KR102283508B1 - Composition for Facilitating Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-2 As Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for promoting osteogenic differentiation of stem cell spheroids and a method for promoting osteogenic differentiation using the same. According to the present invention, the composition for promoting osteogenic differentiation of stem cell spheroids comprising osteogenic protein-2 as an active ingredient has an effect of promoting osteogenic differentiation of stem cell spheroids while maintaining the shape of the stem cell spheroids. The composition for promoting osteogenic differentiation of stem cell spheroids of the present invention may be usefully used to promote osteogenic differentiation of stem cell spheroids.

Description

Composition for Facilitating Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-2 As Active Ingredient comprising osteogenic protein-2 as an active ingredient

The present invention relates to a composition for promoting bone differentiation of stem cell spheroids containing bone morphogenetic protein-2 as an active ingredient, and a method for promoting bone differentiation using the same.

Bone marrow-derived mesenchymal stem cells are known to have multiple lineage differentiation potential and are used for various therapeutic purposes.

Bone morphogenetic protein-2 (BMP-2) has been widely applied for regeneration of destroyed bones. More recently, BMP-2 has been used for osteogenic differentiation of stem cells. Application of BMP-2 enhanced mineralization in vitro. In addition, it has been reported that type 2 diabetes can inhibit the differentiation of bone marrow-derived mesenchymal stem cells. BMP-2 promoted bone marrow-derived mesenchymal stem cell osteogenesis in type 2 diabetic rats via the Wnt signaling pathway.

More recently, three-dimensional human bone marrow-derived mesenchymal stem cells have been tested for osteogenic evaluation.

However, little is known about bone differentiation and mineralization of bone marrow-derived mesenchymal stem cell spheroids by BMP-2.

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

Republic of Korea Patent Registration No. 10-1972988 (Registration Date: 2019.04.22)

The present inventors studied whether bone morphogenetic protein-2 (BMP-2) can be used for osteodifferentiation of bone marrow-derived mesenchymal stem cell spheroids, and BMP-2 was treated with bone marrow-derived mesenchymal stem cells. By doing so, the shape of the stem cell spheroids was well maintained, and when BMP-2 was added to the osteodifferentiation induction medium, it was confirmed that the osteogenic differentiation of the stem cell spheroids was promoted, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for promoting osteodifferentiation of stem cell spheroids containing BMP-2.

Another object of the present invention is to provide a method for promoting osteodifferentiation of stem cell spheroids comprising the step of treating the stem cells with BMP-2.

Other objects and technical features of the present invention are more particularly set forth by the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for promoting bone differentiation of stem cell spheroids comprising bone morphogenetic protein-2 as an active ingredient.

In the present invention, the active ingredient "osteogenesis protein-2" is widely applied for regeneration of destroyed bones (Fan, J., et al., 2015. Tissue Eng Part A 21: 2053-2065) and A substance that is used for bone differentiation (Lee, YH, et al., 2019. J Biomed Mater Res B Appl Biomater 107(2):286-294) and improves mineralization in vitro (Wongwitwichot, P) ., et al., 2017. Adv Med Sci 62: 266-272).

In the present invention, “stem cells” are cells with self-renewal ability that are pluripotent or totipotent capable of differentiating into cells of all tissues of an individual. means, and includes embryonic stem cells, induced pluripotent stem cells and adult stem cells.

The term “embryonic stem cell” is derived from a fertilized egg produced by combining sperm, which is a male reproductive cell, and an egg, which is a female reproductive cell. In addition, embryonic stem cells are obtained from the inner cell mass of the blastocyst before implantation, and have the ability to differentiate into cells of any tissue, but if culture conditions are given, they are undifferentiated cells that can proliferate indefinitely in an undifferentiated state. say In other words, pluripotent cells are expected to be used in the treatment of numerous patients with just one embryonic stem cell line, as cell differentiation is induced in a desired direction in a specialized differentiation culture environment and can be used as a cell treatment for diseases such as nerves, diabetes, and blood. means

The term “induced pluripotent stem cells” refers to cells induced to have pluripotent differentiation capacity through artificial dedifferentiation processes, and is also referred to as induced pluripotent stem cells (iPSCs).

The term “adult stem cell” is a primitive cell just before differentiation isolated from the tissues of mammals including humans, and has the ability to proliferate indefinitely and various types of cells (eg, adipocytes, cartilage It is a stem cell that can differentiate into cells, muscle cells, bone cells, etc.).

In the present invention, a “cell spheroid” is an aggregate of cells formed by aggregating cells in a three-dimensional form during cell culture. Recently, the cultivation of spheroids, which are three-dimensional cell tissues having functions equivalent to those in vivo, has attracted attention. In order to induce cell aggregation to mimic cancer, and to induce normal secretion of insulin for the treatment of diabetes, a method of transplanting aggregated cells is used in transplanting islet cells, so mass production of spheroids this is being requested In addition, as stem cell research matures, various methods have been tried to 3D culture embryonic stem cells and apply them to the study of various differentiation mechanisms.

