KR20090126227A - A method for preparing blood vessel stem cells from women labia minora skin cells by reprogramming method, and use of reprogrammed blood vessel endothelial cells as a cell therapy product - Google Patents

Abstract

PURPOSE: A method for differentiating labium minor-derived adult stem cells to vascular endothelial precursor cells is provided to use for ischemic vascular diseases and other cardiovascular diseases. CONSTITUTION: A method for differentiating labium minor-derived adult stem cells to vascular endothelial precursor cells comprises: a step of culturing labium minor-derived adult stem cells in a medium containing low glucose, DMEM, FBS(fetal bovine serum), L-glutamine and penicillin-streptomycin to obtain pluripotent adult stem cells; and a step of treating the adult stem cells with VEGF in MAPC medium. The MAPC medium contains DMEM, MCDB, insulin, apo-tranferrin, selenium, dexametasone, ascorbic acid. A composition for treating ischemic vascular diseases contains vascular endothelial precursor cells as active ingredient.

Description

Method of preparing vascular stem cells capable of differentiating into vascular cells through reprogramming from adult labia stem-derived adult cells and use as a cell therapeutic agent of the differentiated cells from women labia minora skin cells by reprogramming method, and use of reprogrammed blood vessel endothelial cells as a cell therapy product}

The present invention provides a multi-differentiated adult cell obtained by culturing in low glucose after separating the multi-differentiated adult cells from female labia minora among the easily detachable human skin cells, in vitro culture conditions required for reprogramming using the same. The present invention relates to a method for dramatically increasing the conversion to vascular endothelial progenitor cells, and to the use of vascular endothelial progenitor cells generated through the reprogramming process, for ischemic vascular disease and other cardiovascular treatments.

Stem cells are cells that have the capacity to divide to an infinite extent and to differentiate through suitable stimuli under suitable circumstances to form other types of cells. Stem cells have the potential to develop into cells with characteristic shapes and specialized functions, and many studies have been conducted as effective treatments for various diseases.

So far, there has been a distinction between embryonic stem cells and adult stem cells, based on differences in the potential of stem cells to differentiate into other types of cells. It is a general consensus that stem cell potential decreases as embryos develop from embryonic cells to adult organisms. According to this property, fertilized egg cells are called pluripotent, embryonic stem cells are called pluripotent, and adult stem cells are called multipotent.

Pluripotent stem cells are cells that can develop into complete organisms. Pluripotent cells include early embryonic stage cells with fertilized egg cells. Pluripotent capacity refers to stem cells that are typically capable of differentiating embryonic stem cells obtained from the intermal cell mass of divided blastocysts into all three germ cells of mesoderm, endoderm and ectoderm.

However, according to the known views, adult stem cells are known to be only multipotent and capable of differentiating to a small extent. Thus, tissue-specific stem cells can only differentiate into cells of the same tissue type. Adult stem cells are known as the central nervous system ( Science 255, 1707-1710 1992; Science 287 , 1433-1438 2000), bone marrow ( Science 276 , 71-74, 1997; Science 287 , 1442-1446, 2000; Science 284 , 143-147, 1999), retina ( Science 287 , 2032-2036, 2000) and skeletal muscle ( Proc . Natl Acad . Sci . USA 96 , 14482-14486, 1999; Nature 401 , 390-394, 1999), which can each differentiate into similar lines. For example, neural stem cells can differentiate into neurons and glial cells, and bone marrow mesenchymal stem cells can differentiate into mesodermal cells and hematopoietic stem cells ( Hematopoietic stem cells are capable of differentiating into blood-related cells. Specifically, for example, the outside of the cornea in the blind treatment epidermal stem cells (epithelial stem cells) (Cell 57, 201-209, 1989) are emerging as new methods of corneal transplantation ( N. Engl . J. Med . 343, 86-93, 2000). And hematopoietic stem cells can be used for treatment through autograft ( Blood Cells 20, 15-24, 1994).

However, in recent years, adult stem cells may be differentiated not only from the same lineage but also from other types, so that adult stem cells may be differentiated more than previously known. Studies using adult stem cells obtained from bone marrow tissue (WO 02/064748) or regenerated tissue (WO 02/057430) have led to transplantation into target tissue, culture on the feeder cell layer and / or the presence of specific growth factors. Under the same conditions as under culture, it was demonstrated that differentiation of each stem cell could also occur to form cell types of different germ layers. Therefore, the view that adult stem cells can only differentiate into the same lineage had to be corrected. For example, neural stem cells are in blood in vivo (Science 283 , 534-537, 1999) and skeletal muscle cells ( Nature Neurosci . 3 , 986-991, 2000), and transplanted bone marrow mesenchymal stem cells are known as skeletal muscle cells ( Nature ). 401 , 390-394, 1999; Science 279 , 1528-1530, 1998) and liver cells ( Science 284 , 1168-1170, 1999), and when transplanted into the brain, neuronal markers were expressed ( Science 290 , 1779-1782, 2000; Science ; 290 , 1775-1779, 2000).

