WO2014209013A1 - Novel stem cells having characteristics of endothelial cells and derived from mesenchymal stem cells - Google Patents

Abstract

The present invention relates to stem cells having characteristics of endothelial cells and derived from mesenchymal stem cells, and to a method for producing the stem cells from mesenchymal stem cells. Further, the present invention relates to a pharmaceutical composition for preventing or treating ishemic diseases, the composition containing the stem cells or a culture liquid thereof. The stem cells according to the present invention have characteristics of both endothelial cells and stem cells, and retain a high cell proliferation rate, cell migration ability, and DNA conservation ability, thereby exhibiting active angiogenesis ability in ishemic diseases, and thus can be effectively used to prevent or treat ishemic diseases.

Description

Novel Stem Cells Including Endothelial Cell Characteristics Derived from Mesenchymal Stem Cells

The present invention relates to a method for producing the stem cells from the stem cells and mesenchymal stem cells comprising the characteristics of endothelial cells, derived from mesenchymal stem cells.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of ischemic diseases comprising the stem cells or cultures thereof.

Ischemia is a state of reduced blood flow to body organs, tissues or sites, which ultimately leads to necrosis of cells and tissues, which are irreversible damage. In particular, the brain and heart are the most sensitive organs of the bloodstream, for example, when ischemic tissues are caused by stroke or head injury, triggering a process called ischemic cascade, which permanently damages brain tissue. .

Ischemic disease is a disease related to ischemia caused by an organic disorder of the blood supply. Cardiac diseases (ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.).

Cell therapy products, including stem cell therapies, can be used to proliferate, select, or otherwise invigorate living autologous, allogeneic, or xenogeneic cells in vitro to restore cell and tissue function. It is defined as a drug that is used for the purpose of treatment, diagnosis and prevention through a series of actions, such as changing the biological characteristics. Stem cell therapies are particularly the case of using stem cells, and the current application field is essential, but it does not naturally work well to recover and regenerate lost cells such as neurological disease, heart disease, lung disease, liver disease, cancer If not, development is actively underway.

Stem cells have great potential in cell therapy in that they have differentiation potential and can induce differentiation from damaged tissues to necessary cells.However, at the present development level, the survival rate after transplantation in the body is not high. It is hard to find a successful example.

In particular, fat, bone marrow or umbilical cord blood-derived stem cell therapy can be used to treat ischemic diseases, and it has been found that blood vessels can be regenerated. However, most of the stem cells transplanted into the ischemic site by two-dimensional culture are killed to treat cell therapy. There has been a problem that the efficacy is not great. Rehman J et al. Also reported that adipose derived stem cells secrete factors related to blood vessel regeneration (Circulation 2004; 109 (10): 1292-8). The survival rate of the cells transplanted to the ischemic site was extremely low. Furthermore, Nakagami H et al. Reported the transplantation of adipose-derived stem cells into VEGF and HGF and transplanted them into the lower limb ischemia (Arterioscler Thromb Vasc Biol. 2005; 25 (12): 2542-7). . As a result, the vascular regeneration effect was higher than that of the adipose-derived stem cells transplanted without VEGF and HGF treatment, but growth factors such as VEGF and HGF were likely to cause cancer depending on the concentration, which makes it difficult to apply clinically.

Accordingly, the present inventors have made intensive efforts for the invention of a new stem cell line that can be used as a cell therapy, and as a result, invented a novel stem cell including the characteristics of endothelial cells, and confirmed the effect in its ischemic disease model. Has come to completion.

One aspect of the present invention relates to a method for producing the stem cells from the mesenchymal stem cells and stem cells comprising the characteristics of endothelial cells, derived from mesenchymal stem cells.

In addition, one aspect of the present invention relates to a pharmaceutical composition for preventing or treating ischemic disease comprising the stem cells or a culture thereof.

One aspect of the present invention provides a stem cell comprising the characteristics of endothelial cells, derived from Mesenchymal stem cells.

Hereinafter, the present invention will be described in detail.

In the present invention, the present inventors cultured mesenchymal stem cells under low oxygen partial pressure conditions, and produced a novel cell line containing both new characteristics, that is, endothelial cell characteristics and stem cell characteristics. MTSC (Mesenthelial stem cell) was named for the cell line.

