KR-20260064915-A - Method for isolating and culturing endothelial progenitor cells derived from pig blood

KR20260064915AKR 20260064915 AKR20260064915 AKR 20260064915AKR-20260064915-A

Abstract

The present invention relates to a composition for inducing the differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, and is completed by establishing optimal conditions for isolating and culturing endothelial progenitor cells from porcine blood samples that exhibit the morphology and phenotype of endothelial cells and possess angiogenic ability. In fact, as a result of confirming the characteristics of endothelial progenitor cells isolated and cultured from porcine blood under the above conditions, it was confirmed that the cells not only express endothelial cell-specific markers but also exhibit the tubular formation ability of endothelial cells. Therefore, since the present invention makes it possible to effectively isolate endothelial progenitor cells from blood collected from living pigs and induce differentiation into endothelial cells, it can be utilized for safety verification prior to xenotransplantation, phenotype verification of transgenic pigs, etc.

Inventors

오건봉
김상은
이풍연
이승훈
김석호
노진구
오미애

Assignees

대한민국(농촌진흥청장)

Dates

Publication Date: 20260508
Application Date: 20241030

Claims (14)

A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, comprising EBM-2 medium, FBS, and VEGF, The above FBS is included at a concentration of 3 to 20% relative to the above EBM-2 medium, and A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the VEGF is contained at a concentration of 5 to 100 ng/mL relative to the EBM-2 medium.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above composition further comprises one or more growth factors selected from the group consisting of FGF, IGF, and EGF.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above composition further comprises one or more selected from the group consisting of ascorbic acid and heparin.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above composition does not contain hydrocortisone.
In paragraph 1, The above composition is a composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, which is applied to cells together with fibronectin during porcine peripheral blood mononuclear cell culture.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the porcine peripheral blood mononuclear cells are isolated from the blood of a pig.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above-mentioned endothelial progenitor cells have a cobblestone morphology.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above endothelial progenitor cells express one or more selected from the group consisting of CD31, vWF, CD34, CD133, ESAM, and ICAM2.
In paragraph 1, A composition for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above-mentioned endothelial progenitor cells have the ability to form tubes.
A medium for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, comprising a composition of any one of claims 1 to 9.
A kit for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, comprising a composition of any one of claims 1 to 9; and fibronectin.
A method for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, comprising the step of culturing porcine peripheral blood mononuclear cells using fibronectin in an EBM-2 medium containing 3 to 20% FBS and 5 to 100 ng/mL VEGF.
In Paragraph 12, A method for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, wherein the above fibronectin is coated on a culture dish for culturing the above porcine peripheral blood mononuclear cells.
In Paragraph 12, A method for inducing differentiation of porcine peripheral blood mononuclear cells into endothelial progenitor cells, further comprising, after the above-mentioned culturing step, a step of subculturing the porcine peripheral blood mononuclear cells in an EBM-2 medium containing 0.1 to 2.5% FBS and 0.1 to 1 ng/mL VEGF; and collagen.

Description

Method for isolating and culturing endothelial progenitor cells derived from pig blood The present invention relates to a method for effectively isolating and culturing endothelial progenitor cells from pig blood. Due to their size, anatomical, and physiological similarities, pigs are suitable as model animals for human cardiovascular and metabolic diseases, as well as for providing organs for xenotransplantation. Consequently, transgenic pigs are being developed for various disease models and as source animals for xenotransplantation. As transgenic pigs are expected to serve as high-value resources for the future, standardized data and information, such as biological data, must be secured. Therefore, while molecular biological verification—including gene and protein expression—and physiological, pathological, and functional verification of developed transgenic pigs are required, available verification methods are limited. The vascular endothelium of the donor organ is the first to be exposed to the recipient's blood and immune cells present in the blood upon transplantation, acting as a major target of the recipient's immune system and potentially inducing transplant rejection reactions such as thrombosis and immune responses. Although transgenic pigs that specifically regulate thrombosis and immune rejection have been developed, endothelial cells (ECs) must be isolated from the transgenic pigs to confirm the expression of the introduced genes. In particular, while the expression of the -Gal antigen (galactose-1,3-galactose), which causes hyperacute rejection during xenotransplantation, is confirmed in various tissue cells including endothelial cells, the expression of non-Gal antigens that cause acute rejection, such as N-glycolyneuraminic acid and SDa glycan antigens, can be confirmed in endothelial cells. In other words, transplant rejection by the recipient's immune system can be predicted through the verification of endothelial cells constituting the inner wall of the blood vessels of xenotransplantation donor pigs. As such, while the verification of genetically modified pigs used as source animals is essential for the clinical success of xenotransplantation, methods for such verification are limited. Furthermore, vascular endothelial cells have broad applicability as in vitro model systems not only in the field of xenotransplantation but also in vascular biology and cardiovascular research. As such, the analysis of porcine vascular endothelial cells is an important factor in clinical or research fields of xenotransplantation, but pigs must be euthanized to obtain vascular samples for cell isolation. Therefore, there is a demand for a method to analyze endothelial cells without euthanizing pigs. Meanwhile, endothelial progenitor cells (EPCs) present in the blood are cells that can differentiate into endothelial cells and contribute to the formation of new blood vessels. In 1997, the Asahara research team at Tufts University School of Medicine discovered putative EPCs in human peripheral blood that express CD34 and VEGFR-2, which exhibit endothelial cell characteristics. Subsequent research revealed that these putative EPCs are myelomonocytic progenitors lacking angiogenesis ability and were classified as myeloid angiogenic cells (MACs). In 2004, the Ingram research team at Indiana University School of Medicine discovered endothelial colony-forming cells (ECFCs). ECFCs are progenitor cells capable of developing into highly proliferative endothelial cells with clonal expansion and angiogenesis capabilities. Therefore, it is possible to obtain endothelial progenitor cells from pigs, differentiate them into endothelial cells, and perform analysis. However, there is currently no known method for effectively isolating endothelial progenitor cells from pigs or for culturing them while maintaining their molecular biological and morphological characteristics. Figure 1 shows GTKO porcine peripheral blood mononuclear cells cultured in EGM-2 medium in a collagen-coated culture dish (passage 1, day 12). Figure 2 shows the results of confirming the expression of the endothelial cell marker CD31 in GTKO porcine peripheral blood mononuclear cells cultured in EGM-2 medium in a collagen-coated culture dish. Figure 3 shows GTKO porcine peripheral blood mononuclear cells cultured in mEGM-2 medium in a collagen-coated culture dish (day 7). Figure 4 shows GTKO porcine peripheral blood mononuclear cells cultured in mEGM-2 medium in a collagen-coated culture dish and then subcultured to passage 3. Figure 5 shows GTKO/MCP porcine peripheral blood mononuclear cells cultured in mEGM-2 medium on a fibronectin-coated culture dish and then subcultured to passage 3. Figure 6 shows the results of confirming the myeloid angiogenic cell (MAC) phenotype in GTKO/MCP porcine peripheral blood mononuclear cells cultured in mEGM-2 medium on a fibronectin-coated culture dish and then subcultured to passage 6. Figure 7 shows GTKO/MCP/TBM porc