However, there are many difficulties in separating these stem cells. For example, the separation of the central nervous system, bone marrow, retina, and skeletal muscle cells from the human body involves a lot of difficulties and pain. Especially in the case of the central nervous system (central nervous system) is virtually impossible because of the high risk of separation.

In addition, even when adult stem cells are obtained, it is not sufficient to carry out cell therapy using adult stem cells. For the first reason, cell therapy requires a large number of cells, because the methods presented so far cannot meet the cell count for them. The second reason is economical, and current research suggests that a lot of materials (such as cytokine in the medium) are needed for cell therapy. The materials added to the cell culture are economically inefficient because most of them are expensive materials.

Therefore, the present inventors propose a method that can be used for cell therapy by proliferating a large amount of cells having stem cell characteristics to overcome such problems, and after increasing the number of cells using fewer materials, The purpose of this study is to suggest a way to treat cells more economically.

To this end, the present inventors focused on the labia minora among the most accessible and large area of the organs constituting the human body. Although there are various places where adult stem cells can be separated, it has recently been reported that adult stem cells can be separated from skin. Freda D, Miller, who first isolated adult stem cells with pluripotency from the skin, isolated stem cells using rodents. The glue neurons and glial stem cells (glial cell), smooth muscle cells (smooth muscle cell), was a spontaneous eruption happened to the fat cell (adipocyte), confirm the existence of stem cells in the dermis of the skin (Nature cell was biology 3, 778-784, 2001), demonstrated that adult stem cells present in the skin of rodents that are present in the dermal papilla in the hair follicle (Nature cell biology 6, 1082-1093, 2004). In 2004, Siddharthan Chandran isolated adult stem cells from human skin and differentiated them into neurons, astrocytes and smooth muscle cells. If confirmed the functional imaging of calcium (calcium imaging) fat cells (astrocyte) differentiated through analysis (the Lancet 364 , 172-178, 2004). Adult stem cells were isolated from epidermal tissue following human circumcision and differentiated into neurons, glial cells and smooth muscle cells to confirm the presence of adult stem cells in human skin tissue ( Stem). Cells 23 , 727-737, 2005).

However, a method of extracting adult stem cells from the labia minora and culturing them into adult stem cells having multipotent capacity has not been disclosed. In addition, there have been no studies on the clinical applicability of cell therapeutics that can be mass-produced by inducing differentiation into vascular endothelial progenitor cells using stem cells derived from labia minora which can be easily mass-produced in vitro.

In the meantime, vascular endothelial progenitor cells ( Br J Haematol) are thought to have high therapeutic potential in the treatment of vascular diseases. 115, 186-194, 2001), peripheral blood ( Science 275, 965-967, 1997; Blood 95, 952-958, 2000), umbilical cord blood ( Blood 95, 952-958, 2000; J Clin Invest 105, 1527-1536, 2000). And through numerous animal experiments, the vascular endothelial progenitor cells were found to have peripheral vascular disease ( Blood 105, 1068-1077, 2004; Circ). Res 85, 221-228; 1999; Proc Natl Acad Sci USA 97, 3422-3427, 2000; J Clin Invest 106, 571-578, 2000), coronary artery disease (Circ Res 85, 221-228, 1999; Circ Res 90, e89-93; 2002; Circulation 107, 461-468, 2003), cerebrovascular diseases ( Stroke 33, 1362-1368, 2002; Circ Res 90, 284-288, 2002). However, it is difficult to obtain an adequate number of cells to be used for cell transplantation and treatment, the repeatability of separation is limited, and the production of isolated vascular endothelial progenitor cells in vitro has been limited. Because of this, there has been a continuous search for an alternative source of vascular endothelial progenitor cells that can be an effective cell therapy material for the treatment of vascular diseases.

