KR101406875B1 - Use of casein kinase inhibitor for inducing differentiation of mesenchymal stem cells to endothelial cells - Google Patents

Abstract

The present invention relates to the use of a CK (casein kinase) inhibitor for inducing differentiation of mesenchymal stem cells into vascular endothelial cells and a vascular disease including vascular endothelial cells differentiated from mesenchymal stem cells by a CK (casein kinase) inhibitor There is provided a therapeutic drug composition. Since the mesenchymal stem cells treated with CK (Casein Kinase) inhibitor are specifically induced to differentiate into vascular endothelial cells, they can effectively treat vascular diseases such as vascular endothelial cell damage by balloon dilation and the like.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a CK inhibitor for inducing the differentiation of mesenchymal stem cells into vascular endothelial cells. BACKGROUND ART < RTI ID = 0.0 > CK < / RTI >

The present invention relates to the use of a CK (casein kinase) inhibitor for inducing differentiation of mesenchymal stem cells into vascular endothelial cells and a vascular disease including vascular endothelial cells differentiated from mesenchymal stem cells by a CK (casein kinase) inhibitor To a therapeutic medicinal composition.

Endothelial progenitor cells (EPCs) may be useful for angiogenesis and re-endothelialization. A large number of bone marrow-derived cells have the potential to differentiate into endothelial-like cells. Recent studies in animals and humans have suggested that EPCs, bone marrow-derived progenitor cells, have the potential to stimulate angiogenesis and re-endothelialization, but it is not easy to obtain sufficient numbers of these cells when using blood-derived EPCs . Also, EPCs from adult blood have a limited ability to proliferate compared to umbilical cord blood-derived EPCs. As an alternative to vascular restoration, research on mesenchymal stem cells (MSCs) is under way. Of the various vascular endothelial progenitor cells, MSCs have the advantage of being easy to manufacture and highly flexible for autologous or allogeneic cell therapy. In addition, a sequential analysis of gene expression patterns in MSCs revealed that MSCs are not restricted to the mesodermal lineage in vitro, and that MSCs can be metastasized to other lines such as endothelial cells in Invitro and Invivo . Some of the injected MSCs also appeared to be introduced into new blood vessels or to stimulate collateral vessels by the paracrine mechanism. When cultured in the presence of vascular endothelial growth factor (VEGF), MSCs begin to express endothelial markers, suggesting that MSCs can be differentiated into endothelial-like cells.

The present invention provides a method for inducing differentiation from mesenchymal stem cells into vascular endothelial cells for use as a cell therapy agent for vascular diseases of mesenchymal stem cells.

Reducing the heterogeneity between transplanted cells and tissues may increase the engraftment rate of the transplanted cells. With this in mind, the present inventors have continuously studied a method of inducing mesenchymal stem cells into vascular endothelial cells. As a result, CK (casein kinase) inhibitor can induce differentiation of mesenchymal stem cells into vascular endothelial cells.

In the following examples, it is shown that mesenchymal stem cells derived from bone marrow-derived cells transformed ex vivo by the compound of Chemical Formula 1, which is a CK inhibitor, differentiate into vascular endothelial cells.

Accordingly, the present invention relates to a use of a CK inhibitor for inducing differentiation of mesenchymal stem cells into vascular endothelial cells, a method for inducing differentiation of mesenchymal stem cells into vascular endothelial cells, which comprises treating a mesenchymal stem cell with a CK inhibitor And a composition for inducing differentiation of mesenchymal stem cells into vascular endothelial cells including a CK inhibitor.

In the present invention, the kind of the CK inhibitor used for inducing differentiation of mesenchymal stem cells into vascular endothelial cells is not particularly limited.

In one embodiment, the CK inhibitor may be a compound of formula (I).

[Chemical Formula 1]

In this formula,

R ₁ , R ₂ , R ₃ , R ₄ , R _5, R ₆ and R ₇ are each independently H, C _{1 - 12} is alkoxy, hydroxy, carboxy or halogen atom _{- 12} alkyl, C _1.

"Alkyl" means a straight and branched saturated hydrocarbon group generally having the indicated number of carbon atoms (e.g., 1 to 12 carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t- butyl, pent- Methylbut-2-yl, 2,2,2-trimethylet-1-yl, n-hexyl, n-heptyl and n-octyl, and the like.

