WO2013119045A1

WO2013119045A1 - Neural differentiation of mesenchymal stem cells in thermosensitive hydrogel and composition for neural differentiation

Info

Publication number: WO2013119045A1
Application number: PCT/KR2013/000976
Authority: WO
Inventors: 김문석; 권진선; 김다연
Original assignee: 아주대학교산학협력단
Priority date: 2012-02-09
Filing date: 2013-02-07
Publication date: 2013-08-15
Also published as: KR20130091818A; KR101364624B1

Abstract

The present invention relates to the inducing of neural differentiation of mesenchymal stem cells (MSC) in a thermosensitive hydrogel, and to a composition for neural differentiation needed for inducing neural differentiation. More particularly, the present invention provides a cell delivery vehicle in injection form by culturing MSC and inducing neural differentiation in the thermosensitive hydrogel.

Description

Neuronal Differentiation and Neurodifferentiation Composition of Mesenchymal Stem Cells in Temperature Sensitive Hydrogels

The present invention relates to a neural differentiation composition necessary for inducing neuronal differentiation of mesenchymal stem cells in a temperature sensitive hydrogel and inducing differentiation into neurons. More specifically, the present invention provides an injection-type cell transporter through culturing mesenchymal stem cells and inducing differentiation into neurons in a temperature sensitive hydrogel.

Degenerative neurological disease means a neurological disease that cannot be cured because the underlying cause is unknown. Representative degenerative neurological diseases include Alzheimer's disease and Parkinson's disease. Alzheimer's disease is a disease caused by the accumulation of amyloid protein in brain cells, and there are 27 million patients worldwide. The cause is unknown. In addition, Parkinson's disease is a degenerative neurological disease caused by the breakdown of dopaminergic neurons in the substantia nigra (substantia nigra). There is also a lot of research going on. In addition, degenerative nervous system diseases are caused by central nervous system diseases caused by stroke, industrial accidents and traffic accidents. Recently, the application of stem cells has emerged as a treatment for such degenerative neurological diseases.

Stem cells are cells that continue to replicate in an undifferentiated state and can differentiate into specific cells under specific culture conditions, and are classified into embryonic stem cells and adult stem cells. Embryonic stem cells have infinite differentiation capacity, but there are ethical issues and risks of tumor formation when embryonic stem cells are transplanted. Adult stem cells are stem cells that exist in mature tissues. Has the ability but high safety.

Adult stem cells used in degenerative nervous system diseases typically include neuronal stem cells. However, neuron-derived stem cells exist only in certain parts of the brain, and it is difficult to obtain large amounts of homogeneous cells. In contrast, mesenchymal stem cells (MSCs), which are a type of adult stem cells, are easy to obtain a large amount of homogenous cells by a simple method, and generally by cartilage cells, adipocytes, muscle cells, etc. Differentiation into mesenchymal cells was known to occur, but Woodbury succeeded in inducing differentiation into neurons rather than mesenchymal cells in 2000, and interest in treating degenerative neurological diseases using mesenchymal stem cells increased rapidly.

In order to deliver differentiation-induced mesenchymal stem cells into neurons used in the treatment of degenerative neurological diseases to specific sites, cell delivery technology is required. Cell transfer technology in degenerative neurological diseases is the technique of inducing differentiation of undifferentiated stem cells such as neural progenitor cells, adult stem cells, and embryonic stem cells into neurons, and delivering stem cells induced to differentiation into nerve cells to nerve damage sites. Means. Conventional cell transfer technology is based on direct injection of differentiation-induced stem cells at the site of injury or delivery of cells using a scaffold, but this requires surgical surgery.

Temperature sensitive hydrogel is a polymer that shows a sudden change in solubility according to temperature change. It is an intelligent hydrogel that can be used as an injection without surgical operation by being in a sol state at room temperature and forming a gel near human temperature. Application of the temperature-sensitive hydrogel to cell delivery technology allows neuron differentiation-induced cells to be delivered to the site of injury as an injection without surgical intervention.

The present inventors have found a method for efficiently differentiating neurons into mesenchymal stem cells, and culturing and culturing stem cells in a temperature-sensitive hydrogel for use as a carrier of stem cells induced differentiation into neurons. The present invention was completed through induction of differentiation.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method of preparing an injection for delivering neurons to an injury site without surgical operation by effectively inducing the culture of mesenchymal stem cells and inducing differentiation into neurons in a temperature sensitive hydrogel.