The present inventors have studied in view of the above, as a result, obtained adult cells that can be easily cultured in vitro while multiplying from the labia minora of human skin cells that can be easily separated, and derived from the labia minora which can be mass-produced in vitro Adult cells were briefly grown in high glucose under low glucose DMEM medium culture conditions and then proliferated in large quantities to induce differentiation into vascular endothelial cells. When differentiation was induced under known conditions, differentiated cells were formed into vascular endothelial cells. However, this method has limitations due to difficulty in culturing adult cells in low glucose conditions for a long time. Therefore, the present invention was to provide a method that can more efficiently differentiate adult cells separated from the labia minora into vascular endothelial cells through a process called reprogramming. After inducing differentiation into vascular endothelial progenitor cells in low glucose under EGM-2 medium conditions, differentiation into vascular endothelial cells from in vitro and in vivo was induced. The present invention was completed by confirming the effect of treating the ischemic disease of the vascular endothelial progenitor cells.

An object of the present invention is to provide a method for inducing differentiation of adult stem cells derived from labia minora into vascular endothelial progenitor cells.

It is another object of the present invention to provide a method of significantly improving the acquisition of vascular endothelial cells by inducing differentiation from ectoderm progenitor cells to vascular endothelial progenitor cells through reprogramming of adult labia minora derived adult cells. .

In addition, another object of the present invention is to provide a composition for treating ischemic vascular diseases and other cardiovascular systems including the vascular endothelial progenitor cells differentiated as described above.

In order to achieve the above object, the first aspect of the present invention provides a method for separating multipotent adult stem cells from the labia minora of a woman.

Since women's labia minora tissue has the same origin as the epidermis after men's circumcision, it is possible to treat cells as well as males by identifying stem cells from the adult labia minora and separating adult stem cells. It became. In addition, in the case of labia minora, since the composition ratio of fibers and the like present in the tissue is small, a large number of cells can be obtained compared to the amount of tissue.

In the present invention, the adult stem cell derived from the labia minora of the female, to obtain the dermal tissue by separating the labia minora tissue of the woman, after separating the cells from the dermal tissue, the isolated cells in low glucose (MEM) glucose DMEM medium It can be obtained by culturing.

That is, by separating the labia minora tissue of the woman to obtain the dermis, scraping keratinocytes from the dermis with a blade, and then separating the cells from the dermal tissue obtained by the above method and incubated in low glucose DMEM medium 1 Adult stem cells derived from the labia minora can be obtained.

In a preferred embodiment of the present invention, the low glucose DMEM medium may further comprise 5-15% FBS, preferably 10% FBS.

In one preferred embodiment of the present invention, the low glucose DMEM medium further comprises 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin solution. can do.

In the present invention, the low glucose DMEM medium is characterized in that it is possible to maintain the differentiation capacity of the adult stem cells while culturing the adult stem cells.

In a second aspect of the present invention there is provided an adult stem cell obtained by the above method and exhibiting the following characteristics:

(1) all show positive immunological properties for CD13, CD29, CD44 and CD90;

(2) express dermo-1 but not SHOX-2 gene;

(3) attached to and grown on cell culture dishes, exhibiting linear morphological characteristics; And

(4) Has the ability to differentiate into mesodermal derived cells.

The third aspect of the present invention provides a method for inducing differentiation of adult stem cells derived from labia minora into female vascular endothelial progenitor cells.

In a preferred embodiment of the present invention, a method for inducing differentiation of adult labia minora derived adult cells into vascular endothelial progenitor cells is possible through the following two methods.

First, the method of inducing differentiation of labia minora derived adult cells into vascular endothelial progenitor cells of the present invention may comprise the following steps:

Culturing adult cells isolated from the labia minora in culture conditions consisting of low glucose DMEM, 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin; And

Culturing the adult cells cultured in the above step while treating VEGF with 10 ng / ml in MAPC medium.

Secondly, the method of inducing differentiation of adult labia minora derived adult cells into vascular endothelial progenitor cells of the present invention may comprise the following steps:

Culturing adult cells isolated from the labia minora in culture conditions consisting of low glucose DMEM, 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin; And

Adult cells cultured in the low glucose, EBM-2 base medium, 2% FBS, 0.04% hydrocortisone, 0.4% bFGF, 0.1% VEGF, 0.1% IGF-1, 0.1% Vitamin C, 0.1% EGF, 0.1% Further incubating in EGM-2 medium culture conditions consisting of GA-1000 and 0.1% heparin.

In the present invention, the MAPC medium is 60% DMEM, 40% MCDB, 5 μg / ml insulin (Insulin), 5 μg / ml apo-transferrin, 5 ng / ml selenium, 10 ^-8 It may include M dexamethasone (dexametasone), 10 ^-7 M, ascorbic acid (ascorbic acid).

Cells cultured in the above culture conditions may be differentiated into vascular endothelial cells in a plate coated with a Matrigel.