"Alkoxy" refers to alkyl-O-, wherein alkyl is defined above. Examples of alkoxy groups include, without limitation, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s- do. Alkoxy can be attached to a parent group or substrate at any ring atom provided that attachment does not violate the atomic requirement. Likewise, an alkoxy group may include one or more non-hydrogen substituents if the attachment does not violate the valence requirements.

In one embodiment, R ₁ , R ₂ , R ₃ , R ₄ , R _5, R ₆ and R ₇ are each independently H, C _{1 - 4} alkyl or may be a halogen atom.

In another embodiment, R ₁ , R ₂ , R ₃ , R _4, and R ₅ are each independently H or a halogen atom,

R ₆ and R ₇ are each independently H or C _{1 - 4} alkyl may be.

In another embodiment, R ₁ , R ₂ , R ₃ and R ₄ are each independently H or bromine, R ₅ is H, and R ₆ and R ₇ are each independently H or methyl.

In another embodiment, the compound of formula 1 may be 4,5,6,7-tetrabromo-N, N-dimethyl-1H-benzo [d] imidazol-2-amine.

In addition, in the present invention, the type of mesenchymal stem cells used for inducing differentiation into vascular endothelial cells is also not particularly limited. The mesenchymal stem cells used in the present invention can be used regardless of where it originated. Mesenchymal stem cells can be obtained from known sources of mesenchymal stem cells, such as bone marrow, tissues, embryos, umbilical cord blood, blood or body fluids. The animal to be harvested, such as bone marrow, tissue, etc., may be a mammal. When the animal is a human, the bone marrow, tissue, or the like may originate from a patient's own or other person to be administered as a cell treatment agent, a mesenchymal stem cell in which differentiation into vascular endothelial cells is induced by treatment with the composition of the present invention. Methods for obtaining mesenchymal stem cells from such known mesenchymal stem cell sources are well known in the art.

On the other hand, the method of treating the CK inhibitor with mesenchymal stem cells is not particularly limited. It is sufficient that the mesenchymal stem cells can be induced to differentiate into vascular endothelial cells by contacting the mesenchymal stem cells with the CK inhibitor for a certain period of time. In one embodiment, treatment of the CK inhibitor may be performed by culturing the mesenchymal stem cells in a medium comprising a CK inhibitor.

The concentration of the CK inhibitor to be treated in the mesenchymal stem cells may vary depending on the type of the specific CK inhibitor, the period of treatment to the mesenchymal stem cells, or the degree of differentiation into vascular endothelial cells. In one embodiment, the concentration of the CK inhibitor may be used in the range of 0.01 to 100 [mu] M.

Given the time generally required to induce differentiation of mesenchymal stem cells, culturing in a medium containing a CK inhibitor may be performed for 5 to 15 days, but is not limited thereto. The duration of treatment of the CK inhibitor with mesenchymal stem cells may vary depending on the type or concentration of the compound of formula (I) to be treated.

In the present invention, "vascular endothelial cells" include all the cells that undergo differentiation into vascular endothelial cells or vascular endothelial cells derived from differentiation from mesenchymal stem cells. As used herein, "vascular endothelial cells", "vascular endothelial-like cells" and "endothelial-like cells" are used interchangeably. In the present invention, vascular endothelial cells differentiated from mesenchymal stem cells by treatment with a CK inhibitor exhibit expression of a specific marker associated with vascular endothelial cells. The vascular endothelial cells obtained according to the method of the present invention may have increased expression of these specific markers as compared to mesenchymal stem cells. The specific markers may be selected from the group consisting of, but not limited to, CD34, eNOS, VCAM-1, VE-cadherin, and VEGF-R2.

The present invention also provides a composition for inducing the differentiation of mesenchymal stem cells into a vascular endothelial cell comprising a CK inhibitor. Specific examples of the CK inhibitor and the compound of the formula (1) are as described above. The composition may include a medium generally used for culturing mesenchymal stem cells. Examples of such media include, but are not limited to, MEM-alpha (Minimum Essential Medium alpha), Mesenchymal Stem Cell Growth Medium (MSCGM), and Dulbecco's Modified Eagle's Medium (DMEM). Alternatively, a composition for inducing differentiation of mesenchymal stem cells into a vascular endothelial cell containing a CK inhibitor may be introduced into the body separately from mesenchymal stem cells. That is, a composition containing a CK inhibitor may be separately administered before or after administration of mesenchymal stem cells, or simultaneously with administration of mesenchymal stem cells. In this case, the composition may comprise a known pharmaceutical carrier suitable for administration of a CK inhibitor.