Accordingly, in the present invention, the proliferation of mesenchymal stem cells in the temperature-sensitive hydrogel occurs well and there is no influence on the induction of differentiation into neurons. Therefore, it is possible to use the temperature-sensitive hydrogel as an injection delivery vehicle of cell delivery technology. I want to show

As a means for solving the above problems,

The present invention cell culture substrate; It provides a temperature-sensitive support for the purpose of inducing neuronal differentiation of mesenchymal stem cells comprising a; and gelled temperature-sensitive hydrogel applied to the cell culture substrate. The temperature sensitive hydrogel is in a sol state at room temperature, and may be gelated at 30 ° C. or higher.

The present invention also comprises the steps of dispensing and gelling the temperature-sensitive hydrogel; Seeding the mesenchymal stem cells on the temperature sensitive hydrogel and inducing differentiation into neural progenitor cells; It provides a method for obtaining neurons using a temperature-sensitive hydrogel comprising a; and inducing the neural precursor cells to neurons. The mesenchymal stem cells may be derived from bone marrow, muscle or fat.

Inducing differentiation into neural progenitor cells includes basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), pigment epithelium derived factor, PEDF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), neuronal proliferation At least one or more of a growth factor (NGF) and other neurotrophic factors may be selected and used as a growth factor to induce mesenchymal stem cells to neuronal progenitor cells.

Serum may be contained in the growth factor treatment, and the step of inducing the neural progenitor cells into the neural cells may differentiate the neural progenitor cells into the neural cells using valonic acid. The concentration of valonic acid may be 0.5-4 mmol / L, more preferably 0.5-2 mmol / L.

After the treatment of valonic acid, potassium chloride 10-40 mmol / L, insulin 1-10 g / mL, forskolin 1-30 mol / L, hydrocortisone 0.1-5 mol / L may be added.

The temperature sensitive hydrogel is Pluronic (Poly (ethylen oxide) poly (propylene oxide) poly (ethylene oxide), Pluronic), polycaprolactone (PCL), methoxy polyethylene glycol-polycaprolactone (MPEG-PCL), Methoxypolyethyleneglycol- (polycaprolactone-co-polylactide) (MPEG- (PCL-co-PLLA)), carboxymethylcellulose (CMC), algin, alginic acid or alginate, polypeptide or protein, gelatin or casein, One or two or more selected from chitin derivatives and chitosan may be included.

The present invention also comprises the steps of seeding the mesenchymal stem cells in the gelled temperature sensitive hydrogel to differentiate into neural progenitor cells and induction into neurons; Solvating the temperature sensitive hydrogel; It provides a method for producing a neuron-containing temperature-sensitive hydrogel injection comprising a; and obtaining an injection comprising the obtained neuron and the temperature-sensitive hydrogel.

The present invention also provides a composition for treating neurological diseases comprising a neuron and a temperature sensitive hydrogel obtained by the above obtaining method. The neurological disease may be any one of central nervous system diseases caused by Alzheimer's disease, Parkinson's disease, stroke and spinal cord injury.

Neuronal differentiation of mesenchymal stem cells according to the present invention could be induced relatively simply using a medium having a suitable composition, and even in a temperature sensitive hydrogel through induction of neuronal mesenchymal stem cells in a temperature sensitive hydrogel and induction of neuronal differentiation. Proliferation of mesenchymal stem cells occurs and does not affect the induction of differentiation into neurons, and thermosensitive hydrogel is a neurotransmitter of neuronal differentiation-induced mesenchymal stem cells, which can be used to inject cells in injection form without surgery. There is an advantage that it can.

1 is a diagram showing a process of inducing neuronal differentiation according to the present invention.

Figure 2 is an image showing the phase change behavior according to the temperature of the prepared chitosan hydrogel after performing Experimental Example 1.

3 is a graph showing the result of measuring the viscosity of chitosan hydrogel after performing Experimental Example 2. FIG.

