KR102498202B1 - Method for evaluating a vascular regenerative efficacy of stem cells - Google Patents

Abstract

The present invention relates to a method for evaluating the efficacy of stem cells for vascular regeneration, and more particularly, as biomarkers for evaluating the effectiveness of stem cells for vascular regeneration, angiogenin, IL-8, and MCP-1, which are paracrine factors of stem cells. And by measuring the secretion level of VEGF, it is possible to efficiently evaluate and select stem cells effective for proangiogenic stem cell therapy.

Description

Method for evaluating a vascular regenerative efficacy of stem cells {Method for evaluating a vascular regenerative efficacy of stem cells}

The present invention relates to a method for evaluating angiogenesis efficacy of stem cells using expression levels of angiogenin, IL-8, MCP-1 and VEGF.

Mesenchymal stem/stromal cells (MSCs) have been extensively studied as a promising source of cell therapy for promoting angiogenesis in diseases such as peripheral arterial disease (ischemia), myocardial infarction and stroke (cerebral infarction). The potential for transdifferentiation into endothelial cells has been considered as one of its potential capabilities for treating these diseases, but this issue remains controversial. It has been shown that MSCs can function as perivascular progenitor cells to stabilize nascent blood vessels (Au P et.al. , Blood , 111:4551-4558, 2008). Because MSC engraftment into neo-vasculature is low, other capabilities of MSCs have been suggested to be more important in regenerative medicine (Walter MN et. al. , J. Stem Cells Regen . Med . , 11:18-24, 2015).

It has been reported that the beneficial effects of MSC are mainly due to the secretion of a wide range of physiologically active cytokines and growth factors involved in regulatory and trophic activities (Kinnaird T et. al. , Circulation , 109:1543-1549 , 2004; Boomsma RA et.al. , PLoS One , 7:e35685, 2012). An extensive repertoire of factors secreted by MSCs is known as the MSC secretome (Ranganath SH et.al. , Cell Stem Cell , 10:244-258, 2012). It has been shown that MSC secretome can enhance collateral vessel growth, bone regeneration, cardiovascular repair after myocardial infarction, and wound healing through a paracrine mechanism in ischemic tissue (Linero I et al . Al . , PLoS One , 9:e107001, 2014; Timmers L et.al. , Stem Cell Res. , 6:206-214, 2011; Chen L et.al. , PLoS One , 3:e1886, 2008). Therefore, identification of key factors secreted by MSCs and their functional role in cardiovascular therapy could be a useful approach to designing more potent MSC-based therapies with more predictable clinical outcomes.

Inconsistent stem cell efficacy has long been pointed out as one of the obstacles for clinical application of MSCs (Phinney DG et. al. , J. Cell Biochem . , 113:2806-2812, 2012). It has been reported that differences in the properties of MSCs depend on their tissue origin (Kern S et. al. , Stem Cells , 24:1294-1301, 2006). Cellular heterogeneity among donor populations of MSCs has also been an obstacle to standardization of therapies. Accordingly, several criteria for selecting MSCs with high regeneration potential have been proposed to overcome the problem of intra-population heterogeneity (Du W et. al. , Stem Cell Res. Ther . , 7:49, 2016). Therefore, there is a need to develop reliable protocols for cell preparation procedures and standardized functional assays to improve the clinical utility of MSCs. The functional heterogeneity of MSCs has attracted attention over the past decade, and variations depending on the donor of MSCs have been demonstrated in immunosuppressive potency, bone formation ability, and composition of the MSC secretome. (Salem B et. al. , Cytotherapy , 17:1675-1686, 2015; Siddappa R et. al. , J. Orthop . Res. , 25:1029-1041, 2007). MSC secretome analysis and their correlation with therapeutic effects may provide an opportunity to select more effective MSCs and better MSC expansion conditions to improve the clinical effectiveness of MSC-based therapies.

MSCs can be isolated from several tissues such as bone marrow, adipose tissue and umbilical cord. Among them, mesenchymal stem cells (WJ-MSC) derived from Wharton's jelly of the umbilical cord have no bioethical problems, are young cells, have low immunogenicity, and are used in other adult tissue- Compared to derived MSCs, there are several advantages, such as faster proliferation. WJ-MSCs can secrete growth factors and cytokines related to wound repair, neuroprotection, neurogenesis and neurogenesis. In a previous study, the present inventors demonstrated the revascularizing effect of WJ-MSCs in a mouse lower extremity ischemia model and reported that WJ-MSCs derived from the umbilical cords of different donors of the same age had different secretion levels of some proteins ( Choi M et. al. , Int . J. Biochem . Cell. Biol . , 45:560-570, 2013).

Under this background, the present inventors have made intensive research efforts to evaluate and select stem cells effective for angiogenesis. The migration ability and angiogenesis of vascular endothelial cells are markedly increased by the conditioned medium in which stem cells of a donor with a high secretion level of the above factors are selected and cultured. By confirming that capillary production increases, the present invention was completed.

1. Au P et. al., Blood, 111:4551-4558, 2008 2. Walter MN et. al., J. Stem Cells Regen. Med., 11:18-24, 2015 3. Kinnaird T et. al., Circulation, 109:1543-1549, 2004 4. Boomsma RA et.al., PLoS One, 7:e35685, 2012 5. Ranganath SH et.al., Cell Stem Cell, 10:244-258, 2012 6. Linero I et.al., PLoS One, 9:e107001, 2014 7. Timmers L et. al., Stem Cell Res., 6:206-214, 2011 8. Chen L et. al., PLoS One, 3:e1886, 2008 9. Phinney DG et. al., J. Cell Biochem., 113:2806-2812, 2012 10. Kern S et.al., Stem Cells, 24:1294-1301, 2006 11. Du W et. al., Stem Cell Res. Ther., 7:49, 2016 12. Salem B et.al., Cytotherapy, 17:1675-1686, 2015 13. Siddappa R et. al., J. Orthop. Res., 25:1029-1041, 2007 14. Choi M et. al., Int. J. Biochem. Cell. Biol., 45:560-570, 2013.