Therefore, the stem cells including the characteristics of the endothelial cells of the present invention, CD 44, CD 105, CD 90 and CD 73, which are markers of stem cells, are positive, compared with CD 45, CD 144, CD 31 and mesenchymal stem cells. Characterized by an increased expression of CD 34.

The term stem cell, as used herein, is an undifferentiated cell having the ability to differentiate into various body tissues, which are totipotent stem cells, pluripotent stem cells, and multipotent stem cells. stem cell).

The term mesenchymal stem cell (MSC), as used herein, is a multipotent that has the ability to differentiate into ectoderm cells, such as bone, cartilage, fat, muscle cells, or even ectoderm cells such as neurons. It is a multipotent stem cell. The mesenchymal stem cells may be derived from those selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane, chorion, decidual membrane, and placenta. In addition, the mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans may be more preferably canine, feline, ape, animal, cow, sheep, pig, horse, rat, mouse or guinea pig, and the like, without limitation.

Stem cells comprising the characteristics of the endothelial cells of the present invention can be expressed angiogenesis factors and stem cell factors, the angiogenesis factors are AAMP (angio-associated migratory protein), bFGF (basic fibroblast growth factor) or ANG -1 (Angiopoietin-2), and the stem cell factor may be Sall4 (Sal-like protein, KLF-4 (Kruppel-likefactor, oct (octamer-binding transcription factor) or Nanog. In addition, the endothelial of the present invention) Stem cells comprising the characteristics of the cell may be expressed apoptosis factor, the apoptosis factor may be TNF-b (Tumor necrosis factor-b), BAX, BCL2 (B-cell lymphoma or P53. Or both expression of a protein.

The characteristics of the stem cells of the present invention may be improved cell proliferation, cell mobility or DNA integrity.

In addition, stem cells containing the characteristics of the endothelial cells has been deposited with the Korea Research Institute of Bioscience and Biotechnology, accession number may be KCTC 12404BP.

The stem cells of the present invention can be induced by culturing mesenchymal stem cells at low oxygen partial pressure as described above. The low oxygen partial pressure may be 0.1 to 10%. Preferably from 0.5% to 5%, more preferably 1%.

In addition, the culture may be performed without the addition of a separate growth factor.

Another aspect of the present invention provides a method for producing a stem cell comprising the characteristics of endothelial cells, comprising culturing the mesenchymal stem cells under low oxygen partial pressure conditions.

The low oxygen partial pressure may be 0.1 to 10%. Preferably from 0.5% to 5%, more preferably 1%.

In addition, the culturing can be performed without treatment of other growth factors.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating ischemic disease, comprising the stem cells or a culture thereof.

The stem cells have both endothelial and stem cell characteristics, and have high cell proliferation rate, cell migration ability, and DNA preservation ability, and thus exhibit an active neovascularization ability in ischemic diseases, and thus are effectively used for preventing or treating ischemic diseases. Can be.

The ischemic disease may be ischemic heart disease, myocardial infarction, angina, lower limb ischemic disease, extremity ischemic disease, ischemic neurosis, ischemic pulmonary disease, ischemic colitis, ischemic heart failure, obstructive arteriosclerosis or ischemic cerebrovascular disease. Ischemic cerebral disease can be, for example, thrombosis, embolism, transient ischemic attack, cerebral infarction, cerebral hemorrhage, stroke, subarachnoid hemorrhage, white matter disorder or small infarction.

When the composition is prepared as a pharmaceutical composition for preventing or treating ischemic disease, the composition may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the composition are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The pharmaceutical composition for preventing or treating the ischemic disease may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

Suitable dosages of the pharmaceutical compositions for the prevention or treatment of ischemic diseases include factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response. It can be prescribed in various ways. The preferred dosage of the composition is within the ^{100-100,000,000 (10 2 -10 8) cell} / kg range, based on an adult. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer or to prevent or treat a disease due to angiogenesis.

The composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. In addition, the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered once or additionally if necessary.

Stem cells according to the present invention have both endothelial and stem cell characteristics, and have high cell proliferation rate, cell migration capacity and DNA preservation ability, and thus exhibit an active neovascularization ability in ischemic disease, thereby preventing or treating ischemic disease. Can be used effectively.