4 shows the mesenchyme in DMEM medium containing (a) DMEM medium, (b) DMEM medium containing basic fibroblast growth factor, and (c) basic fibroblast growth factor and fetal calf serum after performing Experimental Example 3. It is an image showing the morphology change of stem cells.

Figure 5 is carried out after Experiment 4, (a) the growth factor and the group was not treated with valphonic acid, and valonic acid (b) 0.5 mmol / L, (c) 1 mmol / L, (d) 2 mmol / L, (e) Image showing morphology change of mesenchymal stem cells at (1) 1 day, (2) 4 day, and (3) 7 day by 4 mmol / L.

6 is a graph showing the results of cytotoxicity evaluation of mesenchymal stem cells according to the concentration of valonic acid after performing Experimental Example 5 (* P <0.05).

7 is an image observing the morphology change of mesenchymal stem cells by 1 mmol / L of valonic acid after performing Experimental Example 6. (a, b) mesenchymal stems at (1) 1 day, (2) 4 day, (3) 7 days by the proliferative factor and the valonic acid-free group, and (c, d) valonic acid The morphological changes of the cells were observed at (a, c) 40 times and (b, d) 100 times.

8 is a graph showing the results of cytotoxicity evaluation in well plates and chitosan hydrogels after Experimental Example 7 (* P <0.001).

9 is an image showing the morphology of (a) the surface and (b) the inside of the chitosan hydrogel after performing Comparative Example 3.

10 is an image of the mesenchymal cells proliferated and differentiated in the chitosan hydrogel after performing Experimental Example 8 at a magnification of (a) to (d) 300 times and (e) to (g) 200 times.

11 is an image of the cell adhesion on the surface of the chitosan hydrogel after the experiment example 9 was observed through green fluorescence.

12 is an image observed after the experiment example 9, the cell adhesion on the surface of the chitosan hydrogel using a confocal laser microscope.

13 is an image showing a protein band through Western blotting after performing Experimental Example 10.

14 is a graph obtained by performing Experimental Example 10, calculating the protein bands for each antibody compared with β-actin, and quantifying the expression levels of each group by the expression levels compared to the control group (* P <0.05, * * P <0.01).

15 is a schematic view showing the preparation of the preparation by injection in the method of Example 8.

Hereinafter, the present invention will be described in more detail with reference to the drawings and examples. The following descriptions are for specific examples of the present invention, but are not intended to limit the scope of the rights set forth in the claims, even if there is an assertive or limited expression.

In one embodiment, the present invention provides an injection-type cell carrier for inducing neuronal differentiation of mesenchymal stem cells made in a temperature sensitive hydrogel. As a specific example, it provides a temperature-sensitive support for the purpose of inducing neuronal differentiation of mesenchymal stem cells comprising a cell culture substrate, and gelled temperature sensitive hydrogel applied to the cell culture substrate. Temperature-sensitive hydrogel is a polymer that shows a sudden change with the change of temperature. For example, a temperature-sensitive hydrogel exists in a sol state at room temperature and forms a gel near human body temperature. Such temperature sensitive hydrogels can be used in the form of injections without surgical intervention. The mesenchymal stem cells may be derived from bone marrow, muscle or fat.

The differentiation of stem cells into cultures and neurons, followed by mixing with a temperature sensitive hydrogel to make injections, firstly requires the neural cells to be separated and separated and mixed with a temperature sensitive hydrogel, and secondly, Differentiated neurons are not preferred because they have a problem of being exposed to a sudden environment different from that of the culture and differentiation.

In the present invention, by applying a temperature-sensitive hydrogel, the above problems were solved by directly seeding, culturing and differentiating stem cells in the hydrogel.

The temperature-sensitive hydrogel is a sol state at room temperature, it can be used that gelled at 30 ℃ or more, more preferably has a property that is gelled at a body temperature of about 37 ℃, biodegradable and biodegradable material is limited Can be used without it. For example, Pluronic (Poly (ethylen oxide) poly (propylene oxide) poly (ethylene oxide), Pluronic), polycaprolactone (PCL), methoxypolyethylene glycol-polycaprolactone (MPEG-PCL), methoxypolyethylene Glycol- (polycaprolactone-co-polylactide) (MPEG- (PCL-co-PLLA)), carboxymethylcellulose (CMC), algin, alginic acid or alginate, polypeptide or protein, gelatin or casein, chitin derivatives and At least one selected from chitosan is preferably included. The polymer may be added to a phosphate buffer solution or the like to be prepared as a hydrogel, but is not limited thereto. Various preparation methods may be used according to the type of the polymer.

