Umbilical cord blood-derived endothelial progenitor cells, and composition for preventing or treating ischemic diseases comprising same

Abstract

The present invention relates to: umbilical cord blood-derived endothelial progenitor cells; and a composition for preventing or treating ischemic diseases, the composition comprising the umbilical cord blood-derived endothelial progenitor cells, and more specifically, to high-purity umbilical cord blood-derived endothelial progenitor cells exhibiting specific surface antigen characteristics, a method for obtaining the umbilical cord blood-derived endothelial progenitor cells through pretreatment of umbilical cord blood, and a composition for preventing or treating ischemic diseases, the composition comprising the umbilical cord blood-derived endothelial progenitor cells. The high-purity umbilical cord blood-derived endothelial progenitor cells according to the present invention exhibit colony formation, high proliferative capacity, and in vivo angiogenesis potential, and thus can exhibit excellent effects in preventing or treating various ischemic diseases caused by vasoconstriction or occlusion.

Description

Cord blood-derived vascular endothelial progenitor cells and compositions containing the same for preventing or treating ischemic diseases

[0001] The present invention relates to umbilical cord blood-derived vascular endothelial progenitor cells and a composition for preventing or treating ischemic diseases containing the same, and more particularly, to highly pure umbilical cord blood-derived vascular endothelial progenitor cells exhibiting specific surface antigen characteristics, a method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells through preprocessing of umbilical cord blood, and a composition for preventing or treating ischemic diseases containing the umbilical cord blood-derived vascular endothelial progenitor cells.

[0002] Ischemic diseases, such as heart disease, cerebrovascular disease, and peripheral vascular disease, have high morbidity and mortality rates. Conventional medical, interventional, and surgical treatments often fail to provide sufficient efficacy. For this reason, novel treatments that promote angiogenesis and improve blood flow to ischemic tissue are needed.

[0003] Endothelial progenitor cells (EPCs) possess the ability to promote angiogenesis and can be derived from bone marrow, peripheral blood, and umbilical cord blood. Umbilical cord blood, in particular, is readily available, has a low risk of immune rejection, and presents relatively few ethical concerns, making it a suitable resource for cell therapy. EPCs express endothelial cell markers such as CD34, CD144, and CD184, and these cells migrate to damaged blood vessels and aid in vascular regeneration.

[0004] Previous studies have proposed various cell therapies using vascular endothelial progenitor cells, but most of these studies have encountered problems with cell purity and proliferation, and have had difficulties in clinical application due to lack of optimization of culture conditions, growth factors, and attachment substrates used in the cell harvesting process.

[0005] Meanwhile, the half-life of heparin in the blood is within 1 to 2 hours in the human body, and it is reported that heparin loses its activity rapidly through binding to numerous proteins present in the blood (Semin Intervent Radiol 2010 Dec; 27(4): 360-367). The binding and dissociation of heparin with blood cytokines and growth factor proteins (J Cell Mol Med. 2018. PMID: 30334335) are affected by temperature (Biochem Physiol. 2018.), and it has also been reported that the interaction with these proteins can affect the cellular function of blood ECFCs or late proliferating vascular endothelial progenitor cells. Therefore, it can be seen that the number of proteins that can bind to heparin is considerable, and the result of the binding between heparin and proteins has a very complex correlation in terms of the cellular effects (Stem Cell Res 2014 May; 12(3):703-15).

[0006] Accordingly, the inventors of the present invention have conducted research and made efforts to obtain high-purity vascular endothelial progenitor cells that can be applied clinically, and as a result, they discovered that by treating umbilical cord blood with heparin to isolate and obtain monocytes and culturing them under specific conditions, high-purity vascular endothelial progenitor cells exhibiting specific surface antigen characteristics can be obtained, thereby completing the present invention.

[0007] The purpose of the present invention is to provide umbilical cord blood-derived vascular endothelial progenitor cells, a method for obtaining the same, and a composition for preventing or treating ischemic disease comprising the umbilical cord blood-derived vascular endothelial progenitor cells.

