KR102533120B1 - Composition for Facilitating the Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-4 as an Effective Ingredient - Google Patents

Abstract

The present invention relates to a composition for promoting bone differentiation of stem cell spheroids and a method for promoting bone differentiation using the same. According to the present invention, a composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-4 as an active ingredient promotes bone differentiation of stem cell spheroids while maintaining the shape of the stem cell spheroids. Since it has the effect of promoting, the composition for promoting bone differentiation of stem cell spheroids of the present invention can be usefully used when trying to promote bone differentiation of stem cell spheroids.

Description

Composition for Facilitating the Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-4 as an Effective Ingredient}

The present invention relates to a composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-4 as an active ingredient and a method for promoting bone differentiation using the same.

Bone morphogenetic protein (BMP) is a potent growth factor in the transforming growth factor-beta (TGF-β) superfamily (He, J., et al., 2019, Exp Ther Med 18: 3333-3340). More than 20 factors with diverse functions such as skeletal formation, hematopoiesis and neurogenesis have already been identified in humans (Bragdon, B., et al., 2011, Cellular Signaling 23: 609-620). This soluble locally acting signaling protein, BMP, can act in an endocrine, paracrine and autocrine manner.

BMP-4 may be involved in various functions, including enhancing the migration ability of mesenchymal stem cells and transition from mesenchymal stem cells to osteogenic and adipocyte cell lines (Modica, S., et al., 2017). , Adipocyte 6: 141-146; Li, Q., et al., 2014, Clin Cancer Res 20: 2375-2387). BMP-4 may serve as an important regulator for proper reproductive tissue development, and it has also been reported that BMP-4 is involved in postnatal tooth cell differentiation (Gluhak-Heinrich, J., et al., 2010, Bone 46: 1533-1545).

Stem cell therapy has generated great interest in recent years. Stem cells are known to have various functions (Tae, J.Y., et al., 2019. Exp Ther Med 18: 326-331). It has been reported that stem cells can differentiate into various tissues and secrete various factors, and that this function, called a paracrine effect, affects surrounding tissues (Lee, H., et al. , 2018. Adv Clin Exp Med 27: 971-977). Recently, stem cells have been applied to tissue regeneration (Kang, S.H., et al., 2019. J Periodontal Implant Sci 49: 258-267). In particular, by applying three-dimensional spheroids, the therapeutic potential can be improved overall by improving cell survival, stem cell function, angiogenic properties, and anti-inflammatory effects (Petrenko, Y., et al., 2017, Stem Cell Res Ther 8:94).

Demand for bone tissue transplantation is very great when treating local bone tissue defects formed after fracture or tumor removal or artificial joint insertion treatment for osteoarthritis. However, bone tissue transplantation has limitations and disadvantages in clinical use, so research on bone tissue substitutes is being actively conducted, and research on regenerative medicine for the treatment of bone loss using stem cells is also being actively conducted. there is. In this regard, as a cell therapy for diseases related to bone defects, promotion and activation of osteoblast differentiation have been studied.

In particular, techniques for differentiating mesenchymal stem cells into osteoblasts using specific proteins or genes have been reported. Mesenchymal stem cells (MSCs) are pluripotent cells capable of differentiating into mesenchymal lineages and can be isolated from various tissues and easily cultured in vitro (Castro-Manrreza, M.E., 2015, Journal of immunology research 394917-394917).

According to previous studies, mesenchymal stem cells derived from gingiva tissue can be a new source for stem cell-based therapy in bone regeneration, such as clinical bone reconstruction. In addition, human gingiva tissue-derived mesenchymal stem cells were found to be superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine (Tomar GB, Srivastava RK, Gupta N, Barhanpurkar AP, Pote ST, Jhaveri HM, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine.Biochem Biophys Res Commun 2010;393:377-383.). As with other sources of stem cells, immunomodulatory properties have been found in gingival tissue-derived mesenchymal stem cells. Stem cells derived from gingival tissue have several advantages, including ease of isolation, accessible tissue sources, and rapid ex vivo expansion.