An object of the present invention is to provide a method for evaluating the efficacy of stem cells for revascularization based on the expression level of biomarkers.

In order to achieve the above object, the present invention provides a method for evaluating the revascularization efficacy of stem cells based on the expression level of biomarkers.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to angiogenin, interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) for stem cells obtained from an individual. It provides a method for evaluating the vascular regenerative efficacy of stem cells, including measuring the expression level of.

In the present invention, the term "stem cell" refers to cells capable of differentiating into various cells constituting biological tissues, and refers to undifferentiated cells capable of regenerating without limitation to form specialized cells of tissues and organs. . Stem cells are pluripotent or multipotent cells capable of development. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one origin (transit) cell, which then proliferate into the mature, complete cell form of the tissue.

In the present invention, the stem cells may be of autologous or allogenic origin, but are not limited thereto.

In addition, the stem cells include adult stem cells derived from umbilical cord, umbilical cord blood, bone marrow, amniotic fluid or adipose tissue, or induced pluripotent stem cells. It can, but is not limited thereto.

Specifically, the stem cells may be mesenchymal stem cells derived from Wharton's jelly of an umbilical cord or bone marrow, or mesenchymal stem cells derived from induced pluripotent stem cells.

In the present invention, the term "mesenchymal stem cell (MSC)" refers to various types of mesenchymal lineages in vivo, such as osteocytes, chondrocytes, tendonocytes, and adipocytes. It refers to stem cells with multipotency capable of differentiating into (Adipocytes), Myocytes, and Fibroblasts, etc., bone marrow, adipose tissue, umbilical cord blood, peripheral blood, and neonatal tissues. , can be isolated from various adult tissues such as the human placenta. Alternatively, it may be derived from induced pluripotent stem cells.

In the present invention, the term "Wharton's jelly" is a major component constituting the umbilical cord (a string-like structure connecting the fetus and placenta, the umbilical cord), and is an embryonic cultured connective tissue generated from the mesoderm and astrocytes has a fibrous network structure, and maintains abundant colloids between them. It is divided into three parts: the subamniotic layer, the perivascular layer, and the central color.

In the present invention, "vascular regeneration" is to repair damaged blood vessels using stem cells, and promote angiogenesis, that is, regeneration of damaged blood vessels using stem cells, and already damaged blood vessels are additionally damaged by a continuous external force. This means preventing damage.

The angiogenesis is a natural healing process of forming new blood vessels to supply blood to an organ or damaged tissue, and can be achieved by inducing proliferation and mobility of vascular endothelial cells. The "endothelial cell" is a layer of cells that line the lumen of a blood vessel and constitute the intima.

The angiogenesis occurs while promoting or inhibiting the proliferation of vascular endothelial cells in close association with various factors. Angiogenesis plays an important role in disease progression and treatment in many diseases. For example, inhibition of angiogenesis is required in the case of cancer or diabetic mania, whereas increased angiogenesis is required in the case of myocardial infarction or cerebral infarction.

In one embodiment of the present invention, angiogenin, IL-8, MCP-1 and VEGF four types of factors as biomarkers for angiogenesis-related factors, that is, evaluation of angiogenesis efficacy, are analyzed for secretion of stem cells and cytokine profiles It was selected through (Figs. 1a to 4).

In addition, the donor's stem cells with high levels of angiogenin, IL-8, MCP-1 and VEGF secretion, specifically Wharton's jedae colloid-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells cultured condition As a result of confirming the migration ability and the degree of angiogenesis of vascular endothelial cells by applying conditioned media to vascular endothelial cells, it was confirmed that the migration ability and angiogenesis of vascular endothelial cells were significantly increased compared to the control group. It was confirmed that capillary production increased even when the stem cells themselves obtained from the conditioned medium were transplanted into the Matrigel plug assay and lower extremity ischemic mouse model (Figs. 2, 5, 6, and 9A to 9). Figure 9 C).

On the other hand, when angiogenin, IL-8, MCP-1, and VEGF 4 types of blocking antibodies and siRNA were applied to the conditioned medium, the migration ability and angiogenesis of vascular endothelial cells were improved compared to the control group. It was confirmed that it was significantly inhibited (Fig. 7 and Fig. 9 D to Fig. 9 G).

Therefore, in selecting effective stem cells for vascular regeneration, the four factors of angiogenin, IL-8, MCP-1, and VEGF can be usefully used as biomarkers, and the expression level of the four factors is It was found that stem cells exhibiting a high expression level compared to a control group or a predetermined reference expression level can be usefully used as a cell therapy agent for vascular regeneration.

In the present invention, the stem cells evaluated and selected as stem cells effective for blood vessel regeneration are not limited thereto, but may be used for treatment of ischemic disease. The ischemic disease is not limited thereto, but ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic stroke and ischemic lower limb disease. can be selected from.

In the present invention, the measurement of the expression levels of the four biomarkers, specifically angiogenin, IL-8, MCP-1 and VEGF, for the evaluation and selection of stem cells effective for vascular regeneration is not limited thereto, but the individual It can be measured using a cell sample obtained from, specifically, a stem cell or tissue sample.

The stem cells can be cultured and proliferated in vitro before measuring the expression levels of angiogenin, IL-8, MCP-1 and VEGF, and the stem cells are not limited thereto, but Wharton's umbilical cord of the umbilical cord It may be glial-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or iPSC (induced pluripotent stem cells)-derived mesenchymal stem cells.

The expression levels of angiogenin, IL-8, MCP-1 and VEGF are not limited thereto, but can be quantified in cell culture medium or cell extract, and by performing ELISA, Western blotting, dot blotting, or PCR. can be measured

The expression levels of the four factors of angiogenin, IL-8, MCP-1 and VEGF can be compared with expression levels of one or more control groups or a predetermined reference. The predetermined reference expression level can be obtained from different tissues from the same individual, or can be derived through an expression level test of the four factors in samples obtained from the same tissue or different tissues from different individuals. The tissue may be, but is not limited to, adipose tissue, cardiac tissue, vascular tissue, or hematopoietic tissue. For example, the predetermined reference expression level may be the expression level of the above four factors measured from an individual not exhibiting symptoms of ischemic disease.

