Decidual placental mesenchymal stem cells and use thereof in the preparation of a pharmaceutical composition for promoting angiogenesis

Abstract

The present application provides a kind of decidual placental mesenchymal stem cell and its culture method, including in existing mesenchymal stem cell culture method, join the induction step, make the decidual placental mesenchymal stem cell highly expresses decoy receptor 3 (Decoy receptor 3;DcR3).The present application also provides a kind of decidual placental mesenchymal stem cell for preparing the use of promoting angiogenesis drug.

Description

TECHNICAL FIELD

[0001] The present application relates to a decidual placental mesenchymal stem cell and a culture method thereof, in particular, a method for culturing a decidual placental mesenchymal stem cell with high expression of decoy receptor 3 (DcR3) by adding an induction step to an existing mesenchymal stem cell culture method. The present application also relates to a use of a decidual placental mesenchymal stem cell for preparing a medicament for promoting angiogenesis. BACKGROUND

[0002] Currently, the biomarkers used to identify mesenchymal stem cells are based on the standards published by the International Society for Cellular and Gene Therapy (ISCT), which require the expression of CD73, CD90, and CD105, and the non-expression of 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 from different tissues from each other. On the other hand, many reports have shown that mesenchymal stem cells from different sources have different behaviors, and therefore, it is necessary to develop specific biomarker molecules that can distinguish mesenchymal stem cells from specific tissues.

[0003] Mesenchymal stem cells are believed to be useful for promoting angiogenesis to treat ischemic diseases because they can express various angiogenic factors. However, the mechanisms by which mesenchymal stem cells from different sources promote angiogenesis can differ due to the different expression of key factors. SUMMARY

[0004] The present application provides a method for culturing decidual placental mesenchymal stem cells (pcMSCs) with high expression of decoy receptor 3 (DcR3), which is by adding an induction step to a serum-free medium formula (MCDB201 medium + EGF (epidermal growth factor) + ITS cell culture supplement (ITS is insulin, transferrin, and selenium)), wherein the induction step is to stimulate the cells with at least one cytokine in the cell culture medium during the culture process, so that the decidual placental mesenchymal stem cells highly express decoy receptor 3 (DcR3).

[0005] The "high expression" of the present application refers to the statistically significant high expression of the expression level of Decoy receptor 3 (DcR3) of the decidual placental mesenchymal stem cells cultured by the culture method of the present application, as compared to the expression level of DcR3 of the decidual placental mesenchymal stem cells cultured by the existing culture method of mesenchymal stem cells.

[0006] In the method of the present application, preferably, the at least one cytokine is selected from tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or a combination thereof.

[0007] In the method of the present application, preferably, the inducing step is performed after the decidual placental mesenchymal stem cells are plated.

[0008] In the method of the present application, more preferably, the effective amount of TNF-alpha is 15-20 ng / ml, and the effective amount of IFN-gamma is 10-20 ng / ml.

[0009] In the method of the present application, more preferably, the TNF-alpha and the IFN-gamma are added to the cell culture medium for 48 hours.

[0010] In the method of the present application, more preferably, the TNF-alpha and the IFN-gamma are added to the cell culture medium together.

[0011] The present application also provides a decidual placental mesenchymal stem cell cultured by the aforementioned method, wherein the decidual placental mesenchymal stem cell highly expresses Decoy receptor 3 (DcR3).

[0012] Preferably, the decidual placental mesenchymal stem cell of the present application expresses Estrogen receptor (ER), Progesterone receptor (PR), and Decoy receptor 3 (DcR3).

[0013] To further explore the application potential of DcR3 in decidual mesenchymal stem cells, the present application also includes a use of a pharmaceutical composition for the preparation of a medicament for promoting angiogenesis in a subject in need thereof, wherein the pharmaceutical composition comprises an effective amount of decidual placental mesenchymal stem cells highly expressing Decoy receptor 3 (DcR3).

[0014] Preferably, in the use of the present application, the subject is a human or a mammal.

[0015] Preferably, in the use of the present application, wherein the effective amount of decidual placental mesenchymal stem cells is from 1 x 10 4 cells to 2 x 10 5 cells. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 shows the characterization of decidual placental mesenchymal stem cells (pcMSCs). (A) The morphology of pcMSCs under serum-free culture condition shows spindle-shaped adherent cells. Scale bar: 100 pm. (B) Immunophenotyping analysis of pcMSCs shows positive for CD29, CD44, CD73, CD90, CD105, and negative for CD14, CD34, CD45, and HLA-DR. (C) In vitro differentiation capacity of pcMSCs in osteogenesis (C2), chondrogenesis (C4), and adipogenesis (C6), respectively. C1, C3, C5 represent the control groups of each in vitro induction condition, which are osteogenesis, chondrogenesis, and adipogenesis, respectively. Scale bar: 200 pm.

