KR102237985B1 - Composition for facilitating osteogenic differentiation of stem cells - Google Patents

Abstract

Disclosed in the present invention are a composition for promoting osteogenic differentiation of stem cells comprising 6,8-diprenylgenistein as an active ingredient, and a method for promoting osteogenic differentiation of stem cells using the same. The composition for promoting osteogenic differentiation of stem cells according to the present invention has a remarkable effect of promoting differentiation of stem cells into osteoblasts compared to conventionally known promoters, and is very useful when trying to induce stem cells into osteoblasts. Therefore, the composition for promoting osteogenic differentiation of stem cells according to the present invention can be used as a preventive and therapeutic agent for osteoporosis by inducing osteogenic differentiation of stem cells.

Description

Composition for facilitating osteogenic differentiation of stem cells}

The present invention relates to a composition for promoting bone differentiation of stem cells, and more particularly, a composition for promoting bone differentiation of stem cells comprising 6,8-diprenylgenistein as an active ingredient, and It relates to a method for promoting bone differentiation of stem cells using the same.

The cells that make up bone are osteocytes that maintain bone homeostasis, osteoblasts that form bones, osteoclasts that absorb bones, and cartilage cells that are responsible for joint function at the ends of bones. (chondrocyte), etc., and even in the bone after growth, the cancellous bone is absorbed by the osteoclasts, and the other surface is remodeled as new bones are formed by the osteoblasts. If the balance between bone formation and bone resorption is broken, it progresses to bone disease. These bone diseases include osteoporosis, non-union fractures, osteonecrosis, osteomalacia, bone defects, and bloodless femurs. There is Avascular necrosis of the Femoral Head.

Traditional treatments for bone diseases include autografting, allografting, and artificial bone grafting, but induce complications such as infection and hematoma at the bone collection site (autologous bone transplant), from donors. There is a problem with the possibility of disease transmission of the disease (allogeneic bone transplantation), and the inability to generate fundamental bones (artificial bone transplantation). Therefore, stem cells are gaining great interest in the field of regenerative medicine using tissue engineering to solve these shortcomings and treat intractable bone diseases.

Stem cells have the property of being able to differentiate into various specific cells in an undifferentiated state, and can be classified into embryonic stem cells and adult stem cells according to their origin. Adult stem cells have limited differentiation ability compared to embryonic stem cells, but are attracting attention in that they can avoid bioethical controversies. In particular, in the case of dedifferentiated stem cells, which are induced pluripotent stem cells obtained by adding foreign genes or specific proteins to somatic cells, they can be differentiated into all cells like embryonic stem cells, and there is an advantage in that the patient's own cells can be used.

Mesenchymal stem cells (MSC) have a fibroblast-like phenotype and are present in various tissues such as muscle, dermis, bone marrow and fat. Mesenchymal stem cells differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. Among them, differentiation into osteoblasts is known to be regulated by various growth factors such as bone morphogenic proteins (BMP) and transforming growth factor-β (TGF-β). In particular, RUNX2 (runt-related transcription factor 2) is an essential transcription factor for osteoblast differentiation, and in addition, various homeobox proteins or transcription factors interact to play an important role in bone development. Proteins such as these transcription factors induce the expression of extracellular matrix proteins and mineralized proteins such as alkaline phosphatase. In particular, type-I collagen (Col Ⅰ), OCN (osteocalcin) and It regulates the expression of the same osteoblast-specific genes.

It can be applied to the treatment of various diseases by promoting the differentiation of stem cells. Recently, some compositions for promoting bone differentiation of stem cells containing Asiasari Radix extract or HVEM inhibitor as an active ingredient (Registration Patent No. 10-1738087, Registration Patent No. 10-1404247) have been reported, but still stem cells There is a demand for discovering new materials that can promote bone differentiation.

Accordingly, as a result of continuous research in order to meet the needs of the prior art, the present inventors have confirmed that 6,8-diprenylgenistine surprisingly promotes bone differentiation of stem cells, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for promoting bone differentiation of stem cells comprising 6,8-diprenylgenistine as an active ingredient.

Another object of the present invention is to provide a method of promoting bone differentiation of stem cells by using the composition.

In order to achieve the object of the present invention, the present invention is the bone differentiation of stem cells containing 6,8-diprenylgenistein having a chemical structure of the following formula (I) as an active ingredient. Provides a composition for promotion:

(I)

The active ingredient of the composition of the present invention, 6,8-diprenylgenistine , may be isolated from the fruit extract of Cudrania tricuspidata , and may also be synthesized by biological or chemical methods.

