Umbilical cord blood-derived vascular endothelial progenitor cell and composition for preventing or treating ischemic disease comprising same

Abstract

The present invention relates to an umbilical cord blood-derived vascular endothelial progenitor cell and a composition for preventing or treating ischemic diseases comprising the same, and more specifically, to an umbilical cord blood-derived vascular endothelial progenitor cell and a composition for preventing or treating ischemic diseases, the present invention relates to a high-purity umbilical cord blood-derived vascular endothelial progenitor cell exhibiting specific surface antigen characteristics, a method for obtaining an umbilical cord blood-derived vascular endothelial progenitor cell by pre-treating umbilical cord blood, and a composition for preventing and treating ischemic diseases comprising the umbilical cord blood-derived vascular endothelial progenitor cell. The high-purity umbilical cord blood-derived vascular endothelial progenitor cell of the present invention exhibits colony formation, high proliferation ability, and high potential for angiogenesis in vivo, and thus can exhibit an excellent effect on the prevention or treatment of various ischemic diseases induced by blood vessel contraction or occlusion.

Description

Technical Field

[0001] This invention relates to umbilical cord blood-derived vascular endothelial progenitor cells and compositions comprising such progenitor cells for the prevention or treatment of ischemic diseases. More specifically, it relates to high-purity 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 by preprocessing umbilical cord blood, and compositions comprising said umbilical cord blood-derived vascular endothelial progenitor cells for the prevention and treatment of ischemic diseases. Background Technology

[0002] Ischemic diseases such as heart disease, cerebrovascular disease, and peripheral vascular disease have high morbidity and mortality rates, and many cases cannot be adequately treated with existing medical, interventional, and surgical methods. For these reasons, new treatments that promote angiogenesis to improve blood flow to ischemic tissues are needed.

[0003] Endothelial progenitor cells (EPCs) possess the ability to promote angiogenesis. These cells can be derived from bone marrow, peripheral blood, umbilical cord blood, etc. In particular, umbilical cord blood is readily available, has low immune rejection rates, and fewer ethical concerns, thus it is considered a suitable resource for cell therapy. EPCs express markers for vascular endothelial cells such as CD34, CD144, and CD184. These cells contribute to angiogenesis by migrating to damaged blood vessels.

[0004] Existing research has proposed various cell therapy methods using vascular endothelial progenitor cells. However, most studies have problems with cell purity and proliferation capacity. Furthermore, the culture conditions, growth factors, and attachment substrates used in the cell acquisition process cannot be optimized, which makes clinical application difficult.

[0005] Furthermore, it has been reported that the half-life of heparin in the human body is within 1-2 hours, and its activity is rapidly lost through binding with many proteins present in the blood (Semin Intervent Radiol 2010 Dec; 27(4): 360-367). It has also been reported that the binding and dissociation of heparin with cytokines and growth factor proteins in the blood (J Cell Mol Med. 2018. PMID: 30334335) are affected by temperature (Biochem Physiol. 2018.), and as a result of interactions with these proteins, it may also affect the cellular function of blood ECFCs or late-stage proliferating vascular endothelial progenitor cells. Therefore, not only are there a considerable number of proteins with the potential to bind to heparin, but the binding of heparin to proteins also has an impact on cells, thus exhibiting a very complex correlation (StemCell Res 2014 May;12(3):703-15). Summary of the Invention

[0006] Technical issues Accordingly, in order to obtain high-purity vascular endothelial progenitor cells that can be used clinically, the inventors discovered through diligent research that if umbilical cord blood is treated with heparin to separate and obtain monocytes, and these monocytes are cultured under specific conditions, high-purity vascular endothelial progenitor cells exhibiting specific surface antigen characteristics can be obtained, thus completing the present invention.

[0007] One object of the present invention is to provide umbilical cord blood-derived vascular endothelial progenitor cells, a method for obtaining the progenitor cells, and a composition comprising the umbilical cord blood-derived vascular endothelial progenitor cells for the prevention or treatment of ischemic diseases.