[0017] Figure 2 shows the karyotype profile of pcMSCs isolated from the placenta of a male neonate. (A) Representative images showing the chromosome analysis of pcMSCs at passage 20 (N100); the analysis shows 46 independent chromosomes of three individuals, including N98 (B), N99 (C), and N100 (D). Fluorescence in situ hybridization of pcMSCs isolated from the placenta of a male neonate shows positive for X chromosome (indicated by red fluorescence) and negative for Y chromosome (indicated by green fluorescence) (F), which is in contrast to the double positive results for X and Y chromosomes of human umbilical vein endothelial cells (HUVECs) from a male neonate (E). The lower left corner is a magnified view of the image in the white box of each panel. Scale bar: 50 pm.

[0018] Figure 3 shows the gene expression analysis of pcMSCs compared to bone marrow mesenchymal stem cells (BMMSCs). Abbreviations: pcMSCs: decidual placental mesenchymal stem cells; BMMSCs: bone marrow mesenchymal stem cells; Adsv: adipose mesenchymal stem cells; hES: human embryonic stem cells; UniRef: gene family database.

[0019] Figure 4 shows the results of hormone receptors of pcMSCs compared to hormone receptors of BMMSCs. Abbreviations: PRA: luteinizing hormone receptor A; PRB: luteinizing hormone receptor B; ER: estrogen receptor a subunit.

[0020] Figure 5The results show that pcMSCs can be induced decidualization by progesterone receptor (PR) and estrogen receptor (ER) in vitro. (A) is the result of PCR quantitative analysis, (B) is the result of Western Blot quantitative analysis, (C) is the result of ELISA quantitative analysis. * means 0.01 < P < 0.05; ** means 0.005 < P < 0.01; *** means P < 0.005; N.D. means not detected.

[0021] Figure 6 The results show the comparison of the expression of tumor necrosis factor (TNF) receptor superfamily related genes of pcMSCs and BMMSCs.

[0022] Figure 7 The results show the expression of DcR3 protein in pcMSCs and BMMSCs.

[0023] Figure 8 The results show that the expression of DcR3 is enhanced by the induction of TNF-α and IFN-γ.

[0024] Figure 9 The results show the experimental procedure of in vitro tube formation assay to demonstrate that pcMSCs promote angiogenesis through DcR3.

[0025] Figure 10 shows the results of in vitro tube formation assay to demonstrate that pcMSCs promote angiogenesis through DcR3. (A) is the result of ELISA assay. (B) is the image of fluorescence microscope. (C) is the statistical result of fluorescence microscope image. The results are expressed as mean ± SD (n = 3), * means 0.01 < P < 0.05; ** means 0.005 < P < 0.01; *** means P < 0.005. DETAILED DESCRIPTION

[0026] It is to be understood that the detailed description of the embodiments is made for the purpose of illustrating the preferred embodiments of the present application and not for the purpose of restricting the present application to certain embodiments. It should be noted that the present application is intended to cover all alternatives, modifications and equivalents included within the spirit and scope of the present application. Some obvious modifications and adjustments can be made by others in light of the idea of the present application, which should be within the scope of the present application.

[0027] Example 1 Identification of decidual placental mesenchymal stem cells (pcMSCs)

[0028] In the research of the present application, we successfully isolated a kind of mesenchymal stem cells from human placental decidua, which is named placenta choriodecidual-derived mesenchymal stromal cells (pcMSCs).

[0029] We isolated a type of mesenchymal stem cell from the decidua of the placenta, which meets the basic international definition of mesenchymal stem cells (Figure 1). Subsequent chromosome identification and fluorescence in situ heterozygous staining analysis confirmed that this cell originated from the maternal site, specifically the decidua (e.g., ...). Figure 2 ).

[0030] Since the basic definition of mesenchymal stem cells cannot distinguish cells isolated from different tissue sources, in order to find sufficient biomarkers to identify decidual mesenchymal stem cells, this invention analyzes the gene expression of bone marrow mesenchymal stem cells and decidual mesenchymal stem cells.

[0031] This invention collected several receptor genes to demonstrate the differences between bone marrow mesenchymal stem cells (BMMSCs) and pcMSCs. For example... Figure 3 As shown, pcMSCs express estrogen receptor (ESR) and luteinizing hormone receptor (PGR) which are not expressed in BMMSCs, while both BMMSCs and pcMSCs express IFN-γ receptor (IFNGR) and TNF-α receptor (TNFRSF1A). The results confirm that estrogen receptor and luteinizing hormone receptor are unique to decidual mesenchymal stem cells.

