KR20080094431A - Method for differentiating, culturing and isolating neural progenitor cells from peripheral blood mononuclear cells - Google Patents

Abstract

A method is provided to isolate and culture effectively neural progenitor cells, which are hard to be isolated from a human adult, maintained and proliferated, from peripheral blood mononuclear cells and supply a large amount of the neural progenitor cells using the peripheral blood mononuclear cells, thereby utilizing the neural progenitor cells as a means for healing various incurable diseases of central nervous system. A method for isolating, culturing and differentiating neural progenitor cells from peripheral blood mononuclear cells comprises the steps of: (a) harvesting mononuclear cells from peripheral blood of a patient; (b) culturing the obtained mononuclear cells in an EGM culture medium including fetal bovine serum(FBS), fibroblast-derived growth factor(FGF), epidermal growth factor(EGF), vascular endothelial cell-derived growth factor and insulin-like growth factor to age precursor cells; (c) culturing adhesive cells and floating cells obtained from the step(b) in the EGM culture medium including a resulting material of the culture medium; and (d) culturing the cells obtained from the step(c) in the EGM culture medium including the FBS. The method further comprises a step of culturing the cells obtained from the step(d) in a culture medium including retinoic acid, Folskolin, nerve growth factor(NGF), and an antibiotics. A composition for treating central nervous system diseases comprises the neural progenitor cells obtained by the method as an effective ingredient. The central nervous system diseases are one or more selected from spinal cord disease, Parkinson's disease, Alzheimer's disease and brain tumor.

Description

Methods for differentiating, culturing and isolating neural progenitor cells from peripheral blood mononuclear cells}

1 is a diagram showing the separation of effective autologous cells from acute cerebral infarction patients with the compositions and methods provided in the present invention.

Figure 2 is a diagram showing a state of proliferating and maintaining the neural progenitor cells by the composition and method provided in the present invention.

Figure 3 is a diagram showing the neuron-related gene expression patterns and proliferation rate of the cells isolated.

Figure 4 is a diagram showing the differentiation state into stable neurons with the compositions and methods provided in the present invention.

5 is a diagram showing the recovery of neurological disorders in mice transplanted with neuroprogenitor cells obtained by the compositions and methods provided in the present invention.

Figure 6 is a diagram showing that the transplanted neural progenitor cells can be effectively differentiated into the neurons in the animal in vivo and seated.

The present invention relates to a method for separating and culturing neuronal progenitor cells from peripheral blood-derived mononuclear cells and to differentiate them into neurons.

Stem cells can be largely divided into embryonic stem cells (ES cells) and adult stem cells (adult stem cells) according to differentiation potency and production time. Embryonic stem cells have the potential to differentiate into cells of most tissues of the human body, but there are ethical challenges in obtaining embryonic stem cells, and it is also easy to control the differentiation capacity of these cells in vivo. It hasn't happened. Adult stem cells, on the other hand, are stem cells that appear during the development of each organ of the embryo or during adult development, and their differentiation capacity is generally limited to cells that make up specific tissues. These adult stem cells remain in most organs after adulthood and play a role in supplementing the loss of normal or pathological cells.

Representative adult stem cells include hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Hematopoietic stem cells differentiate mainly into blood cells such as red blood cells, white blood cells, and platelets, while mesenchymal stem cells are cells such as osteoblasts, chondrocytes, adipocytes, and myoblasts. It is known to differentiate into. Recently, as the method of isolating and culturing embryonic stem cells or adult stem cells is known, many studies are underway for clinical application of cell therapy products. In particular, studies to regenerate damaged neurons by cell replacement therapy in strokes, spinal cord injuries, Parkinson's disease, and intractable brain diseases such as Alzheimer's disease are being actively conducted in all clinical and clinical areas.

