KR100495532B1 - Method of differentiating mesenchymal stem cell into neural cell - Google Patents

Abstract

The present invention provides a method of differentiating and proliferating mesenchymal stem cells into neurons by culturing the mesenchymal stem cells in a medium containing epidermal growth factor and hepatocyte growth factor, and then culture the mesenchymal stem cells after turbid culture. By providing a method for producing neurons from hepatocytes, characterized in that the culturing in the bone marrow-derived mesenchymal stem cells or mononuclear cells including the same compared to the conventional method, the neurons composed of neurons and astrocytes in terms of time, efficiency In addition, the effect of differentiation and multiplication can be obtained more effectively in maturity.

Description

METHOD OF DIFFERENTIATING MESENCHYMAL STEM CELL INTO NEURAL CELL}

The present invention relates to a method for producing neurons from bone marrow-derived mesenchymal stem cells, and to a composition for treating neurological diseases containing neurons prepared by the method as an active ingredient, and more particularly, mesenchymal stem cells present in bone marrow. It is cultured in a medium containing epithelial growth factor, hepatocyte growth factor after 1 day confluent culture to produce neurons and contains nerve cells produced from bone marrow-derived mesenchymal stem cells by different methods as an active ingredient. It relates to a composition for treating neurological diseases.

Recently, after the successful separation of embryonic stem cells from humans, interest in its clinical application has been increasing. Most notable as a field of application of hepatocytes is their use as a complete cell source for cell replacement therapy. In particular, Parkinson's disease, known as intractable disease, neurodegenerative diseases such as Alzheimer's disease, limb paralysis due to spinal cord injury, leukemia, stroke, childhood diabetes, myocardial infarction and liver cirrhosis are caused by the destruction of cells constituting tissue and permanent dysfunction. It is caused, cell replacement therapy that provides cells from the outside destroyed by these diseases has been suggested as an effective treatment.

However, the possibility of cell replacement therapy has many limitations in its practical application. That is, the traditional method of separating fully transplanted cells from a provider's tissue and transplanting them into a patient has a difficulty in obtaining sufficient cells to supply the patient.

In order to solve the problem of cell supply, embryonic hepatocytes may be isolated and used to differentiate into cells of a specific tissue, or tissue specific hepatocytes may be isolated, cultured, expanded, and induced to differentiate and used for cell replacement therapy.

However, embryonic hepatocytes are made from different human bodies, and nerve cells derived from them are difficult to transplant into other individuals, and when transplanted into two male and female individuals that donate the cells used to make an embryo, it is difficult to actually transplant due to the large immunological barrier.

To date, differentiation from embryonic stem cells of mice to hematopoietic cells, cardiomyocytes, insulin-secreting pancreatic cells and neurons has been demonstrated in culture dishes. In addition, differentiation may be induced from embryonic stem cells into oligodendrocytes, myelin producing cells, and then transplanted into mice to increase myelogenesis (Brustle et al ., Science 285: 754-756, 1999). To control blood glucose levels by transplanting insulin-secreting cells differentiated from embryonic hepatocytes into diabetic model mice (Soria et al ., Diabetes 49: 157-162, 2000), or neurons differentiated from embryonic stem cells into spinal cord-damaged mice. Studies that have shown significant improvement in motor impairment due to spinal injury by transplantation (McDonald et al., Nat. Med. 5 (12): 1410-1412, 1999) and others have described the method of transplanting differentiated and proliferated cells from hepatocytes. It is shown to be effective in the treatment of diseases caused by loss.

However, the recent isolation of human embryonic stem cells has been successful, and there have been no reports of differentiation into other specific cells on culture dishes except for differentiation into neurons. Therefore, specific tissue cells differentiated from embryonic stem cells in cell replacement therapy have been reported. Clinical applications are still at the level of potential.

Although the use of cells differentiated from embryonic hepatocytes for cell replacement therapy is considered to be the most effective at present, in addition to immunological barriers, the efficiency of differentiation from embryonic hepatocytes to the specific cells needed is low. There is a risk of side effects from other cells, so the study of sophisticated differentiation methods is required for safer clinical application.

On the other hand, in the case of using tissue-specific hepatocytes for cell replacement therapy, there are problems such as decreased cell proliferative capacity or long-term culture differentiation into unwanted cells due to decreased cell proliferation ability. Furthermore, neurodegenerative diseases such as Parkinson's disease require transplantation of neurons, which are often isolated from fetal brain tissue because it is difficult to obtain neural stem cells directly from the patient. It is obtained by culturing one neural stem cell to differentiate and proliferate into neurons. At this time, the treatment of a single patient requires approximately two fetuses' brains, which is not only problematic for ethical problems and smooth supply, but also for most neural stem cells, rather than differentiating into neural cells. Easy to differentiate into), causing an immune rejection reaction and the like.

