KR20090055691A - Composition for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells, comprising a human umbilical cord blood-derived mesenchymal stem cell as an active ingredient - Google Patents

Abstract

A composition for inducing the differentiation and proliferation of a neural precursor cell or neural stem cell to a nerve cell is provided to treat nerve injury disease using a mesenchymal stem cell isolated from a human umbilical cord blood. A composition for inducing the differentiation and proliferation of neural precursor cell or neural stem cell to a nerve cell comprises a mesenchymal stem cell which is derived from a human umbilical cord blood. The neural precursor cell or neural stem cell is differentiated and proliferated by co-culturing with the mesenchymal stem cell. The ratio of neural precursor cell or neural stem cell is 1:0.1-1:10. The composition is injected on the damaged part of subject such as a mammal. The nerve injury disease comprises stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system diseases, and spinal cord injury disease.

Description

COMPOSITION FOR INDUCING DIFFERENTIATION AND PROLIFERATION OF NEURAL PRECURSOR CELLS OR NEURAL STEM CELLS TO NEURAL CELLS, COMPRISING comprising human cord blood-derived mesenchymal stem cells as an active ingredient A HUMAN UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELL AS AN ACTIVE INGREDIENT}

The present invention comprises differentiation of neural precursor cells or neural stem cells into neurons, including mesenchymal stem cells derived from human umbilical cord blood as an active ingredient. It relates to a composition for inducing proliferation.

Strokes known as incurable diseases, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system diseases, and Spinal cord injury disease is a disorder in which nerve function is caused by nerve cell damage. The method of treating nerve cells damaged by these diseases is generally treated with drug therapy or surgical surgery. . However, this treatment has been pointed out as a problem by damaging many other normal cells.

Recently, cell replacement therapy, which supplies cells destroyed or damaged by diseases from the outside, has been proposed as an effective treatment. In such cell therapies, stem cells capable of proliferation and differentiation into tissues in need of regeneration are in the spotlight.

Stem cells are cells prior to differentiation into cells constituting the tissue, and are capable of infinite proliferation in an undifferentiated state and have a potential of being differentiated into cells of various tissues by a specific differentiation stimulus.

Neural stem cells also have self-replicating capacity and are known to be neurons and / or glia, such as astrocytes, oligodendrocytes and / or Schwann cells. Undifferentiated cells with differentiating ability to differentiate. Neural stem cells are differentiated into neural cells, such as neurons or glia, through the steps of neural precursor cells or glia precursor cells that produce specific nervous system cells.

Meanwhile, mesenchymal stem cells can be differentiated into bone, cartilage, adipose tissue, muscle, tendons, ligaments, and nerve tissues, and thus are attracting attention as cells suitable for cell therapy. Currently, bone marrow is the most representative tissue from which mesenchymal stem cells can be obtained. However, mesenchymal stem cells present in bone marrow have limited application range due to limited differentiation and proliferative capacity. Due to limitations derived from bone marrow, several steps are required, and the procedure is complicated, It is usually accompanied by time, mental and physical pain. In addition, for bone marrow transplantation, there is a problem in that a donor does not show a graft-versus-host response because the antigenic phenotypes are matched through histocompatibility antigen comparison.

In recent years, research has been actively conducted, as it is known that there are a large amount of stem cells in cord blood. Clinically, many attempts have been made to treat blood-related diseases through cord blood transplantation, and cord blood banks are being activated in Korea to freeze cord blood and preserve it for use after several years for autograft treatment.

Umbilical cord blood, unlike the bone marrow, can be obtained through a simple procedure in the umbilical cord that is thrown away during the delivery process, and contains a large number of hematopoietic stem cells and stem cells compared to the amount. In addition, graft-versus-host reactions that can occur during cord blood transplantation are considerably less than in bone marrow transplantation, and studies are being conducted worldwide to confirm the characteristics of stem cells included in cord blood and to expand their applications in clinical practice.

However, it has not been reported that mesenchymal stem cells isolated and cultured from cord blood differentiate and proliferate neighboring neural progenitor cells or neural stem cells into neurons.

Accordingly, an object of the present invention is to provide a composition for inducing differentiation and proliferation of neural progenitor cells or neural stem cells into neural cells, including mesenchymal stem cells isolated from umbilical cord blood.

Another object of the present invention is to provide a method for differentiating and proliferating neural progenitor cells or neural stem cells into neurons using mesenchymal stem cells isolated from umbilical cord blood.

According to the above object, the present invention provides a composition for inducing differentiation and proliferation of neural progenitor cells or neural stem cells into neural cells, including mesenchymal stem cells isolated from umbilical cord blood as an active ingredient.