For neural cell replacement therapy, it is necessary to secure neural progenitors / stem cells. However, only adult tissues capable of obtaining neural precursor cells are in extremely limited areas such as the periventricle and the hippocampus. Recently, attempts have been made to isolate hematopoietic stem cells, mesenchymal (mesenchymal) stem cells, and multipotent adult progenitor cells (MAPCs) from bone marrow and convert them into neurons (Kucia M, et al., Blood Cells Mol Dis 32: 52-57, 2004). However, due to the limited differentiation and proliferative capacity of the cells in the bone marrow, the scope of application is limited, and due to the limitation of bone marrow origin, the procedure for obtaining the cells is complicated, and the harvester suffers from pain. There is a problem in obtaining a cell. In addition, recently known bone marrow-derived multipotent adult progenitor cells have a wide range of use (neural cells, hepatocytes, endothelial cells, etc.) in terms of differentiation capacity, but with the limitation of bone marrow origin, repeat reproducibility for their isolation and culture. There is a problem that cannot be guaranteed (Reyes M, et al., J. Clin. Invest. 109: 337-346, 2002). In addition, recently, cord blood is known to have a large amount of stem cells, and research is being actively conducted. Since umbilical cord blood is known as a rich source of hematopoietic stem cells, many clinical trials have been made to treat blood-related diseases through umbilical cord blood transplantation, and umbilical cord blood can be stored for use after several years by freezing cord blood for autograft treatment. Banks are also active in Korea. However, these resources are also difficult to store in terms of autologous cell transplantation, and are difficult to obtain quickly and obtain large numbers of neurons.

As a result of research showing that vascular stem cells and various tissue stem cells circulate in peripheral blood, efforts have been made to use peripheral blood cells as a means of blood vessel regeneration or tissue regeneration. There have been few attempts to isolate and culture large numbers of neural progenitor cells directly from peripheral blood, and even when successfully isolated and cultured, their repeatability is very low. Moreover, in normal adults, these circulating stem cells are too small and easily aging, making them difficult to use for therapeutic purposes. Unlike bone marrow or umbilical cord blood, blood can be obtained repeatedly through simple procedures, and autologous cells can be collected to separate and proliferate neuronal progenitor cells and use them as a resource for transplantation treatment in brain diseases. Can be said to be very large. Therefore, the present inventors have studied a method for effectively obtaining neural progenitor cells that can be usefully used for cell replacement therapy.As a result, the method of separating and culturing neural progenitor cells from mononuclear cells isolated from peripheral blood and these cells in the laboratory and the brain The present invention has been completed by developing a differentiation induction method that can differentiate into tissue neurons.

It is an object of the present invention to provide a method for efficiently separating, culturing and maintaining neural progenitor cells from peripheral blood-derived mononuclear cells and a method for inducing differentiation of such cells into neurons.

The present invention aims to solve the limitation of autologous neural progenitor cell acquisition in adults, and to provide a reproducible method for separating and culturing neural progenitor cells by effectively acquiring peripheral blood-derived mononuclear cells.

In order to achieve the above object, the present invention

a) harvesting monocytes from peripheral blood of a patient;

b) The obtained monocytes are fetal bovine serum (FBS), fibroblast-derived growth factor (FGF), epidermal growth factor (EGF), vascular endothelial a primary culture step of culturing progenitor cells by culturing in vascular growth medium (EGM) medium containing a cell-derived growth factor, and an insulin-like growth factor;

c) secondary culture of the adherent cells and the floating cells obtained after the primary culture in the vascular proliferation medium of step b) comprising micromolecules derived from monocytes; And

d) isolating and culturing neural progenitor cells from peripheral blood comprising the step of tertiary culturing the cells cultured in step c) in an angioproliferation medium of step b) comprising fetal bovine serum (FBS) And differentiation methods.

In addition, the method of the present invention is the tertiary culture of the progenitor cells obtained after step d) comprising retinoic acid, Folskolin, nerve growth factor (NGF), adjuvant mixture and antibiotics. It provides a method for separating, culturing and differentiating nerve cells from peripheral blood, further comprising culturing in a medium.

In one embodiment of the present invention, the concentration of fetal bovine serum in the primary culture step is preferably 0.1 to 3%, most preferably 0.5-2%.

In one embodiment of the present invention, the micromolecules collectively refer to cell by-products, proteins and minerals of 2 micrometers or less from the primary culture.

In addition, in one embodiment of the present invention, the concentration of fetal calf serum in the tertiary culture step is preferably 5-10%.

In addition, in one embodiment of the present invention, the auxiliary mixture is preferably N2 supplement (N2 supplement) or B27 supplement (B27 supplement).

In addition, in one embodiment of the present invention, the antibiotic is preferably penicillin or streptomycin or a mixture thereof.