In an in vivo assay, in vitro assay, or other functional angiogenesis assay, the predetermined reference expression level is related to the ability of one or more other individuals involved in angiogenesis to promote angiogenesis. It can be a defined expression level or an average expression level. The expression level of the four factors of angiogenin, IL-8, MCP-1 and VEGF of a specific target subject is not limited thereto, but the difference is at least about 1.5 times, 2 times, or 5 times compared to the predetermined reference expression level. It may be one that exhibits a pattern that is 2x, 10x, 20x, or 50x higher. Additional controls may include testing the expression level of one or more factors not associated with angiogenesis.

According to the present invention, stem cells for promoting angiogenesis are measured by measuring secretion levels of angiogenin, IL-8, MCP-1, and VEGF, which are paracrine factors of stem cells, as biomarkers for evaluating the effectiveness of stem cells for angiogenesis. It is possible to efficiently evaluate and select stem cells effective for proangiogenic stem cell therapy.

1a to 1g show comparison of proangiogenic secretome profiles of Wharton's colloidal-derived mesenchymal stem cells (WJ-MSC) among different donor types. Fifty-five angiogenesis-related proteins were relatively quantified based on the reference spot provided by the manufacturer.
Figure 2 shows significant changes in the angiogenesis-promoting secretion profile of WJ-MSCs between different donor types. WJ-MSCs isolated from different donor-derived umbilical cords were cultured in DMEM culture medium containing 20% FBS for 24 hours and then cultured in serum-free EBM-2 medium for 48 hours to form conditioned medium (CM). was collected. (A) Angiogenesis-related proteins contained in each conditioned medium were analyzed with an antibody-based protein array using a Human Angiogenesis Array kit. Here, eight representative factors, angiogenin, angiopoietin-1, FGF-7, IL-8, MCP-1, PDGF-AA, serpin F1 and VEGF Secretion levels are indicated. The secretion level of each protein was normalized by a reference point. (B) Representative images of donor 2 (D2) and donor 6 (D6) among 9 donor types are shown, and arrows indicate reference points. (C-E) Conditioned media of WJ-MSCs from two different donors (D2 and D6) were evaluated in assays for proliferation, migration and tube formation of HUVEC cells. (C) Serum-starved HUVEC cells were cultured for 48 hours in the presence of conditioned medium supplemented with 1% FBS, and then MTS assay was performed. (D) Migration of HUVEC cells was measured in the presence of each conditioned medium in the bottom chamber of the Boyden chamber. (E) Tube formation of HUVEC cells was performed by incubating HUVEC cells on matrigel with the indicated conditioned medium for 12 hours. At this time, VEGF (5ng/ml) was used as a positive control in (D), and EGM-2 was used as a positive control in (C) and (E).
Figure 3 shows a comparison of secretome profiles according to changes in FBS in WJ-MSC culture. (A) D2 or D6 WJ-MSCs were maintained in DMEM medium containing FBS type A or type B, and then cultured in serum-free EBM-2 medium for 48 hours to collect conditioned medium. The collected conditioned medium was analyzed using a human angiogenesis array kit. (B) Eight proangiogenic factors standardized as reference points, angiogenin, angiopoietin-1, FGF-7, IL-8, MCP-1, PDGF-AA, serpin F1 and It is a graph showing the relative secretion level of VEGF.
Figure 4 shows a comparison of secretion levels of four types of angiogenesis-stimulating factors in WJ-MSCs of an additional donor type. (A) Conditioned media collected from WJ-MSCs isolated from additional donors (D10, D11, D12 and D13) were analyzed using a human angiogenesis array kit. The English letter in the box indicates the position of the selected major angiogenesis-promoting factor in the membrane, and the arrow indicates the reference point. (B) The secretion level of each protein was normalized by a reference point.
Figure 5 shows the results showing differential angiogenic activity in vitro and in vivo of WJ-MSCs of different donor types. (A) Serum starved HUVEC cells were allowed to migrate to the bottom chamber with the indicated conditioned media. At this time, VEGF (5ng/ml) was used as a positive control. Representative images (left) and quantification results of migrated cells (right) are shown. The scale bar is 125 μm. (B) Tube formation of HUVEC cells was performed by incubating HUVEC cells on matrigel with the indicated conditioned medium for 12 hours. Representative images (left), tube length (middle) and average number of branches per field (right) are shown. The scale bar is 250 μm. (C) In vivo Matrigel plug assay. Matrigel containing the indicated WJ-MSCs (5Х10 ⁶ cells) or PBS (CTL) was injected into the dorsal region of mice (n=3). After 7 days, the matrigel plug was removed and homogenized, and hemoglobin (Hb) was measured using Drabkin's solution. Representative images (left) and Hb quantification results (right) are shown.
