KR101590722B1 - Pharmaceutical composition for treating nervous system disease comprising human blood derived hematosphere - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the treatment of neurological diseases including hematopoietic cells derived from human blood. More specifically, the present invention relates to a novel cell therapy agent capable of treating neurological diseases by inducing neural progenitor cells and neurons through effective three-dimensional culture using blood mononuclear cells and ultimately enabling efficient differentiation into neurons It is expected to be used for

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for treating a nervous system disease comprising hematopoietic cells derived from human blood,

The present invention relates to a method for treating a neurological disease using a hematopoietic cell derived from human blood, and the like.

Stem cells are the cells that form the basis of cells or tissues that constitute an individual. They are characterized by their ability to self-renew and to differentiate into cells with specific functions depending on the environment Cells. In addition, stem cells are totipotent stem cells that are formed when the embryo begins to divide at first according to the type of the differentiable cells, pluripotent stem cells in which the cells continue to divide, And multipotent stem cells in mature tissues and organs.

A fertile stem cell is a fertilized egg that has sufficient ability to grow into an individual when fertilized in the uterus. It is an omnipotent cell capable of retrieving all the structures (embryo and placenta) necessary for the formation of the embryo. Stem cells are a slightly advanced stage of development than embryos and are the inner cell mass of the blastocyst (ICM). ICM is distinguished from trophoblast, which is a group of cells that will form the body of a fetus later. If ICM is put into the uterus, the placenta is not formed and the fetus can not be formed. However, It means a state in which the ability to differentiate into all kinds of cells is not lost. The cells cultured while maintaining the pluripotency of the ICM are embryonic stem cells.

Duplicate stem cells appear after more progression, and the fate of cells has been determined to some extent to differentiate into specific lines. These cells exist not only in the fetus but also in children and adults, and play a role in continuously supplying the cells in tissues with a rapid cycle of cell replacement. For example, hematopoietic stem cells in the bone marrow, and undifferentiated cells in the epithelial tissues that constitute the digestive wall. It has been found that the adult stem cells of the adult population known to have no regenerative ability have been found to have redundant stem cells, and since the redundant stem cells can also be obtained from adults, they do not cause ethical problems and the types of differentiated cells are already limited compared to embryonic stem cells It is rather easy to obtain cells with specific traits.

Currently, researches for the development of homogeneous human cells and tissues using the characteristics of embryo and pluripotent stem cells are being carried out by world-class biotechnology companies. Hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. Hematopoietic stem cells differentiate into hematopoietic cells such as lymphocytes, leukocytes, and red blood cells after bone marrow transplantation. Neural stem cells become neuronal cells such as neuronal cells, star cells, and rare proliferating cells.

Immortality and pluripotency of stem cells provide a good in vitro model for the study of human development. In addition, drug testing and toxicity tests on homogeneous human tissues or cells obtained from stem cells can be performed to develop new drugs, and a large amount of cells or tissues capable of replacing damaged tissues can be obtained It is expected to be available for the treatment of intractable diseases.

On the other hand, neurodegenerative disease refers to a disease caused by permanent destruction or dysfunction of the nerve cells constituting the central nervous system. Even though it is a serious social problem, neural tissue has a limited ability to recover when damaged, A fundamental treatment for neurodegenerative diseases has not been developed yet. In other words, the theory that central nervous system can not be regenerated until now, the treatment of refractory neurological diseases has been systemically administered medicines and enzymes, and it has been supplemented with deficient neurotransmitters, and brain- There is a limit to the ability to transport to a desired location with a blood-brain-barrier. Recently, various viral vectors have been used for gene therapy in neurons, but they have not been applied to a wide range of lesions and have been limited in reconstructing damaged neurons and circuits.

Stem cell therapy has been actively researched as a new treatment for this intractable brain disease. Replacement of damaged cells is necessary for stem cell therapy to secrete necessary neurotransmitters, regeneration), the interest in therapeutic utility is increasing.

In order to solve the above-mentioned problems, the present inventors have developed a hematopoietic cell derived from human blood to separate the mononuclear cells from human blood to create an environment similar to a human body, and it is possible to differentiate into neural lineage cells by applying it to specific culture conditions And finally completed the present invention.

Accordingly, the present invention provides a method for treating a neurological disease using a hematopoietic cell 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 neurological diseases including 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, when the medium is replaced with a medium for promoting differentiation into neurons after formation of blood cell-derived hematopoietic cells, the cells are induced to neural progenitor cells and / or neurons.

In another embodiment of the present invention, the neurological disease is characterized by including a degenerative neurological disease, an ischemic neurological disease, and a nerve damage disease.

The present invention also provides a method for treating a neurological disease by administering to a subject a pharmaceutically effective amount of the above pharmaceutical composition.

Human blood derived hematopoietic cell, which is formed by separating mononuclear cells from human blood, has the ability to differentiate into cells of different lines in a specific environment. Since the source is human blood, supply is very smooth compared to other stem cell sources And the separation cost is very low. In addition, there is no risk of immunity and teratoma, and it has the greatest merits in practical application of clinical application. It is the most stable autogenous adult stem cell which can give surgical effect without surgical operation. Extracellular matrix, Cytokine , And growth factor.