In another embodiment of the present invention, the step of dispensing and gelling the temperature-sensitive hydrogel, sowing the mesenchymal stem cells to the temperature-sensitive hydrogel and induce differentiation into neuronal precursor cells, and neuronal precursor cells Inducing into neurons. The present invention enables the proliferation of stem cells in the temperature sensitive hydrogel through the culture of the mesenchymal stem cells and differentiation into the neurons in the temperature sensitive hydrogel, and does not affect the induction of differentiation into the neurons. It has been found that temperature sensitive hydrogels can be usefully used.

First, the thermosensitive hydrogel is dispensed and gelled. Dedicated descriptions of temperature sensitive hydrogels are omitted.

Next, the mesenchymal stem cells are sown on the temperature sensitive hydrogel to induce differentiation into neural progenitor cells. Inducing differentiation into neural progenitor cells is characterized in that mesenchymal stem cells are first induced into neural progenitor cells using proliferation factors.

On the other hand, mesenchymal stem cells can be cultured in DMEM medium before differentiating mesenchymal stem cells into neural progenitor cells. For example, it can be passaged in DMEM medium containing 5% fetal calf serum, 5% horse serum and 1% penicillin-streptomycin.

The medium used for differentiation into neuroprogenitor cells is not limited as long as it can achieve the desired differentiation induction. As an example, DMEM medium containing serum may be used, since differentiation of cells may not occur if serum is not present. Preferably, DMEM medium containing 10-30% serum may be used.

Proliferation factors for differentiating mesenchymal stem cells into neural progenitor cells include basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epithelial cell growth factor ( epidermal growth factor (EGF), nerve growth factor (Nerve growth factor, NGF) can be achieved by treatment for 12 to 24 hours in a medium containing at least one selected growth factor. Preferably, basic fibroblast growth factor (bFGF) is preferable.

Next, nerve precursor cells are induced into neurons. To differentiate into neurons, butylated hydroxyanisole (BHA), beta-mercaptoethanol, forskolin, valproic acid (VA), At least one or more of neuronal differentiation inducers such as retinoic acid (RA) may be used. Preferably, valonic acid can be used.

At this time, it was found that the concentration of valonic acid is preferably in the range of 0.5 to 4 mmol / L, particularly in the range of 0.5 to 2 mmol / L (see Examples described later). If less than 0.5 mmol / L induction of differentiation effect to the neurons is less than, if more than 2 mmol / L may be a problem due to the death of neurons due to cytotoxicity. Neuronal differentiation-induced cells by adding potassium chloride 10-40 mmol / L, insulin 1-10 g / mL, forskolin 1-30 mol / L, and hydrocortisone 0.1-5 mol / L after the treatment with valproic acid It is maintained, the differentiation induction time to neurons is preferably 5 hours or more.

The present invention also comprises seeding mesenchymal stem cells into gelled temperature sensitive hydrogels to differentiate into neural progenitor cells and inducing them into neurons, soaking the temperature sensitive hydrogels, and solvated with the obtained neurons. It provides a method for producing a neuron-containing temperature-sensitive hydrogel injection, comprising the step of obtaining an injection comprising a temperature-sensitive hydrogel. The present invention confirmed that the temperature-sensitive hydrogel can be used as a cell transporter through the proliferation and differentiation of stem cells in the temperature-sensitive hydrogel which exists in a sol state at room temperature and forms a gel near human temperature.

After induction into neurons by the above-described method, the neurons distributed in the solvated thermosensitive hydrogel can be obtained by soaking the thermosensitive hydrogel.

