Uses in the manufacture of decidual placental mesenchymal stem cells and angiogenesis-promoting pharmaceutical compositions

Abstract

The present invention provides decidual placental mesenchymal stem cells and a method for culturing the same, which comprises introducing an induction step into a conventional method for culturing mesenchymal stem cells to highly express decoy receptor 3 (DcR3) in the decidual placental mesenchymal stem cells.Furthermore, the present invention provides a use of the decidual placental mesenchymal stem cells in the manufacture of a drug for promoting angiogenesis.

Description

Technical Field

[0001] The present invention relates to decidual placental mesenchymal stem cells and a method for culturing the same. In particular, an induction step is introduced into the conventional method for culturing mesenchymal stem cells to highly express decoy receptor 3 (DcR3) in the decidual placental mesenchymal stem cells. Furthermore, the present invention relates to the use of decidual placental mesenchymal stem cells in the production of drugs that promote angiogenesis.

Background Art

[0002] Currently, for biomarkers used in the identification of mesenchymal stem cells, the criteria published by the International Society for Cellular and Gene Therapy (ISCT) are often referred to, which state that they express CD73, CD90, CD105 and do not express CD14, CD34, CD45 and HLA-DR. However, this combination of biomarkers is based on bone marrow mesenchymal stem cells and lacks tissue specificity, making it difficult to distinguish mesenchymal stem cells derived from different tissues. On the other hand, it has been reported that mesenchymal stem cells exhibit different behaviors depending on their origin. Therefore, it is necessary to develop a biomolecular marker with specificity for distinguishing mesenchymal stem cells derived from specific tissues.

[0003] Since mesenchymal stem cells can express various angiogenesis factors, they are considered applicable to the promotion of angiogenesis for treating ischemic diseases.

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, since the expression of major factors in mesenchymal stem cells varies depending on their origin, the mechanism of promoting angiogenesis may be different.

Means for Solving the Problems

[0005] The present invention provides a method for culturing decidual placental mesenchymal stem cells (pcMSCs) that highly express decoy receptor 3 (DcR3). In this method, an induction step is introduced into the composition of a serum-free medium (MCDB201 medium + EGF (epidermal growth factor) + ITS cell culture additive (ITS being insulin, transferrin, and selenium)). The induction step involves stimulating the cell medium by adding at least one cytokine during the culture process, thereby causing the decidual placental mesenchymal stem cells to highly express decoy receptor 3 (DcR3).

[0006] In the present invention, "high expression" means that the expression level of decoy receptor 3 (DcR3) in decidual placental mesenchymal stem cells cultured using the culture method of the present invention is statistically significantly higher than the expression level of decoy receptor 3 (DcR3) in decidual placental mesenchymal stem cells cultured using conventional mesenchymal stem cell culture methods.

[0007] In the method of the present invention, preferably, the at least one cytokine is selected from tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), or a combination thereof.

[0008] In the method of the present invention, preferably, the induction step is performed after the decidual placental mesenchymal stem cells have adhered to the container.

[0009] In the method of the present invention, more preferably, the effective amount of TNF-α is 15 to 20 ng / ml, and the effective amount of IFN-γ is 10 to 20 ng / ml.

[0010] In the method of the present invention, more preferably, the TNF-α and IFN-γ are stimulated for 48 hours.

[0011] In the method of the present invention, more preferably, the TNF-α and the IFN-γ are added together to the cell culture medium.

[0012] The present invention further provides decidual placental mesenchymal stem cells cultured by the method described above. These decidual placental mesenchymal stem cells highly express decoy receptor 3 (DcR3).

[0013] Preferably, the decidual placental mesenchymal stem cells in the present invention express estrogen receptor (ER), progesterone receptor (PR), and decoy receptor 3 (DcR3).

[0014] Furthermore, in order to explore the potential applications of DcR3 in decidual-placental mesenchymal stem cells, the present invention further includes the use of a pharmaceutical composition in the manufacture of a drug that promotes angiogenesis in a desired individual. The pharmaceutical composition comprises an effective amount of decidual-placental mesenchymal stem cells that highly express decoy receptor 3.