The patents and references mentioned herein are incorporated herein by reference to the same extent as if each document were individually and expressly specified by reference.

Korean Registered Patent No. 10-1565856 (registration date: 2015. 10. 29)

The present inventors studied whether BMP-4 can be used for bone differentiation of gingiva-derived stem cell spheroids, and when BMP-4 is added to the bone differentiation induction medium, gingiva-derived stem cell spheroids The present invention was completed by confirming that it promotes bone differentiation.

Accordingly, an object of the present invention is to provide a composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-4 as an active ingredient.

Another object of the present invention is to provide a stem cell culture medium containing bone morphogenetic protein-4 as an active ingredient.

Another object of the present invention is to provide a method for promoting osteogenic differentiation of stem cell spheroids, comprising the step of treating isolated stem cells with bone morphogenetic protein-4 (bone morphogenetic protein-4).

Another object of the present invention is to provide a composition for preventing or treating bone defects comprising osteoblasts differentiated into the composition for promoting bone differentiation of the present invention as an active ingredient.

Other objects and technical features of the present invention are presented more specifically by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-4 as an active ingredient.

In the present invention, "bone morphogenetic protein-4", an active ingredient, has been reported to act as a regulator for bone differentiation, and also induces intra-membranous and endochondral bone formation (Rahman, M. S., et al., 2015. Bone Res 3: 15005).

According to one embodiment of the present invention, the stem cells may include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized, but are not limited thereto. Preferably, they may be adult stem cells, and more preferably, they may be stem cells derived from gingiva.

In the present invention, the term “stem cell” refers to pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of an individual. means a cell having, and includes embryonic stem cells, induced pluripotent stem cells and adult stem cells.

In the present invention, the term “embryonic stem cell” refers to embryonic stem cells derived from a fertilized egg produced by combining sperm, which is a male reproductive cell, and egg, which is a female reproductive cell. Embryonic stem cells are obtained from the inner cell aggregation of blastocysts before implantation and have the ability to differentiate into cells of all tissues, but are undifferentiated cells that can proliferate indefinitely in an undifferentiated state if culture conditions are given. say In other words, in a special differentiation culture environment, cell differentiation is induced in the desired direction and can be used as a cell therapy for diseases such as nerve, diabetes, and blood, so that only one embryonic stem cell line is expected to be used for the treatment of numerous patients. means

In the present invention, the term "induced pluripotent stem cells" refers to cells induced to have pluripotent differentiation through artificial dedifferentiation process, and is also referred to as iPSC (induced pluripotent stem cells). .

In the present invention, the term “adult stem cell” refers to primitive cells isolated from mammalian tissues, including humans, just before differentiation, which have the ability to proliferate indefinitely and various types of cells (e.g., fat cells). Cells, chondrocytes, muscle cells, bone cells, etc.) are stem cells that can differentiate.

In the present invention, the term “differentiation” refers to a phenomenon in which a less specialized cell develops into a specific cell and changes into a specific type of cell in size or shape, membrane potential, metabolic activity, and response to signals. The differentiation mainly occurs during the process of forming a complex tissue in a single zygote by a multicellular organism, or differentiates adult stem cells into specific cells when a damaged tissue is repaired when a multicellular organism becomes an adult.

According to one embodiment of the present invention, the bone differentiation may be the differentiation of stem cells into osteoblasts.

According to one embodiment of the present invention, the composition for promoting osteogenic differentiation of stem cell spheroids may include the bone morphogenetic protein-4 in an amount of 0.0001 to 100 ng/mL, preferably 0.0005 to 50 ng/mL It may be included in the content of, more preferably may be included in the content of 0.001 to 10 ng / mL, but is not limited thereto.

According to another aspect of the present invention, a stem cell culture medium containing the bone morphogenetic protein-4 as an active ingredient is provided.

The bone morphogenetic protein-4 may be included as a component in a culture medium for stem cells.

The stem cell culture medium includes all media commonly used for stem cell culture, 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), etc. , but not limited thereto.

According to one embodiment of the present invention, the stem cells cultured in the stem cell culture medium may include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized. Not limited to this. Preferably, they may be adult stem cells, and more preferably, they may be stem cells derived from gingiva.

