KR101428852B1 - Pharmaceutical composition for treating ischemic disease comprising human blood derived hematosphere - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the treatment of ischemic diseases including hematopoietic cells derived from human blood. More specifically, the present invention relates to an effective three-dimensional culture using blood mononuclear cells to induce vascular endothelial cells and vascular smooth muscle cells, and ultimately to enable efficient blood vessel formation, thereby being used in the development of new cell therapy agents capable of treating ischemic diseases It is expected to be possible.

Description

[0001] The present invention relates to a pharmaceutical composition for treating ischemic diseases comprising hematopoietic cells derived from human blood,

The present invention relates to a method for treating ischemic diseases using human blood-derived blood cell mass.

Stem cells are pluripotent cells, which can be differentiated into any cell. Stem cells basically include Embryonic Stem Cell (ESC), Induced Pluripotent Stem Cell (IPS), and Adult Stem Cell (ASC).

Embryonic stem cells (ESCs) are proliferative and capable of differentiating into any cell, but there are many problems in using them for actual treatment. For example, constant proliferation can lead to tumor development, immune rejection, and ethical problems.

In order to overcome the problems of embryonic stem cells (ESC), degenerative stem cells (IPS) were developed in 2006 (Kazutoshi Takahashi and Shinya Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. ; 126: 663-676). However, there are various problems that need to be solved in order for IPS to be used for actual treatment, in particular, the use of virus and immune rejection.

On the other hand, adult stem cells (ASCs) have a problem in that the number of cells is small and proliferation is not active. However, these cells have a great advantage that they can be safely used as a cell therapy agent because autotransplantation is possible.

The present inventors overcome the limitations of tumor development, immunodeficiency, and ethical problems, which have been the problems of previously developed stem cell therapy (ESC), degenerative stem cell (IPS), and adult stem cell (ASC) As a result, it has been found that angiogenesis is possible through vascular progenitor cell amplification and vascular endothelial cell and vascular smooth muscle cell differentiation using human blood-derived hemocyte cell mass. Thus, the present invention has been accomplished. The present invention provides a method for treating ischemic diseases using hematopoietic cells derived from human blood.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for the treatment of ischemic diseases comprising hematopoietic cells derived from human blood.

In one embodiment of the present invention, the hematopoietic cell population derived from human blood is formed through three-dimensional coagulation culture by separating mononuclear cells from human blood.

In another embodiment of the present invention, the hematopoietic cell derived from human blood is used after being separated into single cells.

In another embodiment of the present invention, the hematopoietic cell derived from human blood is characterized by being induced into vascular endothelial cells and vascular smooth muscle cells.

In another embodiment of the present invention, the human blood-derived hemocyte cell mass is characterized by forming blood vessels.

In another embodiment of the present invention, the ischemic diseases include ischemic heart disease, myocardial infarction, angina pectoris, and lower limb ischemia.

The present invention also provides a method for treating an ischemic disease by administering to said individual a pharmaceutically effective amount of said pharmaceutical composition.

According to the present invention, it is expected that human blood-derived cells can be effectively cultured to induce vascular progenitor cells and vascular smooth muscle cells and enable angiogenesis, and ultimately be used for the development of a cell therapy agent capable of treating diseases related to ischemia . According to the present invention, it is possible to overcome limitations such as tumor development, immunodeficiency, and ethical problems that have been a problem of conventional stem cell therapeutic agents.