The present invention also provides a pharmaceutical composition for treating a vascular disease comprising vascular endothelial cells differentiated from mesenchymal stem cells by the above method. The medicinal composition for treating vascular diseases is not limited but may be useful for treatment of vascular endothelial cell damage by balloon dilation or the like. The pharmaceutical composition may further include a known carrier used in the art for transplantation of stem cells. The effective amount of the vascular endothelial cells may be 1 x 10 ⁴ to 1 x 10 ⁸ cells / kg. However, their dose may be increased or decreased depending on the patient's weight, age, sex, and degree of lesion. The preparation according to the present invention can be applied to the human body by parenteral or topical administration. For this purpose, the active ingredient is suspended or dissolved in a pharmaceutically acceptable carrier according to the method of the invention, preferably using a water-soluble carrier.

Since the mesenchymal stem cells treated with the CK inhibitor are specifically induced to differentiate into vascular endothelial cells, they can effectively treat vascular diseases such as vascular endothelial cell damage by balloon dilation and the like.

Figure 1 shows the results of immunocytochemistry showing morphological marker expression of MSCs.
Figure 2 shows the results of a sandwich ELISA showing the differentiation into vascular endothelial cells by treatment of compound # 7.
FIG. 3 shows the results of RT-PCR analysis showing that the expression of CD34, eNOS, VCAM-1, VE-cadherin and VEGF-R2 is increased in comparison with normal MSCs in cells induced to differentiate into endothelial cells by the compound.
Figure 4 shows immunocytochemical analysis of endothelial cell-specific markers.
Figures 5 and 6 show re-endothelialization of endothelial-like cells through Evan Blue staining.
FIG. 7 is a morphometric and morphometric analysis result of the left common carotid artery showing that formation of endothelial cell differentiation-induced cells reduces neointimal formation.
FIG. 8 shows the result of cell migration analysis showing that cells induced to differentiate into endothelial cells are less mobile than MSCs.
Figure 9 shows the results of cell attachment tests showing that the adhesion of endothelium-like cells is lower than MSCs and similar to HUVECs.
Figure 10 is a cell spreading analysis showing that endothelium-like cells exhibit cell spreading properties similar to HUVECs.
FIG. 11 shows immunohistochemical analysis results in rats treated with MSCs and endothelial-like cells after balloon dilation.
FIG. 12 shows the results of analysis of vascular relaxation response of carotid arteries in rats treated with MSCs and endothelium-like cells after balloon dilation.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

[ Example ]

Experimental Method

One. MSCs Isolation and Culture

MSCs were isolated from the femur and tibia of donor rats and cultured. Bone marrow-derived MSCs were obtained from the femur and tibia of 4-week-old Sprague-Dawley male rats (approximately 100 g), mixed with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA) and 1% penicillin and streptomycin antibiotic mixture (Gibco) supplemented with Dulbecco's modified Eagle's medium (DMEM) -low glucose. Peokol - it was plated washed twice mononuclear cells (mononuclear cells) obtained from the interface of the separated bone marrow and play a ^{1 x 10 6 cells / 100 cm} 2 to cloudy, and then the flask reproduce the 10% FBS-DMEM. A wet atmosphere containing 5% CO ₂ and maintained for 37 ℃ culture. After 72 hours, the unattached cells were discarded and the adherent cells were washed twice with phosphate buffered saline (PBS). A new complete medium was added and replaced every 3 or 4 days for 10 days. To further purify MSCs, the Isolex Magnetic Cell Selection System (Epic atla, Beckman coulter) was used. The magnetic field was processed into the chamber and the CD34 + cell-bead complex was magnetically separated from the remaining cell suspension. The CD34-fraction was further incubated. The cells were harvested after incubation at 37 ° C for 5 minutes with 0.25% trypsin and 1 mM EDTA (Gibco), and the cells were harvested, replated on plates at 1 × 10 ⁵ /100-cm ² and re-grown for approximately 10 days.

2. MSCs Of endothelial cells

The MSCs endothelial - to induce differentiation into similar cells, the MSCs in 60 mm plates seeded with 2 x 10 ⁵ cells / ml, and a GSK-3β, one of the SB-216763 (Sigma-Aldrich) or CK inhibitors of inhibitors Tetrabromo-N, N-dimethyl-1H-benzo [d] imidazol-2-amine (Compound # 7) was treated to a predetermined concentration. The medium was replaced every 3 days with the culture medium containing SB-216763 or Compound # 7 and cultured until 16 days.