Injectable forms are preferred as the formulation of the cell carrier, but are not limited thereto, and may be prepared in various formulations for treating neurological diseases. Such neurological disease treatment compositions may include neurons and temperature sensitive hydrogels obtained by the method described above. Neurological diseases are not limited but may be any of central nervous system diseases caused by Alzheimer's disease, Parkinson's disease, stroke and spinal cord injury.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

실시예 1: 키토산을 이용한 온도감응성 하이드로젤의 제조Example 1: Preparation of temperature sensitive hydrogel using chitosan

In order to prepare a temperature sensitive hydrogel using chitosan having a viscosity of 200 to 800 cP (medium molecular weight, Sigma Aldrich Co.), 0.4 g of chitosan was added to 18 ml of 0.1 mol / L acetic acid to prepare a chitosan solution. And stir for a day. Beta-glycerol phosphate disodium salt hydrate (β-GP, Sigma Aldrich Co.) was dissolved in phosphate buffer solution so as to be 30 wt% with respect to 4 mL of the prepared chitosan solution, and slowly added to the chitosan solution using a syringe. The prepared chitosan hydrogel was stabilized by storing at 4 ℃ for one day.

실험예 1: 키토산 하이드로젤의 바이알 기울임법 실시Experimental Example 1: conducting a vial tilting method of chitosan hydrogel

The vial tilting method was performed before and after storing the chitosan temperature sensitive hydrogel prepared in Example 1 at 37 ° C.

As a result, it was confirmed that the sol state at room temperature, the gel state in the vicinity of the human body temperature, even if the third distilled water is added to the gel-formed vial was confirmed to maintain the form of the gel (Fig. 2).

실험예 2: 키토산 하이드로젤의 점도 측정Experimental Example 2: Viscosity Measurement of Chitosan Hydrogel

The viscosity of the chitosan hydrogel prepared in Example 1 was measured using a viscometer (Brookfield Engineering Laboratories, Inc., MA USA).

As a result of the experiment, it was confirmed that the viscosity of the chitosan hydrogel is rapidly increased in the vicinity of the human body temperature 37 ℃ (Fig. 3).

실시예 2: 신경분화 유도물질의 농도에 따른 중간엽 줄기세포의 신경분화 유도Example 2 Induction of Neuronal Differentiation of Mesenchymal Stem Cells According to the Concentration of Neuronal Differentiation Inducers

Mesenchymal stem cells were cultured in DMEM medium containing 5% fetal bovine serum, 5% horse serum and 1% penicillin-streptomycin during passage. After seeding the mesenchymal stem cells with a well plate, the cells were incubated for 24 hours in DMEM medium containing 20% fetal bovine serum and containing 20% fetal bovine serum and 10 ng / ml basic fibroblast growth factor. Differentiation into neuroprogenitor cells was induced for 12-24 hours using DMEM medium. Valfuric acid, which is used as a therapeutic agent for neurological diseases, was used to induce neural progenitor cells into neurons, and 0.5, 1, 2, and 4 mmol / L of Valfuric acid were used to determine the concentration of the neuronal differentiation inducers. Neuronal progenitor cells were induced to differentiate into neurons for 5 hours or more using DMEM medium.

비교예 1: 신경분화를 유도하지 않은 중간엽 줄기세포의 배양Comparative Example 1: Cultivation of Mesenchymal Stem Cells Not Inducing Neuronal Differentiation

Mesenchymal stem cells that did not induce neuronal differentiation were seeded in well plates, and then cultured in DMEM medium containing 20% fetal bovine serum for 24 hours, and treated with 12 to 24 hours using the same composition. Next, cultured for 5 hours or more using only DMEM medium.

실험예 3: 증식인자 및 우태아혈청에 의한 중간엽 줄기세포의 몰폴로지 관찰Experimental Example 3: Observation of Morphology of Mesenchymal Stem Cells by Proliferation Factor and Fetal Bovine Serum

In Example 2, the morphology change by the proliferation factor and fetal bovine serum was observed in the process of inducing mesenchymal stem cells into neuronal progenitor cells.

As a result, it was confirmed that a lot of induction into neural precursor cells by the growth factor and fetal bovine serum (Fig. 4).

실험예 4: 신경분화 유도물질의 농도에 따른 중간엽 줄기세포의 몰폴로지 변화 관찰Experimental Example 4 Observation of Morphology Changes of Mesenchymal Stem Cells According to Concentrations of Neuronal Differentiation Inducers

Cell morphologies of Example 2 and Comparative Example 1 were observed using a phase contrast microscope (Nicon ECLIPSETS100, JAPAN).