1 is a diagram showing the cell proliferation of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia) by the number of passages.

Figure 2 is a diagram comparing the cell proliferation of MSC cultured at normal oxygen partial pressure (Normoxia) of passage number 6 and MTSC cultured at hypoxia (Hypoxia).

3 is a diagram showing the morphological characteristics of MSC cultured at normal oxygen partial pressure (Normoxia) at passage number 4 and MTSC cultured at hypoxia (Hypoxia).

4 is a diagram illustrating the cell migration capacity of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia).

5 is a diagram comparing the cell migration capacity of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia).

6 is a diagram comparing gene expression amounts of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at hypoxia (Hypoxia).

7 is a diagram comparing DNA damage of MSCs cultured at normal oxygen partial pressure (Normoxia) and MTSCs cultured at hypoxia (Hypoxia).

8 is a diagram confirming the angiogenic capacity of the MSC and MTSC in the ischemia of the lower limbs.

9 is a diagram illustrating the angiogenic capacity of MSC and MTSC in the ischemic animal model using the immunohistochemistry.

10 is a diagram comparing gene expression patterns of MSC and MTSC in the ischemia of the lower limbs.

FIG. 11 is a diagram confirming that CD31, a surface protein of endothelial cells, is expressed in MTSC cultured at hypoxia (1%) and not expressed in MSC cultured at normoxia (20%).

12 is a diagram confirming the high expression of VEGF affecting cell growth and ischemia treatment in MTSC cultured at Hypoxia (1%).

FIG. 13 shows that the differentiation experiments showed that MTSC was suppressed from adipocyte differentiation compared to MSC, but differentiation into osteoblasts was enhanced.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Culture of Mesenchymal Stem Cells (MSC) and Mesenthelial Stem Cells (MTSC)

2.5 x 10 ⁵ hUCB-MSC2 (human Umbilical Cord blood derived-MSC 2) (purchased from PromoCell (USA): order number: C12972, Lot Number: 1080102.3) in a 100 cm ² Petri dish with DMEM (Dulbecco's modified Eagle medium) Plated in Petri dishes containing low glucose with addition of 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin.

MTSC was obtained by incubating MSC under hypoxia conditions (1, 5 or 10% oxygen partial pressure, in particular 1% oxygen partial pressure and 5% CO ₂ ). Cultures were incubated in low glucose DMEM (Dulbecco's modified Eagle medium) containing low glucose / 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin. At 80% density, cells under each condition were harvested using 0.25% trypsin and replated at 2,000 cells / cm ² in the medium.

In addition, the MTSC was deposited to KRIBB (Korea Research Institute of Bioscience and Biotechnology) dated May 2, 2013, accession number KCTC 12404BP.

Example 2: Confirmation of proliferative properties of MTSC (Mesenthelial stem cell)

Cell proliferation of MSCs cultured under normal oxygen partial pressure conditions and MTSCs cultured under low oxygen partial pressure conditions was analyzed using viable cell density (cells / cm ² ).

Hypoxia conditions were the same as in Example 1, normal oxygen partial pressure (Normoxia) conditions were hUCB-MSC2 (human Umbilical Cord blood derived-MSC 2) in a Petri dish of 100 cm ² 2.0X10 ⁵ cells / 5 plates were plated for each culture period (Day-1, 3, 5, 7) at the concentration of the plate. Incubate at normal and low oxygen partial pressure, and isolate cells from Petri dishes using 0.25% trypsin for each culture period, and count the number of cells three times using a hemocytometer to determine the average cell number.

The results are shown in FIG. 1 and Table 1 and Table 2 below.