3. Sandwich ELISA

Capture antibody was bound to the bottom of each well and the plate was incubated overnight at 4 ° C. Plates were washed twice with PBS (Gibco) and treated with 100 [mu] l of 3% BSA (Sigma, St. Louis, MO, USA) / PBS100 for 2-3 hours at room temperature. After washing the plates twice with PBS, cell lysates were added to each well, and the plates were incubated at room temperature under a humidified atmosphere for at least 2 hours. The plates were washed twice with PBS containing 0.02% Tween-20 (Sigma, St. Louis, Mo., USA). After addition of the detector antibody, the plates were incubated for 2 hours at room temperature in a humidified atmosphere After incubation, peroxidase was added to the conjugated secondary antibody and incubated at 37 [deg.] C for 1 hour. Finally, 100 μl of tetramethylbenzidine (TMB, Sigma, St. Louis, MO, USA) solution (Sigma) was treated as a substrate and 25 μl of 0.1 MH ₂ SO ₄ was added as a final buffer to stop the reaction , And the absorbance at 450 nm was immediately measured with an ELISA plate reader (Bio-Rad).

4. Cell migration analysis

The ability of the endothelium-like cells to migrate was tested by performing a modified Boyden chamber assay using a collagen-treated polycarbonate membrane with 8-μm pores in a 24-well plate. Preconfluent cells were suspended in serum-free DMEM 3.0 × 10 ⁵ cells / ml concentration of the. Serum free DMEM (0.5 ml) with or without bFGF (50 ng / ml) was added to the bottom chamber. 100 μl of cell suspension (final: 30,000 cells / well) was added to the upper chamber, followed by incubation at 37 ° C for 6 hours. The cells were fixed and stained with crystal violet. I filtered the filter and took a picture.

5. Cell attachment cell adhesion )

Cells were harvested by trypsinization, washed once in DMEM containing 10% FBS to stop trypsin activity, and then washed twice with serum-free DMEM to remove serum components. A suspension of 2 x 10 ⁴ live cells was added to the collagen type I-coated wells and allowed to attach for 30 minutes at 37 ° C under 5% CO ₂ . To quantify cell attachment, the plates were carefully washed three times with PBS and then photographed in four sections under a phase contrast microscope. The number of cells attached was estimated by counting the cells under a microscope using a hemocytometer. Each experiment was performed in triplicate wells and repeated 3 times or more. The number of attached cells was calculated by subtracting the number of cells rinsed from the number of seeded cells.

6. Cells On spreading  Analysis for Assay for cell spreading )

As prepared for the cell adhesion assay described above, the cells were added to a collagen type I-coated 24 well cell culture dish to allow cell spreading for 30 minutes. Plates were washed three times with PBS, fixed with 3% formaldehyde, stained with Coomassie blue, destained for control, and then photographed in six sections under a phase contrast microscope.

7. RT - PCR  analysis

Expression levels of various genes were analyzed by RT-PCR (RT-PCR). Total RNA was prepared using UltraspectTM-II RNA system (Biotecx Laboratories Inc., Houston, Tex., USA) and single strand cDNA was synthesized from the separated total RNA by Avian Myeloblastosis virus (AMV) reverse transcriptase. (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1 mM deoxynucleoside triphosphate (dNTPs), 0.5 units of RNase inhibitor, 20 μl of the reverse transcription reaction mixture containing 0.5 μg of oligo (dT) ₁₅ and 15 units of AMV reverse transcriptase was incubated at 42 ° C. for 15 minutes, heated at 99 ° C. for 5 minutes and then incubated at 4 ° C. for 5 minutes Lt; / RTI > PCR was performed for 35 cycles using 3 ' and 5 ' primers based on the sequence of the various genes. GAPDH (5'-CTCCCAACGTGTCTGTTGTG-3 'and 5'-TGAGCTTGACAAAGTGGTCG-3') was used as an internal standard. The signal intensities of the amplification products were normalized for each GAPDH signal intensity.