According to one embodiment of the present invention, bone morphogenetic protein-4 contained in the stem cell culture medium may be included in an amount of 0.0001 to 100 ng/mL, preferably 0.0005 to 50 ng/mL. It may be included, more preferably in an amount of 0.001 to 10 ng / mL, but is not limited thereto.

According to another aspect of the present invention, there is provided a method for promoting bone differentiation of stem cell spheroids, comprising the step of treating the isolated stem cells with bone morphogenetic protein-4 (bone morphogenetic protein-4).

The method for promoting osteogenic differentiation of stem cell spheroids of the present invention includes treating the separated stem cells with bone morphogenetic protein-4 and culturing the isolated stem cells treated with the bone morphogenetic protein-4.

The stem cells to be stimulated for osteogenic differentiation may be in vitro, and the bone morphogenetic protein-4 may be treated with the stem cells in the form of a culture medium for the stem cells. In addition, stem cells can be cultured on a three-dimensional scaffold to promote bone differentiation. Biocompatible materials may be used as components constituting the scaffold, and osteoblasts cultured and differentiated on the scaffold may be transplanted into the living body in a state attached to the scaffold for the treatment of bone diseases without separately separating from the scaffold.

According to one embodiment of the present invention, the stem cells cultured by the method for promoting bone differentiation of stem cell spheroids include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized. It can, but is not limited to this. Preferably, they may be adult stem cells, and more preferably, they may be stem cells derived from gingiva.

According to one embodiment of the present invention, bone morphogenetic protein-4 included in the method for promoting bone differentiation of stem cell spheroids may be included in an amount of 0.0001 to 100 ng/mL, preferably 0.0005 to 50 ng/mL It may be included in the content of, more preferably may be included in the content of 0.001 to 10 ng / mL, but is not limited thereto.

According to one embodiment of the present invention, in the method for promoting bone differentiation of the stem cell spheroid, the stem cell spheroid is preferably cultured on a concave surface, but is not limited thereto.

According to another aspect of the present invention, there is provided a composition for preventing or treating bone defects comprising osteoblasts differentiated into the composition for promoting bone differentiation of stem cell spheroids of the present invention as an active ingredient.

The differentiated osteoblasts of the present invention can be used for various cell therapies. Differentiated osteoblasts can be mixed with various biological supplements such as collagen or hydroxyapatite scaffolds and surgically transplanted into bone tissue. The therapeutic composition of the present invention may further contain collagen or a bone scaffold as an auxiliary component in addition to the differentiated osteoblasts. The number of differentiated osteoblasts in the therapeutic composition of the present invention may be adjusted depending on the type of disease, the route of administration, the age and sex of the patient, and the severity of the disease, preferably 1 to 5×10 ⁷ for an average adult. Administer cell/cm ³ scaffold. In addition, the therapeutic composition of the present invention can be administered once or divided into several times.

In the composition of the present invention, the therapeutic composition is characterized in that it is used for the treatment of bone diseases.

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

The effects and advantages of the present invention are summarized as follows:

(i) The present invention relates to a composition for promoting bone differentiation of stem cell spheroids and a method for promoting bone differentiation using the same.

(ii) The composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-4 of the present invention as an active ingredient has the effect of promoting bone differentiation of stem cell spheroids while maintaining the shape of stem cell spheroids. .

(iii) The composition for promoting osteogenic differentiation of the present invention can be usefully used to promote osteogenic differentiation of stem cell spheroids.