Table 1 Normoxia Day-1 Day-3 Day-5 Day-7 P-4 Normoxia 2143 4762 22321 45015 P-5 Normoxia 2143 7478 21429 43713 P-6 Normoxia 1548 2679 7440 13021 P-7 Normoxia 2500 3333 10789 26972 P-8 Normoxia 1964 5536 15253 34877 P-9 Normoxia 670 982 2500 7440

TABLE 2 Hypoxia Day-1 Day-3 Day-5 Day-7 P-4 Hypoxia 2619 7738 39435 55432 P-5 Hypoxia 2679 12277 68750 109003 P-6 Hypoxia 3571 7321 19940 89286 P-7 Hypoxia 4464 12024 38318 54315 P-8 Hypoxia 3571 8304 31994 46503 P-9 Hypoxia 2098 4196 14167 54613

As shown in FIG. 1, Table 1, and Table 2, MTSC cultured at low oxygen partial pressure of MSC showed higher cell proliferation ability than MSC regardless of the number of passages.

The results of comparing cell proliferation rates of MSC and MTSC in six passages are shown in FIG. 2 and Table 3 below, and p values are shown in Table 4.

TABLE 3 Day-1 Day-3 Day-5 Day-7 Normoxia 1548 2679 7440 13020 Hypoxia 3571 7321 19940 89285

Table 4 P value Day 1-3 Day 1-5 Day 1-7 Day 3-5 Day 3-7 Day 5-7 Normoxia 0.0044 0.000051 0.00074 0.00014 0.001 0.011 Hypoxia 0.000811 0.00023 0.0000027 0.00072 0.0000034 0.0000114 Day 1 Day 3 Day 5 Day 7 by passage 0.00105 0.000282 0.000716 0.0000074

As shown in FIG. 2, Tables 3 and 4, it can be seen that the cell proliferation rate of MTSC at 6 passages is significantly higher than that of MSC.

In addition, the cell morphology at four passages of MSC and MTSC was observed using an OLYMPUS-CKX41 microscope, and images were taken using the Lumenera corporation INFINITY ANALYZE program, and the results are shown in FIG. 3.

As shown in Figure 3, in the case of MTSC cultured at low oxygen partial pressure, the characteristics of the stem cells were more clearly shown.

Example 3 Confirmation of Migration Characteristics of MTSC

The migration of MSCs at normal oxygen pressure (NOrmoxia) and MTSC cells at hypoxia (Hypoxia) was confirmed by analyzing the migrated area using μ-dish 35mm, high culture-insert. .

Specifically, seeding of 3 × 10 ⁴ cells / 70 μl of normal oxygen partial pressure (NOrmoxia) MSC and low oxygen partial pressure (Hypoxia) MTSC medium per 35 μm in each well and incubating at 37 ° C. for 24 hours Afterwards, the culture insert was removed and 2 ml of 10% DMEM was added, followed by microscopy (n = 3). The results are shown in FIGS. 4 and 5.

As shown in FIG. 4, MTSC at low oxygen partial pressure showed significantly higher cell migration capacity than MSC at normal oxygen partial pressure.

FIG. 5 is a photograph analyzing the migration of cells, and as shown in FIG. 5, in a space from which culture-insertion is removed, an empty space without cells is calculated after a predetermined time and is normal. As a result of comparing the cell migration capacity of the cells cultured at the oxygen partial pressure and the low oxygen partial pressure, it was confirmed that the low oxygen partial pressure environmental culture cells show a high mobility.

Example 4: Confirmation of Gene Expression Characteristics of MTSC

RNAs of MSC and MTSC were extracted. Stem cell markers Sall4 (Sal-like protein 4), KLF-4 (Kruppel-likefactor4), OCT-4 (octamer-binding transcription factor4) Nanog and HIF-1 (Hypoxia-inducible factor-1), and blood vessels Angiogenic markers such as AAMP (angio-associated migratory cell protein), bFGF (basic fibroblast growth factor) and ANG-1 (Angiopoietin-1) and apoptosis markers TNF-β (Tumor necrosis factor-β), BAX (Bcl 2-associated X qRT-PCR was performed using protein), BCL2 (B-cell lymphoma 2 and P53 (protein 53 or tumor protein 53)).

Specifically, mRNA was extracted from cells cultured at normal and low oxygen partial pressure and converted to cDNA, followed by qRT-PCR. The qRT-PCR method is as follows. In each muscle tissue, modified cDNA 40ng / ul, primer 10ng, and SYBR Green were added to each muscle tissue, using a qRT-PCR machine for 1 cycle at 95 ° C for 10 seconds, at 5 ° C at 95 ° C and for 34 seconds at 60 ° C. 40 cycles, 15 seconds at 95 degrees, 60 seconds at 60 degrees and 1 cycle at 95 degrees for 15 seconds to amplify and confirm the expression, and the results were compared with the MSC cultured at normal oxygen partial pressure, This is shown in FIG. 6.