8. In vitro angiogenesis In vitro angiogenesis )

Capillary angiogenesis assay was performed according to the manufacturer's instructions using an in vitro angiogenesis kit (Chemicon International Inc, Temecula, CA, USA). 50 microliters of the gel matrix solution was added to each well of 96 wells and the plate was incubated at 37 for 1 hour. The cells were then trypsinized and 5 x 10 ³ cells were suspended in 50 μl of DMEM with or without VEGF, plated on a gel matrix, and incubated for 4 hours. The formation of the capillary structure was counted by eye. Percentage of formed capillaries was calculated based on three independent experiments.

9. Trip plate blue  Cell count after staining

Cells were washed in PBS. The solution was then prepared by mixing 0.5 ml of Trypan blue, 0.3 ml of PBS, and 0.2 ml of cells. After 5 to 15 minutes, a small amount of trypan blue-cell suspension was transferred to both chambers of the hematocytometer. All dyed and undigested cells within a 1 mm center square and four 1 mm corner squares of a hematocytometer were counted.

10. Immune cell chemistry

Cells were grown on a 4-well plastic dish (Nalge Nunc, Rochester, NY, USA). After incubation, the cells were washed twice with PBS and fixed in 0.5 ml PBS containing 4% paraformaldehyde for 30 minutes at room temperature. Cells were washed again with PBS and infiltrated in PBS containing 0.2% Triton for 30 minutes. Cells were then blocked in PBS containing 10% goat serum and incubated with primary antibody for 1 hour. The cells were again washed 3 times for 10 minutes with PBS and incubated with the FITC-conjugated secondary antibody for 1 hour. Cells were photographed under an immunofluorescence microscope (Olympus, Melville, NY, USA). All images were taken under a reflected light fluorescence microscope using an excitation filter and transferred to a computer equipped with MetaMorph software version 4.6 (Universal Imaging Corporation Ltd., Marlow, UK).

11. Vascular injury and cell infusion

Six-week-old male Sprague-Dawley rats (approximately 250 g) underwent balloon injury. All animal experiments were performed according to protocols approved by the Yonsei University Animal Care Committee. 2Fr Forgaty arterial embolization catheter (Edwards Lifesciences, Irvine, CA, USA) was inserted into the left common carotid artery of the rat and infused with 0.3 ml of saline three times. GFP-labeled endothelial-like cells or MSCs (3x10 ⁶ cells / animal) were intravenously injected into the balloon-injured rats.

12. Evan blue  Dyeing and morphological analysis

5% Evan blue dye (Sigma, St. Louis, Mo., USA) was injected into the femoral vein 60 minutes prior to the sacrifice of the rats and the area of the endothelial cells removed and the area recovered were analyzed. The obtained total carotid artery was fixed with 10% formalin, paraffin-cut in longitudinal and lateral directions, and stained with hematocylin and eosin. Morphometric analysis of intimal thickness was performed on cut artery sections cut by light microscopy. This was done to analyze the total morphology of endothelial integrity using Evan blue dye. It is consistent with what is required. Longitudinal longitudinal histological sections obtained from a 20-mm long damaged artery and stained with elastic tissue trichrome were projected onto a digitizing board and used with the techniques described above for the computerized sketching program The technicians who did not know about the treatment plan measured the areas of intima and media. The thickness of the intimal layer of the arterial wall was variable, partially reflecting the dimensions (diameter) of the individual SD rat total carotid artery. Thus, the thickness of the media was used to index the extent of neointimal thickening of the neointima, and is therefore referred to as the I / M ratio. The average perimeter of each arterial sample was 2.3 mm, which allowed for two longitudinal cuts at 500-μm intervals for morphological examination. The I / M ratio was calculated as the mean value measured from these two sections.

13. Immunohistochemistry

The arterial slice (3 탆 thick) was incubated with the primary antibody against rat CD31 (santa cruze biotechnology inc.) And incubated with the FITC-conjugated secondary antibody. Endothelialization after immunohistochemical staining with anti-CD31 was morphologically evaluated. Re-endothelialization was calculated as the ratio of the surface covered by CD31 positive cells to the luminal surface.