[0008] The present invention provides cord blood-derived vascular endothelial progenitor cells in which at least 50% of the cell population expresses the CD34 marker and at least 5% or less of the cell population expresses the CD90 marker.

[0009] The above umbilical cord blood-derived vascular endothelial progenitor cells can express the CD34 marker in at least 80% of the cell population.

[0010] The above umbilical cord blood-derived vascular endothelial progenitor cells can express CD144 marker and CD184 marker in at least 80% of the cell population.

[0011] The above umbilical cord blood-derived vascular endothelial progenitor cells may have been cultured for at least 10 passages.

[0012] The above umbilical cord blood-derived vascular endothelial progenitor cells can increase the expression of one or more angiogenesis stimulating factors selected from ANGPT2, MCP-1, MMP-1, and PIGF.

[0013] In addition, the present invention provides a cell therapy composition for preventing or treating ischemic disease, which comprises the umbilical cord blood-derived vascular endothelial progenitor cells as an active ingredient.

[0014] In addition, the present invention,

[0015] A step of isolating and obtaining monocytes from umbilical cord blood by treating the umbilical cord blood with heparin; and

[0016] A method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells is provided, characterized by including a step of obtaining umbilical cord blood-derived vascular endothelial progenitor cells by culturing the above monocytes in a medium containing fucoidan, oliuropin, and vascular endothelial growth factor as active ingredients.

[0017] The above heparin can be administered at a concentration of 1 to 1000 U per 1 ml of umbilical cord blood.

[0018] The above heparin treatment can be carried out at 1 to 20°C for 1 to 8 hours.

[0019] The method may further include a step of subculturing cord blood-derived vascular endothelial progenitor cells expressing the CD34 marker among the cord blood-derived vascular endothelial progenitor cells obtained above to finally obtain high-purity cord blood-derived vascular endothelial progenitor cells.

[0020] The high-purity umbilical cord blood-derived vascular endothelial progenitor cells of the present invention exhibit colony formation and high proliferation ability and angiogenic potential in the body, and thus can exhibit excellent effects in the prevention or treatment of various ischemic diseases caused by constriction or occlusion of blood vessels.

[0021] In addition, the present invention can secure a sufficient number of colonies of umbilical cord blood-derived vascular endothelial progenitor cells through heparin treatment under specific conditions, thereby contributing to mass production of cell therapy products, etc.

[0022] Figure 1 is a graph comparing the number of colonies obtained after treating umbilical cord blood with the same concentration of heparin and CPDA-1 and then going through the same culture process.

[0023] Figure 2 is a graph comparing the number of colonies obtained after treating umbilical cord blood with heparin three months after initiating use and undergoing the same culture process.

[0024] Figure 3 shows the results of analyzing the surface antigen characteristics of umbilical cord blood-derived vascular endothelial progenitor cells of the present invention.

[0025] Figure 4 shows a photograph of colonies attached to fibronectin, recovered using a triple solution product, and cultured after cell separation.

[0026] Figure 5 is a graph showing blood flow and new capillary density measured after treating a mouse with umbilical cord blood-derived vascular endothelial progenitor cells of the present invention.

[0027] Hereinafter, the present invention will be described in detail. Prior to this, it should be noted that the terms and words used in this specification and claims should not be interpreted as limited to their conventional or dictionary meanings. Rather, they should be interpreted with meanings and concepts that conform to the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concept of the term to best explain his or her invention. Accordingly, the configurations described in the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent the entire technical spirit of the present invention. Therefore, it should be understood that various equivalents and modified examples may exist as of the time of this application.

[0028] The umbilical cord blood-derived vascular endothelial progenitor cells of the present invention are characterized in that, in terms of undifferentiated stem cell-related markers, at least 50% or more of the cell population expresses the CD34 marker, and at least less than 5% of the cell population expresses the CD90 marker.

[0029] Preferably, the umbilical cord blood-derived vascular endothelial progenitor cells can express the CD34 marker in at least 80% of the cell population.