Conventionally, vascular endothelial progenitor cells for the treatment of ischemic vascular disease and cardiovascular disease are separated from bone marrow, peripheral blood, and umbilical cord blood, but the number of cells that can be separated from each source tissue is low, and the efficiency and repetition of separation is low. It was hard to get enough numbers to use for cell therapy.

In the present invention, however, adult cells isolated from the labia minora were cultured under medium conditions consisting of low glucose DMEM, 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, without subdividing pluripotency for a considerable time. Through a large amount of cells required for cell therapy was obtained.

The multipotent adult cells obtained from the labia minora have the characteristics of mesenchymal stem cells, and thus can be differentiated into organs of other lines not the same line. Mesenchymal stem cells are primitive cells that can differentiate into bone, cartilage, fat, nerves, muscles, etc. Specifically, retinal cells, bones that make bones, which have the ability to respond to light and transmit these reactions to the brain. Cells, osteoclasts that absorb bone, or chondrocytes that produce cartilage, myocytes, endothelial cells, epithelial cells, heart, lungs, liver, kidneys or pancreatic cells can be differentiated into various organs. According to the present invention, the cells are separated by small surgery, such as the removal of dermal tissue of labia minora, to obtain cells with the properties of a large number of mesenchymal stem cells with only a few tissues. It becomes possible to use.

It is shown that the immunological phenotype of adult cells derived from the labia minora of FIG. 4 is positive for CD13, CD29, CD44, and CD90 similarly to mesenchymal stem cells. 3 shows that stem cells derived from labia minora actually have differentiation ability to bone, fat, and chondrocytes like mesenchymal stem cells.

In the medium consisting of low glucose DMEM, 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, the cells proliferated while maintaining multipotency and maintaining pluripotency were similar to mesenchymal stem cells. The medium was changed to induce differentiation of the furnace. In other words, when the adult cells were cultured by coating Fibronectin and adding VEGF to MAPC medium, differentiation into vascular endothelial cells was induced (FIGS. 5D and 5E). In addition, another method, reprogramming, adds FBS, hydrocortisone, bFGF, VEGF, IGF-1, vitamin C, EGF, GA-1000, and heparin to cells cultured in low glucose. Differentiation into cells having phenotype and function of vascular endothelial cells was induced in EGM-2 medium culture conditions (FIG. 6 and FIG. 7).

In a fourth aspect of the present invention, there is provided a cell therapeutic agent for treating vascular disease of vascular endothelial progenitor cells obtained by differentiating stem cells derived from labia minora in women.

In the present invention, the vascular disease includes ischemic vascular disease and cardiovascular disease.

In the present invention, specific ischemic vascular diseases that can be treated with vascular endothelial progenitor cells include ischemic muscle necrosis, arrhythmia, angina pectoris, and stroke.

In the present invention, specific cardiovascular diseases treatable with vascular endothelial progenitor cells include atherosclerosis, ischemic reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, ventricular tuning, percutaneous microembolism, tachycardia, bradycardia , Pressure overload, aortic bending, coronary artery ligation, arrhythmia, stroke, angina pectoris, myocardial infarction, heart failure, hypertension.

Vascular endothelial progenitor cells differentiated from stem cells derived from the labia minora of the present invention have been found to prevent necrosis of chronic or acute ischemic leg muscles through neovascularization.

Furthermore, in the present invention, blood and endothelial progenitor cells differentiated from stem cells derived from labia minora may help normal function of myocardium after acute infarction, and in addition, angiogenesis after implantation of stents, artificial organs or other implanted medical devices. This may help to improve the efficiency of the transplant. In addition, the restoration and regeneration of blood vessels may help in the fundamental treatment of other cardiovascular diseases.

Blood and endothelial progenitor cells differentiated from the labia minora adult cells of the present invention may be present in a pharmaceutical composition comprising the blood and endothelial progenitor cells for treatment. Such pharmaceutical compositions may further comprise a physiologically acceptable matrix or a physiologically acceptable excipient in the cell. The type of matrix and / or excipient will depend on others depending on the intended route of administration. The pharmaceutical composition may also optionally include other suitable excipients or active ingredients used together in the treatment of stem cells.

When using stem cells as a therapeutic, the appropriate dosage is 10 ⁴ to 10 ⁶ cells / time per dose.