Figure 1 is a schematic view of the stem cell spheroid culture and analysis method according to the present invention.
Figure 2 shows the morphology of stem cell spheroids on days 1, 3, 5 and 7 of culture, and the scale bar represents 200 μm.
3 is a result of analyzing the diameter of spheroids on days 1, 3, 5, and 7 of culture after treating stem cell spheroids with BMP-4 at 0, 2, 6, and 10 ng/mL.
Figure 4a shows an Optical/Live/Dead/Merged cell image of a stem cell spheroid on day 3 of culture. Scale bar represents 200 μm.
Figure 4b shows an Optical/Live/Dead/Merged cell image of stem cell spheroids on day 7 of culture. Scale bar represents 200 μm.
Figure 4c is a graph showing cell viability by CCK-8 assay on days 1, 3, 5 and 7 of culture.
* A statistically significant difference was shown compared to the group treated with 2 ng/mL of BMP-4 on the first day of culture ( P < 0.05).
5 is a graph showing the activity of alkaline phosphatase on days 1, 3, 5 and 7 of culture.
* A statistically significant difference was shown compared to the group treated with 0 ng/mL of BMP-4 on the 3rd day of culture ( P < 0.05).
6 is a scatter plot showing differential expression of mRNA when BMP-4 was treated with 0, 2, 6, and 10 ng/mL.
The x, y-axes represent relative expression levels. Red means that the expression level of the y-value is higher than that of the x-value, and green means that the expression level of the y-value is lower than the expression level of the x-value.
A. 2 ng/mL/0 ng/mL; B. 6 ng/mL/0 ng/mL; C. 10 ng/mL/0 ng/mL
D. 6 ng/mL/2 ng/mL; E. 10 ng/mL/2 ng/mL; F. 10 ng/mL/6 ng/mL
Figure 7 shows the gene ontology analysis of differentially expressed mRNAs in a Venn diagram (fold change of 1.3 and log2 normalized read count of 4 were applied to the analysis).
Figure 8 shows the results of clustering analysis of differentially expressed mRNAs related to osteoblast differentiation (fold change of 1.3 and log2 normalized read count of 4 were applied to the analysis). .
9A is a graph showing the results of changes in the expression of RUNX2 and CTNNB1 (Log2 normalized read counts).
Figure 9b is a graph showing the results of expression changes of RUNX2 and CTNNB1 (Log2 fold change).
10 shows the PI3K-AKT signaling pathway.

Specific examples described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims.

Example

Experimental Materials and Methods

1. Isolation and culture of gingival-derived stem cells

Healthy gingiva tissues were obtained from healthy patients undergoing periodontal treatment, approved by the Clinical Trial and Research Review Board of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea.

The gingival tissue was immediately placed in sterile phosphate buffered saline (PBS, Welgene) containing 100 U/ml penicillin (Sigma Aldrich, USA) and 100 μg/ml streptomycin (Sigma Aldrich, USA) and placed at 4°C. The gingival tissue was ground by de-epithelialization, digested with collagenase IV (Sigma Aldrich), and cultured at 37°C in a humidified incubator with 5% CO ₂ and 95% O ₂ . After 24 hours of culture, non-adherent cells were washed off and replaced with a medium based on α-MEM medium. The medium contained 15% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2-phosphate (Sigma Aldrich, USA). After that, by changing the medium every 2-3 days, human gingiva-derived adult stem cells were established.

The cells exhibited stem cell characteristics such as colony-forming ability, plastic adherence, and differentiation into various lineages (osteogenesis, adipogenesis, chondrogenesis), and the cells were also CD44, CD73 by flow cytometry. , CD90 and CD105 were expressed, but CD14, CD45, CD34 and CD19 were not expressed.

2. Formation of cell spheroids using gingiva-derived stem cells

Cell spheroids were formed from gingiva-derived stem cells using concave micro-wells made of 600 μm diameter silicone elastomer (H389600, StemFIT 3D; MicroFIT, Seongnam, Korea). 1 x 10 ⁶ cells were placed in each well and the response of the cells was evaluated. The present inventors treated gingival-derived stem cells with BMP-4 to a final concentration of 0, 2, 6, and 10 ng/mL. Morphological changes of stem cell spheroids were observed using an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany). Changes in spheroid diameter were assessed on days 1, 3, 5 and 7. The diameter of the spheroid was measured by referring to the prior literature (Lee, SI, et al., 2016, Biomed Rep 4: 97-101). Figure 1 shows an overview of the stem cell spheroid culture and analysis method according to the present invention.