[0008] Technical solution The present invention provides umbilical cord blood-derived vascular endothelial progenitor cells in which 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.

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

[0010] In the umbilical cord blood-derived vascular endothelial progenitor cells, at least 80% of the cell population can express CD144 and CD184 markers.

[0011] The umbilical cord blood-derived vascular endothelial progenitor cells can be umbilical cord blood-derived vascular endothelial progenitor cells that have been cultured for at least 10 generations.

[0012] The 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] Furthermore, the present invention provides a cell therapy composition for the prevention or treatment of ischemic diseases, comprising the aforementioned umbilical cord blood-derived vascular endothelial progenitor cells as an active ingredient.

[0014] Furthermore, the present invention provides a method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood, characterized by comprising the following steps: separating and obtaining mononuclear cells from umbilical cord blood by treating umbilical cord blood with heparin; and culturing the mononuclear cells in a culture medium containing fucoidan, oleuropein and vascular endothelial growth factor as active ingredients, thereby obtaining vascular endothelial progenitor cells derived from umbilical cord blood.

[0015] The heparin can be processed at a concentration of 1~1000U / 1ml of umbilical cord blood.

[0016] The heparin treatment can be carried out at 1-20°C for 1-8 hours.

[0017] It may also include the following steps: passage culture of the obtained umbilical cord blood-derived vascular endothelial progenitor cells expressing the CD34 marker, thereby finally obtaining high-purity umbilical cord blood-derived vascular endothelial progenitor cells.

[0018] Beneficial effects The high-purity umbilical cord blood-derived vascular endothelial progenitor cells of this invention exhibit colony formation, high proliferative capacity, and high in vivo angiogenesis potential, thus demonstrating excellent effects in the prevention or treatment of various ischemic diseases induced by vasoconstriction or occlusion.

[0019] Furthermore, the present invention can ensure a sufficient number of colonies of vascular endothelial progenitor cells derived from umbilical cord blood through heparin treatment under specific conditions, thereby facilitating the mass production of cell therapy agents, etc. Attached Figure Description

[0020] Figure 1 This is a chart comparing the number of colonies obtained after treating umbilical cord blood with the same concentration of heparin and CPDA-1 and then undergoing the same culture process.

[0021] Figure 2 This is a chart comparing the number of colonies obtained after processing cord blood through the same culture process three months after the start of heparin administration.

[0022] Figure 3 This is a graph showing the analytical results of the surface antigen characteristics of vascular endothelial progenitor cells derived from umbilical cord blood according to the present invention.

[0023] Figure 4 The image shows a photograph of colonies attached to fibronectin recovered using a trypsin analog solution product, separated into cells, and then cultured.

[0024] Figure 5 The graph shows the blood flow and new capillary density measured after mice were treated with umbilical cord blood-derived vascular endothelial progenitor cells of the present invention. Detailed Implementation

[0025] The present invention will be described in detail below. Prior to this, the terms or words used in this specification and the claims should not be interpreted in their ordinary or dictionary sense, but rather should be interpreted in accordance with the principle that the inventor may appropriately define the concepts of the terms to best illustrate their own invention, in a way that conforms to the technical concept of the invention. Therefore, the embodiments described in this specification are merely the most preferred embodiments of the invention and do not represent all the technical concepts of the invention. It should be understood that, at the time of this application, there are various equivalents and modifications that can replace them.

[0026] The umbilical cord blood-derived vascular endothelial progenitor cells of the present invention are characterized in that, among the markers relating to undifferentiated stem cells, 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.

[0027] Preferably, in the umbilical cord blood-derived vascular endothelial progenitor cells, at least 80% of the cell population can express the CD34 marker.

[0028] Furthermore, at least 80% of the endothelial progenitor cells derived from umbilical cord blood can express CD144 and CD184 markers.

[0029] The endothelial progenitor cells derived from umbilical cord blood can be cells that have been cultured for at least 10 generations and can exhibit the aforementioned characteristics within 12 days after being obtained from isolated mononuclear cells.