14. Aortic Ring Preparation and Vasodilation Reactivity Aortic ring preparation and vasodilator  responsiveness)

On the day of the experiment, the animals were euthanized by intraperitoneal injection of pentobarbital sodium (50 mg / kg body weight). Cut the common carotid artery, HEPES-Tyrode solution aerated with a 100% O _2: placed in (mM 10 Glucose, 10 HEPES, 134 NaCl, 5.6 KCl, 1 MgCl 2, 2.5 CaCl 2). Arteries were prepared with ring segments (approximately 3 mm long). The fat and outer membrane were mechanically removed under a binocular microscope. Each arterial ring was horizontally fixed between two parallel stainless steel hooks in a temperature-controlled 3 ml organ bath. One hook was fixed while the other was connected to a force transducer (UFER, Medical Kishimoto, Kyoto, Japan) to measure isometric contraction. After incubation at 37 ° C for 30 min in 100% O ₂ -aerated HEPES-Tyrode solution, the sections were incubated with 10 mM (70 mM high K ⁺ HEPES-Tyrode solution (K ⁺ substitution for Na ⁺ ). Arterial rings were repeatedly shrunk with 70 mM high K ⁺ HEPES-Tyrode solution until a stable response was obtained. Submaximal contraction was obtained with a 50 mM high K ⁺ HEPES-Tyrode (50K) solution. Endothelium-dependent vasorelaxation was induced by the addition of cumulative acetylcholine (A2661, Sigma; 10 ^-8 to 10 ^-4 M).

15. Nitric oxide production analysis

Nitric oxide production analysis was performed according to the method of Dean J et al. In summary, prepare each cell type (HUVECs, MSCs, G-MSCs) in a suitable medium. Cells were then washed three times with warm PBS and stimulated with acetylcholine (5 [mu] M) phenol red-free DMEM for 60 min. The medium was transferred to a new tube and prior to NO production assay, and spun at 2000 xg for 1 minute. Then, the protocol instructions for DAN analysis and fluorescence analysis of NO emission were followed.

16. Statistical analysis

Results are expressed as mean ± SEM. Student's t-test. The correlation was considered statistically significant when the p-value was less than 0.05.

Experiment result

One. MSCs Separation and morphology Marker  Characterization

MSCs were first separated from media mixed with hematopoietic stem cells based on their preferential attachment on culture plates. MSCs retained fibroblastic morphology through repeated passages and their identity was confirmed by immunocytochemistry. The cultured MSCs showed CD71, CD90, CD105, CD106, and ICAM. They were negative for CD34, a marker of hematopoietic stem cells, as can be seen in Fig.

2. MSCs Of GSK-3beta inhibitor against endothelial cell differentiation Wow CK  Effect of inhibitor

We tested the effects of protein kinase inhibitors on the differentiation of MSCs into various cell types and confirmed that GSK-3β inhibitors and CK inhibitors induce MSCs to be endothelial-like cells. In addition, it was shown that the GSK-3? Inhibitor (SB-216763) and the CK inhibitor (compound # 7) induce endothelial cell differentiation without inducing differentiation into different cell lineages. Sandwich ELISA assays were performed to determine the inducible activity of these compounds on the differentiation of MSCs into endothelial cells. Figure 2 shows the results of a sandwich ELISA showing the differentiation into vascular endothelial cells by treatment with compound # 7. When the expression level of CD31, a vascular endothelial cell factor, was evaluated, it was found that the expression was increased similarly to that of SB216763 when compared with the stem cell normal group.

We found that SB-216763 and compound # 7 were induced with similar increases and greatest increases in expression of CD31 in both dose- and time-dependent manner. In addition, SB-216763 and compound # 7 did not show significant cytotoxicity under these experimental concentrations (data not shown).

3. GSK-3 beta inhibitor Wow CK  Induced by inhibitors MSCs Of endothelial cells

Primarily induced by GSK-3? Inhibitors and CK inhibitors Endothelium-like cells were analyzed and characterized based on their morphology under a phase contrast microscope, and endothelial-like cells were identified in the form of gravelly stones. To confirm the GSK-3? Inhibition effect of SB-216763 on endothelial cell differentiation of MSCs, we examined changes in the expression levels of both MSC and endothelial cell-specific markers in SB-216763-treated MSCs . First, we observed that despite the increased expression of CD31, the cell morphology of MSCs cultured for 16 days in the presence of SB-216763 was not significantly changed. SB-216763 reduced the expression level of CD71 mRNA, an MSC-specific marker, in a time-dependent and significant manner in MSCs. We further confirmed these effects by immunocytochemical staining. The fluorescence intensity of another MSC-specific marker, CD90, in SB-216763-treated MSCs was gradually decreased in a time-dependent manner and CD31 protein levels were increased, but no change was observed in control MSCs. Therefore, we have confirmed that GSK-3? Inhibitor-treated MSCs (G-MSCs) are new cell types with a phenotype similar to endothelial cells.