Therefore, if the neurons can be differentiated from the mesenchymal stem cells present in the bone marrow, it is possible not only to solve the cell supply problem but also to use the autologous bone marrow, thereby solving the therapeutic problems including the immune rejection response. However, as mesenchymal stem cells are mesodermal cells, the possibility of differentiation into ectoderm neurons is known to date, but the possibility of differentiation is increasing with the recent discovery that trans-differentiation is possible.

Previously, one type of hepatocyte was considered to differentiate into tissue cells belonging to a particular lineage. In the case of mesenchymal hepatocytes, in vitro colonies were developed in the presence of various growth factors, including serum basic fibroblast growth factor, transforming growth factor-β (TGF-β) or EGF. (Kuznetsov et al., Br. J. Haematol . 97: 561, 1997; van den Bos C et al., Human Cell 10:45, 1997), approximately one third of the initial adherent cells. It is reported that it has differentiation ability and can differentiate into connective tissue cells such as osteoblasts, chondrocytes and adipocytes (Pttenger MF et al., Scienc e 284: 143, 1999). Ferrari et al. Also reported that bone marrow is the source of myogenic precursor cells that can form new muscles (Ferrari G et al., Science 279: 1528, 1998).

However, recent studies have reported that mesenchymal stem cells, which have been thought to only differentiate into connective tissue, can be differentiated into neuronal cells. For example, Sanchez et al. (Sanchez-Ramos et al., Exp. Neurology 164: 247-256, 2000), have been developed from bone marrow mesenchymal stem cells when cultured with retinoic acid and brain-derived neurotrophic factor (BDNF). Nerve cells and astrocytes have been reported to differentiate, and Dale Woodbury et al. (J. Neuro. Res. 61: 364-370, 2000) reported β-mercaptoethanol or DMSO. Antioxidants such as (dimethyl sulfoxide) have been reported to differentiate bone marrow mesenchymal stem cells into neurons.

In the process of differentiating bone marrow mesenchymal stem cells into neurons, when a strong chemical differentiation inducer such as DMSO is used, it is toxic and requires a safer method for actual clinical use due to the possibility of change of affected liver cells. It is becoming.

The present inventors, as part of the results of a method for differentiating liver cells present in the bone marrow into nerve cells while being present in the human body with high safety, differentiates the mesenchymal stem cells into neurons of Korean Patent Application No. 2001-21064. And how to make it. " The technique relates to a method for differentiating and proliferating mesenchymal stem cells into neurons by adding hepatocyte growth factor (HGF) and epidermal growth factor (EGF) to a culture medium.

However, the method of differentiating and proliferating mesenchymal stem cells disclosed in Patent Application No. 2001-21064 into neurons has a disadvantage in that the rate of differentiation into neurons is low and the rate of differentiation is low as 80%. In particular, when growing cells in culture, the differentiation rate is slow and morphological changes appear after 4 weeks of culture.Therefore, there are many problems of contamination during culture, consumption of reagents and vitreous tools, and more than 4 weeks if necessary for transplantation. It has the disadvantage of being necessary.

In addition, Patent Application No. 2001-21064 discloses that neurons differentiated and proliferated from mesenchymal stem cells include Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis and It has been suggested to be useful for degenerative neurological diseases such as ischemic brain disease (stroke), but no in vitro, animal or clinical trials have been conducted. It didn't work.

Therefore, the present inventors have conducted research on methods and pharmacological effects that can improve the differentiation rate and differentiation rate in the process of differentiating mesenchymal stem cells present in bone marrow into neurons by improving the conventional method described above. The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing neurons from bone marrow mesenchymal stem cells that can improve the differentiation rate and differentiation rate in the process of differentiating mesenchymal cells into neurons.

In addition, the present invention has another object to provide a pharmaceutical composition for treating neurological diseases containing neurons differentiated and produced from hepatocytes by the above method as an active ingredient.

The present invention to achieve the above object

In the method of mesenchymal stem cells are cultured in a medium containing epidermal growth factor and hepatocyte growth factor to differentiate and proliferate into neurons,

Provided is a method for producing neurons from hepatocytes, characterized in that the mesenchymal stem cells are cultured in a turbid culture.

In addition, the present invention, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, Amyotrophic lateral sclerosis, Ischemic, containing neurons prepared by the above production method as an active ingredient Provided are pharmaceutical compositions for treating neurological diseases for treating degenerative neurological diseases or spinal injuries, including ischemic brain disease (stroke), and traumatic central nervous system diseases.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for producing neurons by turbid culture of mesenchymal stem cells and then cultured in a medium treated with epidermal growth factor and hepatocyte growth factor.

