figure 2a is a drawing confirming the effect of SB431542 and VEGF on improving MiEC differentiation efficiency according to one embodiment of the present invention (Rosi: rosiglitazone, SB: SB431542).

[0049] Figure 2b is a drawing confirming the effect of improving MiEC differentiation efficiency of the combination of SB431542 and VEGF according to one embodiment of the present invention.

[0050] FIG. 2c is a diagram showing the results of time-dependent evaluation of MiEC differentiation kinetics by continuous flow cytometry according to one embodiment of the present invention.

[0051] Figure 2d is a diagram showing the MiEC differentiation efficiency according to the type of extracellular matrix for coating a culture dish according to one embodiment of the present invention.

[0052] Figure 2e is a diagram showing the MiEC differentiation efficiency by ascorbic acid according to one embodiment of the present invention (AA: ascorbic acid).

[0053] FIG. 2f is a diagram showing the results of confirming an optimized protocol for direct differentiation of EMSCs into functional ECs on a fibronectin-coated dish according to one embodiment of the present invention.

[0054] Figure 3a is a diagram confirming the effect of polybrene on the expression level of ER71 and MiEC differentiation efficiency induced by lentiviral transduction according to one embodiment of the present invention (NT: untreated, PB: polybrene).

[0055] FIG. 3b is a diagram confirming the effect of polybrene treatment on the proliferation ability of MiECs during lentiviral transduction according to one embodiment of the present invention (TD: transduction).

[0056] Figure 3c is a diagram showing the effect of polybrene treatment on the total cell number (left diagram) and MiEC number (right diagram) during lentiviral transduction according to one embodiment of the present invention.

[0057] FIG. 3d is a drawing confirming the MiEC differentiation efficiency according to the presence or absence of polybrene treatment, the type of extracellular matrix for coating, and the presence or absence of chemical treatment according to one embodiment of the present invention (FN: fibronectin, SVA: SB431542).

[0058] Figure 4a is a drawing confirming the differentiation of bone marrow (BM-MSC) and umbilical cord-derived MSC (UM-MSC) into functional ECs according to one embodiment of the present invention (SV: SB431542).

[0059] Figure 4b is a drawing confirming the direct vascular endothelial differentiation efficiency of MSCs of various origins according to one embodiment of the present invention.

[0060] FIG. 5a is a drawing confirming the morphological characteristics of MiEC induced according to an optimized protocol according to one embodiment of the present invention (scale bar = 250 μm).

[0061] Figure 5b is a drawing confirming capillary formation in matrigel of MiEC according to one embodiment of the present invention (scale bar = 250 μm).

[0062] Figure 5c is a drawing showing the results of immunofluorescence staining for a vascular endothelial marker according to one embodiment of the present invention (scale bar = 50 μm).

[0063] FIG. 5d is a diagram showing the results of an acetylated low-density lipoprotein (Ac-LDL) uptake analysis of MiEC according to one embodiment of the present invention (scale bar = 50 μm).

[0064] Figure 5e is a drawing showing the results of a UEA (Ulex europaeus agglutinin)-1 lectin binding analysis of MiEC according to one embodiment of the present invention (scale bar = 50 μm).

[0065] FIG. 5f is a diagram showing real-time PCR results for a vascular endothelial marker according to one embodiment of the present invention.

[0066] Figure 5g is a drawing confirming the limb perfusion and rescue effect by MiEC transplantation in a rat hindlimb ischemia model according to one embodiment of the present invention.

[0067] FIG. 6a is a diagram showing the results of identifying the MiEC, EC, and EMSC cell composition of each sample using XCell according to one embodiment of the present invention.

[0068] FIG. 6b is a diagram illustrating a hierarchical clustered gene expression heatmap of EC-specific and EMSC-specific genes according to one embodiment of the present invention.

[0069] Figure 6c is a diagram showing the results of statistically enriched cell type signature analysis (left diagram) and PaGenBase annotation analysis (right diagram) using differentially expressed genes (DEGs) of each cluster according to one embodiment of the present invention.

[0070] Figure 6d is a diagram showing the results of gene ontology analysis using DEGs of each cluster according to one embodiment of the present invention.

[0071] Figure 6e is a diagram showing the results of network analysis for cluster 1 and cluster 2 genes according to one embodiment of the present invention.

[0072] FIG. 6f is a diagram showing the results of confirming DEG in MiEC using a volcano plot compared to EMSC according to one embodiment of the present invention.

[0073] FIG. 6g is a diagram comparing differentially methylated regions (DMRs) of EMSCs and MiECs according to one embodiment of the present invention.

[0074] Figure 6h is a diagram showing the results of confirming the methylation pattern of DMR confirmed in a comparison of EMSC and MiEC according to one embodiment of the present invention.

[0075] FIG. 6i is a diagram showing the results of analyzing the methylation pattern of genes upregulated in MiECs compared to EMSCs according to one embodiment of the present invention.

[0076] Figure 6j is a diagram confirming the gene-specific methylation level for a vascular endothelial marker gene according to one embodiment of the present invention.

[0077] FIG. 7a is a diagram showing the results of confirming human leukocyte antigen (HLA)-G expression of MiEC according to one embodiment of the present invention using flow cytometry (left diagram) and real-time PCR (right diagram).

[0078] Figure 7b is a diagram showing the results of ELISA for IL-6 secretion in a culture medium of MiEC according to one embodiment of the present invention.

[0079] Figure 7c is a diagram showing the real-time PCR results for the expression of indoleamine 2,3-dioxygenase (IDO) 1 in MiEC 2 days after IFN-γ stimulation according to one embodiment of the present invention.

[0080] Figure 7d is a drawing showing the results of nitric oxide (NO) production analysis of MiEC according to one embodiment of the present invention (scale bar = 250 μm).

[0081] Figure 7e is a diagram showing the results of flow cytometry analysis of HLA-DR expression in MiECs 2 days after IFN-γ stimulation according to one embodiment of the present invention.

[0082] FIG. 8A is a diagram showing the results of screening for transcription factors capable of regulating four major immune suppression molecules according to one embodiment of the present invention.

[0083] Figure 8b is a drawing showing the results of confirming the mRNA expression levels of ER71 and IKZF1 by real-time PCR after transducing ER71 into EMSCs according to one embodiment of the present invention.

[0084] Figure 8c is a diagram showing the results of confirming the mRNA expression levels of 13 transcription factors by real-time PCR according to one embodiment of the present invention.

[0085] FIG. 8d is a drawing showing the results of confirming ER71 and Ikaros protein expression by Western blot after transduction of ER71 according to one embodiment of the present invention.

[0086] Figure 8e is a diagram showing the results of confirming the mRNA expression levels of HLA-G, IL-6, IDO1, and NOS3 by real-time PCR after transduction of ER71 according to one embodiment of the present invention.

[0087] Figure 8f is a drawing showing the results of Western blot analysis of knockdown of Ikaros by shIKZF1 according to one embodiment of the present invention.