Figure 6 shows that the angiogenesis regeneration effect of WJ-MSC in a mouse hindlimb ischemia model depends on the secretion of angiogenesis promoting factors. In mice (BALB/c nu/nu), ischemia was induced by performing femoral artery and phlebectomy. Within 6 hours post-surgery, WJ-MSCs (5Х10 ⁵ in PBS) of different donor types D13 or D10 or PBS (CTL) were injected intramuscularly at lower extremity ischemic sites (n=5). (A) Blood flow was measured using LDPI on days 0, 7, 14 and 21 after surgery (left). The LDPI index was determined as the ischemic ratio for the non-ischemic hindlimb (right). On day 21, the D13 WJ-MSCs-treated group showed significant improvement in perfusion compared to the D10 WJ-MSCs-treated group or the PBS-treated group. Here, * is D10 WJ-MSC vs. (vs.) P < 0.05, ** is PBS vs. P < 0.01. (B) Tissues harvested from ischemic limbs on day 21 after ischemia were stained with CD31 antibody (green) to analyze capillary density. Representative photos (left) and quantification results of blood vessel concentrations (right) are shown. The scale bar is 50 μm.
Figure 7 shows that angiogenin, VEGF, MCP-1 and IL-8 are key mediators of the angiogenesis promoting effect of WJ-MSC. (A) and (B) 5 μg/ml of angiogenin (α-Angiogenin), IL-8 (α-IL-8), MCP-1 (α-MCP-1) and VEGF (α-VEGF) Neutralizing antibodies against each were treated in the prepared conditioned medium for 30 minutes. At this time, IgG1 or IgG2 was used as a control, respectively, and α-4F represents treatment with a combination of the four antibodies (α-Angiogenin, α-IL-8, α-MCP-1 and α-VEGF). (A) HUVEC cells were allowed to migrate to the bottom chamber with antibody-treated conditioned medium. Here, *** is P < 0.001 versus control IgG2 (vs.), and ## is P < 0.01 versus control IgG1+2. The percentage of migrated cells was calculated based on the cell migration of each IgG-treated control. (B) Tube formation of HUVEC cells on Matrigel was performed by incubation for 12 hours in antibody-treated conditioned medium. The percentage of vessel length was calculated based on the tube formation of each IgG-treated control. Here, §§ is P < 0.01 versus control IgG1, * is P < 0.05 versus control IgG2, and ## is P < 0.01 versus control IgG1+2. (C and D) WJ-MSCs were transfected with the indicated siRNAs, cultured and conditioned medium was collected. si4F represents a treatment with a combination of four siRNAs (siAngiogenin, siIL-8, siMCP-1 and siVEGF). (C) HUVEC cells were transferred to the bottom chamber with the indicated conditioned media. The graph shows the percentage of HUVEC cells that migrated to the indicated conditioned media compared to siCTL-treated controls. ** and *** are P < 0.01 and P < 0.001 versus siCTL (100 nM), respectively, and ### is P < 0.001 versus siCTL (400 nM). (D) Vasculogenesis of HUVEC cells on Matrigel was performed by incubation for 12 hours in the indicated conditioned media. The graph shows the percentage of vessel length formed in the indicated conditioned media compared to siCTL-treated controls. ** is P < 0.01 versus siCTL (100 nM), ## is P < 0.01 versus siCTL (400 nM).
8 shows the results of dot blot analysis of the conditioned medium obtained after knockdown by treatment with siRNA for an angiogenesis-stimulating factor. Conditioned medium was obtained from WJ-MSCs transfected with the indicated siRNAs. Knockdown of each factor was confirmed by dot blot analysis using anti-angiogenin, anti-IL-8, anti-MCP-1 and anti-VEGF antibodies.
9 shows that the four selected factors, angiogenin, IL-8, MCP-1, and VEGF, also mediate the angiogenesis-promoting effect of bone marrow-derived mesenchymal stem cells (BM-MSC). (A) Conditioned media collected from BM-MSC culture supernatants from two different donors (BM-D1 and BM-D2, passage 4) were analyzed using a human angiogenesis array kit. Here, a denotes angiogenin, b denotes IL-8, c denotes MCP-1, d denotes VEGF, and four types of angiogenesis-stimulating factors. The secretion level of each protein was normalized by a reference point (bottom). Arrows indicate positive controls. (B) Serum-starved HUVEC cells were transferred to the bottom chamber containing BM-MSC conditioned medium. At this time, VEGF (5ng/ml) was used as a positive control. (C) Vasculogenesis of HUVEC cells was performed by incubating HUVEC cells on Matrigel with the indicated conditioned media of BM-MSC for 12 h. EGM-2 was used as a control. (D and E) Angiogenin, IL-8, MCP-1 and neutralizing antibodies (α-4F) against VEGF were simultaneously co-treated with the conditioned medium of BM-MSC. At this time, IgG1 and IgG2 were used as controls. Migration (D) and angiogenesis (E) assays of HUVEC cells were performed using conditioned media co-treated with antibodies. (F and G) BM-MSCs were transfected with control (siCTL) or combined siRNA (si4F; siAngiogenin, siIL-8, siMCP-1 and siVEGF), cultured and conditioned medium was collected . Migration (F) and angiogenesis (G) assays of HUVEC cells were performed using the collected conditioned media.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Example 1> Preparation of test materials and test method