According to the above another object, the present invention provides a method for differentiating and proliferating neural progenitor cells or neural stem cells into neural cells, including co-culture of cord blood-derived mesenchymal stem cells with neural progenitor cells.

In accordance with another object, the present invention provides a method for treating neuronal cell damage diseases comprising administering cord blood-derived mesenchymal stem cells to a subject in need of recovery of neuronal cell damage.

The mesenchymal stem cells obtained by isolating and culturing from cord blood according to the method of the present invention differentiate and proliferate neural progenitor cells or neural stem cells into neurons, and thus, the mesenchymal stem cells of the present invention and compositions comprising the same include stroke, Parkinson's disease, and Alzheimer's disease. It can be usefully used for cell therapy for neurological damage diseases including disease, peak disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system disease and spinal cord injury disease.

As used herein, the term "umbilical cord blood" refers to blood collected from a umbilical vein connecting the placenta and the fetus in all mammals, including humans. The term "umbilical cord blood-derived mesenchymal stem cells" as used herein refers to mesenchymal stem cells isolated from umbilical cord blood in mammals, preferably humans.

In addition, the term "nerve damage disease" as used herein refers to a disease in which the nerves involved in movement and sensation are damaged and abnormalities occur in behavioral functions including movement and sensation. Stroke, Parkinson, Seed disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system diseases, and spinal cord injury disease It may be illustrated, but is not limited thereto.

In addition, the term "treatment" used in the present invention 1) has not yet been diagnosed as having a neurological damage disease, but the disease or disorder develops in animals, preferably mammals, more preferably humans, which tend to do so Prevention of the thing means 2) suppression of neurologically damaging diseases, i.e. inhibition of development and 3) alleviation of neurologically damaging diseases.

In addition, as used herein, the term "neural cell" refers to neurons and / or glia of the central or peripheral nervous system, for example, asrocytes, oligodendrocytes and / or the like. Or Schwann cell.

In order to separate monocytes including mesenchymal stem cells from umbilical cord blood, a known method such as Ficoll-Hypaque density gradient method can be used, and specifically, the umbilical vein before detachment of the placenta after delivery. Umbilical cord blood collected from the umbilical vein is centrifuged with a Ficoll-Hypaque gradient to obtain monocytes, which are then washed several times to remove impurities.

The mononuclear cells thus separated can be used immediately for isolation and culture of mesenchymal stem cells or can be used after prolonged storage by cryogenic freezing.

In the present invention, in order to isolate and culture the mesenchymal stem cells from the cord blood-derived monocytes isolated as described above, for the culture of animal cells containing 5 to 30% by weight, preferably 5 to 15% by weight of FBS monocytes Suspended in a medium such as DMEM, α-DMEM, Eagle's basal medium, RPMI 1640 medium, and the like, and then dispensed into a medium having the same composition at the appropriate concentration to supply 5% carbon dioxide. Incubate in the incubator. When the cultured cells form a monolayer, the mesenchymal stem cells amplified in the shape of a spindle using a phase contrast microscope are identified, and then the subcultures are repeated until the mesenchymal stem cells are sufficiently amplified. SE et al., Cytotherapy, 6 (5): 476-486, 2004).

The mesenchymal stem cells isolated and cultured from cord blood as described above may induce differentiation and proliferation of neural progenitor cells or neural stem cells into neural cells when cocultured with neural progenitor cells or neural stem cells.

Accordingly, the present invention provides a composition for inducing differentiation and proliferation of neural progenitor cells or neural stem cells into neural cells, including mesenchymal stem cells derived from umbilical cord blood. Compositions containing umbilical cord blood-derived mesenchymal stem cells of the present invention are nerve damage diseases, preferably stroke, Parkinson's disease, Alzheimer's disease, Peak disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system disease and spinal cord injury disease, preferably It can be usefully used for cell therapy for stroke patients or patients with spinal cord injury disease.

The composition of the present invention may further include a pharmaceutically acceptable additive in addition to the active ingredient.

The composition for inducing neuronal differentiation and proliferation of neural progenitor cells or neural stem cells of the present invention comprising the cord blood-derived mesenchymal stem cells as an active ingredient is a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field It can be formulated in the formulation of. Formulations suitable for this purpose are preferably parenteral administration preparations for injection or topical administration. In this case, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants that are generally used can be used together.