[0088] Figure 8g is a drawing showing the results of confirming the inhibitory effect of shIKZF1 on the increase of immune suppression factors by ER71 transduction according to one embodiment of the present invention through real-time PCR.

[0089] Figure 8h is a diagram showing the results of confirming the expression levels of IKZF1, SH2B3, CITA, and HLA-DR mRNA in MiEC and GEAEC by real-time PCR under IFN-γ stimulation according to one embodiment of the present invention.

[0090] Figure 8i is a drawing showing the results of confirming the phosphorylation inhibition signal transduction pathway of STAT1 in MiEC according to one embodiment of the present invention by Western blot.

[0091] Figure 8j is a drawing showing the results of confirming the phosphorylation inhibition signal transduction pathway of STAT1 in MiEC according to one embodiment of the present invention by immunofluorescence staining.

[0092] FIG. 8k is a diagram showing the results of confirming the effect of shIKZF1 on the expression of IKZF1, SH2B3, CITA, and HLA-DR induced by ER71 transduction according to one embodiment of the present invention, using real-time PCR.

[0093] Figure 8l is a drawing showing the results of confirming the signal transduction pathway of STAT1 by shIKZF1 in MiEC according to one embodiment of the present invention by Western blot.

[0094] FIG. 8m is a schematic diagram showing the immune tolerance characteristic mechanism of MiEC induced by ER71 transduction according to one embodiment of the present invention.

[0095] FIG. 9a is a diagram schematically illustrating an in vitro experimental plan for determining whether MiEC according to one embodiment of the present invention can evade the host immune system.

[0096] FIG. 9b is a diagram showing the results of flow cytometry analysis to determine the proportion of cells with reduced CFSE signals among CTV (CellTrace Violet) negative cells according to one embodiment of the present invention.

[0097] FIG. 10a is a diagram schematically illustrating an in vivo experimental plan to determine whether MiEC according to one embodiment of the present invention can evade the host immune system.

[0098] FIG. 10b is a diagram showing the results of flow cytometry analysis of the survival rate of transplanted cells in immunocompromised BALB / c nude mice and immunocompetent C57BL / 6 mice according to one embodiment of the present invention (n=4~5).

[0099] FIG. 10c is a schematic diagram showing the response of the host immune system to MiEC or control EC in an immunocompetent mouse according to one embodiment of the present invention.

[0100] FIG. 11 is a diagram showing the results of confirming the mRNA expression of OCT4 and NANOG by real-time PCR during direct differentiation of EMSCs into MiECs according to one embodiment of the present invention.

[0101] Figures 12a and 12b are diagrams showing the results of comparing the quality and efficiency of EC induction through direct differentiation of EMSCs according to one embodiment of the present invention with that through direct conversion of human fibroblasts.

[0102]

[0103] The present inventors developed a method for directly differentiating vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells through induction by ER71 transduction, and optimized differentiation into vascular endothelial cells by examining the differentiation efficiency according to various culture additives. The vascular endothelial cells produced by the method of the present invention have enhanced immune tolerance characteristics and were confirmed to be capable of saving the lower extremities through angiogenesis in a mouse lower extremity ischemia model. Therefore, they elucidated that this can be applied to a new treatment for regenerative medicine in ischemic diseases, and based on this, the present invention was completed.

[0104]

[0105] Hereinafter, the present invention will be described in detail.

[0106]

[0107] The present invention provides a method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, comprising the step of introducing an ER71 (E26 transformation-specific variant transcription factor 2) gene into embryonic stem cell-derived mesenchymal stem cells.

[0108] In the present invention, the ER71 gene may be a human ER71 gene, and may be a base sequence selected from the group consisting of NCBI Reference Sequence: NM_014209.4, NCBI Reference Sequence: NM_001300974.2, and NCBI Reference Sequence: NM_001304549.2, and 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 75 to 100%, 80 to 100%, 85 to 100%, 90 to 100%, 95 to 100%, 98 to 100%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 It may be composed of or includes a base sequence having a sequence identity of, but is not limited to, 98%, 99%, or 100%.

[0109] In the present invention, “stem cells” are cells that form the basis of cells or tissues that constitute an individual, and their characteristics are that they can self-renew through repeated division and have the ability to differentiate into cells with specific functions depending on the environment. They are produced in all tissues during fetal development, and are also found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissues, even in adults. Depending on the type of cell capable of differentiation, stem cells are classified into totipotent stem cells that are formed when a fertilized egg begins its first division, pluripotent stem cells in the blastocyst lining that are created through continuous division of these cells, and multipotent stem cells that exist in mature tissues and organs. Pluripotent stem cells, at this time, are cells that can differentiate only into cells specific to the tissues and organs in which they are contained. They are involved in the growth and development of each tissue and organ during the fetal, neonatal, and adult stages, as well as in maintaining homeostasis in adult tissues and inducing regeneration in the event of tissue damage. These tissue-specific pluripotent cells are collectively referred to as adult stem cells.

[0110] Mesenchymal stem cells, classified as adult stem cells, are well known as repair cells of various connective tissues, and these cells can differentiate into various types of mesenchymal cells. In addition, because of the advantages of easy acquisition and clinical application, they can complement the shortcomings of neural stem cells, which are located deep in the brain and are difficult to obtain in sufficient quantities and have the risk of brain damage, and are thus attracting attention as a material for the development of therapeutics for nervous system diseases and regenerative medicine. They can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord, and, unlike blood stem cells, have the ability to differentiate into cells that constitute various human tissues, such as adipocytes, osteocytes, chondrocytes, nerve cells, and cardiomyocytes. In the present invention, human embryonic stem cell-derived mesenchymal stem cells were used to directly induce differentiation into vascular endothelial cells.

[0111] In the present invention, "endothelial cell" may refer to a flat cell that forms the layer covering the inner walls of blood vessels and lymphatic vessels. Accordingly, "endothelial cell" may be used interchangeably with "vascular endothelial cell."

[0112] In the present invention, “direct differentiation” means directly differentiating embryonic stem cell-derived mesenchymal stem cells into vascular endothelial cells through transcription factors and an optimized medium, without going through induced pluripotent stem cells (iPSCs).

[0113] In the present invention, the method may further include, but is not limited to, a step of culturing embryonic stem cell-derived mesenchymal stem cells in a culture dish coated with various components including gelatin, fibronectin, collagen, laminin, or poly-L-lysine. In the present invention, the step of introducing the ER71 gene may be performed in a medium containing at least one selected from the group consisting of SB431542, vascular endothelial growth factor (VEGF), and ascorbic acid, but is not limited thereto. According to one embodiment or experimental example of the present invention, when the ER71 gene is introduced into embryonic stem cell-derived mesenchymal stem cells in a medium containing SB431542, VEGF, or ascorbic acid alone or a combination thereof, the differentiation efficiency into vascular endothelial cells can be increased. For example, when the ER71 gene is introduced and cultured in a medium containing at least one selected from the group consisting of SB431542, VEGF, ascorbic acid, SB431542+VEGF, SB431542+ascorbic acid, VEGF+ascorbic acid, and SB431542+VEGF+ascorbic acid, the differentiation efficiency can be increased, but is not limited thereto.