DMEM (Dulbecco's modified Eagle medium), M199, trypsin-EDTA and penicillin-streptomycin were purchased from Gibco BRL (Grand Island, NY), EGM-2MV BulletKit medium (medium) was purchased from Cambrex (Walkersville, MD). Fetal bovine serum was purchased from Gibco, Merck (Darmstadt, Germany) or Wisent Bioproduct (Quebec, Canada). Heparin was purchased from Sigma (St. Louis, MO), and endothelial cell growth supplement (ECGS) was purchased from BD Biosciences (San Jose, CA). Neutralizing antibodies against IL-8 (#MAB331), MCP-1 (#MAB679), VEGF (#MAB293), mouse IgG1 (# MAB002) and IgG2 (#MAB004) were purchased from R&D Systems (Minneapolis, MN). , A neutralizing antibody against angiogenin (#26-2F) was purchased from Millipore (Billerica, MA). HRP-conjugated secondary antibodies (Horseradish peroxidase-conjugated secondary antibodies) were purchased from GeneTex, Inc. (Irvine, CA) and chemiluminescent substrate ECL kits were purchased from GE Healthcare (Piscataway, NJ). .

(cell culture)

Isolation and characterization of WJ-MSCs were performed by Choi M et. al. , Int . J. Biochem . Cell. Biol. , 45:560-570 (2013). Human umbilical cord samples for WJ-MSC were collected according to the Institutional Review Board (IRB) approval procedure (approval number: MC12TISI0156) of the College of Medicine, The Catholic University of Korea. Human BM-MSCs (Catholic MASTER Cells) were obtained from CIC (Catholic Institute of Cell Therapy, Seoul, Korea) and have multidifferentiation potential (adipogenic, chondrogenic and Certified for osteogenic differentiation and cell surface antigens (positive for CD90 and CD73, but negative for CD34 and CD45) MSCs are FBS (20%), penicillin ( 100 units/ml) and streptomycin (100 μg/ml) were added to the DMEM medium at 37°C and 5% CO ₂ in a humidified atmosphere.100mm culture dish (45 cells/mm2) 2.5Х10 were seeded with ⁵ MSCs, and the passaging of MSCs was divided every 2 to 3 days at a ratio of 1: 3. The cells of passages 4 to 5 (PDL 4 to 8) were in vitro Cells of passage 6 (PDL 8-11) were used for in vivo experiments. Primary culture and maintenance of human HUVEC (human umbilical vein endothelial cells) cells were described by Choi M et.al. Int. J. Biochem . Cell. Biol . , 45:560-570 (2013) and Jaffe EA et. al. , J. Clin. Invest. , 52:2745-2756 (1973). Human umbilical cord samples for obtaining HUVEC cells were collected according to IRB-approved procedures at The Catholic University of Korea St. Vincent's Hospital (VC14TIS10208) HUVEC cells were prepared in FBS (20%), ECGS (30 μg/mL, BD Biosciences ) and M199 with heparin (90 μg/mL, Sigma) cultured in medium. HUVEC cells from passages 3 to 6 were used for in vitro assays.

(Preparation of conditioned medium)

2×10 ⁵ MSCs were seeded in a 100 mm culture dish (36 cells/mm ² ) with a culture medium. After 24 hours, cells were washed twice with PBS, serum free EBM-2 was added, and further cultured for 48 hours. After collecting the culture supernatant (conditioned medium), it was centrifuged for 15 minutes at 4°C and 3,200 rpm, and stored at -70°C until use.

(antibody-based protein array analysis)

Analysis of the secretion profile of the conditioned medium was performed using the Human Angiogenesis Array kit (ARY007, R&D system) according to the manufacturer's instructions.

(Cell Proliferation Assay)

HUVEC cells were prepared at a density of 2,000 cells/well in serum-added M199 medium and seeded in a gelatin-coated 96-well plate. After 24 hours, the cells were serum starved for 4 hours in EBM-2 medium, and each conditioned medium supplemented with 1% FBS (Gibco) was added. The cells were cultured for 48 hours, and the degree of proliferation was evaluated using the MTS Cell Proliferation Assay Kit (Promega Corp., Madison, WI).

(Cell Migration Assay)

Cell migration assay was performed by Kim HK et. al. , Mol . Cancer. Ther. , 7:2133-2141 (2008) using a modified Boyden chamber kit. Serum-starved HUVEC cells ( ⁴ cells of 2Х10) were trypsinized and placed in an upper chamber, and conditioned medium from MSC was added to a bottom chamber to induce cell migration. After 5 hours, the migrated cells were fixed and stained with Diff-Quik solution (Sysmex Co., Kobe, Japan). Cells migrating on five randomly selected fields were photographed and counted. To identify key factors related to migration activity, the conditioned medium was pretreated with neutralizing antibodies at 37°C for 30 minutes.

(tube formation assay)

Prechilled matrigel (matrigel, BD Bioscience) was placed in each well of a prechilled 48-well plate (150 μl) or 96-well plate (50 μl) and incubated for 30 minutes. Serum-starved HUVEC cells were trypsinized, suspended in each conditioned medium, and added to the top of a solidified matrigel (30 cells/mm 2 ). After 12 h, the formed vessels were photographed and image J software (http://rsb.info.nih.gov/ij/) was used to determine vessel length and number of branch points. Images were analyzed. To investigate key factors related to angiogenic activity, conditioned medium was pretreated with neutralizing antibodies for 30 minutes at 37°C.

(In Vivo Matrigel Plug Assay)

All procedures were performed in accordance with the policy of the Institutional Animal Care and Use Committee (IACUC) (CUMC-2015-0025-03), The Catholic University of Korea. The dorsal region of 7-week-old BALB/c (nu/nu) mice (SLC, Japan) containing individual WJ-MSCs (5Х10 ⁶ cells in 50 μl of PBS) and without phenol red (phenol red -free), growth factor-depleted, ice-cold 0.4 mL of Matrigel (BD Biosciences) was injected subcutaneously. After 7 days, mice were euthanized and the Matrigel plugs were removed. To quantify the functional blood vessels formed, the Matrigel plug was homogenized in distilled water and centrifuged at 14,000 rpm for 5 minutes. Hemoglobin was measured by mixing the supernatant with Drabkin's reagent (Sigma). After incubation for 30 minutes, the absorbance of the mixture was measured at a wavelength of 540 nm.

(lower extremity ischemia mouse model)

7-week-old male mice (20-25 g, BALB/c nu/nu; SLC) were anesthetized with isoflurane (5% for induction, 2% for maintenance) and injected into the femoral artery ( femoral artery) and phlebectomy were performed. Within 6 hours after surgery, 5Х10 ⁵ WJ-MSCs in PBS obtained from two different types of donors or PBS (control) were intramuscularly injected at the site of lower extremity ischemia (n=5). Blood flow was measured using a laser doppler blood perfusion imager (LDPI; Moor Instruments Ltd. Millwey, Axminster, UK) on days 0, 7, 14 and 21 after surgery. Blood flow in the region from the knee joint to the toes was quantified, and the LDPI index was determined as the ischemic rate for the non-ischemic hindlimb. For histological and immunofluorescent evaluation, mice were sacrificed on postoperative day 21, lower limb muscles were removed, immediately frozen in OCT compound, and cryosections were made. To determine blood vessel formation, tissue sections were immunostained with anti-CD31 antibody (BD Biosciences) and FITC-conjugated anti-rat IgG (Millipore). After staining nuclei with DAPI (Sigma), slides were observed using a fluorescence microscope (Olympus BX51, Middlesex, UK). Blood vessel density was quantified using Image J software. The captured color image was applied to an RGB stack to divide the image into each channel. After segmenting each individual image using thresholding (mouse = 3/group, 30 images for each group), the ratio of vascular area per field was calculated with Image J.