The present invention overcomes the limitations of conventional ischemic vascular diseases and other cardiovascular treatments by obtaining adult stem cells that can be easily cultured in vitro and differentiated into vascular endothelial progenitor cells, while having multipotency from female labia majora as easily detachable human skin cells. It is possible to provide a new stem cell that can be applied as a cell therapy. Peripheral endothelial progenitor cells derived from peripheral blood, bone marrow, and umbilical cord blood, which have been proven effective in animal experiments, are difficult to obtain as many cells as can be used for cell therapy, have low repeatability, and are difficult to mass produce in vitro. Adult stem cells can be easily mass-produced in vitro and can be differentiated into vascular endothelial progenitor cells, which can be the source cells of cell therapies that can be mass-produced. . In addition, since they use their own cells, there are no side effects such as an immune rejection reaction, which may be an effective cell therapy product.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1 Establishment of Reprogramming Conditions for Induction into Vascular Endothelial Progenitor Cells Using Asymptomatic Adult Cells

As a method for inducing differentiation into vascular endothelial cells using adult cells separated from the labia minora, two methods, path I and path II, were used as shown in FIG. 1. First, cells isolated from the labia minora were cultured under low glucose conditions. Such conditions were named condition I. Therefore, Condition I means medium condition containing 10% FBS and 1% penicillin / streptomycin in low glucose DMEM. The first pathway is a representative method of differentiating mesenchymal stem cells in general, and induces differentiation by giving differentiation conditions to cells cultured in condition I. Here, basically, the results of differentiation were induced into adipocytes, chondrocytes, and the like, and differentiation was induced by giving differentiation conditions to vascular endothelial cells. Path I differentiated in this way can be called. The present inventors proposed another method of pathway II. Adult cells isolated from the labia minora induced differentiation into vascular endothelial progenitor cells under EGM-2 medium and confirmed whether they have the ability of blood vessel formation in vitro and in vivo. Pathology II was induced by differentiating vascular cells from condition I to II using EGM-2 medium as condition II. This method means that the adult cells are separated into vascular cells through reprogramming.

Example  2: derived from labia minora Adult cell  detach

The adult labia was obtained from female clinic and obtained labia minoris tissue of patients in the late 40s who underwent pupla and vaginal contraction.

First, sterilized by soaking in 70% EtOH for about 30 seconds to prevent contamination from bacteria, and then washed five times with PBS containing antibiotics. The coagulated blood was removed and the labia minora tissue was cut into 1 cm 2 tall. The tissues were incubated overnight at 4 ° C. in 5 unit / ml of dispase solution. The epidermis was removed using forceps and scraped off with sterile blades to remove keratinocytes and glands present between the epidermis and the dermis. Dermal tissue 1 mm ² Cut to size and incubated for 30 minutes to 1 hour in 5% collagenase (collagenase IV) (Gibco, USA). After incubation, the samples were centrifuged to remove dispase and then suspended in media. The cells were separated from the tissues by pipetting carefully using a pipette. Tissue and single cells were separated using a 70 μm cell strainer (BD sciences, UK). The isolated cells were collected by centrifugation and seeded in 100 mm cell culture dishes by resuspending in basal medium consisting of 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin in low glucose DMEM. . After 24 hours the medium was changed. After 24 hours, most of the cells were attached to the cell culture dish, and the appearance had the form of labia minor dermis, and had the appearance of cells as shown in FIG. 2. The medium was changed every 2 days and passaged at 90% cell density.

Example  3: karyotyping of cells isolated from labia minora

Stem cells derived from labia minora were cultured in large quantities through low glucose DMEM, and then blocked for 1 hour using 0.1 g / ml colsemid in the metaphase step for karyotyping. After trypsin treatment, the cells were resuspended in a hypotonic KCl solution and incubated at 37 ° C for 20 minutes in a 3: 1 solution of methanol: acetic acid. Visualization was performed by inning (G-band staining) (FIG. 3).

Example  4: derived from the labia minora Adult cell Mesenchyme  Confirm similarity with stem cells

The labia minora-derived adult cells have the characteristics of mesenchymal stem cells, and have been found to have a multipotent ability to differentiate into organs of other lines rather than the same line.

First, after analyzing the immunological phenotype through flow cytometry, it was confirmed that adult cells derived from labia minora were expressed with CD13, CD29, CD44, and CD90 like mesenchymal stem cells (FIG. 4). Expression of representative markers was confirmed by RT-PCR by comparing with mesenchymal stem cells. In addition, expression of representative markers was confirmed using immunofluorescence staining. Since the surface expression is not specific for mesenchymal stem cells, the differentiation ability to bone, fat, and cartilage, which are representative characteristics of mesenchymal stem cells, was compared. It was confirmed that RT-PCR and staining specific to bone, fat, and cartilage were similar to mesenchymal-derived adult cells having differentiation ability to bone, fat, and cartilage like mesenchymal stem cells (Figs. 5A-C). Thus, it was confirmed that the cells isolated in the present invention are cells having similar characteristics to the mesenchymal stem cells.