FIG. 1 is a schematic view showing a process of culturing and producing a hematopoietic cell derived from human blood after high-density coagulation culture of a mononuclear cell isolated from human blood through a three-dimensional culture technique.
Fig. 2 is a graph showing that sprouting itself can be performed when human blood hematopoietic cells are cultured on a Matrigel-coated dish.
FIG. 3 is a graph showing the expression of vascular endothelial growth factor receptor 2 (VEGFR-2, KDR, red color, arrow) when human blood-derived hematopoietic cells were cultured on a Matrigel-coated dish.
FIG. 4 shows the expression of vascular endothelial growth factor receptor 2 (VEGFR-2, KDR green) and platelet endothelial cell adhesion molecule (PECAM-1, red) when human blood hematopoietic cells were cultured on Matrigel- , And a tip cell (arrow) is observed.
FIG. 5 shows the expression of Vascular endothelial growth factor (VEGF, green, stomach) and CXC chemokine receptor type 4 (CXCR4, green, and below) in human blood derived hematopoietic cell mass through immunofluorescence staining.
FIG. 6 is a graph showing that the formation of blood cell-derived hematopoietic cells rapidly decreases when VEGF and VEGFR2 (KDR) are inhibited by using a VEGF antibody and its receptor VEGFR2 (KDR) chemical inhibitor (SU1498).
FIG. 7 shows the results of reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for the expression of Cytokine and its receptors, which are known to be important for angiogenesis in human blood- .
FIG. 8 shows the results of confirming the increase of the activity of Matrix metallopeptidase 9 (MMP-9) using the supernatant of human blood-derived blood cell mass through the MMP-9 zymography assay.
FIG. 9 shows that HUVEC migration and tube formation were increased when Human Umbilical Vein Endothelial Cell (HUVEC) was cultured using a supernatant of human blood-derived blood cell mass. Microscopic photographs ) And a graph (below).
FIG. 10 is a graph showing the results of ischemic induction in the Hindlimb of a nude mouse with reduced immune system and perfusion through laser Doppler perfusion imaging (LDPI) Immunofluorescence staining was performed using antibodies specific for human cells and alpha smooth muscle actin (SMA-alpha, red) of Cluster of Differentiation 34 (CD34, green), a vascular endothelial cell marker.

The present invention provides a pharmaceutical composition for the treatment of ischemic diseases comprising hematopoietic cells derived from human blood. The ischemic diseases include ischemic heart disease, myocardial infarction, angina pectoris, lower limb ischemia, and the like.

Blood-born hematopoietic cells (BBHS) used in the invention are aggregates of mononuclear cells present in the blood and specific cells with stem cells, forming a three-dimensional structure, Of the inner cell mass.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include, but is not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate.

The present invention also provides a method for treating an ischemic disease by administering to said individual a pharmaceutically effective amount of said pharmaceutical composition. In the present invention, an 'individual' refers to a subject in need of treatment for diseases, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, And the like. It is to be understood that the term "pharmaceutically effective amount" in the present invention can be variously adjusted depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, .

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. However, it is preferably administered at a daily dose of 0.001 to 100 mg / kg body weight, more preferably 0.01 to 30 mg / kg body weight. The administration can be carried out once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. The method of administration is not limited and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra-cerebroventricular injection.

The pharmaceutical composition of the present invention can be prepared into various pharmaceutical formulations, and there is no limitation on the form of the formulation.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Human hematopoietic stem cells Cell mass  ( blood - born hematosphere , BBHS )

(1) Mononuclear cell separation step in peripheral blood

Peripheral blood was obtained by applying Heparin (approximately 100 ul) to a 50 ml syringe. 10 ml of peripheral blood was poured into 50 ml tubes, and 30 ml of PBS (phosphate-buffered saline) was added and carefully mixed. 10 ml of Ficoll used for the density gradient in the diluted blood was slowly poured into the bottom of the tube using a pipette to separate the clear Ficoll layer and the red blood layer and then the centrifugation was performed at 2500 rpm at 25 ° C for 30 minutes, Respectively. After confirming the separation of the yellow serum layer, the white mononuclear layer, the red erythrocytes and the polynuclear morphology layer at the bottom of the transparent Ficoll layer, the upper yellowish serum layer was inhaled and the white mononuclear layer was carefully transferred to a new tube. The transferred mononuclear layer was divided into two tubes, filled with PBS, and centrifuged at 1800 rpm for 10 minutes at 4 ° C. The cell pellet was vortexed and unfolded to single cells, filled to the end with PBS, and centrifuged at 1700 rpm for 10 min at 4 ° C. The above rinse procedure was repeated three times to remove the substances used in the density gradient and the residues in the blood. Cell counts were measured with a hemocytometer prior to the final centrifugation.

(2) Three-dimensional culture step

The rinsed cells were suspended in an ultra-low-attach culture dish at a density of 10 6 / ml or higher at a density of 10 6 / ml or higher using a culture medium supplemented with 5% FBS in Endothelial basal medium-2 (EBM-2) The cells were incubated at 37 ° C in a 5% CO ₂ -concentrated incubator and the same medium was added for the first two days after incubation.

FIG. 1 is a schematic diagram showing a process of culturing a blood-born hematosphere (BBHS) for 5 days after the separation of human peripheral blood mononuclear cells (PBMC).