As shown in Figure 6, the expression of angiogenesis factors such as AAMP, bFGF, ANG-1 in the cells cultured at low oxygen partial pressure compared to normal oxygen partial pressure is 1.02 times, 2.04 times, 2.66 times higher, respectively, TNF-b, BAX, Apoptosis factor gene expression, such as BCL2 and P53, was also high, confirming high safety. In addition, Sall4, KLF-4, Oct, Nanog was confirmed that the high stem cell high expression amount. The expression level of HIF-1 was confirmed to increase the expression level of HIF-1 in cells cultured in hypoxia. HIF-1 (hypoxia-inducible factor-1) is a transcription factor that is expressed in a hypoxic environment. Highly expressed in hypoxia-induced cells is evidence that the cells were cultured in hypoxic state. When HIF-1 is activated, angiogenesis is activated.

Example 7: Confirmation of DNA Integrity of MTSC

DNA integrity of MSC at normal oxygen partial pressure and MTSC at low oxygen partial pressure was confirmed using p-H2AX (S139). DNA was separated from the two cells and the expression was confirmed by PCR (Polymerase Chain Reaction), and the results are shown in FIG. 7. αp-H2AX is a major indicator of DNA's integrity, and the induction of the genetic stability marker αp-H2AX (S139) means that it is not genetically stable.

As shown in FIG. 7, in normal oxygen partial pressure, αp-H2AX (S139) expression was confirmed as the passage of the cell progressed, indicating that the stability of the cell was reduced. On the other hand, in cells with low oxygen partial pressure, αp-H2AX (S139) is stably maintained even when the number of passages increases, which means that DNA damage was not confirmed despite the increase in the number of passages. High genetic stability could be confirmed.

Example 6: Confirmation of Marker Expression of MTSC

Flow cytometry was performed to confirm cellular characteristics. Cell surfaces were marked with antibodies to CD45, CD73, CD44, CD105, CD90, CD31, CD34 and CD144. Specifically, after culturing and separating MSC and MTSC, CD45, CD73, CD44, CD105, CD90, CD31, CD34, and CD144 were attached to the surface of each cell, using a flow cytometer (FACS: Fluorescence-activated cell sorting). Analyzed.

Expression patterns of the identified markers are shown in Table 5 below.

Table 5 Marker MSC MTSC CD 44 99.96% 99.93% CD 105 99.94% 99.97% CD 90 99.98% 99.94% CD 73 97.69% 99.94% CD 45 0.09% 27.58% CD 144 1.19% 26.42% CD 31 0.26% 20.95% CD 34 1.33% 39.93%

As shown in Table 5, MTSC is more positive than the MSC for the stem cell markers CD44, CD105, CD90 and CD73, and the positive rate is higher for the endothelial cell markers CD144, CD31, and CD34 than the MSC. Could confirm. In addition, it was confirmed that the positive ratio was also high in CD45. In conclusion, MTSC cultured at low oxygen partial pressure includes endothelial cell characteristics while maintaining stem cell characteristics, unlike cell characteristics of conventional MSCs, and cell proliferation ability, cell migration capacity, and DNA preservation. It was confirmed that the capacity is a novel stem cell line containing increased properties compared to MSC.

Example 6: Characterization of MTSC (Mesenthelial stem cell) in the ischemic mouse model

(1) Observation of capillaries in the ischemic mouse

Three to six passages of 1 × 10 ⁶ MSC and MTSC cells were suspended in 60 μl physiological saline and injected into the ischemic lower extremity of mice. Mice were purchased from Orient Bio Co., Ltd., and strain was induced using the 6-week-old Balb-C / nude limb ischemia model. The method of inducing the lower limb ischemia was a method of tying and cutting the lower limb artery of the mouse. Both cells were implanted intramuscularly into the lower limb muscle (femoral artery ligation site) immediately after induction of the ischemia. Four weeks after injecting the cells, the lower extremity muscle was harvested and the angiogenic effect was confirmed.