Conventional hepatocyte culture is carried out while maintaining the proportion of cells occupying the surface of the incubator at about 70% for continuous growth of the cells, but the turbidity culture according to the present invention is continuously cultured after the cells completely occupy the surface of the incubator. . This is to allow the cells to stick together and have differentiation characteristics rather than proliferation. In other words, if the stimulation of differentiation is performed after the turbid culture according to the present invention, more rapid and efficient differentiation proceeds.

At this time, turbid culture, which is a pretreatment step of mesenchymal hepatocytes, is preferably carried out for 1 to 50 hours, and the mesenchymal cells cultured in this manner are epithelial growth factor 1 to 10,000 ng / ml and hepatocyte growth factor 1 to 10,000 ng / ml. It is preferable to culture at least one week in a containing medium.

In particular, turbid culture, which is a pretreatment step of mesenchymal stem cells, is carried out for 24 hours, and after turbid culture, the mesenchymal stem cells are more preferably cultured for at least one week in a medium containing epithelial growth factor 10ng / ml and hepatocyte growth factor 20ng / ml. Do.

When the mesenchymal stem cells are cultured under the above conditions, a neuronal cell population consisting of several cells is formed after 4 days of culture. After about 1 week, the neuronal cell population is continuously grown and proliferated to obtain a large amount of neurons. do.

In particular, after cultured for about two weeks in a medium containing epithelial growth factor and hepatocyte growth factor at the same time and completely differentiated and propagated, the growth of the epithelial growth factor and hepatocyte growth factor in the medium treated with epithelial growth factor alone was maintained while maintaining the characteristics of the neurons. Multiplies.

However, if the cells were treated with hepatocyte growth factor alone and cultured in fully differentiated and propagated neurons in about two weeks of culture, the differentiation proceeded and the number of cells decreased. Thus, after about two weeks of differentiation, the medium containing only epithelial growth factor was used. It is preferable to continue to grow in exchange.

The mesenchymal stem cells used in the present invention may be preferably separated from human bone marrow, and after separating mononuclear cells from the bone marrow for 1 to 2 weeks, all hematopoietic stem cells are easily differentiated to make mature blood cell cells. Therefore, only mesenchymal stem cells can be obtained by separating only hepatocytes, which are infinite proliferating cells.

However, even if the mononuclear cells including mesenchymal stem cells are cultured according to the method of the present invention without separating the mesenchymal stem cells from the monocytes isolated from the bone marrow, the same effect of mass production of neurons is obtained. There is no need to use it.

As described above, when epithelial growth factor and hepatocyte growth factor are simultaneously used to differentiate and proliferate from mesenchymal stem cells, about 90% or more of the total cells differentiate into neurons, and about 70% of the differentiated neurons are neurons, about 30% are composed of astrocytes and do not differentiate into microglial cells.

In the present invention, the term "nerve cell" refers to cells including neurons, astrocytes and microglial cells.

On the other hand, neurons differentiated from mesenchymal stem cells prepared by the method of the present invention can be used as an active ingredient of the cell composition for cell replacement therapy for treating neurological diseases and the like.

Therefore, in the present invention, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, Amyotrophic lateral sclerosis, containing neurons prepared by the method of the present invention as an active ingredient, It provides a pharmaceutical composition for treating neurological diseases for treating degenerative neurological disorders or movement disorders caused by spinal injury, including ischemic brain disease (stroke), and traumatic central nervous system diseases.

The pharmaceutical composition for treating neurological diseases using the nerve cells as an active ingredient may be formulated in a unit dosage form suitable for human administration according to a conventional method in the pharmaceutical field in actual use. For formulations suitable for this purpose, injections and the like are preferred as parenteral administration formulations.

The preparation may include one or more pharmaceutically acceptable conventional inert carriers, in addition to neurons, the active ingredient according to the present invention. For example, in the case of injections, there may be preservatives, analgesics, solubilizers or stabilizers, and the like. In the case of formulations for topical administration, there are bases, excipients, lubricants or preservatives.

Pharmaceutical preparations thus prepared may be parenterally, for example, intravenously, subcutaneously, intraperitoneally, or topically.

For example, a clinical method published by Douglas Kondziolka (Pittsburgh, 1998) can be used. In other words, the skull of the patient may be incised into a pea size of about 1Cm in diameter, and then a method of injecting about 3 neuronal cell solutions mixed with Hanks' balanced salt solution (HBSS) may be used. In this case, the injection of the cell solution is performed using a syringe having a long needle, and a stereotactic frame for inserting the cell solution of interest into the coordinates of the brain. In addition to direct administration to the lesion, it can also be implanted through a vein or an artery that supplies blood to the lesion.

The dose of the neuron is preferably 1 × 10 ^{6 to} 1 × 10 ⁹ cells, and various related factors such as the disease to be treated, the severity of the disease, the route of administration, and the weight, age and sex of the patient. It is possible to increase or decrease in consideration.