[0030] In addition, the umbilical cord blood-derived vascular endothelial progenitor cells may have at least 80% of the cell population express CD144 markers and CD184 markers.

[0031] The above umbilical cord blood-derived vascular endothelial progenitor cells may have been cultured for at least 10 passages, and may exhibit the above characteristics within 12 days after being obtained from isolated mononuclear cells.

[0032] The above umbilical cord blood-derived vascular endothelial progenitor cells can increase the expression of one or more angiogenesis-stimulating factors selected from ANGPT2, MCP-1, MMP-1, and PIGF. Due to the increase in the expression of the angiogenesis-stimulating factors, vascular regeneration, vascular recovery, and vascular differentiation related to the angiogenesis process, including endothelial cell activation, migration, proliferation, matrix remodeling, and cell stabilization, can be induced. In one embodiment of the present invention, the expression level of the angiogenesis-stimulating factors of the umbilical cord blood-derived vascular endothelial progenitor cells is equivalent to or higher than that of HUVEC, indicating that the umbilical cord blood-derived vascular endothelial progenitor cells are an effective candidate for a regenerative therapeutic agent.

[0033] Meanwhile, the present invention features a cell therapy composition for preventing or treating ischemic disease, which comprises the umbilical cord blood-derived vascular endothelial progenitor cells as an active ingredient.

[0034] The above cell therapy products are medicines (as defined by the US FDA) used for the purposes of treatment, diagnosis, and prevention by isolating, culturing, and manufacturing cells and tissues from humans and animals through special manipulation. They refer to medicines used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells through other methods to restore the function of cells or tissues.

[0035] The cell therapy composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means non-toxic to cells or humans exposed to the composition. Any carrier known in the art, such as a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, carrier, excipient, lubricant, or preservative, may be used without limitation.

[0036] The above cell therapy agent can be manufactured using a commonly used technique in various formulations, and can be administered via any route as long as it can induce migration to the diseased site. In some cases, loading the vascular endothelial progenitor cells into a vehicle equipped with a means for directing them to the lesion may also be considered. Therefore, the composition of the present invention can be administered via various routes, including topical (including buccal, sublingual, cutaneous, and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, drip, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid), or transdermal administration.

[0037] The above ischemic disease refers to a condition caused by a reduction in blood supply to an organ, tissue, or area of ​​the body due to the constriction or occlusion of blood vessels. Even after reperfusion of blood to the tissue or area following ischemia, nerve cells are damaged, causing various aftereffects and ultimately leading to irreversible damage, namely, necrosis of cells and tissues. The above ischemic disease may be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic baldness, ischemic stroke, and ischemic lower extremity disease, and more preferably, may be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic stroke, and ischemic lower extremity disease, and most preferably, may be ischemic myocardial infarction or ischemic lower extremity disease.

[0038] Meanwhile, the present invention,

[0039] A step of isolating and obtaining monocytes from umbilical cord blood by treating the umbilical cord blood with heparin; and

[0040] Another feature of the present invention is a method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells, comprising a step of obtaining umbilical cord blood-derived vascular endothelial progenitor cells by culturing the monocytes in a medium containing fucoidan, oliuropine, and vascular endothelial growth factor as active ingredients. The medium may preferably additionally contain 1 to 10% human serum.

[0041] The above umbilical cord blood can be supplied in limited quantities from the mother, and the amount of umbilical cord blood to be processed can be 1 to 1000 ml, preferably 10 to 100 ml, and more preferably 30 to 70 ml.

[0042] The above heparin can be treated at a concentration of 1 to 1000 U per 1 ml of cord blood, preferably 5 to 100 U per 1 ml of cord blood, and most preferably 10 to 50 U per 1 ml of cord blood.

[0043] The above heparin treatment can be carried out at 1 to 20°C for 1 to 8 hours, preferably at 3 to 15°C for 2 to 6 hours, and most preferably at 4 to 10°C for 3 to 4 hours.