The results of confirming the capillary count are shown in FIG. 8.

As shown in FIG. 8, the MTSC-implanted group showed significantly higher neovascularization ability than the ischemic model and the MSC-implanted group.

In addition, the neovascularization ability of MTSC was confirmed through immunohistochemistry (IHC). Specifically, the harvested lower limb muscle tissue was made of paraffin blocks, and then sectioned to 4 μm and hardened on a slide. The slide was labeled with a CD31 or vWF marker that specifically binds to vascular endothelial cells to confirm the count in muscle tissue. The results are shown in FIG.

As shown in FIG. 9, it was visually confirmed that the group transplanted with MTSC exhibited significantly higher neovascularization ability as compared to the ischemic model and the group transplanted with MSC.

(2) Confirmation of gene expression in lower limb ischemic mouse

After injection of MSC and MTSC in the ischemic mouse model, gene expression was confirmed using qRT-PCR (Quantitative Rean-Time Polymerase Chain Reaction). Specifically, mRNA was isolated from muscle tissue harvested 4 weeks after cell treatment, and mixed with CD31, Ang-1 and PIGF primers to perform qRT-PCR. The test method is as follows. In each muscle tissue, modified cDNA 40ng / ul, primer 10ng, and SYBR Green were added to each muscle tissue for 1 cycle at 95 degrees for 10 seconds, 5 seconds at 95 degrees, and 34 seconds at 60 degrees using a qRT-PCR machine. 40 cycles and 15 seconds at 95 degrees, 60 seconds at 60 degrees, and 1 cycle at 95 degrees for 15 seconds were amplified and confirmed. The results are shown in FIG.

As shown in FIG. 10, it was confirmed that the expression levels of angiogenic factors such as CD31, ANG-1, PIGF, AAMP, and PFG were significantly higher in the MTSC-treated group than in the MSC-treated group.

Example 7: Confirmation of expression of CD31, an endothelial cell marker in MTSC (Mesenthelial stem cell)

In order to confirm the expression of CD31, a surface protein of endothelial cells in MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%), the experiment was carried out by the following method.

Seed 1.2 x 10 ⁴ / well cells for 4 days at normal oxygen partial pressure (20%) and low oxygen partial pressure (1%). After 4 days, the medium was washed with PBS and the cells were fixed for 10 minutes with 4% formalin. The fixed cells were washed with PBS and the cell membranes were destroyed with PBS containing Triton X-100. After blocking for 30 minutes with 1% BSA, deodorized with CD31 antibody attached with Texas-Red for 1 hour, washed with PBS, and observed with fluorescence microscope with DAPI. The results are shown in FIG.

As shown in FIG. 11, it was confirmed that CD31, which is used as a marker of endothelial cells, was expressed in MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%), but MSC cultured at normal oxygen partial pressure (20%) Expression of CD31 was not confirmed at all.

Example 8 Confirmation of Expression of VEGF in MTSC (Mesenthelial Stem Cell)

In order to determine whether high expression of VEGF affects cell growth and ischemic treatment in MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%), the experiment was conducted.

1.2 x 10 ⁴ / well cells were seeded and cultured at normal oxygen partial pressure (20%) and hypoxic partial pressure (1%) for 4 days, respectively. After 4 days, the medium was washed with PBS and the cells were fixed for 10 minutes with 4% formalin. The fixed cells were washed with PBS and the cell membranes were destroyed with PBS containing Triton X-100. After blocking for 30 minutes with 1% BSA, VEGF antibody attached with Texas-Red was deodorized for 1 hour, washed with PBS, and observed with a fluorescence microscope with DAPI. The results are shown in FIG.

As shown in FIG. 12, MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%) was found to express abundantly VEGF, but MSC cultured at normal oxygen partial pressure (20%) had very low amount of VEGF. It was confirmed that the expression.

Example 9 Confirmation of Differentiation Characteristics of MTSC (Mesenthelial Stem Cell)

In order to compare the differentiation ability of MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%) and MSC cultured at normal oxygen partial pressure (20%), the experiment was performed by the following method.