[0015] Preferably, in the applications of the present invention, the individual is a human or a mammal.

[0016] Preferably, in the applications of the present invention, the effective amount of decidual-placental mesenchymal stem cells is 1 × 10⁻⁶ 4 ~2×10 5 These are cells. [Brief explanation of the drawing]

[0017] [Figure 1A] Figure 1A shows that decidual placental mesenchymal stem cells (pcMSCs) morphologically transformed into spindle-shaped adherent cells under serum-free culture conditions. The scale is 100 μm. [Figure 1B]Figure 1B shows that in immunophenotypic analysis of decidual placental mesenchymal stem cells (pcMSCs), biomarkers such as CD29, CD44, CD73, CD90, and CD105 were positive, while biomarkers such as CD14, CD34, CD45, and HLA-DR were negative. [Figure 1C] Figure 1C shows the in vitro differentiation potential of decidual-placental mesenchymal stem cells (pcMSCs) in osteogenesis (C2), chondrogenesis (C4), and adipogenesis (C6), respectively. C1, C3, and C5 represent the control groups under each in vitro induction condition, belonging to osteogenesis, chondrogenesis, and adipogenesis, respectively. The scale is 200 μm. [Figure 2] Figure 2 shows the karyotype characteristics of pcMSCs isolated from the placenta of a newborn male infant. A in the figure shows a representative image from chromosomal analysis of the 20th generation pcMSCs (N100). The analysis also shows 46 independent chromosomes from three individuals, including N98 (B), N99 (C), and N100 (D). pcMSCs isolated from the placenta of a newborn male infant showed positive X chromosome (shown in red fluorescence) and negative Y chromosome (shown in green fluorescence) as a result of fluorescence in situ hybridization (F). This contradicts the result (E) that both X and Y chromosomes are positive in cases derived from human umbilical vein endothelial cells (HUVECs) of newborn male infants. The lower left corner shows a magnified image of the image within the white frame in each panel. The scale is 50 μm. [Figure 3] Figure 3 shows the gene expression analysis of pcMSCs compared to bone marrow mesenchymal stem cells (BMMSCs). Abbreviations are as follows: pcMSCs: Decidual-placental mesenchymal stem cells, BMMSC: Bone marrow mesenchymal stem cells, Adsv: Adipose-derived mesenchymal stem cells, hES: Human embryonic stem cells, UniRef: Gene Family Database. [Figure 4]Figure 4 shows the comparison results between the hormone receptors of pcMSCs and those of BMMSCs. Regarding the abbreviations, PRA: progesterone receptor A, PRB: progesterone receptor B, ER: α subunit of estrogen receptor. [Figure 5] Figure 5 shows that pcMSCs can be induced by progesterone receptor (PR) and estrogen receptor (ER) in vitro to undergo decidualization. In the figure, A is the result of PCR quantitative analysis, B is the result of Western Blot quantitative analysis, and C is the result of ELISA quantitative analysis. Also, * means 0.01 < P ≤ 0.05, ** means 0.005 < P ≤ 0.01, *** means P ≤ 0.005, and N.D. means not detected. [Figure 6] Figure 6 shows the comparison of the expression levels of genes related to the tumor necrosis factor (TNF) receptor superfamily in pcMSCs and BMMSCs. [Figure 7] Figure 7 shows the results of the expression levels of DcR3 protein in pcMSCs and BMMSCs. [Figure 8] Figure 8 shows that the expression level of DcR3 increased due to the induction of TNF-α and IFN-γ. [Figure 9] Figure 9 shows the experimental flow when proving that pcMSCs promote angiogenesis by DcR3 in an in vitro tube formation assay. [Figure 10A] Figure 10A is part of the result of proving that pcMSCs promote angiogenesis by DcR3 in an in vitro tube formation assay, showing the result of ELISA measurement. Also, *** means P < 0.005. [Figure 10B] Figure 10B is part of the result of proving that pcMSCs promote angiogenesis by DcR3 in an in vitro tube formation assay, showing the photographed image of a fluorescence microscope. [Figure 10C]Figure 10C shows part of the results demonstrating that pcMSCs promote angiogenesis through DcR3 in an in vitro tube formation assay, presenting the statistical results of fluorescence microscope images. The results are expressed as the mean ± SD (n = 3). Also, * indicates 0.01 < P ≤ 0.05, and ** indicates 0.005 < P ≤ 0.01.