[0030] The 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. This increased expression of angiogenesis-stimulating factors may induce angiogenesis-related processes such as endothelial cell activation, migration, proliferation, matrix remodeling, and cell stabilization, leading to vascular regeneration, vascular recovery, and vascular differentiation. In one embodiment of the present invention, the expression levels of the angiogenesis-stimulating factors in the umbilical cord blood-derived vascular endothelial progenitor cells are at or above the levels of HUVECs, indicating that umbilical cord blood-derived vascular endothelial progenitor cells are a viable candidate for effective regenerative therapeutics.

[0031] In addition, a feature of the present invention is a cell therapy composition for the prevention or treatment of ischemic diseases, the composition comprising the umbilical cord blood-derived vascular endothelial progenitor cells as an active ingredient.

[0032] The cell therapy agent is a drug used for treatment, diagnosis, and prevention by utilizing cells and tissues prepared through isolation, culture, and special operations from humans and animals. (As defined by the US FDA) It refers to a drug used for treatment, diagnosis, and prevention by a series of actions such as in vitro proliferation, screening for surviving autologous, allogeneic, or xenogeneic cells, or altering the biological characteristics of cells using other methods to restore the function of cells or tissues.

[0033] The cell therapy agent may additionally include a pharmaceutically permissible carrier. "Pharmaceutically permissible" means non-toxic to cells or the human body exposed to the composition. The carrier may be used without restriction as long as it is a carrier known in the art (buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, matrix, excipient, lubricant, preservative, etc.).

[0034] The cell therapy agents can be prepared in various dosage forms according to common techniques, and can be administered via any route as long as they can induce migration to the disease site. Depending on the circumstances, loading vascular endothelial progenitor cells into a vehicle with a means of targeting the lesion may also be considered. Therefore, the compositions of the present invention can be administered via a variety of routes, including local administration (including buccal, sublingual, skin, and intraocular administration), non-oral administration (including subcutaneous, intradermal, intramuscular, infusion, intravenous, intra-articular, intra-articular, and intracerebrospinal fluid administration), or transdermal administration.

[0035] The ischemic disease refers to a disease caused by a reduction in blood supply to a body organ, tissue, or site due to vasoconstriction or occlusion. Even after reperfusion occurs following ischemia in the tissue or site, various sequelae can result from nerve cell damage, ultimately leading to irreversible damage (i.e., cell and tissue necrosis). The ischemic disease can 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 alopecia, ischemic stroke, and ischemic lower limb disease. More preferably, it can 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 limb disease. Most preferably, it can be ischemic myocardial infarction or ischemic lower limb disease.

[0036] Another feature of the present invention is a method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood, comprising the following steps: separating and obtaining mononuclear cells from umbilical cord blood by treating the umbilical cord blood with heparin; and culturing the mononuclear cells in a culture medium comprising fucoidan, oleuropein, and vascular endothelial growth factor as active ingredients, thereby obtaining vascular endothelial progenitor cells derived from umbilical cord blood. Preferably, the culture medium may additionally include 1-10% human serum.

[0037] The amount of umbilical cord blood received from the mother is limited. The amount of umbilical cord blood processed can be 1 to 1000 ml, preferably 10 to 100 ml, and more preferably 30 to 70 ml.

[0038] The heparin can be processed at a concentration of 1~1000U / 1ml of umbilical cord blood, preferably at a concentration of 5~100U / 1ml of umbilical cord blood, and more preferably at a concentration of 10~50U / 1ml of umbilical cord blood.

[0039] The heparin treatment can be carried out at 1~20°C for 1~8 hours, preferably at 3~15°C for 2~6 hours, and more preferably at 4~10°C for 3~4 hours.