[0032] Further comparisons were made between hormone receptors in pcMSCs and BMMSCs. Protein expression was confirmed by Western blotting using different receptor-specific antibodies, with breast cancer cell lines MDA-MB-231 and T47D serving as negative and positive controls for luteinizing hormone receptor (PR) and estrogen receptor (ER), respectively. The results showed that, compared to BMMSCs (BM) and adipose-derived mesenchymal stem cells (AdMSCs), only pcMSCs expressed luteinizing hormone receptor (PR) and estrogen receptor (ER) (e.g., ...). Figure 4 ).

[0033] To identify estrogen receptors and luteinizing hormone receptors as unique to decidual mesenchymal stem cells (pcMSCs), this invention further stimulated pcMSCs and bone marrow mesenchymal stem cells (BMMSCs) with 10 nM estrogen and 1 μM luteinizing hormone (E / P). Culture medium and cells were harvested on days 3, 6, and 9 for subsequent quantitative analysis using qPCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) to assess prolactin (PRL) expression. Western blotting used β-actin as the quantitative standard, and all experiments were repeated three times.

[0034] The result is as follows Figure 5Regardless of the quantitative analysis by qPCR (A), Western Blot (B) or ELISA (C), only decidual mesenchymal stem cells were able to produce decidualization differentiation with a significant increase of the marker gene PRL, while bone marrow mesenchymal stem cells were not affected by the hormonal regulation, confirming again that decidual mesenchymal stem cells are able to express their unique behavior derived from the decidual site.

[0035] In addition, we also compared the expression levels of tumor necrosis factor (TNF) receptor superfamily-related genes between pcMSCs and BMMSCs. We collected and analyzed TNF receptor superfamily-related genes to assess the specific expression patterns between pcMSCs and BMMSCs.

[0036] Although several genes showed a distinguishable pattern in pcMSCs (e.g. Figure 6 ), we focused on decoy receptor 3 (DcR3; or TNFRSF6B) for further analysis to confirm that DcR3 is expressed only in pcMSCs but not in BMMSCs. The results are shown in Figure 7 It was found that decidual mesenchymal stem cells express DcR3, while BMMSCs isolated from different genders do not. Therefore, we believe that the estrogen receptor (ER), the progesterone receptor (PR), and the decoy receptor 3 (DcR3) can serve as physiologically meaningful biomarkers for decidual mesenchymal stem cells to distinguish mesenchymal stem cells from different tissues.

[0037] Example 2: Culture method of decidual placental mesenchymal stem cells (pcMSCs) highly expressing decoy receptor 3 (DcR3)

[0038] The present application provides a new culture method for decidual placental mesenchymal stem cells, which is to add inflammatory-related cytokines TNF-α and IFN-γ for stimulation in the existing mesenchymal stem cell culture method.

[0039] Specifically, the decidual placental mesenchymal stem cells are cultured in the existing aseptic operation cell proliferation culture method, and the cells are subcultured using a serum-free stem cell culture medium, and fresh stem cell culture solution is replaced every three days during the culture period, wherein the culture medium comprises MCDB201 formula medium, insulin-transferrin-selenium (ITS) with a concentration of 1%, and epidermal growth factor (EGF) with a concentration of 10 ng / ml.

[0040] Unlike the existing culture method, the present application adds an induction step after the decidual placental mesenchymal stem cells are plated.

[0041] The present application divides the cells into four groups, which are (1) negative control group, (2) 20 ng / mL cytokine TNF-α induction group, (3) 20 ng / mL cytokine IFN-γ induction group, and (4) 20 ng / mL cytokine TNF-α and 20 ng / mL cytokine IFN-γ induction group.

[0042] First, 1 x 10 5 The decidual placental mesenchymal stem cells were cultured in a six-well culture plate, and after overnight culture in the incubator, the culture medium was removed and (1) complete culture medium without stimulating factors, (2) complete culture medium containing 20 ng / mL cytokine TNF-α, (3) complete culture medium containing 20 ng / mL cytokine IFN-γ, and (4) complete culture medium containing 20 ng / mL cytokine TNF-α and 20 ng / mL cytokine IFN-γ were added according to the conditions of each group, and cultured for 48 hours. The induced decidual placental mesenchymal stem cells were identified by Western Blot, and the results showed that all the induction groups showed that the expression of DcR3 in decidual mesenchymal stem cells was enhanced (such as Figure 8 ).

[0043] The present application also further tests the suitable dose of cytokine for inducing decidual placental mesenchymal stem cells to enhance the expression of DcR3, and the results show that 15 ng / mL cytokine TNF-α can also enhance the expression of DcR3 in decidual placental mesenchymal stem cells, and 10 ng / mL cytokine IFN-γ can also enhance the expression of DcR3 in decidual placental mesenchymal stem cells, and similarly, the addition of both for induction also observed the results of enhancing the expression of DcR3 in decidual placental mesenchymal stem cells.

[0044] Example 3 Decidual placental mesenchymal stem cells (pcMSCs) have the potential to promote angiogenesis

[0045] Further explore the application potential of DcR3 in decidual mesenchymal stem cells, the present application uses the decidual placental mesenchymal stem cells (pcMSCs) for research.