As a result, when 0.5 mmol / L of valonic acid was included, differentiation into neurons did not occur much, whereas in 1, 2 and 4 mmol / L, differentiation into neurons occurred well. However, in the case of 2, 4 mmol / L, the morphology of the cells killed by the toxicity of neuronal differentiation inducers was observed (FIG. 5).

실험예 5: 신경분화 유도물질의 농도에 따른 중간엽 줄기세포의 세포독성 평가Experimental Example 5: Evaluation of cytotoxicity of mesenchymal stem cells according to the concentration of neuronal differentiation inducer

WST-1 assay was performed to evaluate the cytotoxicity according to the neuronal differentiation inducer concentration of Experimental Example 2. WST-1 reagent was added at a ratio of 1/10 of the volume of the medium, and after treatment, the WST-1 reagent was incubated for 4 hours in an incubator at 37 ° C. and 5% CO ₂ . Four hours later, cytotoxicity was evaluated by dispensing 100 μl into 96-well plates and measuring the absorbance at 450 nm through an EL808 ultra microplate reader (Bio-tek instrument, USA).

When the results of the WST-1 assay for various concentrations of valphonic acid and the results of Experimental Example 4 (FIG. 6), in the case of valphonic acid, a concentration of 0.5 to 2 mmol / L, in particular, 1 mmol / L Was found to be suitable for inducing neuronal differentiation of mesenchymal stem cells.

실시예 3: 1 mmol/L의 발프론산을 이용한 중간엽 줄기세포의 신경분화 유도Example 3 Induction of Neuronal Differentiation of Mesenchymal Stem Cells Using 1 mmol / L of Valproic Acid

In the process of Example 2 to induce differentiation of neural progenitor cells into neurons, neuronal differentiation of mesenchymal stem cells was induced using only 1 mmol / L of valonic acid.

실험예 6: 1 mmol/L의 발프론산에 의한 중간엽 줄기세포의 몰폴로지 변화 관찰Experimental Example 6 Observation of Morphology Changes of Mesenchymal Stem Cells by 1 mmol / L Valproic Acid

Morphology changes of mesenchymal stem cells according to Comparative Example 1 and Example 3 were observed using a phase contrast microscope (FIG. 7).

실시예 4: 키토산 하이드로젤 내에서의 중간엽 줄기세포의 신경분화 유도Example 4 Induction of Neuronal Differentiation of Mesenchymal Stem Cells in Chitosan Hydrogels

Chitosan hydrogel prepared by the method of Example 1 was dispensed on a well plate to form a gel at 37 ° C, and mesenchymal stem cells were seeded therein to induce neuronal differentiation of mesenchymal stem cells in the same manner as in Example 3. It was.

비교예 2: 키토산 하이드로젤 내에서 신경분화를 유도하지 않은 중간엽 줄기세포의 배양Comparative Example 2: Cultivation of Mesenchymal Stem Cells Not Induced Neuronal Differentiation in Chitosan Hydrogel

Chitosan hydrogel prepared by the method of Example 1 was dispensed on a well plate to form a gel at 37 ° C. Herein, mesenchymal stem cells that did not induce neuronal differentiation were cultured in the same manner as in Comparative Example 1.

실험예 7: 신경분화 유도물질 및 키토산 하이드로젤에 의한 세포독성 평가Experimental Example 7: Evaluation of cytotoxicity by neuronal differentiation inducer and chitosan hydrogel

In order to evaluate the cytotoxicity of Comparative Example 1, Example 3, Comparative Example 2, Example 4 was carried out WST-1 assay in the same process as Experimental Example 5.

When neuronal differentiation was induced in chitosan hydrogels using 1 mmol / L of valonic acid, it was confirmed that stem cells proliferated in chitosan hydrogels due to the increase in absorbance toward day 1, day 4, and day 7 (FIG. 8).

실험예 8: 온도감응성 하이드로젤 내에서의 중간엽 줄기세포의 증식 확인Experimental Example 8: Confirmation of proliferation of mesenchymal stem cells in a temperature sensitive hydrogel

Chitosan hydrogel was dispensed into the well plate and hardened at 37 ° C., and then cultured by seeding mesenchymal stem cells. The well plate cultured by inducing neuronal differentiation in the same manner as in Example 2 was rapidly cooled with liquid nitrogen and lyophilized and observed through a scanning electron microscope (SEM).