In another aspect, the present invention provides a composition for treating a central nervous system disease, the neuroprogenitor cells produced by the above method as an active ingredient.

In the present invention, the disease includes, but is not limited to, one or more diseases selected from the group consisting of stroke, spinal cord disease, Parkinson's disease, Alzheimer's disease, epilepsy, and brain tumors.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for screening subjects with easy acquisition of neural progenitor cells and a method for effectively separating and culturing neural progenitor cells by culturing peripheral blood-derived mononuclear cells sequentially in a medium for culturing animal cells, and a medium composition therefor. to provide.

In addition, the present invention provides a method for proliferating and maintaining the neural bulb / stem cells separated by the above method, and inducing differentiation into neurons.

In the present invention, the "primary culture medium" is a medium for culturing mammalian cells comprising inorganic salts, amino acids, vitamins and / or cofactors, and the medium commonly used for culturing animal cells in the animal cell culture art. Means, commercially available

One animal cell culture medium is EBM (Endothelial basal medium) and EGM medium containing low concentration of FBS, FGF, EGF, VEGF, IGF-1.

In the present invention, "secondary culture medium" is a medium that effectively separates neural progenitor cells from the mononuclear cell group cultured in the primary culture medium. Secondary culture medium means a medium to which microparticles derived from monocytes are added in the composition of the primary culture medium.

In the present invention, "tertiary culture medium" is a medium for proliferating cells cultured in the secondary culture medium to homogeneous neuroprogenitor cells. Tertiary culture medium means a medium to which a high concentration of FBS is added in the primary culture medium.

The method of the present invention for separating and culturing neural progenitor cells from peripheral blood-derived mononuclear cells comprises: a primary culture medium in which peripheral blood-derived mononuclear cells are added to a medium for conventional animal cell culture with low concentration of FBS and various growth factors; A secondary culture medium in which monocyte-derived microparticles are added to the primary culture medium; And sequentially culturing the primary culture medium in a tertiary culture medium to which a high concentration of FBS is added.

In the present invention, "a subject easily acquiring neural progenitor cells" means a person having high yield of neural progenitors / stem cells through peripheral blood monocyte culturing. The origin of progenitor / stem cells circulating in peripheral blood is likely to be cells in the bone marrow, and substances or signals from damaged biological tissues are known to induce their escape into peripheral blood. Therefore, the damage of acute nerve tissue is expected to promote the escape signal of the neural progenitor / stem cells from the bone marrow, and thus, the targets that can easily acquire the neural progenitor cells can be selected and managed according to the extent of the injury.

In order to separate monocytes from peripheral blood, a known method such as a histopack density gradient method can be used.

The mononuclear cells thus separated can be used immediately for isolation and culture of neural progenitors / stem cells or can be used after prolonged storage by cryogenic freezing. In order to isolate and culture neuronal progenitor cells from the peripheral blood-derived mononuclear cells separated as described above, mononuclear cells are sequentially cultured in primary, secondary and tertiary culture media. In the present invention, the culture medium is based on EGM and may be used by adding antibiotics and necessary components according to the purpose of each culture. Antibiotics that can be added to the medium may be any antibiotic commonly used for animal cell culture in the animal cell culture art, for example penicillin, streptomycin, kanamycin , Antibiotics for animal cell culture, such as ampicillin, may be added alone or in combination.

Primary culture is the step of maturing progenitor cells among peripheral blood mononuclear cells. Primary cultures were thawed fresh mononuclear cells or cryogenic cryopreserved mononuclear cells directly isolated from peripheral blood, and then concentrations of 5x10 ⁶ to 1x10 ⁷ cells / well (6 well plates) in 6 well plates coated with 2% gelatin. Inoculate with and incubate primary culture medium. After 7 days, all the adherent cells and the floating cell group are collected and newly inoculated in a 6 well plate, and the micromolecules dissolved in the medium are added by ultracentrifugation to make a secondary culture medium. Subsequently, the secondary culture medium is replaced every 3-5 days while incubating at 37 ° C. in a 5% carbon dioxide incubator for 1-2 weeks. Secondary culture is a step of separating neural progenitor cells from a variety of mononuclear cells contained in peripheral blood, inducing neural progenitor cells to form a multicellular cell population that grows while attaching to the bottom of the culture vessel (see FIG. 1). .