Example 5 of adult cells derived from labia minora pathway Induction of Differentiation into Vascular Endothelial Progenitor Cells Using the II Method

Cells seeded in 100 mm cell culture dishes by resuspending the cells cultured in condition I, a medium consisting of 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin in low glucose DMEM After growing, the cells were stabilized and grown to 90% or more, followed by subculture and cultured in vitro with as many cells as desired. Then, after 70% to 80% of the cells are filled in the culture vessel, EGM-2 medium containing FBS, hydrocortisone, bFGF, VEGF, IGF-1, vitamin C, EGF, GA-1000, and heparin added to the EBM-2 base medium. The medium was replaced with. The badge was changed every two days. Occasionally, the cell growth was observed and passaged when cells were 90% or more of the culture vessel for continued proliferation.

Check whether the adult cell derived from the labia minoris differentiated for more than a week has been induced to differentiate into vascular endothelial cells. First, the shape of the cell changes to a pebble-like shape (Fig. 6A), and RNA is extracted from the cells at this time, and the method of RT-PCR Expression of CD31 (PECAM), CD34, VE-Cadherin, vWF, eNOS, which are markers specific for vascular endothelial progenitor cells, was confirmed at mRNA level (Fig. 6C). In addition, the cells at this time were confirmed using the FACS analysis method to confirm whether the expression of CD31 and CD34. Finally, it was confirmed at the protein level that the proteins of CD31 (PECAM) and vWF were expressed on the cell surface and cytoplasm through immunofluorescence staining (Fig. 6D).

In addition, it was confirmed that adult stem cells derived from the labia minoris were differentiated under medium culture conditions to induce vascular endothelial progenitor cells, thereby actually forming network structures similar to blood vessels (FIG. 7). First, the 4-well cell culture dish was coated with matrigel (matrigel, 8 mg / ml), and then 0.5 ml of 1 × 10 ⁵ cells / ml of cell suspension was seeded onto the plate. After 12 h and 24 h, they were observed to have a network structure similar to that of blood vessels. As a result, it was found that the cells (b) differentiated in condition II had about 100 times the number of complete tubule structures compared to the cells (a) that did not differentiate (FIGS. 7A and 7B). .

Absorption of Dil-AC-LDL, a representative function of vascular endothelial cells, was confirmed by fluorescence microscopy observation (FIG. 7C). FIG. 7C shows that cells (b, c) absorbed the cells when they were cultured for about 6 hours in a cell differentiated for 1 week in condition II at 10 μg / ml with a fluorescent LDL analog of Dil-AC-LDL. Is showing.

Example  6: confirm the ischemic disease treatment effect of the vascular endothelial progenitor cells of the present invention

In Example 5, the cells obtained by the differentiation in the culture medium for inducing vascular endothelial progenitor cells were injected into a disease animal model with ischemic limbs to form blood vessels through immunological fluorescent staining using human cell specific antibodies. It was confirmed (FIG. 8). In addition, by confirming that muscle necrosis can be prevented through the formation of blood vessels, vascular endothelial progenitor cells differentiated from the labia minora-derived adult cells of the present invention have an effective and direct therapeutic effect on ischemic disease (FIG. 8). . As shown in FIG. 8A, the injection rate in condition I and II shows that foot necrosis rate or limb loss rate is significantly lower in condition II than condition I. In addition, it was confirmed that the cells that induced differentiation by the pathway II method play a function of blood vessel formation in vivo.

For animal testing, 200-250 g of Sprague-Dawley rats were injected subcutaneously with ketamine and xylazine (80 mg / kg + 10 mg / kg). , SC) Anesthesia was produced by femoral aortic dissection after anesthesia. The branch of the skin from the external iliac artery to the femoral artery and the branch from the femoral aorta to the saphenous artery and popliteal artery after incision of the skin for femoral aortic incision Was ligated using a 5-0 silk thread and then removed by incision between the ligation sites. Cyclosporin-A (20 mg / kg) was injected daily into the abdominal cavity for postoperative immunosuppression. Three hours after surgery, the rats were randomly divided into two groups, 1 × 10 ⁶ differentiated cells were suspended in 50 μl of serum-free EGM-2 medium (condition II), and the femoral aortic dissection was performed. The injection was divided into several places. As a control, PBS or undifferentiated cells (cells cultured in condition I) were injected into the leg muscles subjected to femoral aortic dissection.