[0114] In the present invention, the method for producing vascular endothelial cells from the embryonic stem cell-derived mesenchymal stem cells includes a step of introducing an ER71 (E26 transformation-specific related protein 71) gene into the embryonic stem cell-derived mesenchymal stem cells, and the step of introducing the ER71 gene may be a step performed in a medium containing at least one selected from the group consisting of SB431542, VEGF, and ascorbic acid in a culture dish coated with fibronectin, but is not limited thereto.

[0115] In the present invention, “introduction” means a phenomenon in which DNA of a certain bacterium is transferred to another bacterium, and may be, for example, transduction using a virus such as lentivirus or adeno-associated virus (AAV) or transformation using a plasmid, but is not limited thereto.

[0116] In the present invention, the manufactured vascular endothelial cells may have immune tolerance characteristics, and the immune tolerance characteristics may be mediated by the Ikaros protein, but are not limited thereto.

[0117] In the present invention, “immune tolerance” means a state of non-responsiveness of the immune system to a substance or tissue capable of eliciting an immune response.

[0118] Vascular endothelial cells produced by the method of the present invention have enhanced immune tolerance characteristics compared to mesenchymal stem cells, and thus have the characteristic of being able to be transplanted into tissues and organs of other people.

[0119] In the present invention, the manufactured vascular endothelial cells may be double positive cells for vascular endothelial cell (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM1), which are indicators of functional endothelial cells, but are not limited thereto.

[0120] In the present invention, the manufactured vascular endothelial cells can induce blood vessel formation and have an angiogenic effect, but are not limited thereto.

[0121] In the present invention, "angiogenesis" refers to the physiological process by which new blood vessels are formed from existing ones, occurring in the early stages of angiogenesis. Angiogenesis primarily sustains the growth of vasculature through sprouting and branching processes, but processes such as adhesive angiogenesis, vascular elongation, and vascular fusion also play a role. Angiogenesis is a normal and important process in growth and development, wound healing, and granulation tissue formation.

[0122]

[0123] In addition, the present invention provides a composition for preventing, treating, or improving cerebrovascular and cardiovascular diseases, comprising vascular endothelial cells produced through the above method as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0124] In the present invention, the composition may be a pharmaceutical composition or a food composition, and the food composition may be a health functional food composition, but is not limited thereto.

[0125] The present invention provides a pharmaceutical composition for preventing or treating cerebrovascular and cardiovascular diseases, comprising vascular endothelial cells produced by the above method as an active ingredient, characterized in that the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0126] The pharmaceutical composition for preventing or treating cerebral and vascular diseases of the present invention refers to a composition comprising vascular endothelial cells produced by the method of the present invention so that they can be injected or transplanted into a living animal and taken root.

[0127] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0128] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0129] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0130] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0131] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0132] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0133] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0134] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0135] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0136] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0137] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0138] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0139] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0140] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0141] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0142] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0143] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0144] In the present invention, the cerebrovascular disease may be at least one selected from the group consisting of lower extremity vascular disease, ischemic cerebral disease, and heart disease, but is not limited thereto.

[0145] In the present invention, the lower extremity vascular disease includes, but is not limited to, lower extremity arterial occlusive disease, lower extremity arterial stenotic disease, or lower extremity arteriosclerosis.

[0146] In the present invention, the ischemic brain disease includes, but is not limited to, ischemic stroke, vascular dementia, Alzheimer's dementia, Huntington's disease, or Parkinson's disease.

[0147] In the present invention, the heart disease includes, but is not limited to, coronary artery disease, congestive heart failure, chronic heart failure, cardiomyopathy, or myocardial infarction.

[0148] In the present invention, the cardiomyopathy refers to a group of diseases in which abnormalities occur in the heart muscle, causing symptoms such as dyspnea, chest pain, and palpitations, without other heart diseases such as congenital, valvular, hypertension, coronary artery, and pericardial diseases, and includes, but is not limited to, ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and valvular cardiomyopathy.

[0149] In the present invention, "ischemia" refers to a condition in which blood vessels supplying blood to a bodily organ, tissue, or area become narrowed or constricted, or normal angiogenesis is insufficient, resulting in a disruption in blood supply and localized tissue necrosis. In particular, the heart and brain are the organs most sensitive to insufficient blood flow. When ischemia occurs in a tissue, a series of processes known as the ischemic cascade is triggered, resulting in permanent tissue damage.

[0150]

[0151] In addition, the present invention provides a method for preventing or treating cerebrovascular and cardiovascular diseases, comprising a step of administering a pharmaceutically effective amount of vascular endothelial cells produced by the above method or a composition containing the same as an active ingredient to a subject in need thereof, wherein the vascular endothelial cells are characterized in that they are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0152] In addition, the present invention provides a use for preventing or treating cerebrovascular and vascular diseases using vascular endothelial cells produced by the above method or a composition containing the same as an effective ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0153] In addition, the present invention provides a use for manufacturing a preparation for preventing or treating cerebrovascular and cardiovascular diseases using vascular endothelial cells manufactured through the above method or a composition containing the same as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

[0154] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0155] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0156] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0157] In the present invention, when the term "comprising" is used, it means that other components can be included rather than excluding other components unless specifically stated otherwise. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0158]

[0159] Hereinafter, preferred examples or experimental examples are presented to aid in understanding the present invention. However, the following examples or experimental examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples or experimental examples.

[0160]

[0161] [Example]

[0162] Example 1. Cell culture

[0163] Embryonic stem cell (ESC)-derived mesenchymal stem cells (EMSC) were prepared as previously reported (Tissue Eng Part A. 2010 Feb;16(2):705-15.) and cultured in microvascular endothelial cell growth medium MV2 (EGM-2MV) (Promocell, C-22121) in tissue culture-treated plates (Falcon, 353046). 293T cells were cultured in DMEM medium (Gibco, 11995-092) containing 10% fetal bovine serum (FBS) (Gibco, 16000-044). Human umbilical vein endothelial cells (HUVECs) (Lonza, C2519A) or gastroepiploic endothelial cells (GEAECs), which were primarily isolated from adult donors, were cultured in EGM-2MV on 1.5% gelatin-coated plates and served as a positive control. Bone marrow-derived MSCs (BM-MSCs) (Promocell, C-12974) were cultured in minimal essential medium supplemented with Glutamax (Gibco, 41090-036), and umbilical cord stromal-derived MSCs (UM-MSCs) (Promocell, C-12971) were cultured in mesenchymal stem cell growth medium-2 (Promocell, C-28009). Adult human dermal fibroblasts (Gibco, C-013-5C) were cultured in DMEM containing 10% FBS and 2 mM L-glutamine (Gibco, 25030-081), and 100 U / mL penicillin-streptomycin (Gibco, 15140-122) was added to all culture media. Cells were cultured at 37°C with 5% carbon dioxide, and the medium was replaced the day after seeding and every other day thereafter.