Mode for Carrying Out the Invention

[0018] It should be understood that the detailed description of the embodiments is for explaining the preferred embodiments of the present invention and is not intended to limit the present invention to any of the embodiments. It should be noted that the present invention intends to cover all alternative embodiments within the same spirit and scope of the present invention. Therefore, any non-essential modifications and adjustments made by others based on the concept of the present invention also fall within the scope of protection of the present invention.

Examples

[0019] Identification of Decidual Placenta Mesenchymal Stem Cells (pcMSCs)

[0020] In the research of the present invention, we successfully isolated mesenchymal stem cells from human placental decidua and named them placenta choriodecidual-derived mesenchymal stromal cells (pcMSCs).

[0021] We isolated mesenchymal stem cells that meet the basic international definition of mesenchymal stem cells from the chorionic and decidual parts of the placenta (see Figure 1). Subsequently, in chromosome identification and fluorescence in situ hybridization staining analysis, it was identified that these cells are derived from the maternal site, that is, from the decidual site (see Figure 2).

[0022] The basic definition of mesenchymal stem cells does not allow for the distinction of cells isolated from different tissues. Therefore, in order to find a biomarker that can adequately identify decidual mesenchymal stem cells, this invention analyzes the gene expression of bone marrow mesenchymal stem cells and decidual mesenchymal stem cells.

[0023] In this invention, several receptor genes were collected to demonstrate the differences between bone marrow mesenchymal stem cells (BMMSCs) and pcMSCs. As shown in Figure 3, pcMSCs expressed estrogen receptor (ESR) and progesterone receptor (PGR), which are not expressed in BMMSCs. Furthermore, both BMMSCs and pcMSCs expressed IFN-γ receptor (IFNGR) and TNF-α receptor (TNFRSF1A). These results demonstrate that estrogen receptor and progesterone receptor are unique to decidual mesenchymal stem cells.

[0024] Furthermore, we compared the hormone receptors of pcMSCs and BMMSCs. Protein expression was verified using Western blotting with different receptor-specific antibodies, and breast cancer cell lines MDA-MB-231 and T47D were used as negative and positive controls, respectively, for progesterone receptor (PR) and estrogen receptor (ER). The results showed that only pcMSCs expressed progesterone receptor (PR) and estrogen receptor (ER) compared to BMMSCs (BM) and adipose-derived mesenchymal stem cells (AdMSCs) (see Figure 4).

[0025] To identify that estrogen and progesterone receptors are specific to decidual-mesenchymal stem cells, this invention further stimulated decidual-mesenchymal stem cells (pcMSCs) and bone marrow-mesenchymal stem cells (BMMSCs) with 10 nM estrogen and 1 μM progesterone (E / P). The culture medium and cells were collected on days 3, 6, and 9, and prolactin (PRL) expression was evaluated by subsequent quantitative analysis using qPCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA). β-actin was used as the quantitative standard in Western blotting. All experiments were repeated three times.

[0026] As a result, as shown in Figure 5, regardless of whether qPCR (A), Western blot (B), or ELISA (C) quantitative analysis was performed, only decidual mesenchymal stem cells underwent decidual differentiation, and the indicator gene PRL was significantly elevated. On the other hand, bone marrow mesenchymal stem cells were not regulated by hormones. This further demonstrates that decidual mesenchymal stem cells can exhibit specific behaviors originating from the decidual site.

[0027] In addition, we compared the expression levels of tumor necrosis factor (TNF) receptor superfamily-related genes in pcMSCs and BMMSCs. We evaluated specific phenotypes in pcMSCs and BMMSCs by collecting and analyzing TNF receptor superfamily-related genes.