[0044] The above heparin can be used by diluting it in physiological saline or DPBS (Dulbecco's phosphate-buffered saline), and it is preferable to process the umbilical cord blood within 1 month from the start of using heparin. If the umbilical cord blood is processed more than 3 months after the start of using, there is a problem that colonies of vascular endothelial progenitor cells are not secured after culture. In this specification, the start of use means the time when heparin is first exposed to the external environment and becomes usable, which is generally the same as the day when heparin is opened from the storage container or packaging.

[0045] After heparin treatment of the above umbilical cord blood, monocytes are separated and obtained. Monocytes can be obtained by density gradient centrifugation using Ficoll.

[0046] The above-described isolated monocytes can be cultured in a medium containing fucoidan, oliuropine, and vascular endothelial growth factor as active ingredients to obtain umbilical cord blood-derived vascular endothelial progenitor cells. The medium may preferably additionally contain 1 to 10% human serum.

[0047] The above fucoidan may be included in the medium at a concentration of 0.05 to 20 μg / ml, preferably at a concentration of 0.1 to 10 μg / ml, and more preferably at a concentration of 0.1 to 5 μg / ml.

[0048] Additionally, the olieurophin may be included in the medium at a concentration of 0.01 to 10 μM, preferably at a concentration of 0.1 to 5 μM, and more preferably at a concentration of 0.2 to 0.75 μM.

[0049] Additionally, the vascular endothelial growth factor may be included at a concentration of 1 to 500 ng / ml, preferably 10 to 300 ng / ml, and more preferably 10 to 100 mg / ml.

[0050] Additionally, the human serum may be included in the medium at 1 to 10%, preferably 1 to 5%, and more preferably 1 to 3%.

[0051] The above medium refers to a culture solution that can support stem cell growth and survival under in vitro culture conditions, and includes all media commonly used in the art suitable for stem cell culture. The above culture solution is a cell culture minimum medium (CCMM), which generally contains carbon source, nitrogen source, and trace element components. For example, ECGM (Endothelial Cell Growth Medium), DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, MEM (Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc. can be used, and may include an endothelial cell growth medium, but is not necessarily limited thereto.

[0052] The above culture was performed with fibronectin at 0.5 to 6 μg / cm 2 At a concentration of , preferably 0.5 to 5 μg / cm 2 It can be carried out in a well-plate coated with a concentration of .

[0053] Colonies of the above-mentioned cultured cells can be obtained and subcultured to finally obtain high-purity umbilical cord blood-derived vascular endothelial progenitor cells. At this time, among the vascular endothelial progenitor cells, umbilical cord blood-derived vascular endothelial progenitor cells expressing the CD34 marker can be subcultured to finally obtain high-purity umbilical cord blood-derived vascular endothelial progenitor cells.

[0054] At this time, the colonies attached to the fibronectin, etc. can be separated and subcultured using a proteolytic enzyme reagent containing 1 to 5 mM EDTA, preferably a proteolytic enzyme reagent containing 1.5 to 2 mM EDTA. The EDTA chelates calcium and magnesium ions, thereby weakening intercellular and cell-matrix bonds, thereby assisting the efficient action of the proteolytic enzyme.

[0055] Hereinafter, the present invention will be described in detail using examples and experimental examples. However, the examples according to the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the examples described below. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0056]

[0057] Experimental Example 1: Confirmation of colony count by heparin treatment

[0058] Heparin was added at a concentration of 20 U per ml of cord blood to 50 ml of the mother (YBG-2022-012), reacted at 5°C for 4 hours, and then monocytes were obtained by density gradient centrifugation using Ficoll.

[0059] The above monocytes were obtained and treated with fibronectin (2.5 μg / cm 2 ) were seeded in 25T flasks coated with cells (1.8 to 2.0 x 10 7(cells / well) and cultured for 5 days in a medium containing 0.1 μg / ml fucoidan, 0.5 μM olieurophin, 100 ng / ml vascular endothelial growth factor, and human serum (2%). Next, colonies attached to fibronectin, etc. were separated using a proteolytic enzyme reagent containing 2 mM EDTA, and subculture was performed, and the number of colonies was confirmed after 12 days (10 passages or more).