When the medium is exchanged in the first and second culture stages, a partial medium exchange method is used to preserve the state of the existing medium as much as possible. When only floating cells or adherent cells are isolated and cultured, the neuroprogenitor cell separation yield is reduced by 80%, and the separation yield of the whole medium exchange method that discards the whole medium and replaces it with a new medium is reduced by 50%. In the partial medium exchange method, the culture medium is carefully aspirated and discarded with a micropipette while maintaining the existing culture state as much as possible so that there is no cell loss during medium exchange.

When the formation of the multilayered cell population is observed by the secondary culture, the medium is changed to the tertiary culture medium, in which the high concentration FBS is added to the primary culture medium. The tertiary culture is replaced after the secondary culture and incubated at a temperature of 37 ° C. in a 5% carbon dioxide incubator for 1-2 weeks, at which time fresh medium is supplied every 3-5 days. Tertiary culturing removes cells that do not adhere to the bottom of the culture vessel, and transforms the multilayered cell population consisting of spindle-shaped cells into a single cell population showing neuroblast-like cells. Inducing proliferation.

When the cells forming the mononuclear cell population showing the neuroblast morphology by the third culture grow to 80-90% adhesion, the medium is removed, washed with PBS, and treated with trypsin / EDTA solution to recover the cells. The recovered cells were inoculated in a tertiary culture medium at a concentration of 1 × 10 ⁴ to 5 × 10 ⁴ cells / cm ² , incubated at 37 ° C. in a 5% carbon dioxide incubator for 1-2 weeks, and supplied with fresh medium at intervals of 3 to 5 days.

While inducing growth and proliferation into neural progenitor cells (see FIG. 2).

According to the immunofluorescence staining method, the neural progenitor cells responded positively to CD133, Nestin, and Vimenin antibodies, which are markers of neural stem cells, and negative to CD34, CD31, and CD45 antigens of hematopoietic stem cells and hematopoietic system. Positive and partial positive responses to doublecortin and Tuj-1, markers of immature neurons, and negative and partial negative responses to VE-cadherin and DiI-acLDL and ulex-lectin stains, respectively. See FIG. 2). On the other hand, gene expression profile analysis using RT-PCR showed strong expression for Nestin, Tuj-1, and NF, and genes for Flt-1 and VE-cadherin expressed in vascular stem cells were not expressed at all ( 3). That is, from the results of the immunophenotyping analysis, the neural progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells according to the method of the present invention are different from the known vascular stem cells, hematopoietic stem cells or mesenchymal stem cells. It can be seen that.

The cultured neural progenitor cells show a doubling time of about 3 to 5 days, and continue to proliferate for more than 12 weeks to produce about 10 ⁴ times more cells (see FIG. 3). Moreover, when followed for 12 weeks at 4 week intervals, there is little change in immunophenotype until 12 weeks. From the above facts, it can be seen that the neural progenitor cells of the present invention show a typical characteristic of progenitor cells capable of continuously proliferating without losing its characteristics even in prolonged culture to produce many homogeneous cells.

The present invention also provides a method for differentiating neuronal progenitor cells cultured by the above method into neurons and a medium composition therefor. The medium for differentiation into neurons includes retinoic acid, Folskolin, nerve growth factor (NGF), co-mixture, and EGM medium with high concentration of FBS as defined above. Antibiotic for animal cell culture may be included alone or in combination. Commercially available auxiliary mixtures that can be used in the present invention include N2 supplement or B27 supplement. The neural progenitor cells were inoculated in the differentiation-inducing medium at a concentration of ¹ × 10 ⁴ to 5 × 10 ⁴ cells / cm ² , and 5% carbon dioxide was cultured in a culture vessel for 1 to 2 weeks in a 37 ° C. incubator. Cells cultured as described above exhibit microscopic characteristics of typical neurons with well-expanded neuronal projections under light microscope observation (see FIG. 4), and fluorescence- or immunocytochemistry. ), Western blotting and reverse transcription polymerase chain reaction (RT-PCR), which are known to specifically express neurons in neurons (neuron specific enolase (NSE), neurons) The expression of the genes and proteins of filament (Nurofilament-L; NF), microbutule associated protein-1 (MAP-1) is shown (see FIG. 4).