[0046] The present application further tests the difference between the decidual placental mesenchymal stem cells without cytokine treatment and the decidual placental mesenchymal stem cells stimulated by cytokines TNF-α and IFN-γ in the angiogenesis test.

[0047] According to the culture method described above, the decidual mesenchymal stem cells of the present application are cultured in a hanging thin film cell culture dish (Transwell), and the experimental procedure is shown in Figure 9 , and the number of cells used is adjusted according to the different pore sizes of the hanging thin film cell culture dish.

[0048] The present application first cultured 1 x 105 4 The present application's decidual mesenchymal stem cells were cultured in a 24-well cell culture plate using a suspended film cell culture dish (Transwell) and induced with 15 ng / mL of TNF-α and 10 ng / mL of IFN-γ for 48 hours. The induced decidual mesenchymal stem cells were then co-cultured with SVEC in a culture dish for tube formation assay. The SVEC cell line was cultured on a cell culture matrix gel (matrigel) and co-cultured for 6 hours. The number of total branch points was calculated from images taken by fluorescence microscopy.

[0049] The present application's decidual mesenchymal stem cells were cultured in a 24-well cell culture plate using a suspended film cell culture dish (Transwell) and induced with 15 ng / mL of TNF-α and 10 ng / mL of IFN-γ for 48 hours. The induced decidual mesenchymal stem cells were then co-cultured with SVEC in a culture dish for tube formation assay. The SVEC cell line was cultured on a cell culture matrix gel (matrigel) and co-cultured for 6 hours. The number of total branch points was calculated from images taken by fluorescence microscopy. Figure 10A

[0050] The present application's decidual mesenchymal stem cells were cultured in a 24-well cell culture plate using a suspended film cell culture dish (Transwell) and induced with 15 ng / mL of TNF-α and 10 ng / mL of IFN-γ for 48 hours. The induced decidual mesenchymal stem cells were then co-cultured with SVEC in a culture dish for tube formation assay. The SVEC cell line was cultured on a cell culture matrix gel (matrigel) and co-cultured for 6 hours. The number of total branch points was calculated from images taken by fluorescence microscopy. Figure 10B

[0051] The present application also cultured 2 x 105 5 The present application's decidual mesenchymal stem cells were cultured in a 24-well cell culture plate using a suspended film cell culture dish (Transwell) and induced with 15 ng / mL of TNF-α and 10 ng / mL of IFN-γ for 48 hours. The induced decidual mesenchymal stem cells were then co-cultured with SVEC in a culture dish for tube formation assay. The SVEC cell line was cultured on a cell culture matrix gel (matrigel) and co-cultured for 6 hours. The number of total branch points was calculated from images taken by fluorescence microscopy.

[0052] The results showed that pcMSCs induced with TNF-α and IFN-γ significantly induced more branch points than the normal culture condition group (Normal control), and this phenomenon was reversed by the use of anti-DcR3 antibody (0.5 μg / mL) (as shown in Figure 10C ). The number of branch points in the anti-DcR3 antibody (DcR3 antibody) neutralization group was the lowest, even lower than the normal culture condition group, reflecting the constant expression of DcR3 affecting angiogenesis. Therefore, decidual mesenchymal stem cells can promote angiogenesis by expressing DcR3.​​

Claims

1. A method of culturing placental chorionic membrane decidua mesenchymal stem cells, characterized by, The method comprises adding an induction step in a serum-free medium formula, wherein the induction step is adding at least one cytokine to stimulate the placental chorionic decidua mesenchymal stem cells to highly express the decoy receptor 3 during the culture process; wherein the at least one cytokine is selected from tumor necrosis factor-α, interferon-γ or a combination thereof, and the serum-free medium formula comprises MCDB201 medium, epidermal growth factor and ITS cell culture additives.

2. The method of claim 1, wherein, The induction step is adding the at least one cytokine after the placental chorionic decidua mesenchymal stem cells are plated.

3. The method of claim 1, wherein, The effective amount of the tumor necrosis factor-α is 15-20 ng / ml, and the effective amount of the interferon-γ is 10-20 ng / ml.

4. The method of claim 1, wherein, The tumor necrosis factor-α and the interferon-γ are used to stimulate for 48 hours.

5. The method of claim 1, wherein, The tumor necrosis factor-α and the interferon-γ are added together into the cell culture medium.

6. The placental chorionic membrane decidua mesenchymal stem cell cultured by the method according to claim 1, characterized in that, The placental chorionic decidua mesenchymal stem cells highly express the decoy receptor 3.

7. The placental chorionic mesodermal stem cell of claim 6, wherein, The placental chorionic decidua mesenchymal stem cells express estrogen receptor, luteinizing hormone receptor and decoy receptor 3.

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