[0060] The number of colonies was compared with that of a comparative example that underwent the same culture process after treating CPDA-1, known as an anticoagulant, at the same concentration as heparin instead of the above-mentioned heparin, and this is shown in Figure 1.

[0061] As shown in the above Figure 1, it was confirmed that colonies of vascular endothelial progenitor cells could hardly be obtained from cord blood and mononuclear cells that interacted with CPDA-1, and that at least 50 times more colonies could be obtained when heparin was treated under specific conditions.

[0062]

[0063] Experimental Example 2: Confirmation of the number of colonies according to heparin treatment conditions

[0064] The experiment was conducted in the same manner as Experimental Example 1 above, except that the temperature and reaction time of heparin were changed as shown in Table 1 below, and the final number of colonies generated was confirmed.

[0065] Reaction temperature (℃) 10102323 Reaction time (hours) 4848 Number of colonies 289.31.253

[0066] As a result of the above experiment, it was confirmed that when the reaction temperature and reaction time were different from those in Experimental Example 1, the number of colonies produced was relatively smaller.

[0067]

[0068] Experimental Example 3: Confirmation of the number of colonies according to the heparin dilution period.

[0069] Culture was performed in the same manner as in Experimental Example 1, except that the umbilical cord blood was treated 3 months after the start of heparin use, and the results are shown in Fig. 2.

[0070] In general, heparin's anticoagulant activity is maintained for at least one month after initiation of use. However, as shown in the experiment above, after three months of use, almost no endothelial progenitor cell colonies were formed.

[0071]

[0072] Experimental Example 4: Analysis of Surface Antigens of Vascular Endothelial Progenitor Cells

[0073] In the above experimental example 1, the surface antigens CD34 and CD90 were analyzed through antibody reaction using flow cytometry during subculture, and the results are shown in Figure 3.

[0074] As shown in the above Figure 3, it was confirmed that at least 50% of the vascular endothelial progenitor cells expressed the CD34 marker, and at least less than 5% of the cell population expressed the CD90 marker.

[0075] CD34-positive cells are present at levels of 1.1 ± 0.9% in peripheral blood, 0.1 to 1% in umbilical cord blood, and 1.7 ± 0.5% in bone marrow. However, there has been no report of such a ratio of CD34-positive cells in late proliferating vascular endothelial progenitor cells.

[0076]

[0077] Experimental Example 5: Obtaining vascular endothelial progenitor cells using a proteolytic enzyme reagent.

[0078] Instead of using the protein decomposition enzyme reagent containing 2 mM EDTA in the above Experimental Example 1, cell separation was performed using a TrypLE solution product containing 1 mM EDTA, and then subculture was performed, and the results are shown in Fig. 4.

[0079] As shown in Fig. 4, when the triple-action product was used, cell separation was not performed normally, resulting in MSC-like morphological changes in the cultured cells, and the changed cells did not regain their previous cellular appearance.

[0080]

[0081] Experimental Example 6: Analysis of angiogenic factors in vascular endothelial progenitor cells

[0082] The vascular endothelial progenitor cells (XEPC), umbilical cord blood-derived mesenchymal stem cells (CBMSC), and fully differentiated vascular endothelial cells (HUVEC) cells obtained in the above Experimental Example 1 were cultured in 25T each and cultured in the same growth medium until 80% confluency, washed 2-3 times using serum-free basal media, and cultured for 3 days with serum-free media and the supernatant was recovered, centrifuged to remove debris, and then stored at -80°C.

[0083] The expression levels of ANGPT2, MCP1, MMP-1, and PIGF were quantitatively analyzed using the Angiogenesis Array Q1000 kit (Ray Biotech) for the above samples, and the results are shown in Table 2 below.