[0164]

[0165] Example 2. Construction of lentiviral vectors for candidate factors

[0166] The coding sequences of human ER71, KLF2, and TAL1 were each cloned into the pLenti6.3 / V5-DEST vector, and pLenti6.3 / V5-GW / lacZ was used as a mock vector. To produce lentiviruses, 10 μg each of pLP1, pLP2, pLP / VSVG, and the destination vector were mixed with 80 μg of polyethylenimine (Polysciences, 23966) in DMEM. The mixture was then incubated at room temperature for 30 minutes and added dropwise to 293T cells, which were then cultured for 1 day at 37°C under 5% CO2 conditions. The culture medium was replaced with fresh DMEM containing 10% FBS. After 24 hours, the supernatant was collected, filtered through a 0.45 μm filter, and centrifuged at 25,000 rpm for 90 minutes. Then, the virus particles were resuspended in DMEM and added dropwise to the target cells in EGM-2MV containing 10 μg / mL polybrene (Sigma-Aldrich, H9268).

[0167]

[0168] Example 3. Cell culture dish coating

[0169] Bovine skin gelatin (Sigma-Aldrich, G9391), human fibronectin (Gibco, 33016015), type IV collagen (Sigma-Aldrich, C6745), mouse laminin (Gibco, 23017015), and poly-L-lysine (Sigma-Aldrich, P2636) were used to coat culture dishes. Gelatin or type IV collagen was dissolved in phosphate-buffered saline (PBS), and fibronectin, poly-L-lysine, or laminin was dissolved in distilled water. Each solution was then placed on a 100 mm cell culture dish for coating. After incubation at 37°C for 1 h, the solution was aspirated, and the dish was dried for 30 min.

[0170]

[0171] Example 4. Flow cytometry

[0172] Cells were detached with accutase (Merck Millipore, SCR005) and blocked with PBS containing 1% fetal bovine serum albumin (FBS) and 0.5% bovine serum albumin (BSA). Antigen-presenting cell (APC)-conjugated antibodies to human CD44, CD73, CD90, CD105, CD34, platelet endothelial cell adhesion molecule-1 (PECAM1), human leukocyte antigen (HLA)-G, HLA-DR, and mouse PECAM1, human vascular endothelial (VE)-cadherin (eBioscience, 17-1449-42), and FITC-conjugated antibodies to human PECAM1 were used to stain the cells. Appropriate isotype antibodies were used as negative controls. 10 5 Cells were stained with 1–5 μl of antibodies for 1 h on ice and washed twice with PBS. Samples were analyzed using a fluorescence-activated cell sorting (FACS) Aria cell sorter (BD biosciences), and data were analyzed using FlowJo software (BD biosciences).

[0173]

[0174] Example 5. Immunofluorescence

[0175] 2 × 10 seeded on 1.5% gelatin or fibronectin-coated confocal dishes 5Dog cells were fixed with 4% paraformaldehyde for 10 min and washed three times with PBS. Then, after blocking with PBS containing 1% BSA, cells were stained with primary antibodies against human PECAM1 (1:400, Cell signaling, s3528), VE-cadherin (1:400, Cell signaling, 2500s), vascular endothelial growth factor receptor (VEGFR)-2 (1:200, Cell signaling, 2479s), Ulex europaeus agglutinin (UEA)-1 (1:100, Sigma Aldrich, L9006), or phosphorylated signal transducer and activator of transcription 1 (pSTAT1) (1:100, Cell signaling, s7649). Cells were then cultured overnight at 4°C and washed with Tris-buffered saline containing 0.5% Tween-20. For samples requiring secondary antibody staining, Alexa-Fluor 488-conjugated donkey anti-mouse IgG antibody (for PECAM1 or pSTAT1, 1:200, Invitrogen, A21202) or Alexa-Fluor 555-conjugated donkey anti-rabbit IgG antibody (for VE-cadherin, VEGFR-2, 1:200, Invitrogen, A31572) were stained for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:1000) for 15 min, and fluorescence images were captured using an LSM 710 fluorescence microscope (Zeiss, LSM710).

[0176]

[0177] Example 6. Quantitative real-time polymerase chain reaction (PCR)

[0178] RNA from harvested cells was isolated using the RNeasy Mini Kit (Qiagen, 74104), and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix (Toyobo, FSQ-201). Quantitative real-time PCR was performed using FastStart Universal SYBR Green Master (Roche, 4913914001). Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences used in the present invention are shown in Table 1 below.

[0179]

[0180]

[0181]

[0182]

[0183] Example 7. Matrigel tube formation

[0184] Matrigel basement membrane matrix (Corning, 356234) was coated on a 24-well plate and incubated at 37°C for 30 minutes. Then, 10 5 The cells were resuspended in 500 μl of EGM-2MV and seeded on plates. The cells were cultured for 24 h at 37°C in 5% CO2. Tube formation was evaluated using an inverted microscope (Olympus, IX71).

[0185]

[0186] Example 8. Uptake of DiI-labeled acetylated-low-density lipoprotein (DiI-Ac-LDL)

[0187] 2×10 5Dog cells were seeded in 4-well plates coated with 1.5% gelatin, and the following day, the culture medium was replaced with EGM-2MV containing 10 μg / mL DiI-Ac-LDL. After culturing for 5 h at 37°C in 5% CO2, the cells were washed twice with PBS and fixed with 4% paraformaldehyde. After washing twice with PBS, the nuclei were stained with DAPI. The cells were imaged using a fluorescence microscope.

[0188]

[0189] Example 9. Western Blot

[0190] Cells were lysed on ice with lysis buffer (Cell Signaling Technology, #9803) supplemented with protease and phosphatase inhibitors (Gendepot, P3100 and P3200). The lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4°C, and then with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The primary antibodies used in the present invention were as follows: ER71 (ab181847) from Abcam; VE-Cadherin (#2500), PECAM1 (#3528), phospho-signal transducer and activator of transcription 1 (Tyr701)(pSTAT1)(#7649), STAT1 (#9172), and Ikaros (#9034) from Cell Signaling Technology, and GAPDH (G9545) and β-Actin (A2228) from Sigma-Aldrich. Novex ECL (WP20005) and WestGlow DURA (BWD0100) were used as detection reagents.

[0191]

[0192] Example 10. Nitric oxide (NO) production

[0193] 2×10 5Dog cells were seeded in 4-well plates coated with 1.5% gelatin and cultured in EGM-2MV containing 5 μM 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (Invitrogen, D-23842). After 30 min of incubation, cells were washed with PBS and imaged using a fluorescence microscope.