After 4 weeks, cryosections of the muscles of the hind limbs were made, and immunological fluorescent tissue staining and H & E staining were used to determine whether blood vessels formed and the necrosis of the leg muscles. The frozen section photograph of A of FIG. 10A, which was injected with undifferentiated cells as a control, shows that the necrosis of the leg muscles occurred due to the inability to move the cells and the formation of blood vessels. However, the frozen section photograph of B of FIG. 8, injected with the cells differentiated in condition II, shows that the blood vessels form and thereby prevent the necrosis of surrounding muscles, thereby showing a therapeutic effect. In addition, as a result of the immunofluorescence staining method, it was confirmed that the representative markers, CD31 and vWF are expressed (Fig. 8C).

Figure 1 shows briefly how to induce differentiation of adult cells isolated from labia minora into vascular endothelial cells. pathway I represents a pathway, or method, that induces differentiation by culturing adult cells in a culture condition containing VEGF in MAPC medium. In pathway II, reprogramming is induced through condition II in pathway II. The method of differentiating into cells is shown. Condition II is EGM-2 medium. EGM-2 medium is hydrocortisone, GA-1000, 2% FBS, 0.4% bFGF, 0.1% VEGF, 0.1% IGF, 0.1% vitamine C, 0.1% EGF in EBM-2 basal medium. It includes. Through this process, adult cells isolated from labia minora are induced to differentiate into vascular endothelial cells through reprogramming process.

Fig. 2 shows the results of observing the shape of the cells separated from the labia minor under a microscope. a is 40 times, b is 100 times, c is 200 times the result of observation, and it was confirmed that the isolate | separated cell has the same shape as a fibroblast.

Figure 3 shows the results confirming that the cells isolated in the labia minora maintained a normal karyotype.

Figure 4A shows the results of analyzing the immunological phenotype of cells isolated from labia minora by flow cytometry. 4B shows the results of confirming that the mesenchymal cells separated from the bone marrow as a control, and the representative marker whether the cells isolated from the labia minora have the same characteristics as the mesenchymal stem cells. In FIG. 4B, hLMDCs represent adult cells isolated from labia minora, and hMSCs represent cells of a control group. 4C shows the results of confirming whether markers are expressed at the protein level by immunofluorescence staining.

AC of FIG. 5 shows that adult stem cells of the present invention isolated from labia minora have differentiation capacity similar to that of mesenchymal stem cells with multipotency, and differentiate into bone (A), fat (B), and chondrocyte (C) It shows the result confirmed through. On the left side, markers expressed when differentiated into bone, adipose, and chondrocytes were identified by RT-PCR, where-marks indicate undifferentiated cells. The right side shows the result of confirming the differentiation using the appropriate stain method.

FIG. 6 is an experimental result of induction into vascular endothelial progenitor cells using the reprogramming method mentioned in FIG. 1, and adult cells separated from the labia minora are changed into a morphological form similar to vascular endothelial progenitor cells under the condition II. To show that. At this time, the left picture of A shows the cells incubated in condition I, and the right picture shows the pictures of cells cultured in condition II for 7 days. FIG. 6B shows the results of confirming that the cells cultured in condition II for 7 days are expressed by CD31 and CD34 using FACS analysis. In addition, FIG. 6C shows the results of confirming the expression of markers expressed in vascular endothelial progenitor cells in condition II by comparing with condition I and condition II by RT-PCR. 6D shows the results of confirming the expression of CD31 and vWF by immunofluorescence staining in the cells at this time, and the adult cells used at this time were EGFP-expressing cells. In the figure, DAPI means the result of staining the nucleus.

Figure 7 shows the results confirmed in vitro whether the differentiation into vascular cells through the pathway II process. A shows the result of confirming that the tubule formation is significantly higher in the cells undergoing the conditio II stage compared to condition I (the result after 12 hours of differentiation), and B is the graph of the number of tubular structures at this time. The comparison results are shown. C shows that the differentiated cells can absorb Dil-AC-LDL, which is a representative function of vascular endothelial progenitor cells, in medium culture conditions that induce vascular endothelial progenitor cells. In this case, a represents cells that did not differentiate in EGM-2 medium (condition II), and b and c were cells that were absorbed after incubating for 6 hours with LDL analog in cells differentiated for 1 week in EGM-2 medium (condition II). Indicates the appearance.