As a result, it was confirmed that mesenchymal stem cells and differentiated mesenchymal stem cell morphology in chitosan hydrogels were induced, thereby inducing the growth and differentiation of mesenchymal stem cells in chitosan hydrogels ( 10).

비교예 3: 주사전자현미경을 통한 키토산 하이드로젤의 몰폴로지 관찰Comparative Example 3: Observation of Morphology of Chitosan Hydrogel by Scanning Electron Microscopy

In order to see the morphology of the chitosan hydrogel only in which the cells did not proliferate, the chitosan hydrogel prepared by the method of Preparation Example 1 was dispensed on the well plate and hardened at 37 ° C., then rapidly cooled with liquid nitrogen and freeze-dried. Observation was made by scanning electron microscopy (SEM) (FIG. 9).

실험예 9: 키토산 하이드로젤 표면에서의 세포부착 확인Experimental Example 9: Confirmation of cell adhesion on the surface of chitosan hydrogel

After putting the sterilized cover glass in the well plate, chitosan hydrogel was dispensed and hardened at 37 ° C., and then cultured by seeding mesenchymal stem cells labeled with green fluorescence with PKH67. After 4 days of incubation, the green fluorescence was confirmed by fixation with paraformaldehyde (Carl Zeizz, Germany) (FIG. 11), and confirmed by confocal laser microscopy (OLYMPUS, Japan) (FIG. 12).

실시예 5: 신경분화 유도된 줄기세포의 단백질 분리Example 5 Protein Isolation of Neuronal Differentiation-Induced Stem Cells

In Example 4, protein was isolated on day 1, day 4 and day 7 to confirm neuronal specific protein expression of neuronal differentiation induced stem cells. First, the cell medium was removed, washed with phosphate buffer solution, and 0.05% trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) was added thereto, followed by treatment for 3 minutes at 37 ° C. and 5% CO ₂ . It was. The separated cells were centrifuged at 1500 rpm for 3 minutes and proteins were separated using Liparisis buffer (AMRESCO, USA).

실시예 6: 단백질을 이용한 웨스턴 블로팅Example 6: Western Blotting with Protein

Western blotting was performed using the protein separated using Example 5. In order to prevent nonspecific binding of the blotting membrane, it was treated with 0.1% tween-phosphate buffer solution (blocking buffer solution) containing 5 wt% skim milk. Next, the block buffer solution containing the primary antibody (Table 1) was treated at room temperature for 2 hours or at 4 ° C. overnight, and washed with 0.1% Tween-phosphate buffer solution at least 5 times, followed by attaching the secondary antibody at room temperature for 2 hours. . Finally, it was washed six times with 0.1% twin-phosphate solution.

Table 1

name	Dilution ratio	host
β-actin	1: 10000	mouse
NSE	1: 7000	Rabbit
GFAP	1: 50000	Rabbit
Tuj-1	1: 1000	mouse
Olig2	1: 5000	Rabbit
Neun	1: 1000	mouse

Dilution Ratio of Western Blotting Primary Antibody

실험예 10: 웨스턴 블로팅한 단백질의 발현량 확인Experimental Example 10 Confirmation of Expression of Western Blot Protein

In order to confirm the results of Example 6, it was measured using ImageQuant LAS 4000 mini (GE healthcare Bio-science AB, Japan), the program was used ImageQuant LAS 4000.

In addition, the protein band was quantified by using a program called Image J. After calculating the expression amount relative to β-actin, the expression amount of each group was divided by the expression amount compared to the control group.

As a result, when treated with valproic acid, the expression levels of NSE, GFAP, Tuj-1, Olig2, NeuN, etc. were similar on day 1 and day 4, and it was confirmed that expression levels increased with 7 days (FIG. 14). .

실시예 7: 신경분화 유도된 중간엽 줄기세포를 포함하는 하이드로젤을 이용한 주사제형의 제조Example 7 Preparation of Injectable Formulation Using Hydrogels Including Neuronal Differentiation-Induced Mesenchymal Stem Cells

Example 3 was performed after chitosan hydrogel was dispensed into the well plate. The gelated chitosan hydrogel was collected in a centrifuge tube using a scraper, and then centrifuged at 4 ° C. and 2000 rpm for 5 minutes. Chitosan hydrogel dozed at 4 ° C. was taken using a syringe (FIG. 15).