As a method of extracting 6,8-diprenylgenistine, a known extraction method such as a solvent extraction method, an ultrasonic extraction method, a filtration method and a reflux extraction method may be used, and preferably a solvent extraction method or a reflux extraction method may be used. As an example, there is a method of obtaining the active ingredient of the present invention by extracting from the pulverized Cudrania fruit with an organic solvent, concentrating under reduced pressure, then suspending in an organic solvent, fractionating it with a solvent such as hexane, and separating it by liquid chromatography However, it is not limited thereto.

In the present invention,'stem cell' refers to a cell having a self-renewal capable of being pluripotent or totipotent capable of differentiating into cells of all tissues of an individual, Embryonic stem cells, induced pluripotent stem cells, and adult stem cells are included, preferably adult stem cells, and most preferably periosteum-derived adult stem cells.

In the present invention,'adult stem cells' are primitive cells isolated from tissues of mammals including humans, just before differentiation, and are stem cells capable of proliferating indefinitely and differentiating into various types of cells. .

In the present invention,'differentiation' refers to the development of less specialized cells into specific cells, and the size and shape of cells, membrane potential, metabolic activity, and response to signals are changed by differentiation. In this case, adult stem cells differentiate into specific cells. 'Bone differentiation' means developing stem cells into osteoblasts.

6,8-diprenylgenistine, an active ingredient of the composition according to the present invention, exhibits an effect of promoting the differentiation of stem cells into osteoblasts. Specifically, stem cells are induced to differentiate into osteoblasts, and differentiated osteoblasts exhibit high alkaline phosphatase (ALP) activity and form mineralization (calcium deposition) (Figs. 3 and 4).

The composition for promoting bone differentiation of stem cells of the present invention may contain 6,8-diprenylgenistine in an amount of 10 nM to 10 μM, and preferably in an amount of 10 nM to 1 μM.

If 6,8-diprenylgenistine is out of the above range, the effect of promoting stem cell bone differentiation may be inhibited. In addition, when 6,8-diprenylgenistine is contained in excess of 10 μM, the shape of stem cells may change from a fusiform to a round shape, and the number of dead cells may increase.

When the composition according to the present invention was treated with periosteum-derived adult stem cells, the rate at which stem cells differentiate into osteoblasts was improved, indicating ALP activity in a short time compared to the positive control group (Fig. 3), and it was confirmed that mineralization proceeded. (Fig. 4).

According to another object of the present invention, it provides a medium for bone differentiation of stem cells containing 6,8-diprenylgenistine.

The medium includes all media commonly used for culturing stem cells, for example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F -12, MEM (Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), such as a basic medium, or a medium containing a component capable of inducing bone differentiation.

According to another object of the present invention,

i) treating stem cells with a composition for promoting bone differentiation of stem cells containing 6,8-diprenylgenistine as an active ingredient, and

ii) culturing stem cells

It provides a method for promoting bone differentiation of stem cells comprising a.

In the present invention, the stem cells to be subjected to bone differentiation promotion may be in an in vitro state, and the composition may be treated on the stem cells in the form of a stem cell culture medium. In addition, stem cells to be subjected to bone differentiation promotion can be cultured on a three-dimensional support. Components constituting the scaffold may use a biocompatible material, and osteoblasts cultured and differentiated from the scaffold may be transplanted into a living body in a state attached to the scaffold for the treatment of bone diseases without being separated from the scaffold.

In the present invention, when the cultivation is performed in an in vitro state, it may be performed in a _{CO 2 incubator.} The cultivation in the CO _{2 incubator may be performed in an environment where the concentration of CO 2} is 5 to 15%, preferably 5 to 10%, more preferably 5 to 8%. Not limited. In addition, the inside of the _{CO 2} incubator may be filled with O _{2 in addition to the CO 2.}

In the present invention, the cultivation may be performed at a temperature range of 25 to 45°C, preferably 30 to 40°C, more preferably 34 to 37°C, but is not limited thereto. When the culture temperature is out of the above temperature range, there is a problem in that the differentiation of stem cells is not promoted efficiently, and the number of stem cells that are killed increases.

In the present invention, the cultivation may be performed continuously for 1 to 35 days, preferably 1 to 30 days, more preferably 1 to 25 days, but is not limited thereto. When the culture period exceeds 35 days, as the density of cells in the culture vessel increases, nutrients are rapidly depleted, cell characteristics change, and differentiation into osteoblasts is not induced. In addition, during the culture period, subculture may be performed to divide the cell group and culture.

According to another object of the present invention, it provides a composition for preventing or treating osteopathy, comprising osteoblasts differentiated by the composition of the present invention.