[0028] While several genes showed distinguishable forms in pcMSCs (see Figure 6), we focused particularly on decoy receptor 3 (DcR3 or TNFRSF6B) and conducted further analysis to confirm that DcR3 is expressed only in pcMSCs and not in BMMSCs. As a result, as shown in Figure 7, we found that decidual mesenchymal stem cells express DcR3, but BMMSCs isolated from different sexes do not. Therefore, we hypothesized that the estrogen receptor (ER), progesterone receptor (PR), and decoy receptor 3 (DcR3) could serve as biomarkers for decidual mesenchymal stem cells that have physiological significance in distinguishing mesenchymal stem cells from different tissue origins. [Examples]

[0029] A method for culturing decidual placental mesenchymal stem cells (pcMSCs) that highly express decoy receptor 3 (DcR3).

[0030] This invention provides a novel method for culturing decidual placental mesenchymal stem cells. This method involves stimulating conventional mesenchymal stem cell culture methods by adding inflammation-related cytokines TNF-α and IFN-γ.

[0031] Specifically, for decidual placental mesenchymal stem cells, conventional aseptic cell proliferation and culture methods were used, and cell subculture was performed using serum-free stem cell medium. During the culture period, the stem cell culture medium was replaced with fresh medium every three days. The medium contained MCDB201 composition medium, 1% insulin transferrin selenium (ITS), and 10 ng / ml epidermal growth factor (EGF).

[0032] Unlike conventional culture methods, the present invention introduces an induction step after the decidual placental mesenchymal stem cells have adhered to the container.

[0033] In this invention, cells were divided into four groups: (1) a negative control group, (2) a 20 ng / mL cytokine TNF-α induction group, (3) a 20 ng / mL cytokine IFN-γ induction group, and (4) a group induced by simultaneous addition of 20 ng / mL cytokine TNF-α and 20 ng / mL cytokine IFN-γ.

[0034] First, 1 x 10 5 Individual decidual placental mesenchymal stem cells were cultured in a 6-well culture plate. Next, after culturing overnight in an incubator, the culture medium was removed, and according to the conditions for each group, (1) complete medium without stimulating factors, (2) complete medium containing 20 ng / mL of cytokine TNF-α, (3) complete medium containing 20 ng / mL of cytokine IFN-γ, and (4) complete medium containing both 20 ng / mL of cytokine TNF-α and 20 ng / mL of cytokine IFN-γ were added, and the cells were cultured for 48 hours. Western blotting of the induced decidual placental mesenchymal stem cells showed that the expression level of DcR3 in decidual mesenchymal stem cells was increased in all induction groups (see Figure 8).

[0035] This invention further tested appropriate cytokine dosages to induce increased DcR3 expression in decidual placental mesenchymal stem cells. The results showed that while 15 ng / mL of the cytokine TNF-α could increase DcR3 expression in decidual placental mesenchymal stem cells, 10 ng / mL of the cytokine IFN-γ could also increase DcR3 expression. Similarly, when these were added together, increased DcR3 expression in decidual placental mesenchymal stem cells was also observed. [Examples]

[0036] The potential of decidual placental mesenchymal stem cells (pcMSCs) to promote angiogenesis

[0037] Furthermore, in order to explore the potential applications of DcR3 in decidual-placental mesenchymal stem cells, this invention conducted research using the aforementioned decidual-placental mesenchymal stem cells (pcMSCs).

[0038] The present invention further tested the differences between decidual placental mesenchymal stem cells that were not treated with cytokines and decidual placental mesenchymal stem cells stimulated with the cytokines TNF-α and IFN-γ in angiogenesis studies.

[0039] Based on the culture method described above, decidual mesenchymal stem cells according to the present invention were cultured in a suspended thin-film cell culture dish (Transwell). The experimental flow is shown in Figure 9. In addition, the number of cells used was adjusted according to the difference in the pore diameter of the suspended thin-film cell culture dish.