The thermosensitive hydrogel according to the present invention is a cell transporter of neuronal differentiation-induced mesenchymal stem cells, which is industrially useful because it can be used to inject cells in an injection form without surgical operation.

Claims

Cell culture substrates; And

A temperature sensitive support for use in inducing neuronal differentiation of mesenchymal stem cells, comprising; gelated temperature sensitive hydrogel applied to the cell culture substrate.
The method of claim 1,

The temperature sensitive hydrogel is in a sol state at room temperature, temperature sensitive support for induction of neuronal differentiation of mesenchymal stem cells, characterized in that the gelation at 30 ℃ or more.
Dispensing and gelling the temperature sensitive hydrogel;

Seeding the mesenchymal stem cells on the temperature sensitive hydrogel and inducing differentiation into neural progenitor cells; And

Inducing neuronal progenitor cells into neurons; A method of obtaining neurons using a temperature sensitive hydrogel comprising a.
The method of claim 3,

Induction of differentiation into neuronal progenitor cells includes basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), pigment epithelium derived factor (PEDF). ), Brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), neuronal growth factor (nerve growth factor, NGF) and other neurotrophic factors (neurotrophic factors) is selected from at least one of the neurons using a temperature-sensitive hydrogel, characterized in that it is used as a growth factor to induce mesenchymal stem cells to neuronal precursor cells Obtaining method.
The method of claim 4, wherein

A method for obtaining nerve cells using a temperature sensitive hydrogel, characterized in that serum is contained during the growth factor treatment.
The method of claim 3,

Inducing the neural progenitor cells into the neural cells is a method of obtaining neural cells using a temperature sensitive hydrogel, characterized in that the neural progenitor cells are differentiated into neural cells using valonic acid.
The method of claim 6,

A method for obtaining nerve cells using a temperature sensitive hydrogel, characterized in that the concentration of valonic acid is 0.5 to 4 mmol / L.
The method of claim 7, wherein

A method for obtaining nerve cells using a temperature sensitive hydrogel, characterized in that the concentration of valonic acid is 0.5 to 2 mmol / L.
The method of claim 6,

After the treatment of valonic acid, potassium chloride 10-40 mmol / L, insulin 1-10 g / mL, forskolin 1-30 mol / L, hydrocortisone temperature sensitivity, characterized in that the addition of 0.1-5 mol / L Method for obtaining nerve cells using a hydrogel.
The method of claim 3,

The temperature sensitive hydrogel is Pluronic (Poly (ethylen oxide) poly (propylene oxide) poly (ethylene oxide), Pluronic), polycaprolactone (PCL), methoxypolyethylene glycol-polycaprolactone (MPEG-PCL), Methoxypolyethyleneglycol- (polycaprolactone-co-polylactide) (MPEG- (PCL-co-PLLA)), carboxymethylcellulose (CMC), algin, alginic acid or alginate, polypeptide or protein, gelatin or casein, A method for obtaining nerve cells using a temperature-sensitive hydrogel, characterized in that the preparation comprises at least one selected from chitin derivatives and chitosan.
The method of claim 3,

The mesenchymal stem cell is a method of obtaining a nerve cell using a temperature-sensitive hydrogel, characterized in that derived from bone marrow, muscle or fat.
Seeding mesenchymal stem cells on the gelled temperature sensitive hydrogel to differentiate into neural progenitor cells and inducing them into neurons;

Solvating the temperature sensitive hydrogel; And

A method of preparing a neuron-containing temperature sensitive hydrogel injection comprising a step of obtaining an injection comprising the obtained neuron and a temperature sensitive hydrogel.
A composition for treating neurological diseases comprising a neuron and a temperature sensitive hydrogel obtained by the method of obtaining any one of claims 3 to 11.
The method of claim 13,

The neurological disease is a composition for treating neurological disease, characterized in that any one of central nervous system diseases caused by Alzheimer's disease, Parkinson's disease, stroke and spinal cord injury.