As described above, it has been found in the previous studies that differentiation and proliferation of neural cells from mesenchymal stem cells by using hepatocyte growth factor and epithelial growth factor at the same time, but epithelial growth factor and hepatocyte growth factor at the same time after turbid culture Therefore, the differentiation and proliferation of neurons from mesenchymal stem cells can accelerate the differentiation into neurons within a week, and it is first revealed by the present invention that the proliferation rate and the maturity of neurons are high.

In the present invention, only mononuclear cells are separated from the bone marrow and then treated with epithelial growth factor and hepatocyte growth factor at the same time for 24 hours turbid culture, and when treated with bone marrow-derived mononuclear cells without turbid culture. The degree of differentiation and proliferation of was investigated. As a result, when co-treatment with the above factors was performed, the neuronal cell population began to appear after about 1 week and continued to proliferate until 2 weeks, but only epithelial growth factor was treated. In case of neural cell differentiation, only hepatocyte growth factor was treated.

Therefore, in the differentiation and proliferation of mesenchymal stem cells or bone marrow-derived mononuclear cells including the same, the 24-hour turbidity culture has an effect of allowing the effect of growth factors to be rapid, and epithelial growth factor is mainly a cell. It is judged that the proliferation stimulating effect of, the hepatocyte growth factor has an effect of stimulating differentiation into neurons, and only one of these factors can not obtain the desired sufficient amount of neurons.

In the present invention, the mesenchymal cells or the bone marrow-derived mononuclear cells including the same are cultured for 24 hours after addition of epithelial growth factor and hepatocyte growth factor after turbid culture, and then differentiated and propagated cells into single cells. As a result, it was confirmed that the neurons were composed of long projections such as axons and short projections such as neurites and astrocytes composed of only short neurites.

In addition, in the present invention, as a result of immunocytochemical staining of the differentiated and expanded cells, it was shown that the cells are positively stained for the neuronal markers NSE, NeuN, MAP-2 and the astroglia marker GFAP. It was clear that it is composed of astrocytes. It was found that OX-42, a microglial cell marker, showed a negative response and did not differentiate into microglial cells.

On the other hand, mesenchymal cells or bone marrow-derived mononuclear cells containing the same were treated with epithelial growth factor and hepatocyte growth factor after 24 hours turbid culture, and after about two weeks, about 90% of them differentiated into neurons. About 70% of neurons and about 30% of astrocytes. After about two weeks, after the differentiation and proliferation of the neurons was sufficient, the epithelial growth factor was treated, and the cells proliferated continuously while maintaining the neuronal morphology. However, when only hepatocyte growth factor was treated, only differentiation proceeded and continued to proliferate.

As described above, only epithelial growth factor and hepatocyte growth factor were used to separate mesenchymal stem cells from the mononuclear cells in order to determine whether the neurons differentiated from the monocytes in the bone marrow are differentiated from the mesenchymal stem cells. Among the bone marrow-derived mononuclear cells, there are two types of hepatocytes, hematopoietic stem cells and mesenchymal stem cells. Since hematopoietic stem cells easily differentiate into blood cells under normal culture conditions, they can be cultured continuously for about 1 to 2 weeks. Hepatocytes that appear to be mesenchymal stem cells. After separating only mesenchymal stem cells that can be passaged more than 20 times in this manner, differentiation experiments were performed on various connective tissues to identify mesenchymal stem cells with differentiation ability to various connective tissue cells. As a result, it was confirmed that the mesenchymal stem cells have differentiation ability into osteoblasts, chondrocytes, and adipocytes.

In the present invention, the isolated mesenchymal stem cells, as in the case of the bone marrow-derived mononuclear cells, through the differentiation and proliferation experiments into neurons, optical microscopy, immunocytochemical staining, etc., by epidermal growth factor and hepatocyte growth factor Mesenchymal stem cells were effectively differentiated into neurons and astrocytes and continued to proliferate.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

Example 1 Isolation of Mononuclear Cells in Bone Marrow

About 10 ml of bone marrow was collected from the pelvis and transferred to a glass tube containing heparin. 30 ml of phosphate buffered saline (PBS) was added to 10 ml of bone marrow, and 20 ml of the mixed solution was slowly flowed over 10 ml of Ficoll-Paque ^TM plus (1.077 g / ml, Amersham Pharmacia Biotech) solution at 2,000 rpm. Density gradient centrifugation was performed for 20 minutes. Next, the monocyte layer at the top layer and the Ficoll-Paque ^TM plus interface was recovered and centrifuged at 1,800 rpm for 5 minutes to recover only monocytes.