[0194]

[0195] Example 11. Hindlimb ischemic mouse model

[0196] 5×10 5 Cells were resuspended in 30 μL of PBS. Eight-week-old male BALB / c nude mice were anesthetized, and the left femoral artery was surgically ligated. PBS or cells were then injected into two sites on the left thigh. Hindlimb surface blood flow was continuously recorded using laser Doppler perfusion imaging (LDPI) (Moor Instruments) on days 0, 7, and 14 postoperatively.

[0197]

[0198] Example 12. In vivo immune evasion model

[0199] Cells were transduced with lentivirus-GFP. Eight-week-old male BALB / c nude mice and C57BL / 6 mice were anesthetized, and the left femoral artery was surgically ligated. Then, 10 6Cells were resuspended in 50 μL of PBS and injected into three sites on the left thigh. The limbs of euthanized mice were obtained 14 days after surgery. For whole-cell lysis analysis, thigh muscles were minced with scissors and incubated with prewarmed digestion buffer consisting of DMEM and 0.5% collagenase II (Gibco, 17101015) at 37°C and 5% CO2 for 30 minutes. DMEM containing 10% FBS was then added to the buffer. After filtering through a 40 μm cell strainer, the cells were washed twice with PBS. GFP-expressing cells were counted by flow cytometry.

[0200]

[0201] Example 13. Chemical substances

[0202] The chemicals used in the present invention are as follows: Rosiglitazone (5 μM, Sigma-Aldrich, R2408), transforming growth factor-β (TGF-β) inhibitor SB431542 (10 μM, Sigma-Aldrich, S4317), recombinant human VEGF (10 ng / mL, RnD Systems, 293-VE-010), ascorbic acid (50 μg / mL, Sigma-Aldrich, A4544), and interferon (IFN)-γ (10 ng / mL, Sigma-Aldrich, SRP3058).

[0203]

[0204] Example 14. ELISA

[0205] 10 5 The cells were seeded in 6-well culture plates coated with 1.5% gelatin. The following day, the cells were washed once with EGM-2MV, and 1 mL of fresh EGM-2MV was added. After 24 hours, the supernatant was collected, and IL-6 was quantified using the Bio-Plex Pro Cytokine Assay according to the manufacturer's protocol.

[0206]

[0207] Example 15. Mixed lymphocyte reaction (MLR) analysis

[0208] Twenty milliliters of peripheral blood was collected from a healthy donor. The blood was diluted with an equal volume of phosphate-buffered saline (PBS) and added to a 10-mL Ficoll layer in a conical tube. The PBMC layer was obtained by centrifugation at 2,500 rpm for 30 minutes and washed twice with PBS. T cells and non-T cells were then separated by Magnetic Associated Cell Sorting (MACS) using a Pan T Cell Isolation Kit (Miltenyi Biotec, 130-096-535). T cells were stained with the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, C34571), and non-T cells were stained with the CellTrace™ Violet Cell Proliferation Kit and then treated with mitomycin C (Sigma Aldrich, M4287). EMSCs, MiECs, and GEAECs 10 4 The dogs were seeded in 96-well round-bottom plates (Falcon, 353077), and the medium was replaced the next day with fresh RPMI containing 10% FBS and 1% P / S. 10 cells from different donors 5 T cells and 10 5 Non-T cells from the dog were mixed and added to the seeded cells. After 6 days of reaction, floating cells were collected and analyzed by flow cytometry. CTV (CellTrace Violet)-negative cells (=T cells) were gated, and reduced CFSE cells (=proliferated T cells) were quantified.

[0209]

[0210] Example 16. RNA sequence analysis

[0211] Bioinformatic analysis was performed using the computing server of the Research Service Center of the Korea Institute of Genomic Medicine. Quality control of the sequenced raw files was performed using FastQC (version 0.11.9), and adapter trimming was performed using TrimGalore (version 0.6.7) using default parameters. The STAR alignment algorithm (version 2.7.9a) was used for mapping to the human genome GRCh38em.p13. After determining the read strand using the infer_experiment.py script of the RSeQC package (version 4.0.0), gene-level quantification of the reads was performed using the RSEM package (version 1.3.3). Subsequent analyses were performed within the R programming environment (version 4.1.3). Differentially expressed genes (DEGs) were identified among various sample groups using the DESeq2 package (version 1.34.0), and p-values ​​were adjusted using the Benjamini-Hochberg method. The sets of "EC-specific genes" and "EMSC-specific genes" were identified through differential gene expression analysis between EMSCs and GEAECs. DEGs with adjusted p-values ​​≤ 0.05 were sorted according to the (log2FC) x (1 / Adj.Pval) scoring method, and the top 500 DEGs based on the scoring system were set as EC-specific genes and EMSC-specific genes, respectively. To annotate the functions of DEGs, GO (Gene Ontology) and network analysis of enriched pathways were performed using Metascape. In addition, xCell 6 We performed cell type enrichment analysis from gene expression data using , and inferred efficient induced endothelial cell (iEC) generation.

[0212]

[0213] Example 17. Methylation sequencing analysis

[0214] FastQC (version 0.11.9) was used to visualize the sequencing quality of raw reads trimmed with TrimGalore (version 0.6.7) using default parameters. Sequences were aligned to the reference genome (hg38 assembly) using Bismark (version 0.23.0 using Bowtie2 alignment software version 2.3.5). After deduplication, the bismark_methylation_extractor command was used to extract the number of methylated reads and the coverage of each cytosine in CpG contexts. Subsequent analyses were performed within the R programming environment (version 4.1.3). The extracted methylation information was processed with DMRichR (version 1.7.4) using the dmrseq (version 1.14.0) and bsseq (version 1.30.0) algorithms for downstream enrichment analysis, as well as for differentially methylated regions and block calling. Visualization of differentially methylated regions (DMRs) was performed using the dmrseq Bioconductor v.3.8 package.

[0215]

[0216] Example 18. Statistical Analysis

[0217] All data are expressed as mean ± standard error of the mean. For between-group comparisons, Student's t-test or one-way ANOVA with post-hoc analysis (Bonferroni) was performed. Data were analyzed using SPSS version 23.0 (IBM SPSS Statistics), and a value of p < 0.05 was considered statistically significant. In the bar graphs, * indicates p < 0.05, ** p < 0.01, and *** p < 0.001.

[0218]

[0219] [Experimental Example]

[0220] Experimental Example 1. Establishment of a protocol for direct differentiation of MSCs into functional ECs using defined factors.

[0221] An efficient and reliable protocol for obtaining MSCs from human EMSCs has been reported (Tissue Eng Part A. 2010 Feb;16(2):705-15.). As shown in Fig. 1a, these EMSCs strongly expressed MSC markers such as CD44, CD73, CD90, and CD105, but did not express hematopoietic or vascular endothelial markers such as CD34, CD45, PECAM1, and VE-cadherin.