In the present invention, "bone defect" refers to a disease related to bone that requires bone differentiation and regeneration, and includes traumatic bone damage such as fracture and bone defects that occur after surgery.

The differentiated osteoblast cells of the present invention can be used for various cell treatments. Differentiated osteoblasts can be transplanted into bone tissue through surgical surgery by mixing with various bio-adjuvants such as collagen or hydroxyapatite scaffold.

The composition for preventing or treating bone defects according to the present invention may further contain collagen or bone scaffolds as auxiliary components in addition to the differentiated osteoblasts.

In the composition for preventing or treating bone defects according to the present invention, the number of differentiated osteoblasts can be adjusted according to the type of disease, route of administration, age and sex of the patient, and the degree of the disease, preferably 1 to It is a 5×10 ⁷ cell/cm ³ scaffold, which can be administered once or several times.

The composition for promoting bone differentiation of stem cells according to the present invention has a remarkable effect of promoting the differentiation of stem cells into osteoblasts, compared to conventionally known promoters, and is very useful when attempting to induce stem cells into osteoblasts. Therefore, the composition for promoting bone differentiation of stem cells according to the present invention can be used as a preventive and therapeutic agent for bone defects by inducing bone differentiation of stem cells.

1 is a schematic diagram showing an example of a process of extracting 6,8-diprenylgenistine, which is an active ingredient of the composition for promoting bone differentiation of stem cells according to the present invention.
Figure 2 is a result of HNMR and Mass Spectrum analysis of 6,8-diprenylgenistine, an active ingredient of the present invention.
3 is according to the treatment of the composition for promoting bone differentiation of stem cells according to the present invention. This is a graph showing the level of activity of stem cells ALP (alkaline phosphatase; bone differentiation marker).
4 is a graph showing the degree of mineralization (calcium deposition) of stem cells according to the treatment of the composition for promoting bone differentiation of stem cells according to the present invention.

Hereinafter, the configuration and effects of the present invention will be described in more detail through specific examples to aid understanding of the present invention. However, the following examples are merely illustrative in order to more clearly understand the present invention, and the scope of the present invention is not limited by the following examples.

Reference Example 1: Method for inducing stem cell cell culture and bone differentiation

Periosteal mesenchymal stem cells (POMSCs) are isolated by acquiring human periosteal tissue with patient consent in accordance with the regulations of the Ethics Committee of Gyeongsang National University Hospital (GNUH 2014-05-012). I did. Cell culture of the isolated POMSCs was performed using DMEM culture solution (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5%. It was carried out in the culture conditions of _{CO 2.}

In order to induce differentiation from POMSCs into bone cells, an osteogenic differentiation induction medium (OM culture) containing 50 ㎍/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate in DMEM culture medium. ) Was used to culture the cells. In the cell culture for bone differentiation, the OM culture medium was changed every 3 days.

Reference Example 2: Extraction of 6,8-diprenylgenistine

Cudrania fruit was harvested and dried to obtain 7 kg (dry weight). Cudrania fruit was subjected to immersion extraction 3 times using 20 L of 90% methanol. The extracted solution was filtered using a No.2 paper filter, and concentrated using a large rotary vacuum concentration to obtain about 1.09 kg of crude extract.

About 300 g of the crude extract was suspended in 1 L of 5% methanol, and the solvent fractions were repeatedly performed in the order of hexane, chloroform, and ethyl acetate, and the fractions were collected and concentrated using a rotary vacuum concentrator. As a result, 35 g of an ethyl acetate fraction (fraction E) in which a number of active substances were identified could be obtained.

The obtained fractions were subjected to preparative liquid chromatography using a 40 g ODS-25 (YMC-DispoPack AT) column, and the samples were divided by 1 g at a time and injected, and repeated under gradient elution conditions from 10% methanol to 100% methanol. To obtain a small fraction. Compound 10 (Comp. 10) was isolated from the small fraction E9 by preparative liquid chromatography using a Kinetex 5μ Biphenyl 100 (250 × 21.2 mm) column (which has the effect of promoting bone differentiation of stem cells), and this compound 10 6,8-diprenylgenistine was confirmed through HNMR and Mass Spectrum (FIGS. 1, 2A, and 2B).

Example 1: Alkaline phosphatase (ALP) activity assay

In order to find out the effect of the composition according to the present invention on bone differentiation and osteoblast activity, the activity of ALP (alkaline phosphatase), which is an early differentiation marker of osteoblasts, was analyzed.