3. Cell viability measurement

For the qualitative analysis of stem cell spheroids on days 3 and 7 of culture, we used a commercially available two-color assay based on plasma membrane integrity and esterase activity. A kit (Live/Dead Kit assay, Molecular Probes, Eugene, OR, USA) was used. In the Live/Dead Kit assay, live cells exhibit strong, single green fluorescence and dead cells exhibit red fluorescence. Briefly, the analysis method is described, spheroids are washed twice with growth medium, 2 μL of 50 mM calcein acetoxymethyl ester working solution and 4 μL of 2 mM ethidium homodimer-1 (ethidium homodimer-1) was loaded and incubated for 30 minutes at room temperature. Spheroids stained with calcein acetoxymethyl ester working solution and ethidium homodimer-1 were observed under a fluorescence microscope (Axiovert 200; Zeiss, Germany). For quantitative analysis of cell viability of stem cells on days 1, 3, 5 and 7 of culture, the present inventors used a water-soluble tetrazolium salt-based, cell counting kit-8 (CCK-8, Dojindo , Tokyo, Japan) assay kits were used to evaluate viability. Cell counting kit is an analysis method to check cell viability. It uses the principle that water-soluble tetrazolium salt is reduced by cell dehydrogenase in the culture medium to become formazan, and the amount of formazan produced is Since it is proportional to the number of cells, the viability of the cells can be confirmed by checking the amount of formazan produced by colorimetric assays. Briefly explaining the analysis process, the cells were incubated with a water-soluble tetrazolium salt (WST-8) solution at 37 ° C for 45 minutes, and the spectrophotometer absorbance at 450 nm was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA).

4. Alkaline phosphatase (ALP) activity evaluation

Alkaline phosphatase (ALP) activity was evaluated to evaluate the osteogenic potential of gingival-derived stem cell spheroids.

ALP is known as a marker of osteoblast activity and acts as a regulator of inorganic phosphate transport and cell division or differentiation during the calcification process. Therefore, ALP activity was measured to confirm whether BMP-4 induces the differentiation of gingival-derived stem cell spheroids into osteoblasts.

The present inventors cultured stem cell spheroids in an osteogenic differentiation induction medium, and then analyzed alkaline phosphatase activity on days 1, 3, 5, and 7 of culture. A commercially available kit (K412-500, BioVision, Inc., Milpitas, USA) was used for evaluation of the level of alkaline phosphatase activity. Briefly explaining the analysis process, the cell lysate was incubated with a p-nitrophenyl phosphate substrate for 40 minutes at 25°C. Absorbance was measured at 405 nm using a microplate reader (BioTek, Winooski, VT, USA).

5. mRNA sequencing, gene ontology and pathway analysis

Construction of the RNA library was performed using the SENSE mRNA-Seq Library Prep Kit (Lexogen, Greenland, NH, USA). RNA was incubated with magnetic beads containing oligo-dT and a washing solution was used to remove all other RNA except mRNA. Random hybridization of starter/stopper heterodimers applied to poly(A) RNA still coupled to magnetic beads to generate a library. These starter/stopper heterodimers have Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction was applied to extend the starter into the next hybrid heterodimer. Then, the newly synthesized cDNA insert was ligated to the stopper. Second strand synthesis was applied to remove the library from the beads and the library. The library was then amplified and barcodes were introduced. High-throughput sequencing was performed using a HiSeq 2500 (Illumina, San Diego, CA, USA) for paired-end 100 sequencing.

RNA-Seq reads were mapped using a software tool (TopHat, Toronto, Canada). Assembling transcripts and detecting differential expression of genes or isoforms was performed in alignment files using cufflinks. Gene classification was performed using the Medline database (http://www.ncbi.nlm.nih.gov/), DAVID (http://david.abcc.ncifcrf.gov/), and GenMAPP (http://www.genmapp.org/ ) and BioCarta (http://www.biocarta.com/). Pathway analysis was performed for differentially expressed genes. A fold change of 1.3 and a log2 normalized read count of 4 were applied for analysis.