[0040] In this invention, first, decidual mesenchymal stem cells of the present invention are cultured in a suspended thin-film cell culture dish (Transwell) for 24-well cell culture plates at a rate of 1 × 10⁶ 4 Individual cells were cultured. Next, they were induced with 15 ng / mL TNF-α and 10 ng / mL IFN-γ for 48 hours. Then, the suspended thin-film cell culture dish (Transwell) was transferred to a dish containing endothelial cell lines (SVEC), and the induced decidual mesenchymal stem cells of the present invention and SVEC were co-cultured for tube formation measurement. The SVEC cell line was cultured in Matrigel for cell culture. After co-culture for 6 hours, the total number of branching points was calculated from images taken with a fluorescence microscope.

[0041] In this invention, decidual mesenchymal stem cells were induced with cytokines for 48 hours, and then the DcR3 concentration in the culture medium was measured by ELISA. It was found that the DcR3 expression level of pcMSCs was significantly increased in the induced group compared to the control group (see Figure 10A).

[0042] After co-culturing decidual mesenchymal stem cells and SVECs in this invention for 6 hours, the total number of branching points was calculated from images taken with a fluorescence microscope. Figure 10B shows representative images of each group.

[0043] In this invention, decidual mesenchymal stem cells of the present invention are cultured in a suspended thin-film cell culture dish (Transwell) for 6-well cell culture plates in a 2 × 10⁶ container. 5 The same results were obtained when individual cells were cultured and the same experiment was performed (the results are not shown).

[0044] Statistical results showed that pcMSCs induced with TNF-α and IFN-γ significantly induced more branching compared to the normal control group. Furthermore, this phenomenon was the opposite of that observed when using anti-DcR3 antibody (0.5 μg / mL) (see Figure 10C). The number of branching points in the anti-DcR3 antibody neutralization group was the lowest, even lower than that of the normal control group. This reflected that steady-state expression of DcR3 affects angiogenesis. Therefore, decidual mesenchymal stem cells can promote angiogenesis through DcR3 expression.

Claims

1. A method for culturing decidual placental mesenchymal stem cells that highly express decoy receptor 3, A step of culturing decidual placental mesenchymal stem cells in a culture dish containing serum-free medium until they adhere to the culture dish, The step includes adding an effective amount of at least one type of inflammatory cytokine to the culture dish containing the serum-free medium, The aforementioned at least one inflammatory cytokine is selected from tumor necrosis factor-α, interferon-γ, or a combination thereof, and stimulates the expression of decoy receptor 3 in the decidual placental mesenchymal stem cells for 48 hours. The present invention provides decidual placental mesenchymal stem cells with increased decoy receptor 3 expression levels compared to decidual placental mesenchymal stem cells cultured without stimulation of at least one of the aforementioned inflammatory cytokines, wherein the increase in expression level is statistically significant with a P value of less than 0.

05. The method is characterized in that the serum-free medium comprises MCDB201 medium, epidermal growth factor, and ITS cell culture additives.

2. The method according to claim 1, characterized in that the effective amount of tumor necrosis factor-α is 15 to 20 ng / ml, and the effective amount of interferon-γ is 10 to 20 ng / ml.

3. The method according to claim 1, characterized in that the tumor necrosis factor-α and the interferon-γ are added together to the cell culture medium.

4. Decidual placental mesenchymal stem cells cultured by the method of claim 1, Decidual placental mesenchymal stem cells characterized by high expression of decoy receptor 3.

5. The decidual placental mesenchymal stem cells according to claim 4, characterized in that the decidual placental mesenchymal stem cells express estrogen receptors, progesterone receptors, and decoy receptor 3.

6. Uses of pharmaceutical compositions in the manufacture of drugs that promote angiogenesis, The use of the pharmaceutical composition is characterized by containing an effective amount of decidual placental mesenchymal stem cells as described in claim 4.

7. The use according to claim 6, characterized in that the decidual placental mesenchymal stem cells express estrogen receptors, progesterone receptors, and decoy receptor 3.

8. The effective amount of decidual-placental mesenchymal stem cells is 1 × 10 ４ ~2 x 10 ５ The use according to claim 6, characterized in that it is an individual cell.

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