In the present invention, the isolated neuronal progenitor cells migrate to the lesion, survive, and differentiate into neurons when transplanted into the brain in a cerebral infarction injury model, thereby improving brain function. Neural progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells according to the present invention can be useful for the treatment of various intractable central nervous system diseases such as stroke, spinal cord disease, Parkinson's disease, Alzheimer's disease, epilepsy, brain tumors.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

[ Example 1 Isolation of Autologous Proliferation Cells from Peripheral Blood of Adults Using the Methods and Compositions of the Present Invention

(1-1) Target person selection

20 healthy volunteers with no history of hypertension, hyperlipidemia, diabetes, obesity and smoking history, and no risk factors for atherosclerosis such as heart failure, angina pectoris, myocardial infarction and hypertension in the family history and the risk factors. 20 risk factors with at least one of them, and 75 patients with acute cerebral infarction. Patients with acute cerebral infarction were first diagnosed, and peripheral blood was collected 5-7 days after symptom onset. This was based on previous experiments in which CD34-positive cells in peripheral blood continued to increase up to 7 days after stroke, stayed up to 14 days and then decreased (Taguchi A et al., Circulation 109: 2972-2975, 2004). The isolation yields of neural progenitor cells were compared in the three groups, and factors influencing the isolation of neural progenitor cells were examined and analyzed. Neuroprogenitor cells were not isolated from 20 normal volunteers and 20 risk factors, but neuroprogenitor cells were isolated from 33 of 44 stroke patients (44%). In particular, the more severe the symptoms, the larger the size of the lesions tended to separate better. This may be a subject selection criterion.

(1-2) Separation Method

Peripheral blood was collected from five 10 cc heparin tubes collected from the brachial vein. In order to separate mononuclear cells from peripheral blood, dispense 15cc of peripheral blood within 24 hours into 50ml conical tube, and slowly superimpose Histopaque-1077 15cc on the bottom of conical tube. Centrifugation was performed for a minute to separate the erythrocyte layer at the bottom, the monocyte layer at the middle, and the serum layer at the top. Of these, only the mononuclear cell layer was transferred to a new tube using a Pasteur pipette, mixed well by adding 20 cc of PBS, and washed by centrifugation at room temperature for 10 minutes at a speed of 2,000 rpm. Thereafter, the supernatant was removed, and the mononuclear cell precipitate was further suspended by treatment with 40 cc of PBS, followed by centrifugation at 2,000 rpm for 10 minutes at room temperature after measuring the viability and cell number of monocytes. The mononuclear cell precipitate obtained through the above procedure was immediately suspended in a basal medium for use in isolation and culture of neural progenitor cells, or stored at cryogenic freezing. For cryogenic cryopreservation, the mononuclear cell precipitate was suspended in autologous serum containing 10% dimethyl sulfoxide (DMSO) and then aliquoted at a concentration of 2 × 10 ⁷ to ⁴ × 10 ⁷ cells in a 1.8 cc freezing tube. The tubes were packaged in anti-contamination and frozen at a temperature lowered to -100 ° C. in a program-controlled freezer and stored in cryogenic freezing tanks containing liquid nitrogen.

If cryogenic cryopreserved mononuclear cells are used, the frozen tubes loaded with mononuclear cells are thawed rapidly in a 37 ° C. constant temperature water bath and then transferred to conical tubes and treated with a basal medium containing 10 times the volume of 10% BSA. It was. The tube at room temperature 2,000

After centrifugation for 10 minutes at rpm, the supernatant was removed and the obtained cell precipitate was used for the subsequent separation and culture of neural progenitor cells. After the primary culture medium in a 6 well plate mononuclear cells prepared as above were inoculated at a concentration of 1x10 ⁷ to 2x10 ⁶ cells / well. At this time, the primary culture medium is 2% fetal bovine serum (FBS), fibroblast-derived growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth Commercial vascular growth medium (EGM-2; Clonetics, San Diego) medium containing vascular endothelial cell-derived growth factor and insulin-like growth factor was used. This was placed in a cell incubator maintained at 37 ° C. with 5% carbon dioxide and incubated for 1 week. Subsequently, the culture medium was replaced with a secondary culture medium containing microparticles in the primary culture medium and cultured again for 2 weeks. When the formation of the multilayered cell population was confirmed, the secondary culture medium was 10% FBS in the primary culture medium. The culture was replaced with a third culture medium containing 2 weeks again. At this time, the formation of monolayer cell colonies growing on the bottom surface of the culture vessel was observed every day. The cells forming the monolayer cell population were grown by 80-90% adhesion, and then the monolayer cell population was washed with PBS and treated with 0.25% trypsin / EDTA solution. The cells obtained through the above procedure were inoculated in a fresh T25 culture flask at a concentration of ¹ × 10 ⁴ to 5 × 10 ⁴ cells / cm ² and cultured in a 3% culture medium in a 5% carbon dioxide, 37 ° C. incubator for 1 week. 1 is a microscopic observation of neural progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells according to the above method. Neuroprogenitor cells were observed about 13.0 ± 3.3 days after mononuclear cell culture.