[0040] The heparin can be used after dilution in physiological saline or Dulbecco's phosphate-buffered saline (DPBS). Preferably, cord blood can be processed within one month of starting heparin use. Processing cord blood more than three months after starting heparin use may result in the inability to ensure colony formation of vascular endothelial progenitor cells after culture. In this specification, "start of use" refers to the point in time when heparin is initially exposed to the external environment and becomes usable, which is generally the same date as the opening of the heparin from its storage container or packaging.

[0041] After treating the umbilical cord blood with heparin, mononuclear cells can be separated and obtained by centrifugation using the density difference of a lymphocyte separation medium (ficoll).

[0042] The isolated mononuclear cells are cultured in a medium containing fucoidan, oleuropein, and vascular endothelial growth factor as active ingredients to obtain umbilical cord blood-derived vascular endothelial progenitor cells. Preferably, the medium may additionally include 1-10% human serum.

[0043] The fucoidan can be contained in the culture medium at a concentration of 0.05~20 μg / ml, preferably at a concentration of 0.1~10 μg / ml, and more preferably at a concentration of 0.1~5 μg / ml.

[0044] Furthermore, the oleuropein can be contained in the culture medium at a concentration of 0.01~10 μM, preferably at a concentration of 0.1~5 μM, and more preferably at a concentration of 0.2~0.75 μM.

[0045] Furthermore, the concentration of the vascular endothelial growth factor can be 1~500 ng / ml, preferably 10~300 ng / ml, and more preferably 10~100 ng / ml.

[0046] Furthermore, the human serum can be contained in the culture medium at 1-10%, preferably at 1-5%, and more preferably at 1-3%.

[0047] The culture medium refers to a culture medium capable of supporting the growth and survival of stem cells under in vitro culture conditions, including all conventional culture media used in the art suitable for stem cell culture. The culture medium, as a minimum cell culture medium (CCMM), typically includes a carbon source, a nitrogen source, and trace elements. For example, endothelial cell growth medium (ECGM), Dulbecco's Modified Eagle's Medium (DMEM), minimal essential medium (MEM), basal medium Eagle (BME), RPMI 1640, F-10, F-12, and Iscove's Modified Dulbecco's Medium can be used, and may include, but are not limited to, endothelial cell growth medium.

[0048] The culture can be carried out on a substrate coated with a concentration of 0.5~6 μg / cm³. 2 The procedure is performed in well plates containing fibronectin, preferably coated with a solution of 0.5–5 μg / cm³. 2 The fibronectin was used in the well plate.

[0049] By obtaining colonies of the cultured cells and passage them, high-purity umbilical cord blood-derived vascular endothelial progenitor cells can be obtained. At this time, umbilical cord blood-derived vascular endothelial progenitor cells expressing the CD34 marker in vascular endothelial progenitor cells are passaged to obtain high-purity umbilical cord blood-derived vascular endothelial progenitor cells.

[0050] At this point, a proteolytic enzyme reagent containing 1-5 mM EDTA can be used to isolate and passage colonies attached to fibronectin, etc. Preferably, a proteolytic enzyme reagent containing 1.5-2 mM EDTA can be used to isolate and passage colonies attached to fibronectin, etc. The EDTA chelates calcium and magnesium ions to weaken cell-to-cell and cell-matrix binding, thereby facilitating the effective action of the proteolytic enzyme.

[0051] In the following detailed description, embodiments and experimental examples will be provided to illustrate the invention in detail. However, embodiments of the invention can be modified into various other forms, and the scope of the invention should be interpreted as not being limited to the embodiments described below. The embodiments of the invention are provided to provide a more complete explanation of the invention to those skilled in the art.

[0052] Experimental Example 1: Confirmation of Colony Count After Heparin Treatment A concentration of 20 U heparin / 1 ml cord blood was added to 50 ml of cord blood from the mother (YBG-2022-012). After reacting at 5°C for 4 hours, mononuclear cells were obtained by centrifugation using the density difference of the lymphocyte separation medium (ficoll).