4. Endothelial cell specific Marker  Expression RT - PCR  analysis

To further confirm that these MSCs could differentiate in response to SB-216763, changes in endothelial cell specific markers were analyzed by RT-PCR. As can be seen in FIG. 3, the expression of CD34, eNOS, VCAM-1, VE-cadherin and VEGF-R2 was found to increase in comparison with normal MSCs after 16 days of culture in the presence of SB-216763 Respectively. These results show that GSK-3? Inhibition by SB-216763 effectively induces the differentiation of MSCs into endothelial cells.

5. Endothelial cells On the marker  Immunocytochemical analysis

MSC and G-MSC were treated with Vascular Cell Adhesion Molecule-1 (VCAM-1) and Vascular Endothelial Growth Factor-Receptor 2 (VEGF-R2) for immunocytochemical staining after incubation for 16 days in the presence of GSK- Lt; / RTI > As can be seen in Fig. 4, the monolayer of G-MSC was positively stained for VACM-1 and VEGF-R2.

6. Formation of capillary-like structures by endothelial-like cells

To further confirm that differentiated MSCs could function as endothelial cells, we investigated the ability of these cells to form capillary vessels on semi-solid medium in the presence and absence of VEGF using an angiogenesis assay . When the undifferentiated MSCs were irradiated 4 hours later, almost no capillaries were observed, and most of the cells were rounded in the absence of VEGF. However, undifferentiated MSCs formed a large number of capillary structures under VEGF stimulation. Interestingly, in pre-differentiated MSCs, the number of capillary-like cells was significantly increased by 85% and 75%, respectively, in the presence and absence of VEGF. These results demonstrate that endothelial-like cells obtained from GSK-3? Inhibitor-induced differentiation of MSCs have angiogenic and endothelial properties (data not shown)

7. Enhanced by endothelial-like cells Re-endothelialization

To assess the effect of endothelium-like cells on re-endothelialization, we performed planimetric analysis with damage and evan blue staining at designated time points up to 21 days after cell injection. Evan blue dye was administered to stain non-endothelialized areas at 5 and 14 days after injury. Figures 5 and 6 show re-endothelialization of endothelial-like cells through Evan Blue staining. The negative control, BI CCA (total carotid artery), was white on day 21 after injury. Non-endothelialized lesions showed blue color, while re-endothelialized areas showed white color. ~ 60% of neointimal formation was observed in the lumen area. The results of Evan Blue staining showed that the formation of neointima after 4 days of injury showed a significant decrease in endothelial-like cell-injected rats compared to the MSCs-injected rat control, - is associated with a significant decrease in neovascular endothelialization in pseudocyte-injected rats. Only non-living nappies were stained with only Evan blue dye. A larger, unstained, re-endothelialized area was observed at day 5 in the rat injected with endothelium-like cells. A similar undyed area was observed in control rats injected with MSCs at day 14. Evan blue staining showed rapid re-endothelialization in endothelial-like cell-injected rats compared to control rats injected with undifferentiated MSCs. Significant differences were observed after 5 days of injury; An ~ 40% increase in re-endothelialization was observed in endothelial-like cell-injected rats.

8. Neovascular endothelial membrane  Inhibition of formation and Morphometrical  analysis

Morphological and morphometric analyzes of the left common carotid artery were performed on day 21 after balloon dilatation and treatment with endothelium-like cells or MSCs. Each carotid artery was compared with the uninvolved artery on the opposite side and the uninjured area on the distal side. On day 21 after balloon dilatation, immunohistochemical studies were performed. FIG. 7 is a morphometric and morphometric analysis result of the left common carotid artery showing that formation of endothelial cell differentiation-induced cells reduces neointimal formation. The luminal area surrounded by IEL (internal elastic laminar) was significantly reduced in MSC treated rats as compared to rats treated with endothelial-like cells, indicating that treatment with endothelial-like cells resulted in a narrowing of the lumen in restenosis which is a major determinant of luminal narrowing, is significantly reduced.

9. Cell migration analysis

MSCs showed greater mobility on collagen-coated membranes than G-MSCs. The number of migrated cells in G-MSCs was similar to that of HUVECs. Data were expressed as the number of transferred cells at 2 x 10 ⁴ cells per well, and images were randomly photographed three times. The average number of cells calculated was used. FIG. 8 shows the result of cell migration analysis showing that cells induced to differentiate into endothelial cells are less mobile than MSCs.