Example 2 Mononuclear Cell Culture

Mononuclear cells obtained in Example 1 were inoculated into a culture flask at 1 × 10 ⁶ cells / cm ² , and after 4 hours, washed with fresh basal medium to remove nonadherent cells. In the basal medium, 3.5 μM of hydrocortisone (Sigma), fatty acid-free bovine serum albumin (fatty acid free bovine serum albumin, Gibco BRL) mixed with the same molar ratio of 50ng / ml linoleic acid (linoleic acid, Sigma Co.), CuSO ₄ · 5H ₂ O (0.1 μM, Sigma), ZnSO ₄ · 7H ₂ O (50 pM, Sigma), H ₂ SeO ₃ (3 ng / ml, Sigma), NaHCO ₃ (1.05 mg / ml, Sigma Co.), HEPES (1.19 mg / ml, Sigma), penicillin (100 U / ml, Gibco BRL), streptomycin (10 mg / ml, Gibco BRL), amphotericin (25 μg / ml, Gibco BRL) Williams E medium (Gibco BRL) was used.

Example 3 Differentiation of Neurons into Neurons Without Turbidity

In the basal medium, the mononuclear cells of Example 2 were added to neurons using differentiation medium containing 10ng / ml of epithelial growth factor (Gibco BRL) and 20ng / ml of hepatocyte growth factor (R & D Systems) without turbid culture. Check for differentiation. At this time, the differentiation medium was cultured twice a week.

When mononuclear cells were cultured using the differentiation medium, there was no morphological change after about 1 week, and after 4 weeks, neural cell colonies began to appear and continued to grow. After about 8 weeks of incubation in differentiation medium, neurons composed of long and short neurites, such as axons and dendrites, were formed of astrocytes. Only cells with were observed. In addition, it was confirmed that after 8 weeks, even if treated only with epidermal growth factor, it proliferated while maintaining its form.

However, unlike the combination treatment of EGF and HGF as described above, EGF-only treatment did not differentiate into neurons, and only HGF treatment resulted in early differentiation and growth and proliferation.

Table 1 shows the number of cells proliferated after treatment with bone marrow-derived mononuclear cells with EGF and HGF for 4 weeks without turbid culture. Table 2 shows the cells pretreated with EGF and HGF for 4 weeks. And proliferation behavior of the cells after the 8-week treatment (initial inoculation cell number was 1 × 10 ⁵ ).

Example 4 Differentiation into Neurons after Turbid Culture of Mononuclear Cells

After 24-hour turbid culture of mononuclear cells of Example 2 in basal medium, the cells were cultured using differentiation medium containing 10ng / ml of epithelial growth factor (Gibco BRL) and 20ng / ml of hepatocyte growth factor (R & D Systems). It was confirmed to differentiate into cells. At this time, the differentiation medium was cultured twice a week.

When mononuclear cells were cultured using the differentiation medium, neuronal cell colonies began to appear after about 1 week and continued to proliferate (FIG. 1). And about 2 weeks after incubation in differentiation medium, forms of neurons consisting of long and short neurites and long neurites such as axons and dendrites. Only cells with were observed (FIGS. 2 and 3). In addition, it was confirmed that after 2 weeks, the cells proliferated while maintaining their morphology even when treated only with epidermal growth factor.

However, unlike the combination treatment of EGF and HGF as described above, EGF-only treatment did not differentiate into neurons, and only HGF treatment resulted in early differentiation and growth and proliferation.

Table 1 shows the number of cells proliferated after treatment with bone marrow-derived mononuclear cells for 24 hours after 2-hour incubation with EGF and HGF, and in Table 2, the cells pre-treated with EGF and HGF for 2 weeks as the growth factors. Cell proliferation behavior (initial inoculated cell number was 1 × 10 ⁵ ) after 4 and 8 weeks of treatment.

Pretreatment term Initial inoculation monocyte count HGF only treatment EGF only processing Combined treatment with EGF and HGF No treatment (non turbid culture) 4 Weeks 7.5 × 10 ⁷ Do not multiply 1 × 10 ⁵ 2.0 × 10 ⁵ Turbidity culture 2 weeks 7.5 × 10 ⁷ Do not multiply 1.2 × 10 ⁵ 1.8 × 10 ⁵

Pretreatment term HGF only treatment EGF only processing Combined treatment with EGF and HGF No treatment (non turbid culture) 4 weeks later Do not multiply 2 × 10 ⁵ 2 × 10 ⁵ 8 weeks later Do not multiply 5 × 10 ⁵ 1 × 10 ⁵ Turbidity culture 4 weeks later Do not multiply 2 × 10 ⁵ 1.7 × 10 ⁵ 8 weeks later Do not multiply 5 × 10 ⁵ 1.3 × 10 ⁵