After seeding the POMSCs prepared in Reference Example 1 on a ^{24-well plate at a concentration of 2×10 4} cells/well, 6,8-diprenylgenistine prepared in Reference Example 2 was added to 10 μM, 1 μM, 100 nM, and 10 nM. After treatment at the concentration, the cells were cultured in OM culture solution under culture conditions of _{37°C and 5% CO 2.} The OM culture solution was changed every 3 days.

ALP activity was analyzed on the 14th day of bone differentiation-induced cell culture. Specifically, cell lysis was first performed using NP40 Cell Lysis Buffer (Life Technologies, Carlsbad, USA). After adding a buffer solution and p-nitrophenyl phosphate (P-nitrophenyl phosphate; Thermo Fisher Scientific, Waltham, USA), a buffer solution and ALP substrate, to some of the cell lysates obtained here, enzymes at 37° C. for 20 minutes. Reacted. After the reaction was terminated by adding a 0.9 N NaOH solution, ALP activity was analyzed by measuring absorbance at a wavelength of 405 nm using a microplate reader (Molecular Devices, San Jose, USA). In order to correct the absorbance according to the number of cells, the absorbance per protein of the same amount in the cell lysate was calculated to determine the final ALP activity, and the results are shown in FIG. 3.

For comparison, POMSCs were treated in the same manner as above using estradiol, which is known as a bone differentiation promoter as a positive control, to determine ALP activity in the same manner as above on the 14th day of bone differentiation-inducing cell culture. Fig. 3 shows.

As shown in Figure 3, when the composition according to the present invention was treated at concentrations of 10 μM, 1 μM, 100 nM, and 10 nM, the ALP activity was significantly (about 1.7 times) increased compared to the normal bone cell differentiation medium (OM medium), and the positive control group Compared to estradiol, it was confirmed that the ALP activity increased by about 1.5 times.

Therefore, it can be seen that the composition according to the present invention remarkably promotes bone differentiation of stem cells.

Example 2: Analysis of mineralization (calcium deposition)

Alizarin red S is deposited by binding to minerals such as calcium. Because osteoblasts form mineral deposits, the degree of differentiation into osteoblasts can be determined in proportion to the degree of alizarin red staining.

In order to check whether stem cells differentiate into osteoblasts by the composition according to the present invention, treatment was performed by concentration as in Example 1. Specifically, after seeding the POMSCs prepared in Reference Example 1 on a ^{24-well plate at a concentration of 2×10 4} cells/well, 6,8-diprenylgenistine prepared in Reference Example 2 was added to 10 μM, 1 μM, and 100 nM. , After treatment at a concentration of 10 nM, cells were cultured in OM culture solution under culture conditions of _{37° C. and 5% CO 2.} The OM culture solution was changed every 3 days. Induction of bone differentiation was analyzed for mineralization on days 21-28 of cell culture.

The cultured POMSCs were stained with Alizarin Red. Specifically, the cultured POMSCs cells were washed with phosphate saline, fixed with 4% formaldehyde for 10 minutes, stained with Alizarin Red S (Sigma Aldrich, USA) solution for 30 minutes to 1 hour, and washed with demineralized water to remove excess staining solution. It was removed and the staining pattern was observed. Then, in order to quantify alizarin red bound to Ca ²⁺ , a 100 mM cetylpyridinium chloride solution was added and reacted at room temperature for 2 hours to dissolve and extract the dyed alizarin red. The concentration of dissolved Alizarin Red was calculated by measuring absorbance (570 nm) with a plate reader, and the results are shown in FIG. 4.

For comparison, an alizarin red concentration was determined by performing the same manner as above using estradiol as a positive control, and the results are shown in FIG. 4.

As shown in FIG. 4, when the composition according to the present invention was treated, mineralization (calcium deposition) was increased than that of normal bone cell differentiation medium (OM medium), and it was confirmed that it was comparable or increased compared to mineralization of the positive control estradiol. .

Therefore, it can be seen that the composition according to the present invention remarkably promotes the differentiation of stem cells into osteoblasts.

Claims (7)

A composition for promoting bone differentiation of periosteum-derived adult stem cells comprising 6,8-diprenylgenistine having the structure of the following formula (I) as an active ingredient:

(I).
delete delete delete The composition for promoting bone differentiation of periosteum-derived adult stem cells according to claim 1, comprising 6,8-diprenylgenistine in an amount of 10 nM to 1 μM.
A medium for bone differentiation of adult stem cells derived from periosteum comprising the composition according to claim 1.
i) treating the composition according to claim 1 to periosteal-derived adult stem cells in vitro, and
ii) culturing adult stem cells derived from periosteum
A method for promoting bone differentiation of periosteum-derived adult stem cells in in vitro comprising a.

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