[0053] The monocytes were obtained and coated with fibronectin (2.5 μg / cm). 2 Cells (1.8~2.0 μL) were seeded in 25T wells. 10 7 Cells (cells / well) were cultured for 5 days in a medium containing 0.1 μg / ml fucoidan, 0.5 μM oleuropein, 100 ng / ml vascular endothelial growth factor, and 2% human serum. Then, colonies attached to fibronectin and other proteins were isolated using a proteolytic enzyme reagent containing 2 mM EDTA and passaged. Colony numbers were confirmed after 12 days (more than 10 passages).

[0054] The colony counts were compared with those of a comparative example in which CPDA-1, a known anticoagulant, was used instead of heparin and treated at the same concentration as heparin, followed by the same culture process, and are shown in the figure. Figure 1 .

[0055] As mentioned above Figure 1 As shown, it can be confirmed that vascular endothelial progenitor cell colonies are almost impossible to obtain in umbilical cord blood and monocytes that interact with CPDA-1, but at least 50 times more colony numbers can be obtained when heparin is treated under specific conditions.

[0056] Experimental Example 2: Confirmation of Colony Numbers Based on Heparin Treatment Conditions Except for changing the temperature and reaction time of heparin as shown in Table 1 below, the experiment was conducted in the same manner as in Experimental Example 1, and the final number of colonies produced was confirmed.

[0057] [Table 1]

[0058] The experimental results confirm that when the reaction temperature and reaction time are set differently from those in Experiment 1, the number of colonies produced becomes relatively smaller.

[0059] Experimental Example 3: Confirmation of Colony Count During Heparin Dilution Except for processing the cord blood 3 months after the start of heparin administration, it was cultured in the same manner as in Experimental Example 1, and the results are presented in... Figure 2 .

[0060] Typically, the anticoagulant effect of heparin is maintained after one month or more of initiation. However, as shown in the experiments above, it can be confirmed that almost no colonies of vascular endothelial progenitor cells are formed after three months of initiation of heparin.

[0061] Experimental Example 4: Analysis of Surface Antigens in Vascular Endothelial Progenitor Cells In Experiment 1, during passage culture, CD34 and CD90, as surface antigens, were analyzed by flow cytometry via antibody reactions, and the results are presented below. Figure 3 .

[0062] As mentioned above Figure 3 As shown, it can be confirmed that at least 50% of the cell population in the vascular endothelial progenitor cells express the CD34 marker, and at least less than 5% of the cell population expresses the CD90 marker.

[0063] CD34-positive cells were present at a level of 1.1 ± 0.9% in peripheral blood, 0.1–1% in cord blood, and 1.7 ± 0.5% in bone marrow. No CD34-positive cell percentage as described above was reported in late-stage proliferating vascular endothelial progenitor cells.

[0064] Experimental Example 5: Obtaining Vascular Endothelial Progenitor Cells Using Proteolytic Enzyme Reagents Cell isolation was performed using a trypsin analogue (TrypLE) solution containing 1 mM EDTA instead of the proteolytic enzyme reagent containing 2 mM EDTA in Example 1. Cells were then passaged, and the results are presented below. Figure 4 .

[0065] like Figure 4 As shown, when using protein-degrading enzyme solution products, normal cell separation cannot be performed, resulting in phenotypic changes in cultured cells similar to those of MSCs, and the altered cells cannot regain their previous cellular characteristics.

[0066] Experimental Example 6: Analysis of Angiogenesis-Stimulating Factors in Vascular Endothelial Progenitor Cells The vascular endothelial progenitor cells (XEPC), umbilical cord blood-derived mesenchymal stem cells (CBMSC), and fully differentiated vascular endothelial cells (HUVEC) obtained in Experimental Example 1 were cultured in 25T medium and cultured to 80% confluence. After washing 2-3 times with serum-free basal media and culturing in serum-free medium for 3 days, the supernatant was recovered, centrifuged, and the residue was removed. The cells were then stored at -80°C.

[0067] The expression levels of ANGPT2, MCP1, MMP-1, and PIGF in the sample were quantitatively analyzed using the Angiogenesis Array Q1000 kit (Ray Biotech), and the results are shown in Table 2 below.