[0222] Furthermore, we constructed lentiviruses encoding three transcription factors, ER71, KLF2, and TAL1, which are key combinations for direct differentiation of adult human fibroblasts into ECs. VE-cadherin and PECAM1 are the only two EC markers that are highly specific and constitutively expressed in all endothelial beds, and double positivity for VE-cadherin / PECAM1 has been reported to be an essential and ideal indicator for defining functional ECs. Two weeks after lentiviral infection, direct differentiation of MSCs into ECs was assessed by confirming VE-cadherin / PECAM1 double positivity by flow cytometry. This process is schematically depicted in Figure 1b. As a result, as shown in Figure 1c, the combination of ER71, KLF2, and TAL1 (EKT) induced differentiation of 3.3±1.2% of MSCs into functional ECs. In particular, transduction of ER71 single factor (E) showed an improved differentiation efficiency (27.5±2.0%). The generated VE-cadherin / PECAM1 double-positive cells were sorted, and they exhibited a cobblestone shape, a morphological characteristic of ECs, as shown in Fig. 1d. In the present invention, cells induced to differentiate from MSCs into ECs were named MiEC (MSC-derived inducible endothelial cells).

[0223]

[0224] Experimental Example 2. Optimization of a Protocol for Efficient Vascular Endothelial Differentiation

[0225] We tested whether peroxisome proliferator-activated receptor (PPAR)-γ agonists, mesenchymal-to-epithelial transition inducers rosiglitazone, TGF-β inhibitors SB431542, or VEGF could enhance the vascular endothelial differentiation efficiency of EMSCs. As shown in Fig. 2a, SB431542 and VEGF significantly enhanced the differentiation efficiency, and as shown in Fig. 2b, the combination of these two resulted in a synergistic increase in efficiency up to 57.4±3.0%. Although EGM-2MV medium contains a small amount (0.5 ng / mL) of VEGF, the addition of a high concentration (10 ng / mL) of VEGF enhanced the differentiation efficiency. As shown in Figure 2c, the time-dependent evaluation results confirmed that the differentiation rate reached its peak 1-2 weeks after ER71 transduction, which may reflect the overgrowth of undifferentiated EMSCs after 2 weeks. Therefore, the differentiation protocol period was shortened to 1 week thereafter. As a result of examining the differentiation efficiency according to the type of extracellular matrix, such as gelatin, fibronectin, type IV collagen, laminin, and poly-L-lysine for culture dish coating, it was found that among the extracellular matrices, fibronectin was most suitable for enhancing vascular endothelial differentiation, as shown in Figure 2d.

[0226] Ascorbic acid has been reported to directly convert adipocyte progenitor cells into neurons or fibroblasts into cardiomyocytes, but the underlying mechanisms remain unclear. Therefore, we tested whether adding ascorbic acid to EGM-2MV medium containing 1 μg / mL ascorbic acid could increase differentiation efficiency. As shown in Figure 2e, ascorbic acid dose-dependently enhanced differentiation efficiency, and it was confirmed that differentiation efficiency was significantly enhanced at concentrations above 50 μg / mL. Furthermore, as shown in Figure 2f, when ER71 was forcedly induced and cultured in EGM-2MV supplemented with SB431542, VEGF, and ascorbic acid on fibronectin-coated dishes, 75.7±4.5% of EMSCs directly differentiated into functional ECs.

[0227]

[0228] Experimental Example 3. Further Optimization of the Protocol for Preserving the Proliferative Capacity of MiECs

[0229] Polybrene is used to improve the transduction efficiency of lentivirus, but it is known to negatively affect the proliferation capacity of human MSCs. As shown in Fig. 3a, polybrene (PB) increased the expression level of ER71 induced by lentivirus transduction (left figure in Fig. 3a) and improved the differentiation efficiency of MiECs (right figure in Fig. 3a). However, the resulting proliferation capacity of MiECs was severely affected when polybrene was used in lentivirus transduction, as shown in Fig. 3b, and when polybrene was not used during lentivirus transduction, as shown in Fig. 3c, the total cell counts (left figure in Fig. 3c) and the final number of MiECs (right figure in Fig. 3c) on day 7 after the initiation of direct differentiation were significantly higher. In addition, each of the 6 × 10 5When sorted MiECs were plated on 100 mm cell culture dishes, as shown in Fig. 3d, after 7 days, 10.5 times more cells were generated when induced according to the final protocol without polybrene compared to when induced according to the initial experimental plan consisting of ER71 transduction using only polybrene.

[0230]

[0231] Experimental Example 4. Confirmation of the effectiveness of the MiEC differentiation protocol on MSCs of different origins.

[0232] MSCs can be isolated from various tissues. In this experimental example, we examined whether the MiEC differentiation protocol could be applied to MSCs of different origins. As shown in Figure 4a, BM-MSCs and UM-MSCs were confirmed to differentiate into functional ECs using the protocol of the present invention. Furthermore, as shown in Figure 4b, among the three different types of MSCs, EMSCs showed the highest direct vascular endothelial differentiation efficiency, suggesting that EMSCs are the most suitable candidates for therapeutic purposes.

[0233]

[0234] Experimental Example 5. Confirmation of vascular endothelial properties of MiEC

[0235] MiEC derived using the optimized protocol exhibited a characteristic cobblestone morphology of ECs, as shown in Fig. 5a. As shown in Fig. 5b, MiEC formed capillaries in Matrigel, and as shown in Fig. 5c, immunofluorescence staining demonstrated that MiEC expressed vascular endothelial-specific markers, such as VE-cadherin, PECAM1, and VEGFR-2, at the protein level.

[0236] Furthermore, MiECs absorbed Ac-LDL, as shown in Fig. 5d, and bound to UEA-1 lectin, as shown in Fig. 5e, both of which are characteristics of the vascular endothelium. Real-time PCR results showed high expression of vascular endothelium markers at the RNA level, as shown in Fig. 5f. Furthermore, when MiECs were transplanted into the ischemic limb in a rat hindlimb ischemia model using BALB / c nude immunodeficient mice, limb perfusion increased, and limb salvage was as good as that of the positive control, GEAEC transplantation, as shown in Fig. 5g.