(3) Cell mass  Disassembly step into single cell

The cell mass obtained through the culturing process was separated into single cells for use in characterization and treatment. The suspended cells were collected by centrifugation at 4 ° C for 10 minutes at 1,700 rpm. After the cells were collected by centrifugation, the cells were collected by centrifugation. The cell pellet was lightly loosened with 1 ml of Accutase, a cell disassembly solution, and cultured in a 37 ° C incubator for 2 to 3 minutes. After incubation, 1 ml of the cell culture was added, followed by several pipetting, followed by centrifugation at 1,700 rpm at 4 ° C for 10 minutes.

Example  2. Angiogenesis-related genes and Marker  Expression Study in vitro  Experiment)

The results of examining the expression of genes, receptors, and markers related to angiogenesis in the cell mass (BBHS) formed in Example 1 are shown in FIGS. 2 to 9.

(1) human hematopoietic stem cells Of cell mass (BBHS)  Self-germination confirmation

GFR Matrigel (BD Biosciences) was thickly coated on a 35 mm Confocal dish (ibidi) on ice at approximately 200 ul and then incubated at 37 ° C in an Incubator. After that, the human blood-derived hematopoietic cell (BBHS) cultured for 5 days was observed with a microscope and only the spheres were collected and carefully raised on a 35 mm thick confocal dish coated with Matrigel. The culture medium was replaced with EGM-2MV (Lonza). After 24 hours and 72 hours, germination of human blood-derived blood cell mass (BBHS) was confirmed by a microscope, and it was confirmed that human blood-derived blood cell mass (BBHS) germinated by itself 2).

(2) VEGFR2  And PECAM -1 expression and self-germination confirmation

Human blood hematopoietic cell mass was placed on a 35 mm Confocal dish coated with GFR Matrigel in the same manner as in Example 2 (2), cultured for 24 hours, fixed, and washed three times with PBS And washed. Immunofluorescence was performed with VEGFR-2 (KDR) and VEGFR-2 (red arrow) as shown in FIG. 3. As a result, .

In addition, as shown in FIG. 4, when the human blood-derived hemocyte cell mass was cultured on a Matrigel-coated dish, the platelet endothelial cell adhesion molecule (PECAM-2), as well as VEGFR- 1, red), and a tip cell (arrow) was observed.

(3) VEGF  And CXCR4  Confirmation of expression

The human blood-derived hematopoietic cell (BBHS) cells were cultured for 5 days, and then immunofluorescent staining revealed that VEGF, green, and stomach) and CXC chemokine receptor type 4 (CXCR4, green, Below) was expressed.

(4) VEGF  - VEGFR2  ( KDR ) loop signaling this BBHS - Angiogenic niche Confirmation of importance to

VEGF and VEGFR2 (KDR) were inhibited by using a VEGF antibody and its receptor, a chemical inhibitor of VEGFR2 (SU1498), before the cell mass was formed (0 hrs). VEGF neutralizing antibody (R & D) was treated with 10 ug / ml, and VEGFR2 chemical inhibitor SU1498 (Calbiochem) was treated with 10 uM. As a result, as shown in Fig. 6, it was confirmed that the formation of human blood-derived blood cell mass (BBHS) was abruptly decreased.

(5) RT - PCR  , Which is important for angiogenesis Cytokine  And Receptor  Confirmation of expression

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was performed by separating RNA from Fresh Human PBMC and the human blood derived hematopoietic cell mass (BBHS) at 3 and 5 days respectively. We have identified IL-9, CXC chemokine receptor type 1 (CXCR1), CXCR2, VEGF, KDR, hepatocyte growth factor (HGF), c-Met and Matrix metalloproteinases 9 (MMP-9), which are known to be important for angiogenesis , And the primers used at this time are shown in Table 1 below.