[0068] [Table 2]

[0069] The vascular endothelial progenitor cells (XEPCs) obtained in Experiment 1 showed high expression of ANGPT2, a characteristic gene of vascular endothelial progenitor cells, and the expression levels of MCP1, MMP-1, and PIGF were also at or above the levels of HUVECs.

[0070] Experiment Example 7: Analysis of the Injection Effect of Vascular Endothelial Progenitor Cells in Vivo Diabetes was induced in nude mice by injecting them with vascular endothelial progenitor cells (STZ) obtained in Experiment 1. After one month, femoral artery ligation was performed and Doppler imaging was performed to confirm that blood flow was blocked.

[0071] One day after ligation, a cell therapy agent containing vascular endothelial progenitor cells from Experiment 1 was injected into the muscles on both sides of the site of vascular injury. Blood flow was observed for 6 weeks, followed by an autopsy. The density of neovascularization per unit area was measured using IHC with α-SMA antibody, and the results are presented below. Figure 5 .

[0072] G1: Normal control group; G2: Disease-induced and untreated group; G3: Disease-inducing and low-concentration cell therapy treatment group (0.5) 10 4 Cells / head (n=5); G4: Disease-inducing and medium-concentration cell therapy treatment group (1) 10 5 (cells / head, n=5) G5: Disease-inducing and high-concentration cell therapy treatment group (5 10 5 Cells / head, n=5).

[0073] As described Figure 5 As shown, the results of blood flow observation showed that there was no difference in blood flow between the experimental substance administration group (G3-G5) and the untreated group (G2) up to the first week, but an improvement in blood flow was observed from the third week onwards.

[0074] Furthermore, the results of observing the density of neovascularization per unit area in IHC using α-SMA antibody confirmed that in the experimental substance administration groups (G3-G5), there was a tendency for the neovascularization of capillaries to increase according to the concentration of cell therapy agent.

Claims

1. A type of vascular endothelial progenitor cell derived from umbilical cord blood, characterized in that, At least 50% of the cell population expressed the CD34 marker, and at least less than 5% of the cell population expressed the CD90 marker.

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

3. The vascular endothelial progenitor cells derived from umbilical cord blood according to claim 1, characterized in that, In the umbilical cord blood-derived vascular endothelial progenitor cells, at least 80% of the cell population expressed CD144 and CD184 markers.

4. The vascular endothelial progenitor cells derived from umbilical cord blood according to claim 1, characterized in that, The umbilical cord blood-derived vascular endothelial progenitor cells are umbilical cord blood-derived vascular endothelial progenitor cells that have been cultured for at least 10 generations.

5. The vascular endothelial progenitor cells derived from umbilical cord blood according to claim 1, characterized in that, The umbilical cord blood-derived vascular endothelial progenitor cells increase the 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 diseases, comprising umbilical cord blood-derived vascular endothelial progenitor cells selected from any one of claims 1 to 5 as an active ingredient.

7. A method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood, characterized in that, Includes the following steps: Mononuclear cells were isolated and obtained from the umbilical cord blood by treating it with heparin; and The mononuclear cells were cultured in a culture medium containing fucoidan, oleuropein and vascular endothelial growth factor as active ingredients to obtain vascular endothelial progenitor cells derived from umbilical cord blood.

8. The method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood according to claim 7, characterized in that, The heparin was processed at a concentration of 1~1000U / 1ml of umbilical cord blood.

9. The method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood according to claim 7, characterized in that, The heparin treatment was carried out at 1-20°C for 1-8 hours.

10. The method for obtaining vascular endothelial progenitor cells derived from umbilical cord blood according to claim 7, characterized in that, It also includes the following steps: High-purity umbilical cord blood-derived vascular endothelial progenitor cells were obtained by passage culture of umbilical cord blood-derived vascular endothelial progenitor cells expressing CD34 markers.

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