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[0238] Experimental Example 6. RNA Transcript and DNA Methylation Analysis

[0239] To assess the similarity of MiECs to functional ECs at the transcriptome level, RNA-seq profiling of MiECs, EMSCs, and GEAECs was performed. Using XCell to identify the cellular composition of each sample, as shown in Figure 6a, the MiEC samples were predominantly composed of vascular endothelial-like cells, with a much smaller proportion of MSC-like cells. To further investigate how MiECs transcriptionally differentiate from MSCs into ECs, we selected the most differentially expressed genes between EMSCs and GEAECs and examined the expression dynamics of these genes in MiECs. We then identified four distinct gene clusters reflecting shared characteristics between MiECs and controls. As a result, as shown in Fig. 6b, we confirmed that cluster 1 (n=290) was upregulated in both MiEC and GEAEC, cluster 2 (n=210) was upregulated specifically in GEAEC, cluster 3 (n=123) was upregulated specifically in EMSC, and cluster 4 (n=376) was upregulated in both MiEC and EMSC. Although MiEC still shared transcriptome similarity with MSC, gene set analysis showed clear indications that MiEC had vascular endothelial transcriptome characteristics. Through the Metascape database, cell type signature analysis (left figure in Fig. 6c) and PaGenBase annotation analysis (right figure in Fig. 6c) results showed that cluster 1 genes upregulated in both MiEC and GEAEC were the most vascular endothelial cell-specific. Gene ontology enrichment analysis, as shown in Fig. 6d, also showed that cluster 1 genes were associated with vascular endothelial function.

[0240] Furthermore, network analysis was performed to measure the differences between cluster 1 and cluster 2 genes, demonstrating that the EC-specific gene set was composed of cluster 1 genes rather than cluster 2 genes, as shown in Fig. 6e, and as shown in Fig. 6f, the differentially expressed genes between MSCs and MiECs were further confirmed to be upregulated in MiECs, including canonical EC cell marker genes such as cadherin 5 (CDH5) encoding VE-CADHERIN, endothelial cell-selective adhesion molecule (ESAM), and von Willebrand factor (VWF).

[0241] In addition, for epigenetic profiling, we investigated the differential DNA methylation patterns of the three groups using targeted methylation sequencing. Comparing the differentially methylated regions (DMRs) of EMSCs and MiECs, hypomethylated DMRs were significantly (q<0.05) enriched within CpG islands, as shown in Figure 6g, whereas hypermethylated DMRs were not significantly enriched within CpG islands. Then, as a result of confirming the methylation patterns of the DMRs identified in the comparison between EMSCs and MiECs, most of the identified DMRs (n=3,168) were hypomethylated in MiECs (n=2,453), as shown in Figure 6h, and 1,666 of them were also hypomethylated in GEAECs compared to EMSCs.

[0242] To determine how the identified DMRs are reflected in the differentially expressed gene analysis, we analyzed the methylation patterns of genes upregulated in MiECs compared to EMSCs. As shown in Fig. 6i, DMRs were mapped to genes using Ensembl, and most of the genes upregulated in MiECs were associated with hypomethylation of their corresponding DMRs. As shown in Fig. 6j, gene-specific methylation differences were observed among all three groups for known vascular endothelial marker genes, such as CDH5, KDR, ERG, and FLT4, which were also identified as differentially methylated regions (q<0.05).

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[0244] Experimental Example 7. Immune Tolerance Characteristics of MiEC

[0245] The immune tolerance properties of MSCs are mainly mediated by HLA-G, interleukin (IL)-6, indoleamine 2,3-dioxygenase (IDO) 1, and NO. In addition, HLA-DR has been reported to be downregulated in MSCs. Therefore, in this experimental example, we examined whether the immune tolerance properties were maintained after MiEC differentiation. As a result, as shown in Figure 7a, flow cytometry and real-time PCR showed that MiECs highly expressed HLA-G. Enzyme-linked immunosorbent assay (ELISA) results using the culture medium supernatant showed that MiECs secreted IL-6 well, as shown in Figure 7b. Real-time PCR results showed that MiECs highly expressed IDO1 2 days after IFN-γ stimulation, as shown in Figure 7c. As a result of NO production analysis, MiECs also produced NO, as shown in Figure 7d. Under IFN-γ stimulation for two days, HLA-DR expression was significantly reduced in MiECs, as shown in Figure 7e. In particular, HLA-G and IL-6 expression were significantly higher in MiECs, while HLA-DR expression was significantly lower in MiECs compared to EMSCs. These results suggest that immune tolerance characteristics are not only maintained but also enhanced in MiECs.

[0246]

[0247] Experimental Example 8. Immune Tolerance Mechanism of MiEC

[0248] To investigate the mechanism of immune tolerance characteristics of MiEC, we used GeneCards (https: / / www.genecards.org) to identify transcription factors that can bind to the promoters of four major immunosuppressive molecules: HLA-G, IL-6, IDO1, and nitric oxide synthase 3 (NOS3). As shown in Figure 8a, among the 125 transcription factors that bind to the promoter of HLA-G, 230 for IL-6, 700 for eNOS, and 75 for IDO1, 13 are cross-reactive transcription factors, and the promoters of these 13 transcription factors each have binding sites for E26 transformation-specific (ETS) mutant transcription factors (ETV)1, ETV4, or ETV6. For reference, ER71, known as ETV2, belongs to the ETS family, which includes other ETV subtypes, and ETS family members share a very similar consensus sequence.

[0249] Therefore, we speculated that the expression of 13 transcription factors could be regulated by ER71. After transducing ER71 into EMSCs, the mRNA level of ER71 was confirmed, which increased after 12 hours, as shown in Fig. 8b. Subsequently, the level of IKZF1 (Ikaros family zinc finger protein 1), a gene encoding Ikaros, significantly increased after 12 hours. In contrast, the expression levels of the other 12 transcription factors were confirmed, which showed no change, as shown in Fig. 8c. The Western blotting results showed that after transduction of ER71, ER71 protein expression increased followed by Ikaros protein expression, as shown in Fig. 8d. The mRNA levels of HLA-G, IL-6, IDO1, and NOS3 increased about 72 hours after ER71 transduction, as shown in Fig. 8e.

[0250] In addition, as shown in Fig. 8f, Ikaros expression was confirmed by knocking down IKZF1 using short hairpin RNA (shRNA), and knocking down IKZF1 by shIKZF1 suppressed the increase in this immunosuppressive factor caused by ER71 transduction, as shown in Fig. 8g.

[0251] HLA-DR induction by IFN-γ is mediated by its interaction with the class II transactivator (CIITA) and regulatory factor X (RFX) family. STAT1 is one of the mechanisms that upregulates CIITA expression, and Ikaros, as a transcription factor, induces SH2B3 (Src homology 2-B adaptor protein 3) gene expression. SH2B3 is known to negatively regulate Janus kinase activity and inactivate the STAT signaling pathway by recruiting phosphatases to dephosphorylate pSTAT1. Therefore, we speculated that Ikaros may also be involved in reducing the HLA-DR response to IFN-γ through the SH2B3-mediated STAT1-CIITA pathway. As shown in Fig. 8h, IKZF1 mRNA was highly induced under IFN-γ stimulation, confirming that the expression of SH2B2 was further increased in MiECs compared to GEAECs. Western blotting and immunofluorescence staining showed that phosphorylation of STAT1 was suppressed in MiECs, as shown in Fig. 8i and Fig. 8j, respectively, and the expression of CIITA and subsequent HLA-DR under IFN-γ stimulation was suppressed in MiECs compared to GEAECs, as shown in Fig. 8h. shIKZF1 reversed the expression patterns of all components in this signaling pathway, as shown in Fig. 8k and Fig. 8l, confirming that Ikaros plays a crucial role in the immune tolerance characteristic associated with the reduced HLA-DR response to IFN-γ in MiECs. A schematic diagram showing the mechanism of the immune tolerance characteristic of MiECs induced by ER71 transduction is shown in Fig. 8m.