The formulation of the present invention thus prepared is administered parenterally, for example, directly to the lesion, in addition to the cerebrospinal fluid, for example, to spinal cord or cerebral parenchyma, or to supply blood flow to the vein or lesion according to a conventional method. It may also be administered through an artery, and may be directly administered to or around the damaged area of the brain or spinal cord. For direct administration to the site of injury, for example, a clinical method published by Douglas Kondziolka, Pittsburgh, 1998 can be used. That is, the skull of the subject is first incised into a pea size of about 1 cm in diameter, and then HBSS using a syringe with a long needle and a steeotactic frame for inserting the desired cell solution into the coordinates inside the brain. Injection of mesenchymal stem cell suspension mixed with Hank's balanced salt solution may be used.

The dose of the mesenchymal stem cells is 1 × 10 ⁵ to 1 × 10 ⁷ cells / kg body weight, preferably 5 × 10 ⁵ to 5 × 10 ⁶ cells / kg body weight, but divided once or several times May be administered. However, it is to be understood that the actual dosage of the active ingredient should be determined in view of several related factors such as the amount of neurons to be differentiated and expanded, the route of administration, the patient's weight, age and gender, and thus the dosage should be It is not intended to limit the scope of the invention in any aspect.

The present invention also provides a method for differentiating and proliferating neural progenitor cells or neural stem cells into neural cells, including co-culture of cord blood-derived mesenchymal stem cells with neural progenitor cells or neural stem cells. In the co-culture, cord blood-derived mesenchymal stem cells are mixed with neural progenitor cells or neural stem cells in a ratio of cells in a ratio of 1: 0.1 to 1:10, preferably 1: 1 to 1: 2, and DMEM, α-MEM, α It can be cultured in a general cell culture medium such as DMEM, Eagle's basal medium, RPMI 1640 medium. In this case, antibiotics such as gentamicin and / or 5 to 15% by weight of FBS may be further added to the culture medium. The incubation period is preferably 5 to 10 days.

In another aspect, the present invention provides a method for treating a neurological damage disease by administering cord blood-derived mesenchymal stem cells to the damaged area of the subject in need of treatment of neuronal cell disease. In this case, the subject may be a mammal including a human.

Umbilical cord blood-derived mesenchymal stem cells, when administered in a therapeutically effective amount to the damaged area, differentiate and proliferate neuronal progenitor cells or nerve stem cells of the peripheral central nervous system or peripheral nervous system into neurons to restore impaired nerve function and to treat nerve injury diseases. Can be.

The therapeutic effect of umbilical cord blood-derived mesenchymal stem cells of the present invention is significantly enhanced and lasts for a long time due to its ability to proliferate regenerated neurons.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Isolation and Culture of Cord Blood-derived Mesenchymal Stem Cells

Step 1) Obtaining Umbilical Cord Blood (UCB)

Umbilical cord blood samples were obtained from umbilical veins at birth with the consent of the mother. Specifically, a 16-gauge needle in a UCB collection bag containing 44 ml of CPDA-1 anticoagulant (green cross) was inserted into the umbilical vein to collect the UCB by gravity collection. All cord blood harvests were treated within 48 hours after harvesting and the overall cell viability was at least 90%.

Step 2) Isolation and Amplification of Mesenchymal Stem Cells

The cord blood obtained in step 1 was centrifuged with a Ficoll-Hypaque gradient (density: 1.077 g / ml, Sigma) to obtain monocytes, and washed several times to remove impurities. Cells were suspended by addition of minimal basal medium (α-MEM, Gibco BRL) containing 10% FBS (HyClone). The cells were dispensed in a minimum basal medium containing 5-15% by weight FBS at an appropriate concentration, and then cultured in a 37 ° C. cell incubator supplied with 5% carbon dioxide, changing medium twice a week. When the cultured cells formed a monolayer, the mesenchymal stem cells amplified in the shape of spindles were identified using a phase contrast microscope, and the subculture was repeated until the mesenchymal stem cells were sufficiently amplified (Yang SE). et al., Cytotherapy, 6 (5): 476-486, 2004).

Example 2: Incubation of NG108-15

Neuroblastoma X glioma hybrid (Neuroblastoma X glioma hybrid) (ATCC, Cat. No. ATCC-CRL-HB-12317) derived from mouse brain exhibiting physiologically and morphologically similar properties to neuronal progenitor cells were obtained from Dulbecco's modified Eagle's medium. ) (4 mM / L glutamine, 4.5 g / L glucose, 4.0 mg / L pyridoxine-HCl, 0.1 mM hypoxanthin-guanine, 400 nM aminopterin, 0.016 mM thymidine, 5-15 wt% fetal bovine serum) Incubated.