(RNA interference)

Small interfering RNA (siRNA) targeting angiogenin, IL-8, MCP-1, VEGF and control siRNA (siCTL) were obtained from Bioneer Co. (Daejeon, Korea). siRNA sequences targeting angiogenin, IL-8, MCP-1, and VEGF and siRNA sequences of siCTLs that do not show homology to mammalian genes used as negative controls are shown in Table 1 below. After culturing MSCs at a density of 36 cells (36 cells/mm2) for 24 hours, siRNA at a concentration of 100 nM was added to the cells using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. was transfected into After 4 hours, the cells were washed twice with PBS to remove the transfection medium and then cultured with fresh medium. After 48 hours of culture, cells were washed with PBS and cultured with fresh EBM-2 medium. After 48 hours, the conditioned medium was collected and clarified by centrifugation at 3,200 rpm for 15 minutes. The conditioned medium was then used for in vitro angiogenesis assays or blotting onto nitrocellulose membranes using a 96-well Bio-dot apparatus (Bio-Rad Laboratories). . After blocking the membrane with 5% skim milk, it was treated with specific primary antibodies followed by HRP-conjugated secondary antibodies and incubated. Immunoreactive dots were visualized using the ECL kit (GE Healthcare).

siRNA type base sequence sequence number angiogenin sense 5′-ACG UUG UUGUUG CUU GUG A dTdT-3′ One antisense 5'-U CAC AAG CAA CAACAA CGU dTdT-3' 2 IL-8 sense 5'-GCC AAG GAG UGC UAA AGA A dTdT-3' 3 antisense 5'-U UCU UUA GCA CUC CUU GGC dTdT-3' 4 MCP-1 sense 5'-GUC ACC UGC UGU UAU AAC U dTdT-3' 5 antisense 5'-A GUU AUA ACA GCA GGU GAC dTdT-3' 6 VEGF sense 5'-GGA GUA CCC UGA UGA GAU C dTdT-3' 7 antisense 5'-G AUC UCA UCA GGG UAC UCC dTdT-3' 8 siCTLs sense 5'-GUU CAG CGU GUC CGG CGA G dTdT-3' 9 antisense 5'-C UCG CCG GAC ACG CUG AAC dTdT-3' 10

<Example 2> Analysis of changes in angiogenesis-promoting factor profile and activity between different donor-derived Wharton's umbilical cord-derived mesenchymal stem cells

Warton's jelly mesenchymal stem cells (WJ-MSC) were isolated from human umbilical cords of 9 different donor types. Their conditioned medium (CM) was collected after culturing in serum-free medium. To compare angiogenesis-promoting factor profiles, CM was analyzed using a Human Angiogenesis Array kit according to the manufacturer's instructions (FIGS. 1A to 1G). The secretion level of each protein was normalized by a reference point.

Among the 55 factors, 8 factors, angiogenin, angiopoietin-1, FGF-7, IL-8, MCP-1, PDGF-AA, serpin F1, and VEGF, showed diversity in secretion according to the donor. Although visible, it was confirmed that it was secreted at a fairly high level (Fig. 2A). At this time, the selection criterion for the 8 types of factors is that 3 to 8 donor types among 9 donor types must have a secretion level at a relative pixel intensity higher than 0.5. The secretion profile of these eight factors varied between donors. Using the human angiogenesis array kit, it was confirmed that the above 8 factors were significantly secreted from some MSCs regardless of tissue origin, and it was also confirmed that there was no clear difference according to the donor's gender type (Table 2). To test the correlation between the secretome profile and the angiogenic activity, two WJ-MSC types with higher and lower secretion levels were selected among 9 donor types. All of the 8 cytokines were secreted at much higher levels in WJ-MSCs from donor 6 (D6) compared to WJ-MSCs from donor 2 (D2) (Fig. 2B). The angiogenic activity of the conditioned media obtained from donor D6 and donor D2 was evaluated in cell proliferation, cell migration and angiogenesis assays using HUVEC cells. Interestingly, compared to donor D2, the conditioned medium of donor D6 induced proliferation, migration and angiogenesis of HUVEC cells at significantly higher levels (Fig. 2C to Fig. 2E). The results indicated that the pro-angiogenic cytokine profile and potency of different donor-derived MSCs varied from one another. Based on this, it was inferred that there was a correlation between the angiogenesis-promoting activity and the secreted cytokine profile according to the donor type.

gender male WJ-MSCs (n=6) BM-MSCs (n=6) D2, D3, D6, D7, D9, D12 6 donors female WJ-MSCs (n=7) BM-MSCs (n=8) D1, D4, D5, D8, D10, D11, D13 BM-D1, BM-D2, 6 donors

<Example 3> Comparative analysis of the secretome profile of WJ-MSC under different FBS conditions

In Example 2, it was confirmed that different donor types affect the secretion cytokine profile of MSCs. In addition to this, we investigated whether different fetal bovine serum also affect the secretion profile of MSCs. FBS is known to provide growth factors and essential nutrients necessary for the cultivation of MSCs, but its composition is not always the same as a limitation of biological products. We hypothesized that efficient biomarkers should be consistently secreted even if the cells were cultured in media containing different FBS. Therefore, the levels of secretion of cytokines promoting angiogenesis between MSCs of donors D2 and D6 cultured in DMEM medium containing FBS from two different companies before preparation of the conditioned medium were compared.

There were some changes in the level of secretion, but the main pattern did not change (FIG. 3). Compared to the conditioned medium from donor D2, the conditioned medium from donor D6 was more potent in promoting angiogenesis in vitro, so the conditioned medium from donor D6 compared to the conditioned medium from donor D2 in both FBS conditions. Cytokines that were higher in the medium and differentially secreted were selected. In addition, the present inventors considered the secretion level of the factor at a relative pixel intensity higher than 0.5 in order to select an efficient biomarker. The present inventors confirmed that among the eight factors, angiopoietin-1, FGF-7, PDGF-AA, and serpin F1 did not satisfy the above conditions. Accordingly, the present inventors finally selected four factors, angiogenin, IL-8, MCP-1, and VEGF, as possible biomarkers of MSC for angiogenesis-promoting efficacy.