[0084] Cells ANGPT2 (pg / ml) MCP1 (pg / ml) MMP-1 (pg / ml) PIGF (pg / ml)

[0085] It was confirmed that the vascular endothelial progenitor cells (XEPCs) obtained in the above experimental example 1 showed a high expression level of ANGPT2, a signature gene of vascular endothelial progenitor cells, and that MCP1, MMP-1, and PIGF also showed expression levels equivalent to or higher than those of HUVECs.

[0086]

[0087] Experimental Example 7: Analysis of the Effects of In Vivo Endothelial Progenitor Cell Injection

[0088] Diabetes was induced in nude mice by injecting endothelial progenitor cells (STZ) obtained in Experimental Example 1, and after one month, femoral artery ligation was performed and Doppler imaging was performed to confirm that blood flow was blocked.

[0089] One day after ligation, the cell therapy agent containing the vascular endothelial progenitor cells of Experimental Example 1 was injected into the muscles on both sides of the injured blood vessel area, and blood flow was observed for 6 weeks. Afterwards, an autopsy was performed, and the density of new blood vessels per unit area was measured by IHC using α-SMA antibody, and the results are shown in Figure 5.

[0090] G1, normal control group;

[0091] G2, disease-induced and untreated group;

[0092] G3, disease-inducing and low-concentration cell therapy treatment group (0.5 x 10 4 cells / head, n=5);

[0093] G4, disease-inducing and medium-dose cell therapy treatment group (1 x 10 5 cells / head, n=5);

[0094] G5, disease-inducing and high-concentration cell therapy treatment group (5 x 10 5 cells / head, n=5)

[0095]

[0096] As shown in the above Figure 5, the blood flow observation results showed that there was no difference in blood flow in the test substance administration group (G3-G5) compared to the untreated group (G2) until the first week, but an improvement in blood flow was observed from the third week.

[0097] In addition, when examining the density of new blood vessels per unit area in IHC using α-SMA antibody, it was confirmed that there was a tendency for new capillaries to increase depending on the concentration of cell therapy treatment in the test substance administration groups (G3-G5).

Claims

1. Cord blood-derived vascular endothelial progenitor cells characterized in that at least 50% of the cell population expresses the CD34 marker and at least less than 5% of the cell population expresses the CD90 marker.

2. In paragraph 1, The above umbilical cord blood-derived vascular endothelial progenitor cells are umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that at least 80% of the cell population expresses the CD34 marker.

3. In paragraph 1, The above umbilical cord blood-derived vascular endothelial progenitor cells are umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that at least 80% of the cell population expresses CD144 markers and CD184 markers.

4. In paragraph 1, The above umbilical cord blood-derived vascular endothelial progenitor cells are characterized in that they have been cultured for at least 10 passages.

5. In paragraph 1, The above umbilical cord blood-derived vascular endothelial progenitor cells are characterized by increased expression of one or more angiogenesis stimulating factors selected from ANGPT2, MCP-1, MMP-1, and PIGF.

6. A cell therapy composition for the prevention or treatment of ischemic disease, comprising umbilical cord blood-derived vascular endothelial progenitor cells selected from any one of claims 1 to 5 as an active ingredient.

7. A step of isolating and obtaining monocytes from the umbilical cord blood by treating the umbilical cord blood with heparin; and A method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that it comprises a step of obtaining umbilical cord blood-derived vascular endothelial progenitor cells by culturing the above monocytes in a medium containing fucoidan, oliuropin, and vascular endothelial growth factor as active ingredients.

8. In paragraph 7, A method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that the above heparin is treated at a concentration of 1 to 1000 U per 1 ml of umbilical cord blood.

9. In paragraph 7, A method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that the above heparin treatment is performed at 1 to 20°C for 1 to 8 hours.

10. In paragraph 7, A method for obtaining umbilical cord blood-derived vascular endothelial progenitor cells, characterized in that it further comprises a step of subculturing umbilical cord blood-derived vascular endothelial progenitor cells expressing the CD34 marker among the obtained umbilical cord blood-derived vascular endothelial progenitor cells to finally obtain high-purity umbilical cord blood-derived vascular endothelial progenitor cells.

Images