Example 5 Immunocytochemistry I

The cells differentiated in Examples 3 and 4 were attached at 1 × 10 ⁴ cells / cm ² onto a 1 cm ² cover glass. After washing for 5 minutes with 0.1M phosphate buffer, it was fixed for 15 minutes with 0.1M phosphate buffer containing 4% paraformaldehyde, twice for 5 minutes with 0.1M phosphate buffered saline (PBS). Washed. After treatment for 5 minutes with 0.1M PBS containing 1% BSA and 0.2% Triton X-100, the reaction was added for 16 hours by adding a primary antibody. The primary antibodies include anti-human NSE (neuron-specific enolase; Chemicon Inc.), anti-human NeuN (Chemicon Inc.), anti-human β-tubulin III (Sigma Co.), anti-human GFAP (glial) fibrillary acidic protein (Sigma Co.) and anti-human microtubule-associated protein-2 (MAP-2) antibodies were used. After the reaction with the primary antibody was terminated, the primary antibody that did not participate in the reaction was removed and washed twice with 0.1M PBS containing 0.5% BSA for 15 minutes. Secondary antibody was added and incubated for 30 minutes, and then washed twice with 0.1M PBS containing 0.5% BSA for 5 minutes. Then, the reaction was performed for 30 minutes using a kit containing avidin-biotin (Vectastain Elite ABC kit; Vector Laboratory Inc.). After washing twice with 0.1 M phosphate buffer solution for 5 minutes, DAB (3,3'-diaminobenzidine tetrahydrochloride dehydrate, Sigma Co.) was added as a coloring substrate and reacted for 5 minutes. The reaction was stopped by treatment with 0.1 M phosphate buffer for 5 minutes and washed twice with the buffer for 5 minutes. The reaction was dried and washed with distilled water for 5 minutes. Then dehydrated with distilled water, 70, 80, 95 and 100% ethanol in turn and fixed.

As shown in FIG. 4, the cells differentiated with EGF and HGF from bone marrow mononuclear cells were shown to be neuronal markers NeuN, NSE, MAP-2 and β-tubulin III and astrocyte marker GFAP. Staining positive for, it was confirmed that not only morphological characteristics but also biochemically differentiated into both cells of neurons and astrocytes. However, it was found that OX-42, a microglia marker, was negatively stained and did not differentiate into microglia.

The mononuclear cells in the bone marrow were treated with EGF alone, EGF and HGF, or co-treated with EGF and HGF for 24 hours and then cultured for 2 weeks and then neurons (positive staining of NSE, NeuN, MAP-2). neuron) and the ratio of astrocytes stained positively in GFAP is shown in Table 3 below.

term NSE Neun GFAP Map 2 Voice cell EGF only treatment 4 Weeks 0.9% 0.8% 1.2% . 89% EGF + HGF Treatment 2 weeks 10% 25% 4% . 70% EGF + HGF Treatment 4 Weeks 56% 75% 24% . 20% EGF + HGF treatment after 24 hours turbid culture 2 weeks 62% 88% 31% 11% 10%

As can be seen from the results of Table 3, when EGF and HGF were treated for about two weeks, 90% of all cells differentiated into neurons, and about 70% of differentiated neurons were neurons and about 30% were astrocytes. Appeared.

Example 6 Isolation and Culture of Bone Marrow Mesenchymal Stem Cells

In order to determine whether the mesenchymal stem cells differentiated from the bone marrow-derived mononuclear cells, the mononuclear cells were cultured, and only the mesenchymal stem cells were isolated and examined for differentiation into various cells.

The mononuclear cells of Example 2 were inoculated into the culture flask at 1 × 10 ⁶ cells / cm ² using a low glucose DMEM (Gibco BRL) medium to which 10% FBS (fetal bovine serum) was added as a culture medium. The flask was incubated at 37 ° C. in the presence of CO ₂ , and the cells proliferated to allow passage in about 1 to 2 weeks after the culture, but continued to grow even after passage at least 20 times.

Mononuclear cell populations obtained from the bone marrow include mature white blood cells, lymphocytes, osteoblasts, chondrocytes, muscle cells, fibroblasts, adipocytes, as well as liver cells that can differentiate into these cells, including hematopoietic stem cells and mesenchymal stem cells. They are. Hematopoietic stem cells that produce blood cells such as red blood cells, white blood cells, and lymphocytes do not continue to proliferate in a general culture medium, and all are differentiated into mature cells, and thus the cells proliferated and cultured continuously are mesenchymal stem cells.

In order to confirm this more clearly, various cytokines and reagents are treated on the cells obtained above, and characteristics of mesenchymal stem cells, that is, various connective tissues such as osteoblasts, chondrocytes, and fat cells. It was confirmed whether there was a possibility of differentiation into cells.