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[0253] Experimental Example 9. Mixed Lymphocyte Reaction (MLR)

[0254] We investigated whether MiECs could actually evade the host immune system in vitro. T cells and non-T cells were isolated from the blood of healthy donors, and CFSE-stained T cells and CTV-stained non-T cells from different donors were co-cultured with EMSCs, MiECs, or GEAECs, respectively. This process is schematically shown in Figure 9a. The proportion of cells with reduced CFSE signals among CTV-negative cells was measured, and as shown in Figure 9b, in contrast to the proliferation of T cells co-cultured with GEAECs (36.7±4.6%), the proliferation of T cells co-cultured with EMSCs (20.2±4.9%) or MiECs (24.1±4.1%) was inhibited.

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[0256] Experimental Example 10. In vivo viability of MiECs in a hindlimb ischemic mouse model.

[0257] We examined whether MiECs could evade the host immune system in vivo. To this end, the hindlimbs of BALB / c nude or C57BL / 6 mice were ligated, and RFP-labeled MiECs, EMSCs, or GEACEs were transplanted intramuscularly. Two weeks later, the mice were euthanized, and the resulting limbs were dissociated into single cells by enzymatic digestion. The dissociated cells were stained with human CD105-PE and mouse PECAM1-PE antibodies, and then analyzed by flow cytometry. This process is schematically depicted in Figure 10a.

[0258] CD105 antibody was used to label human MiEC, EMSC, and GEAEC. GFP of each group was used to determine how well the transplanted cells survived and were incorporated into the blood vessels. + Dae PE +The cell ratio was calculated. As a result, as shown in Figure 10b, there was no difference in the ratio among the three groups in immunocompromised BALB / c nude mice. In contrast, in C57BL / 6, more transplanted cells survived in the MiEC or EMSC groups compared to the GEAEC group. This suggests that MiEC or EMSC successfully evaded the host immune system of immunocompetent mice. A schematic diagram showing the host immune system response to MiEC or control EC in immunocompetent mice is shown in Figure 10c.

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[0260] Experimental Example 11. Confirmation of MiEC induction without passing through a pluripotent state.

[0261] As shown in Figure 11, daily real-time PCR results showed that pluripotent markers such as OCT4 and NANOG were not significantly induced during the direct differentiation of EMSCs into MiECs. These results suggest that the MiEC induction process does not involve the induction of a pluripotent state.

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[0263] Experimental Example 12. Comparison of induction efficiencies between direct conversion of fibroblasts and direct differentiation of EMSCs into ECs.

[0264] The quality and efficiency of EC induction through direct differentiation of EMSCs were compared with those through direct conversion of human fibroblasts. As shown in Fig. 12a, direct differentiation of EMSCs induced a higher proportion of VE-cadherin / PECAM1 double-positive functional ECs (76.0±2.0%) compared to direct conversion of fibroblasts (19.6±3.0%). Notably, as shown in Fig. 12b, most of the induced cells were double-positive for VE-cadherin / PECAM1 (84.1±1.6%) in the direct differentiation of EMSCs, whereas they were single-positive for VE-cadherin in the direct conversion of fibroblasts (25.2±2.8%). Overall, direct differentiation of EMSCs was more efficient in generating high-quality ECs than direct conversion of fibroblasts.

[0265]

[0266] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0267]

[0268] The method for producing vascular endothelial cells according to the present invention is a method for inducing direct differentiation of embryonic stem cell-derived mesenchymal stem cells into vascular endothelial cells using ER71. In the present invention, differentiation into vascular endothelial cells was optimized by confirming the differentiation efficiency according to various culture additives. In addition, the vascular endothelial cells obtained through this have enhanced immune tolerance characteristics and are capable of saving the lower extremities through angiogenesis in a mouse lower extremity ischemia model. Therefore, the vascular endothelial cells produced by the method of the present invention are expected to be useful in the treatment of cardiovascular and vascular diseases without showing an immune rejection response when injected in vivo, and thus have industrial applicability.

Claims

1. A method for producing vascular endothelial cells from embryonic stem cell-derived mesenchymal stem cells, comprising the step of introducing an ER71 (E26 transformation-specific variant transcription factor 2) gene into embryonic stem cell-derived mesenchymal stem cells.

2. In paragraph 1, A method characterized in that the above vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

3. In paragraph 1, A method characterized in that the step of introducing the ER71 gene is performed in a medium containing at least one selected from the group consisting of SB431542, vascular endothelial growth factor (VEGF), and ascorbic acid.

4. In paragraph 1, A method characterized in that the manufactured vascular endothelial cells are vascular endothelial cell (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM1) double positive cells.

5. A pharmaceutical composition for preventing or treating cerebrovascular and cardiovascular diseases, comprising vascular endothelial cells produced by the method of any one of claims 1 to 4 as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

6. In paragraph 5, A pharmaceutical composition, characterized in that the above cerebrovascular disease is at least one selected from the group consisting of lower extremity vascular disease, ischemic cerebral disease, and heart disease.

7. In paragraph 6, The above lower extremity vascular disease is at least one selected from the group consisting of lower extremity arterial occlusive disease, lower extremity arterial stenotic disease, and lower extremity arteriosclerosis; The above ischemic cerebral disease is at least one selected from the group consisting of ischemic stroke, vascular dementia, Alzheimer's dementia, Huntington's disease, and Parkinson's disease; or A pharmaceutical composition, characterized in that the heart disease is at least one selected from the group consisting of coronary artery disease, congestive heart failure, chronic heart failure, cardiomyopathy, and myocardial infarction.

8. In paragraph 7, A pharmaceutical composition, characterized in that the cardiomyopathy is at least one selected from the group consisting of ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and valvular cardiomyopathy.

9. A method for preventing or treating cerebrovascular and cardiovascular diseases, comprising administering to a subject in need thereof a pharmaceutically effective amount of vascular endothelial cells manufactured by the method of any one of claims 1 to 4 or a composition containing the same as an active ingredient, wherein the vascular endothelial cells are characterized in that they are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

10. A use for preventing or treating cerebrovascular and cardiovascular diseases using vascular endothelial cells manufactured by the method of any one of claims 1 to 4 or a composition containing the same as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

11. A use for producing a preparation for preventing or treating cerebrovascular and cardiovascular diseases using vascular endothelial cells produced by the method of any one of claims 1 to 4 or a composition containing the same as an active ingredient, wherein the vascular endothelial cells are directly differentiated from mesenchymal stem cells derived from embryonic stem cells.

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