< Example  2> Peripheral Blood Derivation From monocytes  Characterization of isolated and cultured neuronal progenitor cells

In order to analyze the immunophenotype of neuronal progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells in Example 1, the cultured cells were treated with 0.05% trypsin solution, separated from the culture vessel, and washed twice with PBS.

Protein expression by immunocytostaining and gene expression by RT-PCR were examined. For immunophenotyping, Cy3- or FITC-conjugated, CD34, CD31, KDR, VE-cadherin, DiI-acLDL and Ulex-lectin binding, Nestin, vimentin, Dcx, Tuj-1 antibodies were used. As a result, as shown in Figure 2, the neural progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells according to the method of the present invention is positive for antibodies to CD133, Nestin, vimentin, Dcx, and Tuj-1 markers Negative responses to antibodies against, CD34, CD31, CD45, KDR, VE-cadherin, DiI-acLDL and Ulex-lectin antigens were shown. In addition, in order to observe the change in the immunophenotype according to the culture time of the cultured neural progenitor cells, each immunophenotype was examined by the same method as described above for cells cultured up to 12 weeks at intervals of 4 weeks. It was confirmed to keep. On the other hand, the RT-PCR test strongly expressed genes of neuronal markers such as Nestin, β-tubulin III, and NF, whereas there was little expression of Flt, VE-cadherin, and GFAP.

< Example  3> Differentiation of Peripheral Blood-derived Neuroprogenitor Cells into Neurons

(3-1) Induction of differentiation into neurons

In order to check whether neural progenitor cells isolated and cultured from peripheral blood-derived mononuclear cells according to the present invention can be differentiated into neural cells, first differentiate the neural progenitor cells cultured in Example 1 to a concentration of 1 × 10 ⁴ cells / cm 2. Neuronal differentiation was induced by incubation for 1-2 weeks after inoculation into induction medium. At this time, DMEM added with 10% FBS, 0.10 μM retinoic acid, 10 μM foscholine, 100 ng / mL NGF, B27 supplement, 100 U / mL penicillin and 100 μg / mL streptomycin was used as a neuronal differentiation medium. .

(3-2) Confirmation of Differentiation into Neurons

Microscopic observation of neural progenitor cells cultured according to the method differentiated into neural cells, and they had typical neuronal morphology such as forming neurites over time of differentiation induction (FIG. 3).

In addition, fluorescence- or immunocytochemical staining was performed to confirm whether the cells differentiated into neurons express typical markers of neurons. First, 4% of the slides in which the cells were cultured to perform immunocytochemical staining

It was soaked in paraformaldehyde (pH 7.4) for 10 minutes and washed three times with PBS for about 5 minutes. After washing, about 30-50 μl of blocking solution was added dropwise to the cell stained surface, and then reacted at 37 ° C. for about 1 hour to block non-specific reactions, and the primary antibody was reacted at 37 ° C. After reacting for about 1 to 2 hours at 3 washes with PBS for about 5 minutes. After washing, the secondary antibody was reacted at 37 ° C. for about 1 hour, and then washed three times with PBS for about 5 minutes. When the antibody reaction is terminated, the dehydration reaction is induced in the cell by using a sequential ethanol reaction at low concentration, and then substituted with xylene, and then mounted using a poly-mount solution. Observed. For fluorescent immunocytochemical staining, the sample was prepared using 90% glycerol after the completion of the antibody reaction and observed by fluorescence microscopy. NSE (Snata Cruz Biotechnology), MAP-2 (Snata Cruz Biotechnology), NF-L (Snata Cruz Biotechnology), and GFAP (Snata Cruz Biotechnology) antibodies were used for fluorescence- or immunocytochemical staining. As an antibody, Cy3- or FITC-conjugated secondary antibody (Santa Cruz Biotechnology) was used. As a result, cells induced by differentiation from neural progenitor cells of the present invention strongly expressed neuron specific markers such as MAP-2, NF-L, NSE, etc., and confirmed that the differentiation of neurons was effectively induced by the above method. (FIG. 3).