6. Statistical analysis

Experimental data are presented as mean ± standard deviation. Normality test was performed, and one-way analysis of variance (ANOVA) including Tukey's post hoc test was performed using a commercially available program (SPSS 12 for Windows, SPSS Inc., USA) to determine differences between groups. was performed to confirm The significance level is 0.05.

Experiment result

1. Formation of cell spheroids using human gingiva-derived stem cells

Spheroids were well formed in each microwell on day 1 of culture (see Figure 2). In addition, when BMP-4 was added at concentrations of 2, 6, and 10 ng/mL, no significant morphological changes in cell spheroids were observed. Morphological results on days 3, 5 and 7 of culture show that no significant morphological changes were observed at longer incubation times. The analysis results of the spheroid diameter are shown in Figure 3. It was analyzed that the spheroid diameter generally decreased with incubation time.

2. Evaluation of cell viability after BMP-4 treatment

Qualitative results on the viability of cell spheroids were obtained by analyzing the Live/Dead Kit assay on days 3 and 7 of culture (FIG. 4a; day 3 of culture, FIG. 4b; day 7 of culture). In all cases, most cells within cell spheroids showed intense green fluorescence. In cell spheroids on day 3 of culture, red fluorescence was partially observed around the spheroid border. In the comparison between the groups on the 7th day of culture, no significant difference was found (see Fig. 4b).

In addition, the quantitative analysis results for cell viability on days 1, 3, 5, and 7 of culture are shown in FIG. 4c. Quantitative analysis revealed no significant differences between groups on day 1 of culture ( P > 0.05). Generally, there was no statistically significant difference in cell viability between groups at longer incubation times.

3. Evaluation of alkaline phosphatase activity after BMP-4 treatment

The results of the alkaline phosphatase activity assay on days 1, 3, 5 and 7 of culture are shown in FIG. 5 . In general, alkaline phosphatase activity was found to increase as the culture time increased, up to 7 days. In addition, when BMP-4 was treated at 2 ng/mL on the 3rd day of culture, a significantly higher value was shown when compared to the untreated control group (0 ng/mL) ( P < 0.05).

4. Gene ontology analysis

As a result of the analysis, a total of 25,737 mRNAs were analyzed to be differentially expressed. Scatter plots differentially expressing mRNA are shown in FIG. 6 . Gene ontology analysis of differentially expressed mRNAs is shown in the Venn diagram of FIG. 7 . Compared to the results of the control group (0 ng/mL), 1,270 mRNAs were up-regulated and 1,070 mRNAs were down-regulated in the group treated with BMP-4 at 2 ng/mL. Compared to the control group (0 ng/mL), 1,536 mRNAs were up-regulated and 1,889 mRNAs were down-regulated in the group treated with BMP-4 at 6 ng/mL. Compared to the control group (0 ng/mL), 1,525 mRNAs were up-regulated and 1,533 mRNAs were down-regulated in the group treated with BMP-4 at 10 ng/mL.

The results of clustering analysis of differentially expressed mRNAs related to osteoblast differentiation are shown in FIG. 8 . Expression changes of RUNX2 and CTNNB1 are shown in FIG. 9 . The expression of CTNNB1 was analyzed to increase as BMP-4 was treated, and the expression of RUNX2 was analyzed to increase in a dose-dependent manner with up to 6 ng/mL of BMP-4 (see FIGS. 9a and 9b). Phosphoinositide-3-kinase-protein kinase B/Akt (PI3K/AKT) signaling pathway was analyzed to be involved in target genes selected for osteoblast differentiation (see FIG. 10).

Claims (15)

delete delete delete delete delete delete delete delete delete In the method for promoting bone differentiation of stem cell spheroids,
Including; treating stem cell spheroids formed from the separated stem cells with bone morphogenetic protein-4;
The stem cells are human gingiva-derived adult stem cells,
The human gingival-derived adult stem cells are CD44, CD73, CD90 and CD105 positive and CD14, CD45, CD34 and CD19 negative,
The bone morphogenetic protein-4 is included in an amount of 2 to 6 ng/mL,
The stem cell spheroid is a method for promoting bone differentiation of stem cell spheroids, characterized in that for culturing on a concave surface. delete delete delete delete delete

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