primer Sequence TM Access Number Product size (bp) IL-8 Forward 5'-GGCCGTGGCTCTCTTGGCAG-3 ' 60 ° C NM_000584.3 178 Reverse 5'-TGTGTTGGCGCAGTGTGGTCC-3 ' CXCR1 Forward 5'-GAGCCCCCGAATCTGACATTA-3 ' 60 ° C NM_000634.2 222 Reverse 5'-AGTGCCTGCCTCAATGTCTCC-3 ' VEGF Forward 5'-GGGCAGAATCATCACGAAGT-3 ' 60 ° C NM_001025366.2 211 Reverse 5'-TGGTGATGTTGGACTCCTCA-3 ' KDR Forward 5'-ATGCTGGACTGCTGGCACGG-3 ' 64 ° C NM_002253.2 289 Reverse 5'-TCACAGGCCGGCTCTTTCGC-3 ' HGF Forward 5'-CTGGTTCCCCTTCAATAGCA-3 ' 60 ° C NM_00601.4 168 Reverse 5'-CTCCAGGGCTGACATTTGAT-3 ' c-Met Forward 5'-CCAATGGCCTGCAGCCGTGA-3 ' 59 ℃ NM_001127500.1 199 Reverse 5'-CTGTTCTGGGGCTGCCGCTC-3 ' MMP-9 Forward 5'-CAACATCACCTATTGGATCC-3 ' 56 ℃ NM_004994.2 482 Reverse 5'-CGGGTGTAGAGTCTCTCGCT-3 ' GAPDH Forward 5'-GAGTCAACGGATTTGGTCGT-3 ' 60 ° C NM_002046.3 185 Reverse 5'-GACAAGCTTCCCGTTCTCAG-3 '

As shown in FIG. 7A, when BBHS was cultured for 3 days (3D) and 5 days (5D) in contrast to Fresh Human PBMC (0D), cytokines known to be important for the angiogenesis increased.

In addition, as shown in FIG. 7 (b), enzyme-linked immunosorbent assay (ELISA) was performed to confirm whether secretion of molecules known to be important for RNA as well as angiogenesis was actually secreted. BBHS) increased the secretion of VEGF, HGF, and IL-8.

(6) MMP - 9 of  Check for increased activity

As shown in FIG. 8, the activity of Matrix metallopeptidase 9 (MMP-9) was increased by performing the MMP-9 zymography assay using the supernatant of the hematopoietic stem cells derived from the liver.

(7) Of HUVEC  Confirm movement and tube formation increase

When Human Umbilical Vein Endothelial Cell (HUVEC) was cultured using the supernatant of human blood-derived blood cell culture (BBHS) cultured for 5 days, migration and tube formation of HUVEC ) Was increased.

Example  3. Verification of angiogenic effect in vivo  Experiment)

The present inventors have demonstrated that blood vessel-derived hematopoietic cell (BBHS) formed in Example 1 is tested in pre-clinical phase to enable angiogenesis.

Specifically, the human blood-derived hematopoietic cell of the present invention is induced by inducing ischemic action in the Hindlimb of the Nude mouse with reduced immune system, and then the ischemic preconditioning is performed through Laser Doppler perfusion imaging (LDPI) Perfusion was measured at 3, 7, and 14 days after ischemia. As a result, as shown in FIG. 10A, when human blood hematopoietic cell masses formed in Example 1 were directly injected or disintegrated and cell debris was injected as compared to the group injected directly with phosphate buffered saline (PBS) or human blood mononuclear cells Perfusion was found to be better.

Immunofluorescence staining was performed using antibodies specific for human cells and alpha smooth muscle actin (SMA-alpha, red) of the vascular endothelial cell marker Cluster of Differentiation 34 (CD34, green) As shown, it was confirmed that human hematopoietic stem cells from the blood can actually cause vasculogenesis.

In addition, BS-1 Lectin was stained for blood vessels. As shown in FIG. 10C, it was confirmed that more blood vessels were formed when the cells were injected.

This result suggests that efficient induction of vascular endothelial cells and vascular smooth muscle cells through effective three-dimensional culture of blood mononuclear cells enables blood vessel formation to be efficiently performed. Therefore, it is expected that a therapeutic agent for ischemic diseases can be developed using the human hematopoietic cell mass (BBHS) formed in Example 1 above.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (7)

A pharmaceutical composition for treatment of lower limb ischemia comprising a vascular endothelial cell and a vascular smooth muscle cell differentiation-inducing human blood-derived blood cell mass,
Wherein said human hematopoietic cell mass is formed through three-dimensional coagulation culture by separating mononuclear cells from human blood. delete The method according to claim 1,
Wherein the human blood-derived hemocyte cell mass is separated and used as a single cell. delete The method according to claim 1,
Wherein said hematopoietic cell derived from human blood forms blood vessels. delete delete

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