<Example 4> Analysis of angiogenin, IL-8, MCP-1 and VEGF secretion levels as angiogenesis-promoting biomarkers for the efficacy of Wharton's colloidal-derived mesenchymal stem cells (WJ-MSC)

In Example 3, four factors, angiogenin, IL-8, MCP-1, and VEGF were selected as angiogenesis-promoting biomarkers, and secretion of the four factors for the angiogenesis-promoting efficacy of WJ-MSC To validate the level, WJ-MSCs derived from different umbilical cords of 4 additional donors were further investigated. After obtaining the conditioned medium, secretome profiles were analyzed to compare secretion levels of the above four factors.

As shown in Figure 4, both MSCs from donors D12 and D13 secreted higher levels of the four factors, angiogenin, IL-8, MCP-1 and VEGF, compared to the two MSCs from donors D10 and D11. It was confirmed that Overall, it was confirmed that donor D13 had a higher level of secretion compared to cells of other donor types. In order to compare the angiogenesis-promoting activity of conditioned media derived from different MSCs according to these donors in vitro, the degree of migration and tube formation of HUVEC cells induced in individual conditioned media was confirmed. Although there were some differences in the in vitro results of angiogenic activity between donors D10 and D11 or between donors D12 and D13 depending on the assay, the conditioned medium of donor D12 and the conditioned medium of donor D13 were approximately the same as those of donor D10 and D13. It showed higher activity than the conditioned medium of D11 (FIG. 5A and FIG. 5B). In terms of the angiogenesis promoting activity tested, the conditioned medium from donor D13 showed significantly more potent activity than the conditioned medium from donors D10 and D11.

Finally, the angiogenic activity was tested in vivo using MSC instead of the conditioned medium. The Matrigel plug assay was performed in vivo by transplanting mice with WJ-MSCs mixed with cold Matrigel. After 7 days, the Matrigel plug was removed from the mouse, and the weight and hemoglobin (Hb) content of the Matrigel plug was measured (FIG. 5C).

Since the Matrigel plug is initially avascular, all blood vessels formed on the Matrigel plug can be easily observed. All Matrigel plugs containing WJ-MSCs were red in contrast to the control Matrigel plugs. In addition, it was visually observed that the Matrigel plugs of donors D12 and D13 were more vascularized than those of donors D10 and D11. Accordingly, it was confirmed that the hemoglobin content of the Matrigel plug of donor D13 was significantly higher than that of the Matrigel plug of donor D10. Through the above results, WJ-MSCs derived from different donor umbilical cords secreted different levels of angiogenin, IL-8, MCP-1 and VEGF, and the secretion levels of the four factors were found to be vascular in vitro and in vivo. It was found that there was a high correlation with angiogenesis promoting activity.

<Example 5> Correlation analysis between angiogenin, IL-8, MCP-1 and VEGF secretion levels and blood vessel regeneration efficacy of Wharton's gelatin-derived mesenchymal stem cells (WJ-MSC)

In Example 4, it was confirmed that the secretion levels of angiogenin, IL-8, MCP-1, and VEGF factors had a high correlation with angiogenesis promoting activity in in vitro and in vivo models, and based on this, in disease models It was confirmed whether the secretion level of the four factors had a great effect on the blood vessel regeneration effect. Specifically, in order to determine whether the secretion level of the four factors affects the revascularization effect, a mouse model of lower extremity ischemia induced by femoral artery and venous resection was used. Within 6 hours after surgery, WJ-MSCs from donors D10 or D13 or PBS (control) were injected locally at four injection points in the ischemic femoral muscle area.

Continuous laser-Doppler perfusion imaging (LDPI) analysis of lower extremity blood flow showed the ischemic/non-ischemic lower extremity blood flow injected with WJ-MSCs from donor D13 compared to the experimental group injected with WJ-MSCs from donor D10 or the control group injected with PBS. It was found that the ratio increased significantly (Fig. 6 A). To evaluate MSC-mediated angiogenesis in ischemic tissues, tissues were collected on day 21 after surgery and stained by immunohistochemical staining with an anti-CD31 antibody (green fluorescence) to measure capillary density. Compared to the group treated with WJ-MSC or PBS from donor D10, it was confirmed that the number of CD31-positive capillaries was increased in the group treated with WJ-MSC from donor D13 (FIG. 6B). Through the above results, it was found that the secretion levels of angiogenin, IL-8, MCP-1, and VEGF factors of WJ-MSC in ischemia were highly correlated with the therapeutic efficacy of MSC for revascularization.

<Example 6> Verification of angiogenesis-promoting activity of WJ-MSC derived from Wharton's Jedae Colloid according to secretion levels of angiogenin, IL-8, MCP-1 and VEGF

In order to determine whether the four factors selected in Example 3, angiogenin, IL-8, MCP-1 and VEGF, are the main factors mediating the angiogenesis promoting effect of WJ-MSC, blocking antibodies or siRNA It was confirmed whether or not the angiogenesis-promoting effect of WJ-MSC was blocked by treatment. Specifically, prior to the assay, neutralizing antibodies against angiogenin, IL-8, MCP-1 and VEGF were pretreated in the conditioned medium of donor D13 in which the above four factors were secreted at relatively high levels.

As expected, it was confirmed that the migration of endothelial cells induced into the conditioned medium of donor D13 was significantly inhibited by single treatment with anti-IL-8, anti-MCP-1 or anti-VEGF neutralizing antibodies. In addition, migration of endothelial cells induced by the conditioned medium of donor D13 was also inhibited by a single treatment with an anti-angiogenin neutralizing antibody, but the inhibition was not statistically significant. It was confirmed that the migration of endothelial cells (EC) was most effectively inhibited when the above four antibodies (α-Angiogenin, α-IL-8, α-MCP-1 and α-VEGF; α-4F) were treated in combination. (FIG. 7 A). Also, in the vasculature of HUVEC cells induced by the conditioned medium of donor D13, a similar inhibitory pattern was observed when the blocking antibodies were treated singly or in combination (FIG. 7B).

Next, the effects of four factors, angiogenin, IL-8, MCP-1, and VEGF, on the angiogenesis-promoting effect of WJ-MSC by treatment with siRNA were evaluated. WJ-MSCs were individually or simultaneously treated with siRNAs targeting the above four factors. After obtaining the conditioned medium, the degree of deficiency of the above four factors was confirmed through dot blot analysis.