Figure 8 shows the results confirmed by immunofluorescence staining that the blood vessels formed when the cells differentiated in the culture medium conditions inducing vascular endothelial progenitor cells are injected into a disease animal model with ischemic limbs. In this case, A shows the result of comparing the rate of necrosis in each group, and when the cells cultured in condition II are injected, the rate of necrosis is low. In addition, B shows that the differentiated cells in the medium culture conditions inducing vascular endothelial progenitor cells prevent muscle necrosis through the formation of blood vessels when injected into a disease animal model with ischemic limbs. At this time, the control (-) group shows a sliced picture after injection of undifferentiated cells, and the condition II group below shows a sliced picture after injection of cells differentiated under condition II. At this time, the result of confirming the tissue through immunofluorescence staining method is Figure 8C. FIG. 8C shows the results of staining cells of CD31 (a) and vWF (b), which are markers specifically expressed in vascular endothelial progenitor cells, and EGFP was expressed by inserting a vector called pBabe puro EGFP into the labia minora-derived cells. It shows that it becomes. Here, DAPI shows the result of staining the nucleus, and the picture labeled merge shows the result of showing the expression of each specifically expressed marker and EGFP together.

Claims (11)

Adult stem cells isolated from labia minora were cultured under low glucose DMEM, FBS (fetal bovine serum), L-glutamine (L-glutamine) and penicillin-streptomycin (multiple) to show multi-differentiation characteristics. Steps to prepare adult cells: (1) all show positive immunological properties for CD13, CD29, CD44 and CD90; (2) express dermo-1 but not SHOX-2 gene; (3) attached to and grown on cell culture dishes, exhibiting linear morphological characteristics; (4) having the ability to differentiate into endoderm, ectoderm and mesoderm derived cells; And (5) under cultivation of cytokines-free medium, having multipotent capacity and having the ability to multiply; Through the step of culturing the adult stem cells prepared in the above while processing VEGF in MAPC medium, differentiated vascular endothelial progenitor cells. The method of claim 1, wherein the MAPC medium comprises DMEM, MCDB, insulin (Insulin), apo-transferrin (apo-transferrin), selenium (selenium), dexamethasone (dexametasone), and ascorbic acid (ascorbic acid) Differentiated vascular endothelial progenitor cells. Culturing adult stem cells isolated from the labia minora in culture conditions consisting of low glucose DMEM, FBS, L-glutamine and penicillin-streptomycin; And Adult stem cells cultured in the low glucose were added in EGM-2 medium culture conditions consisting of EBM-2 base medium, FBS, hydrocortisone, bFGF, VEGF, IGF-1, vitamin C, EGF, GA-1000 and heparin. Through the step of culturing, differentiated vascular endothelial progenitor cells. According to claim 1 or claim 3, The adult stem cells isolated in the labia minora dermal tissue obtained from the isolated labia minora tissue, the cells are separated from the obtained dermal tissue, and then the isolated cells are cultured in low glucose DMEM Differentiated vascular endothelial progenitors, characterized in that obtained by. 4. The differentiated vascular endothelial progenitor cells according to claim 1 or 3, wherein the differentiation-induced vascular endothelial progenitor cells exhibit the following characteristics: (1) differentiated from adult cells of the labia minora in women; (2) expresses CD31, CD34, VE-Cadherin, vWF, and eNOS; (3) has a tubule structure; And (4) Absorbs Dil-AC-LDL. A composition for treating vascular diseases comprising the differentiated vascular endothelial progenitor cells of claim 1 or 3 as an active ingredient. The composition for treating vascular diseases according to claim 6, wherein the differentiated vascular endothelial progenitor cells exhibit the following characteristics: (1) differentiated from adult cells of the labia minora in women; (2) expresses CD31, CD34, VE-Cadherin, vWF, and eNOS; (3) has a tubule structure; And (4) Absorbs Dil-AC-LDL. The method of claim 6, wherein the vascular disease is ischemic vascular disease or cardiovascular disease, characterized in that the composition for treating vascular diseases. The method of claim 8, wherein the ischemic vascular disease is ischemic muscle necrosis, arrhythmia, angina, or stroke. The method of claim 8, wherein the cardiovascular disease is atherosclerosis, ischemic reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, ventricular tuning, coronary microembolism, tachycardia, bradycardia, coronary artery ligation, arrhythmia, Stroke, angina pectoris, myocardial infarction, heart failure or high blood pressure composition for the treatment of vascular disease. Adult stem cells isolated from labia minora were cultured under low glucose DMEM, FBS (fetal bovine serum), L-glutamine (L-glutamine) and penicillin-streptomycin (multiple) to show multi-differentiation characteristics. Composition for treating vascular disease comprising adult cells as an active ingredient: (1) all show positive immunological properties for CD13, CD29, CD44 and CD90; (2) express dermo-1 but not SHOX-2 gene; (3) attached to and grown on cell culture dishes, exhibiting linear morphological characteristics; (4) having the ability to differentiate into endoderm, ectoderm and mesoderm derived cells; And (5) under cultivation of cytokines-free medium, having multipotent capacity and having the ability to multiply; And (6) Differentiate into vascular endothelial progenitor cells.

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