Example 3: Cultivation of Neural Stem Cells

Mouse fetal brain cortical neural stem cells (Chemicon, Cat. No. SCR029) were cultured in Neural stem cell basal medium (20 ng / ml FGF-2, 20ng / ml EGF and 2mg / ml heparin).

Example 4 Coculture of Cord Blood-derived Mesenchymal Stem Cells with NG108-15 I

Human cord blood-derived mesenchymal stem cells (hUCB-MSCs) obtained in Example 1 were 1: 1 in the medium of Example 2 using NG108-15 and a transwell chamber (FIG. 1) cultured in Example 2. Co-culture was carried out at the ratio of. Only NG108-15 was cultured under the same conditions as a control. The transwell chamber used for the culture is divided into a lower compartment and an upper compartment by a microporous membrane having pores of 1 μm, as shown in FIG. 1. Human umbilical cord blood-derived mesenchymal stem cells were cultured in the upper part and NG108-15 in the lower part based on the microwells of the transwell chamber.

Differentiation of NG108-15 was observed using a phase contrast microscope (× 100), respectively, after 4 and 7 days of culture. As shown in FIG. 2, it can be seen that NG108-15 (NG108) co-cultured with cord blood-derived mesenchymal stem cells have been differentiated into mature neuron-like cells that elongate and differentiate into spindle forms. have.

In addition, after 7 days of culture, immunostaining for tubulin-beta III, an early marker of neuronal development, was performed as follows to confirm that the differentiated cells were neuron-like cells.

hUCB-MSCs, NG108-15 and 1: 1 mixed cells of hUCB-MSC and NG108-15 were incubated in the cover slide, respectively, and then placed in 10% normal goat serum containing 0.3% Triton X-100 for 1 hour at room temperature. Blocked. Anti-tubulin-beta III mouse monoclonal antibody (Chemicon) with phycoerythrin attached as a primary antibody was added at a dilution of 1: 100, followed by overnight reaction at 4 ° C, followed by 0.01 M PBS. Washed three times with 5 minutes each.

As shown in FIG. 3, NG108-15 (NG108) incubated with mesenchymal stem cells of Example 1 showed distinct response to immunostaining for tubulin-beta III, demonstrating that differentiated cells are neuron-like cells. It was.

Example 5 Coculture of Cord Blood-derived Mesenchymal Stem Cells with NG108-15 II

Mesenchymal stem cells (hUCB-MSCs-1 and hUCB-MSCs-2) having different origins obtained by the method of Example 1 were incubated with NG108-15 for 7 days in the same manner as in Example 4, and a phase contrast microscope (× 100) was used. The differentiation and proliferation of the cells were observed. Further, while culturing only NG108-15 under the same conditions, as a comparative example, a group to which 1 mM of cAMP (NeuroReport 9, 1261-1265, 1998), which was previously known to differentiate NG108-15 into cells such as neurons, was added, and The differentiation and proliferation of the cells were also observed in the group in which only NG108-15 was cultured in the same medium. The results are shown in FIG.

As shown in FIG. 4, it can be seen that NG108-15 co-cultured with cord blood-derived mesenchymal stem cells were differentiated in the form of cells such as mature neurons. In addition, there was no significant difference in the differentiation-inducing activity of two different cord blood-derived mesenchymal stem cells.

Example 6: Co-culture of Cord Blood-derived Mesenchymal Stem Cells and Neural Stem Cells I

Mesenchymal stem cells hUCB-MSCs-1 and hUCB-MSCs-2 of Example 5 were co-cultured in the medium of Example 3 using the mouse fetal brain cortical neural stem cells of Example 3 and a transwell chamber. Human cord blood-derived mesenchymal stem cells were cultured in the upper part and mouse fetal cerebral cortical neuronal stem cells in the lower part based on the fine sclera of the transwell chamber, and the mesenchymal stem cells were 500, 1000, 2000, 4000 and 6000 cells / cm 2 Concentration was added to the medium, and neural stem cells were added to the medium at a concentration of 2000 cells / cm 2, respectively. In addition, only mouse fetal brain cortical neural stem cells were cultured under the same conditions as a control. After 7 days, the differentiation and proliferation of the cells were confirmed using a phase contrast microscope (× 100) (FIG. 5).

As shown in FIG. 5, it can be seen that the mouse fetal brain cortical neural stem cells co-cultured with cord blood-derived mesenchymal stem cells were differentiated into mature neuronal forms. In addition, there was no significant difference in the differentiation-inducing activity of two different cord blood-derived mesenchymal stem cells. In addition, it was confirmed that the differentiation and proliferation of neural stem cells by cord blood-derived mesenchymal stem cells were proportional to the concentration of mesenchymal stem cells co-cultured.