As a result, it was confirmed that the above four factors were significantly absent in the conditioned medium (FIG. 8). As a result of applying the conditioned medium for endothelial cell migration and tube formation, compared to the control conditioned medium transfected with siCTL, siAngiogenin-, siMCP-1-, siIL-8-, or siVEGF transfected It was confirmed that the conditioned medium from the conditioned MSCs reduced migration and angiogenesis of HUVEC cells (Fig. 7C and Fig. 7D). It was confirmed that when HUVEC cells were treated with the conditioned medium from MSC transfected with the combined siRNA (siAngiogenin, siMCP-1, siIL-8, and siVEGF; si4F), their migration and angiogenesis were most reduced. Through the above results, it was found that angiogenin, IL-8, MCP-1 and VEGF are the main factors for the angiogenesis promoting activity of WJ-MSC.

<Example 7> Analysis of availability of angiogenin, IL-8, MCP-1 and VEGF factors as biomarkers for predicting angiogenesis promotion effectiveness

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are a major cell source for autologous and allogeneic cell therapy. Therefore, it was investigated whether the four factors selected in Example 3, angiogenin, IL-8, MCP-1, and VEGF, could be applied as biomarkers for predicting the angiogenic activity of MSCs derived from various tissue origins. To do this, it was tested whether the four factors selected above play an important role in the angiogenesis promoting effect of BM-MSC. To identify these major angiogenesis-promoting factors, secretion profiles of BM-MSCs obtained from 14 donors were analyzed using the same human angiogenesis array kit.

As a result, the two representative BM-MSC types also showed differences in secretion levels of the four factors among the donor types, whereas other factors showed similar secretion patterns (FIG. 9A). Levels of IL-8 and MCP-1 in these BM-MSC types were found to be relatively low compared to WJ-MSC, but Roubelakis MG et. al. , PLoS One , 8: in the case of BM-MSC reported in e54747 (2013), the secretion level of IL-8 was higher than that of the BM-MSC type tested in the present invention. This suggests the potential importance of this factor as a biomarker.

As in the experiment using WJ-MSC, in order to confirm the correlation between the secretome profile and the angiogenesis-promoting activity, HUVEC cells were used for cell migration and angiogenesis assays to differentiate secretion levels of angiogenesis-promoting factors. Two BM-MSC types with were tested for angiogenic activity.

As a result, the conditioned medium of the BM-MSCs of donor BM-D2 secreting high levels of the above four factors was different from the conditioned medium of BM-MSCs of donor BM-D1 secreting low levels of the above four factors. In comparison, it was confirmed that HUVEC cell migration and angiogenesis were remarkably high (FIG. 9B and FIG. 9C). The conditioned medium pretreated with the combined blocking antibody (α-4F) targeting the above four factors significantly inhibited migration and angiogenesis of HUVEC cells (Fig. 9D and Fig. 9E). In addition, compared to the conditioned medium obtained from BM-MSC transfected with siCTL, the conditioned medium obtained from BM-MSC transfected with siRNA (si4F) targeting the above four factors was more It was found to significantly reduce migration and vasculature (FIG. 9F and FIG. 9G). Through the above results, it was found that the above four factors, angiogenin, IL-8, MCP-1 and VEGF can be usefully used as biomarkers for predicting the angiogenic activity of BM-MSC.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Method for evaluating a vascular regenerative efficacy of stem cells <130> P17U11C0739 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for angiogenin, sense <400> 1 acguuguugu ugcuugugad tdt 23 <210> 2 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for angiogenin, antisense <400> 2 ucacaagcaa caacaacgud tdt 23 <210> 3 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for IL-8, sense <400> 3 gccaaggagu gcuaaagaad tdt 23 <210> 4 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for IL-8, antisense <400> 4 uucuuuagca cuccuuggcd tdt 23 <210> 5 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for MCP-1, sense <400> 5 gucaccugcu guuauaacud tdt 23 <210> 6 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for MCP-1, antisense <400> 6 aguuauaaca gcaggugacd tdt 23 <210> 7 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for VEGF, sense <400> 7 ggaguacccu gaugagaucd tdt 23 <210> 8 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for VEGF, antisense <400> 8 gaucucauca ggguacuccd tdt 23 <210> 9 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for siCTL, sense <400> 9 guucagcgug uccggcgagd tdt 23 <210> 10 <211> 23 <212> RNA <213> artificial sequence <220> <223> siRNA targeting for siCTL, antisense <400> 10 cucgccggac acgcugaacd tdt 23

Claims (9)

Expression levels of angiogenin, interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were measured in bone marrow-derived mesenchymal stem cells obtained from individuals. including measuring
The expression levels of angiogenin, IL-8, MCP-1, and VEGF are high compared to a predetermined reference expression level, indicating the revascularization efficacy of bone marrow-derived mesenchymal stem cells ( vascular regenerative efficacy evaluation method.
delete According to claim 1,
The bone marrow-derived mesenchymal stem cells are autologous or allogenic origin, bone marrow-derived mesenchymal stem cells blood vessel regeneration efficacy evaluation method.
delete delete According to claim 1,
The bone marrow-derived mesenchymal stem cells are used for ischemic disease (ischemic disease) treatment, a method for evaluating the efficacy of bone marrow-derived mesenchymal stem cells for revascularization.
According to claim 6,
The ischemic disease is selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic stroke and ischemic lower limb disease, A method for evaluating the efficacy of bone marrow-derived mesenchymal stem cells for revascularization.
According to claim 1,
The expression levels of angiogenin, IL-8, MCP-1 and VEGF are measured by ELISA, Western blotting, dot blotting, or PCR, a method for evaluating the efficacy of bone marrow-derived mesenchymal stem cells for revascularization .
According to claim 1,
The bone marrow-derived mesenchymal stem cells are cultured in vitro before measuring the expression levels of angiogenin, IL-8, MCP-1 and VEGF to proliferate.

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