10. Cell adhesion test

GSK-3? Inhibitors inhibited cell adhesion on collagen-coated culture dishes. GSK-3? Inhibitors have been shown to reduce the expression of? -Catenin. The cytoplasmic domains of type I cadherins (N- and E-cadherin) bind to beta -catenin. This indicates that the adhesion of endothelial-like cells is lower than that of MSCs and is similar to HUVECs. As can be seen in FIG. 9, G-MSCs exhibit adhesion properties similar to HUVECs. MSCs showed the greatest adherence to invert.

11. Cells Spreading  analysis

The fuzzy area around the cell nucleus was counting criteria. As can be seen in FIG. 10, MSCs exhibited the greatest spreading characteristics on collagen coated 24 well culture dishes, and G-MSCs exhibited nearly the same% spread cells as HUVECs . .

12. Balloon expansion  Immunohistochemical evaluation of posterior carotid artery

To clarify the involvement of injected cells in models with endothelial cell depletion, the co-expression of endothelium-specific markers in GFP-labeled cells was examined. As can be seen in Fig. 11, paraffin-embedded sections immunostained against CD31 and the whole-mounted specimen showed the formation of the endothelial layer of the 1-cell layer in the re-endothelialized area. Immunostaining of total carotid artery to CD31 resulted in the presence of more GFP-positive cells in re-endothelialized areas than in MSCs-injected rats injected with endothelium-like cells. GFP-positive cells were not identified in normal arteries without balloon dilatation. We have found that most GFP-positive cells are located in the neovascular endothelial area of the artery.

13. Vasodilatory response of carotid artery

To evaluate the endothelium-dependent vascular relaxation of the carotid artery, the presence (Fig. 12C) or absence (Fig. 12D) of the GSK-3β inhibitor after the angioplasty was compared with the normal group (Fig. 12A), the angioplasty group ACh-induced vasorelaxation in MSCs treated group. The 50K-induced contraction was stable and Ach was added in a dose-dependent manner. In all groups, Ach induced dose-dependent vasorelaxation and maximum relaxation was observed at 10 ^-4 M ACh. ACh-induced maximal vascular relaxation was 43.21 ± 4.2% (n = 5) in the normal carotid artery, which was calculated by inverse relaxation of 50K-induced contractions. However, in carotid arteries under balloon dilation, ACh-induced vascular relaxation was significantly reduced (28.69 ± 2.09%, n = 6). There was no significant change (27.09 ± 4.07%, n = 5) in the MSC-treated carotid artery after angioplasty compared with the balloon dilatation group. In contrast, in MSC-treated carotid arteries in the presence of GSK-3β inhibitors, ACh-induced vascular relaxation was significantly increased (41.92 ± 2.85%, n = 5) Similar to vascular relaxation.

Claims (16)

Mesenchymal stem cell endothelial cells comprising treating 4,5,6,7-tetrabromo-N, N-dimethyl-1H-benzo [d] imidazol- Lt; / RTI >
delete delete delete delete The method according to claim 1,
Wherein said mesenchymal stem cells are obtained from bone marrow, tissue, embryo, umbilical cord blood, blood or body fluids, and inducing differentiation of mesenchymal stem cells into vascular endothelial cells.
The method according to claim 1,
Treatment of 4,5,6,7-tetrabromo-N, N-dimethyl-1H- benzo [d] imidazol-2-amine allows mesenchymal stem cells to be treated with 4,5,6,7-tetrabromo- N, N-dimethyl-1H-benzo [d] imidazol-2-amine in the medium.
8. The method of claim 7,
Wherein the culturing is carried out for 5 to 15 days. The method for inducing differentiation of mesenchymal stem cells into vascular endothelial cells.
The method according to claim 1,
A method for inducing differentiation of mesenchymal stem cells into vascular endothelial cells, wherein the vascular endothelial cells have increased expression of CD34, eNOS, VCAM-1, VE-cadherin or VEGF-R2 compared to stem cells.
A composition for inducing differentiation of mesenchymal stem cells into vascular endothelial cells comprising 4,5,6,7-tetrabromo-N, N-dimethyl-1H-benzo [d] imidazol-2-amine.
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