After seeding 2 x 10 ⁴ / well cells, the cells were incubated at normal oxygen partial pressure (20%) and hypoxic partial pressure (1%) for 4 days, respectively, and then on day 4, Adipogenesis differentiation medium (StemPro Adipogenesis Differnetiation Kit-Gibco) and Osteogenesis differentialtion medium Differentiation was induced at each oxygen partial pressure using (StemPo Osteogenesis Differentiation Kit-GIBO).

Adipogenesis was stained at 14 days and Osteogenesis at 21 days after induction of differentiation. Staining proceeded as follows.

In the case of Adipogenesis staining, the medium was removed, washed with PBS, and the cells were fixed for 30 minutes with 10% formalin. After 5 minutes of 60% isopropanol, Oil Red O was added and allowed to stand for 5 minutes. Oil Red O was washed and counted with Hematoxylin and observed under a microscope.

In the case of oesteogenesis staining, the medium was removed, washed with PBS, the cells were fixed with 10% formalin for 30 minutes, and treated with 2% Alzarin Red S for 5 minutes, washed and observed under a microscope.

The results are shown in FIG.

As shown in FIG. 13, in the MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%), differentiation into adipocytes was suppressed, but differentiation into osteocytes was increased than MSC cultured at normal oxygen partial pressure (20%). It was confirmed.

Claims (17)

Stem cells comprising the characteristics of endothelial cells, derived from mesenchymal stem cells. The method according to claim 1, wherein the stem cells are CD 44 ⁺ , CD 105 ⁺ , CD 90 ⁺ and CD 73 ⁺ ; Stem cells, characterized in that the expression of CD 45, CD 144, CD 31 and CD 34 increased compared to the mesenchymal stem cells. The stem cell of claim 1, wherein the stem cell is expressed by angiogenesis factor and stem cell factor. The stem cell of claim 3, wherein the angiogenesis factor is selected from the group consisting of an anio-associated migratory protein (AAMP), a basic fibroblast growth factor (bFGF), and angiopoietin-1 (ANG-1). The method according to claim 3, wherein the stem cell factor is selected from the group consisting of Sall4 (Sal-like protein 4), KLF-4 (Kruppel-like factor 4), OCT-4 (octamer-binding transcription factor-4), and Nanog Characterized in stem cells. The stem cell of claim 1, wherein the stem cell is expressed by an apoptosis factor. The method according to claim 6, wherein the apoptosis factor is selected from the group consisting of Tumor necrosis factor-b (TNF-b), Bcl 2-associated X protein (BAX), B-cell lymphoma and P53 (protein 53). Stem cells. The stem cell of claim 1, wherein the stem cell has a feature selected from an improvement of cell proliferation ability, an improvement of cell mobility, and an improvement of DNA integrity. The stem cell of claim 1, wherein the stem cells are induced by culturing mesenchymal stem cells at low oxygen partial pressure. The method of claim 9, wherein the hypoxic partial pressure is characterized in that 0.1 to 10%, stem cells. The stem cell of claim 1, wherein the mesenchymal stem cells are derived from one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic membrane, decidual membrane, and placenta. The stem cell of claim 1, wherein the stem cell is accession number KCTC 12404BP. Method for producing a stem cell comprising the characteristics of endothelial cells, comprising the step of culturing the mesenchymal stem cells under low oxygen partial pressure conditions. The method of claim 13, wherein the hypoxic partial pressure is 1 to 10%. 13. A pharmaceutical composition for preventing or treating ischemic disease, comprising the stem cell of claim 1 or a culture thereof. The ischemic disease, ischemic heart disease, myocardial infarction, angina pectoris, lower limb ischemic disease, extremity ischemic disease, ischemic neurosis, ischemic pulmonary disease, ischemic colitis, ischemic heart failure, obstructive atherosclerosis and ischemic cerebrovascular disease The pharmaceutical composition for the prevention or treatment of ischemic diseases, characterized in that selected from the group consisting of. The method of claim 16, wherein the ischemic brain disease is selected from the group consisting of thrombosis, embolism, transient ischemic attack, cerebral infarction, cerebral hemorrhage, stroke, subarachnoid hemorrhage, white matter dystrophy and small infarction. Pharmaceutical composition for.

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