According to the present invention, it is expected that it will be possible to develop a cell therapy agent capable of inducing neural progenitor cells and / or neurons using the hemocyte cell line derived from human blood and ultimately treating diseases related to nerve damage. Also, according to the present invention, it is possible to overcome limitations such as tumor generation, immunodeficiency, and ethical problems that have been a problem of conventional stem cell therapy agents.

FIG. 1 is a schematic diagram showing a process of culturing and producing a hematopoietic cell derived from human blood after dense coagulation culture of a mononuclear cell isolated from a human blood using a three-dimensional culture technique.
FIG. 2 is a schematic diagram showing a process of inducing differentiation into neurons after formation of human blood-derived blood cell mass.
FIG. 3 is an image of cells after inducing differentiation into neurons as in the schematic diagram shown in FIG. 2. FIG.
FIG. 4 shows the result of immunofluorescence staining for inducing neural progenitor cells after induction of neuronal differentiation. Green means Nestin and red means Musashi.
FIG. 5 shows the result of immunofluorescence staining for neuronal differentiation induced by neuronal differentiation. Green means Sox2 and red means beta-III tubulin.

The present inventors have identified the nerve cell-friendly microenvironment through the blood-born hematosome (BBHS) derived from human blood and the ability of mononuclear cells to differentiate into neurons. The nerve cell-friendly microenvironment due to the formation of blood cell-derived hematopoietic cell mass is effective in preventing cell death protection in neural stem cells, cell death protection effect in differentiated neural cell lines, inducing differentiation into neural cells in neural stem cells, , And peripheral nerve regeneration effects. The present invention has the effect of promoting the survival and differentiation of neurons in stem cell therapy and gene therapy using hematopoietic stem cells derived from human blood, and is useful for prevention of various types of degenerative neuronal diseases, ischemic neuronal diseases or neuronal injury diseases such as dementia and cerebral ischemia It is expected to be used in the development of stem cell therapy.

Accordingly, the present invention provides a pharmaceutical composition for the treatment of neurological diseases including hematopoietic cells derived from human blood. The hematopoietic cell derived from human blood is induced into neural progenitor cells or nerve cells, and the nervous system diseases include degenerative nerve diseases, ischemic nerve diseases, nerve damage diseases and the like.

The term "blood-born hematopoietic cells (BBHS)" as used in the present invention refers to an aggregate of mononuclear cells existing in the blood and specific cells having stem cells, and forms a three- It means that it forms a spherical shape like the inner cell masses of aeration.

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 a neurological disease disorder or an abnormal disease by administering to a subject a pharmaceutically effective amount of the above 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 a 50 ml tube, 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 the yellow serum layer, the white mononuclear layer, the red erythrocytes and the polynuclear morphology layer were separated at the upper part and the lower part of the transparent monocyte layer, the yellow mononuclear 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 into single cells, then filled to the end with PBS and centrifuged at 1,700 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. Human hematopoietic stem cells In the cell mass  Identification of neuronal differentiation

The cell mass (BBHS) formed in Example 1 was induced to differentiate into neurons as shown in the schematic diagram of FIG.

Specifically, human peripheral blood mononuclear cells were three-dimensionally cultured for 10 days to form human blood-derived hematopoietic cell mass. Then, the cell mass was transferred to a general culture dish instead of the ultra-low adhesion culture dish, and hFGF, hEGF, NSF-1, and GA were added, and the cells were cultured for 7 days. An image taken by an Olympus IX2 inverted fluorescence microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP50 CF CCD camera is shown in FIG. As a result, most of the hematopoietic cells derived from human blood are well differentiated into nerve cells.

Example  3. Protein characterization after induction of neuronal differentiation

After the formation of human blood-derived hematopoietic cells, the neuronal differentiation was induced by the method of Example 2, and whether it was actually induced into neural progenitor cells was confirmed by immunofluorescence staining. The results are shown in FIG.

As shown in Fig. 4, Nestin (green), a marker of neural progenitor cells, was partially stained and Musashi (red), another neuro progenitor marker, was mostly stained. DAPI (blue) was used for nuclear staining.

In addition, immunofluorescence staining was performed to confirm whether the cells differentiated into neurons through induction of neuronal differentiation after formation of human blood-derived hematopoietic cells. The results are shown in FIG.

As shown in FIG. 5, Sox2 (green), which maintains the undifferentiated state of neural stem cells, expresses only a part of it. In the case of beta-Ⅲ tubulin (red), a representative marker of neuron cells, It is shown that most of them are expressed in nerve cells.

The above results indicate that it is possible to induce neural progenitor cells and differentiate into neurons in the hematopoietic stem cells derived from human blood. Ultimately, it is expected that the neuronal cells can be used to treat neurological diseases. Therefore, It is expected to be used for the development of cell therapy.

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 (2)

A method for differentiating human blood-derived mononuclear cells into neurons comprising the steps of:
A step of three-dimensionally coagulating and culturing monocyte cells isolated from human-derived blood to form a hemocyte-derived human blood-derived cell mass; And
And culturing the human blood-derived hemocyte cell mass in a neuron differentiation medium.
The method according to claim 1,
Wherein the neural cell differentiation medium comprises human fibroblast growth factor (hFGF), human epidermal growth factor (hEGF), neural survival factor (NSF) -1 and GA (glutamic acid).

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