Example 7: Co-culture of Cord Blood-derived Mesenchymal Stem Cells and Neural Stem Cells II

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) obtained in Example 1 were co-cultured at a ratio of 1: 1 in the medium of Example 3 using the mouse fetal brain cortical neural stem cells of Example 3 and a transwell chamber (co -culture). Human umbilical cord blood-derived mesenchymal stem cells were cultured in the upper part and mouse fetal cerebral cortical neural stem cells in the lower part based on the fine sclera of the transwell chamber. As a control, only mouse fetal brain cortical neural stem cells were cultured under the same conditions.

After 4 and 7 days of culture, immunostaining for the initial neuronal markers of tubulin-beta III and MAP2 (microtubule-associated protein 2) was carried out in the same manner as described in Example 4, respectively. It was confirmed that the neurons.

As shown in FIG. 6, neural stem cells cultured with mesenchymal stem cells of Example 1 showed distinct responses to immunostaining for tubulin-beta III and MAP2, demonstrating that differentiated cells were neurons.

Example 8: Co-culture of Cord Blood-derived Mesenchymal Stem Cells and Neural Progenitor Cells or Neural Stem Cells

After NG108-15 and mouse fetal brain cortical neural stem cells were co-cultured with human cord blood-derived mesenchymal stem cells (hUCB-MSCs) for 7 days according to the method of Examples 5 and 6, trypan blue staining ) Was used to measure the number of viable cells (FIGS. 7 and 8).

As shown in Figs. 7 and 8, the number of NG108-15 and neurons was increased with the concentration of co-cultured mesenchymal stem cells, and these results indicate that mesenchymal stem cells isolated and cultured from umbilical cord blood are neuronal cells of neural progenitor cells. In addition to the differentiation of the furnace as well as proliferation at the same time indicating that it is very effective in increasing the number of neurons.

1 is a schematic diagram showing a transwell chamber used for co-culture of cord blood-derived mesenchymal stem cells and neural progenitor cells of the present invention,

2 shows the results of observing the differentiation and proliferation of cells using phase contrast microscopy (× 100), respectively, after 4 and 7 days of NG108-15 cells alone or co-cultured with cord blood-derived mesenchymal stem cells of the present invention. ,

FIG. 3 shows immunostaining of tubulin beta-III, an early marker for neurons, after 7 days of NG108-15 cells alone or co-culture with cord blood-derived mesenchymal stem cells of the present invention. Result,

Figure 4 shows the differentiation and proliferation of cells using phase contrast microscopy (x100) after 7 days of NG108-15 cells alone and coculture with two cord blood-derived mesenchymal stem cells with different cAMP or different origins. One result,

5 is a result of co-culture of mouse fetal cerebral cortical neuron stem cells and cord blood-derived mesenchymal stem cells in a transwell chamber for 7 days by concentration, and then observed the differentiation and proliferation of cells using a phase contrast microscope (× 100).

Figure 6 shows immunity to tubulin beta-III (TubIII) and MAP2 (MAP2), which are early neurons markers, after 7 days of mouse fetal brain cortical neural stem cells alone or co-culture with cord blood-derived mesenchymal stem cells of the present invention. The result of staining (immunostaining),

7 is a result of measuring the number of living cells using trypan blue staining after co-culturing NG108-15 cells and cord blood-derived mesenchymal stem cells in a transwell chamber for 7 days by concentration.

8 is a 7-day co-culture of mouse fetal brain cortical neural stem cells and umbilical cord blood-derived mesenchymal stem cells in a transwell chamber, and measured the number of living cells using trypan blue staining. to be.

Claims (6)

Composition for inducing differentiation and proliferation of neural progenitor cells or neural stem cells into neural cells, comprising mesenchymal stem cells derived from umbilical cord blood. A method of differentiating and proliferating neural progenitor cells or neural stem cells into neural cells, comprising co-culturing mesenchymal stem cells derived from umbilical cord blood with neural progenitor cells or neural stem cells. The method of claim 2, The cord blood-derived mesenchymal stem cells and nerve precursor cells or neural stem cells ratio of 1: 0.1 to 1:10 characterized in that the ratio. The method of claim 1, A composition for administration to a site of injury in a subject in need of recovery of nerve cell damage. The method of claim 4, wherein Stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system diseases and spinal cord injury diseases (spinal cord injury disease) A composition for treating a nerve injury disease selected from the group consisting of. The method of claim 4, wherein A composition characterized in that the subject is a mammal.

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