First, in order to differentiate into osteoblasts, 100 mM dexamethasone, 10 mM β-glycerol phosphate and 50 nM ascorbic acid 2-phosphate and 10% fetal bovine serum were treated. In addition, to investigate the possibility of differentiation into chondrocytes, the cultured cells were centrifuged at 1500 rpm for 10 minutes to form pellets, and then treated with 100 nM dexamethasone and 10 ng / ml TGF-β3 under serum-free conditions. On the other hand, differentiation into adipocytes was treated with 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM dexamethasone, 10 g / ml insulin and 10 nM indomethacin (above Pittenger). et al., Science 284: 143-147, 1999).

Differentiation into individual cells was performed by alkaline phosphatase staning for osteoblasts (Jaiswal et al., J. Cell Biochem . 64 (2) 143-147,1999) and collagen type II for chondrocytes. Staining reaction (type II collagen staining RT-PCR; Mackay et al., Tissue Eng. 4 (4): 415-428, 1998), stained with toluidine blue, and oil red O for adipocytes. It was confirmed by staining (oil red O staining).

As a result, as shown in Figs. 5A, 5B and 5C, all of the mesenchymal stem cells that appeared positive in culture in vitro and still have the characteristics of hepatocytes that can be differentiated into various connective tissues such as osteoblasts, chondrocytes and adipocytes. It could be confirmed.

Example 7 Differentiation of Bone Marrow-derived Mesenchymal Hepatocytes into Neurons without Turbid Culture

To confirm whether the mesenchymal stem cells isolated in Example 6 can be differentiated into neurons, bone marrow cultured and expanded ex vivo with a low glucose concentration of DMEM containing 10% FBS (fetal bovine serum) Mesenchymal stem cells were cultured for 8 weeks in differentiation medium to which 10ng / ml of EGF (Gibco BRL) and 20ng / ml of HGF (R & D Systems) were added without turbid culture in the same manner as in Example 3.

As a result, as in the case of neurons differentiated from bone marrow mononuclear cells, there was no morphological change after about 1 week, and after 4 weeks, neural cell colonies began to appear and continued to proliferate. It continued to grow and proliferate for up to 5 weeks. Eight weeks later, EGF-only treatment continued to proliferate while maintaining its morphology as a neuron.

Example 8 Differentiation of Bone Marrow-derived Mesenchymal Hepatocytes into Neurons after Turbid Culture

In order to determine whether the mesenchymal stem cells isolated in Example 6 can rapidly differentiate into neurons after turbid culture as in Example 4, in vitro with low glucose concentration of DMEM containing 10% FBS (fetal bovine serum) (Ex vivo) cultured and propagated bone marrow mesenchymal stem cells in the same manner as in Example 4, and then cultured for one week in a differentiation medium to which 10ng / ml of EGF (Gibco BRL) and 20ng / ml of HGF (R & D Systems) were added. Incubated.

As a result, as in the case of neurons differentiated from bone marrow mononuclear cells, neuronal cell populations were formed after about 1 week, and continued to grow and proliferate for 2 weeks (FIGS. 6 and 7). Two weeks later, EGF-only treatment continued to proliferate while maintaining its morphology as a neuron.

Example 9 Immunocytochemistry

Immunohistochemical staining was performed on the differentiated cells of Experimental Examples 7 and 8 in the same manner as in Example 5, and the immunocytochemical markers were similar to those of the neuronal tests derived from the mononuclear cells in the bone marrow. It was shown to be positively stained for NeuN, NSE, MAP-2, β-tubulin III and GFAP, an astrocyte marker (Figs. 8A, 8B, 8C, 8D). Therefore, mesenchymal stem cells were identified as differentiated into both cells, neurons and astrocytes, and two times faster than the differentiation without pretreatment when the culture was turbid by pretreatment. It can be seen that it improved to 90%.

<Example 10>

In order to check whether the neurons differentiated from the mesenchymal stem cells of Example 8 can perform functionally the same as those found in the human body, transplanted neurons differentiated to animal diseases to reach the damaged sites well. Experiment was conducted to determine whether to settle.

To this end, in order to form ischemic brain disease, which is the most common neurological disease in humans, the brain of the mouse is incised, the middle cerebral artery is tied for 1 hour, and then loosened again to make a local ischemic disease in the cerebral cortex, and then through the vein. 3 x 10 ⁵ neurons obtained at 8 were injected. The injected cells were subjected to LacZ staining to distinguish them from surrounding cells.

One week after intravenous injection, the brain of the rat was dissected and observed under a microscope and shown in FIG. 9. FIG. 9A is a photograph showing the state of the brain before inducing local ischemic disease, and FIG. 9B is a diagram showing focal ischemic disease. Two weeks after intravenous injection of differentiated neurons after induction, it is a photograph showing the state of the brain, and FIG. 9C is an enlarged photograph of the local hypotension region of FIG. 9B.