< Example  4> Transplantation of Cells Derived from the Invention in Stroke Animal Models

(4-1) Change of function

After brain injury in a cerebral infarction animal model, neuronal precursor cells of the present invention were implanted. An animal model was used in rats with a transient cerebral ischemia model that blocked the middle cerebral aorta for 90 minutes. The transplantation was performed directly to the cerebral cortex 4 days after brain injury. There was a statistically significant improvement in impaired function after 2 weeks on the neuroscopic test, and the improvement lasted up to 5 weeks. Therefore, the composition of the present invention can be seen to have the effect of reducing the neurological deficit caused by cerebral ischemic injury.

(4-2) In Vivo Differentiation of Neural Progenitor Cells Derived from the Present Invention

After neuronal progenitor cell transplantation, migration, differentiation, and survival patterns were observed in chronological order. Neuroprogenitor cells pretreated with a membrane membrane dye called DiO were followed through fluorescence cell staining. The neural progenitor cells migrated from the transplanted cerebral cortex to the lesion and were mainly located around the lesion to survive. The survival rate was about 25%, and most of the living cells were positive for neuronal markers, Tuj-1 (39.1%) and NF (26.1%), and some differentiated into vascular endothelial cells and glial cells. Transplantation of neural progenitor cells in a cerebral ischemic environment was able to regenerate damaged neurons and induce functional recovery.

According to the isolation and culture method of the present invention, neuronal progenitor cells can be isolated from their peripheral blood-derived mononuclear cells while maintaining high viability and purity and cultured in large quantities, and differentiated into neurons by a differentiation induction method. . In addition, it is effective to restore brain dysfunction through damaged intracellular grafts, and it is possible to differentiate into stable neurons. The neural progenitor cells obtained by the method of the present invention can replace embryonic stem cells which are divided into bioethical and controversial targets for embryos, which are living organisms, and have limitations of cord blood or bone marrow cells in that their neurons can be obtained in large quantities. It can be usefully used to overcome.

Claims (8)

a) harvesting monocytes from peripheral blood of a patient; b) The obtained monocytes are fetal bovine serum (FBS), fibroblast-derived growth factor (FGF), epidermal growth factor (EGF), vascular endothelial a primary culture step of culturing progenitor cells by culturing in vascular growth medium (EGM) medium containing a cell-derived growth factor, and an insulin-like growth factor; c) secondary culture of the adherent cells and the suspended cells obtained after the primary culture in the vascular growth medium of step b) containing the resultant material of the primary culture solution; And d) isolating and culturing neural progenitor cells from peripheral blood comprising the step of tertiary culturing the cells cultured in step c) in the angiogenesis medium of step b) comprising fetal bovine serum (FBS) And differentiation method. The method of claim 1, wherein the method comprises the step of d) the cell culture comprising the retinoic acid (retinoic acid), Folskolin (Folskolin), nerve growth factor (NGF), and antibiotics in the tertiary culture medium Method for isolation, culture and differentiation of neural progenitor cells from peripheral blood further comprising the step of culturing. The method of claim 1, wherein the concentration of fetal bovine serum in the primary culture step is 0.1 to 3%. The method of claim 1, wherein the resultant material of the primary culture is a cell by-product, a protein, and a mineral of 2 micrometers or less that occur during the primary culture. The method of claim 1, wherein the concentration of fetal bovine serum in the tertiary culture step is 5 to 10%. 2. The method of claim 1, wherein the existing medium is preserved upon medium exchange during said culturing step. A composition for treating central nervous system diseases comprising neuroprogenitor cells prepared by the method of claim 1 as an active ingredient. The composition of claim 8, wherein the disease is one or more diseases selected from the group consisting of stroke, spinal cord disease, Parkinson's disease, Alzheimer's disease, epilepsy, and brain tumors.

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