As shown in FIG. 9A and FIG. 9B, the difference between a normal brain having no ischemic disease and a brain having an ischemic disease can be clearly identified. In particular, in FIG. 9B and FIG. 9C, the deposition of cells stained with dark color by LacZ staining can be found on the lightly stained area, which selectively reaches only the damaged area of neurons derived from human mesenchymal stem cells. Eventually, it can be seen that it started neuronal differentiation normally and found a place where it could exist well. Therefore, it can be seen that mesenchymal-derived neurons prepared by the present invention can be usefully applied to the treatment of the damaged nervous system.

As described above, the present invention can more effectively differentiate and proliferate bone marrow-derived mesenchymal stem cells or mononuclear cells comprising the same, in the time, efficiency, and maturity of neurons composed of neurons and astrocytes. It can provide a large amount of nerve cells for producing a composition for treating cells for the treatment of neurological diseases. In addition, proliferation and differentiation using natural substances present in the cells is effective in terms of safety and less problems in practical clinical applications. In addition, the patient's own bone marrow can sufficiently obtain the neurons necessary for treatment, there is less side effects such as immune rejection reaction, there is an advantage that can supply the neurons smoothly.

Figure 1 shows the adherent cells in bone marrow mononuclear cells cultured for one week by adding 10 ng / ml epithelial growth factor and 20 ng / ml hepatocyte growth factor to the medium. Observed photograph (* 100, same as below),

FIG. 2 is an optical microscope of bone marrow mononuclear cells cultured for 24 hours in cultured bone marrow mononuclear cells for 2 weeks in culture medium containing epithelial growth factor 10ng / ml and hepatocyte growth factor 20ng / ml. One Photo,

FIG. 3 is an optical microscope photograph of the differentiated neurons of FIG. 2, wherein FIG. 3a is a neuron and FIG. 3b is an astrocyte.

Figure 4 is a photograph observed by staining the differentiated neurons of Figure 2 by immunocytochemical staining method, Figure 4a is NSE positive staining cells, Figure 4b is NeuN positive staining cells, Figure 4c is GFAP positive staining cells, Figure 4d Photo showing MAP-2 positive staining cells,

FIG. 5 shows that mononuclear cells isolated from bone marrow are cultured in a medium of low glucose concentration to separate mesenchymal stem cells and differentiated into osteoblasts (FIG. 5A), chondrocytes (FIG. 5B) and adipocytes (FIG. 5C), respectively. The microscopic images of the differentiated cells,

6 is an optical micrograph of mesenchymal stem cells cultured for 1 week in a medium containing epidermal growth factor 10ng / ml and hepatocyte growth factor 20ng / ml.

7 is an optical micrograph of neurons differentiated and expanded by culturing mesenchymal stem cells for 2 weeks in a medium containing epidermal growth factor 10ng / ml and hepatocyte growth factor 20ng / ml,

Figure 8 is a photograph observed by staining neuronal cells differentiated from Figure 6 by immunocytochemical staining method, Figure 8a is NSE positive staining cells, Figure 8b is NeuN positive staining cells, Figure 8c is a GFAP positive staining cells, Figure 8d Photo showing MAP-2 positive staining cells,

FIG. 9 is a 3 × 10 ⁵ injection of mesenchymal stem cell-derived neurons into a vein of a rat induced local ischemic disease, and two weeks later, the brain of the rat was dissected and the result of microscopic observation was shown. FIG. Figure 9b is a picture showing the state of the brain before inducing ischemic disease, Figure 9b is a picture showing the state of the brain 2 weeks after intravenous injection of differentiated neurons after inducing local ischemic disease, Figure 9c Magnified image of focal scarring at 9b.

Claims (7)

Pretreatment step of turbid culture of mesenchymal stem cells; And culturing the turbidly cultured mesenchymal stem cells in a medium containing epidermal growth factor and hepatocyte growth factor. The mesenchymal stem cells turbidly cultured for 1 to 50 hours are cultured for 1 week or more in a medium containing epithelial growth factor 1 to 10,000 ng / ml and hepatocyte growth factor 1 to 10,000 ng / ml. A method of differentiating and proliferating mesenchymal stem cells into neurons. 2. The mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells cultured for 24 hours are cultured in a medium containing 10 ng / ml of epidermal growth factor and 20 ng / ml of hepatocyte growth factor for at least one week. How to multiply. The method according to any one of claims 1 to 3, wherein after incubating for 2 weeks in a medium containing epithelial growth factor and hepatocyte growth factor, the growth is continued by replacing with a medium treated only with epithelial growth factor. How to differentiate and proliferate mesenchymal stem cells into neurons delete The method of claim 4, wherein the mesenchymal stem cells use mononuclear cells including mesenchymal stem cells isolated from bone marrow. delete