In the present invention, "differentiation" refers to a phenomenon in which a less specialized cell develops into a specific cell, and the cell size, shape, membrane potential, metabolic activity, and response to a signal change into a specific type of cell. The differentiation mainly occurs during the process of forming a complex tissue in a multicellular organism from a single zygote, or adult stem cells are differentiated into specific cells when the damaged tissue is repaired in adulthood.

According to one embodiment of the invention, the composition for promoting osteodifferentiation of the stem cell spheroids may contain the osteogenic protein-2 in an amount of 0.0001 to 500 ng/ml, preferably 0.0005 to 300 ng/ml. It may be included in the content, and more preferably, may be included in an amount of 0.001 to 100 ng/ml, but is not limited thereto.

According to one embodiment of the invention, the stem cells may include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized. Preferably, it may be an adult stem cell, and more preferably a bone marrow-derived mesenchymal stem cell.

According to another aspect of the present invention, there is provided a method for promoting osteodifferentiation of stem cell spheroids, comprising the step of treating the osteogenic protein-2 to the stem cells.

The method for promoting osteodifferentiation of stem cell spheroids of the present invention includes treating the osteogenic protein-2 to stem cells and culturing the osteogenic protein-2-treated stem cells.

According to one embodiment of the invention, in the method for promoting osteodifferentiation of stem cell spheroids of the present invention, the osteogenic protein-2 may be included in an amount of 0.0001 to 500 ng/ml, preferably 0.0005 to 300 ng/ml It may be included in a content of, more preferably, may be included in a content of 0.001 to 100 ng/ml, but is not limited thereto.

According to one embodiment of the invention, in the method for promoting osteodifferentiation of stem cell spheroids of the present invention, the stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells (ips cells) in which various differentiated cells are initialized. can, but is not limited thereto. Preferably, it may be an adult stem cell, and more preferably a bone marrow-derived mesenchymal stem cell.

According to one embodiment of the invention, in the method for promoting osteodifferentiation of stem cell spheroids of the present invention, the stem cell spheroids are preferably cultured on a concave surface, but are not limited thereto.

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

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

(ii) The composition for promoting osteodifferentiation of stem cell spheroids comprising osteogenic protein-2 of the present invention as an active ingredient has an effect of promoting osteodifferentiation of stem cell spheroids while maintaining the shape of stem cell spheroids .

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

1 is a schematic diagram of a 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, 7, 14 and 21 of culture. Scale bars indicate 200 μm.
Figure 3a shows a Live/Dead/Merged cell image of stem cell spheroids on day 1 of culture. Scale bar represents 50 μm.
Figure 3b shows Live/Dead/Merged cell images of stem cell spheroids on day 3 of culture. Scale bar represents 50 μm.
Figure 3c shows Live/Dead/Merged cell images of stem cell spheroids on day 5 of culture. Scale bar represents 50 μm.
Figure 3d shows Live/Dead/Merged cell images of stem cell spheroids on day 7 of culture. Scale bar represents 50 μm.
4 is a graph showing cell viability by Cell Counting Kit-8 analysis of culture days 1, 3, 5 and 7;
* A statistically significant difference was observed compared to the control group on day 3 of culture (P < 0.05).
5 is a graph showing the activity of alkaline phosphatase on the 14th day of culture.
* A statistically significant difference was observed compared to the control group (P < 0.05).
6A is an image of Alizarin Red S staining results observed under a microscope on days 7, 14 and 21 of culture, and the scale bar indicates 200 μm.
6B is a graph showing the quantitative analysis of Alizarin Red S staining on day 21 of culture.
* A statistically significant difference was observed compared to the control group (P < 0.05).
7A is a graph showing the quantitative analysis of Runx2 expression by quantitative polymerase chain reaction on day 7 of culture.
* A statistically significant difference was observed compared to the control group (P < 0.05).
Figure 7b is a graph showing the quantitative analysis of collagen I (Col1) expression by the quantitative polymerase chain reaction on the 7th day of culture.
* A statistically significant difference was observed compared to the control group (P < 0.05).

The specific examples described herein 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 modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.

Example

Experimental materials and methods

1. Cell spheroid formation using bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (Catholic MASTER cells) were obtained from the Catholic Cell Therapy Center (CIC, Seoul, Korea). The Catholic Cell Therapy Project Group confirmed that the human bone marrow-derived mesenchymal stem cells expressed more than 90% of CD73 and CD90. The culture medium was changed every 2 or 3 days, and the bone marrow-derived mesenchymal stem cells were cultured at 37° C. _{in 95% O 2} and 5% CO _{2 .} grown in an incubator.

1 shows an overview of the stem cell spheroid culture and analysis method according to the present invention. For stem cell spheroid formation, a commercially available concave micro-well (H389600, StemFIT 3D; MicroFIT, Seongnam, Korea) was used. A total of 1 x 10 ⁶ cells were placed in each well and the response of the cells was evaluated. The present inventors treated bone marrow-derived mesenchymal stem cells with BMP-2 at predetermined concentrations of 0, 10 and 100 ng/ml. Morphological characteristics of stem cell spheroids were evaluated using an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany). Morphological evaluation of stem cell spheroids was performed on days 1, 3, 7, 14 and 21 of culture.

2. Measurement of Cell Viability

The present inventors cultured stem cell spheroids in an osteodifferentiation induction medium.

For the qualitative analysis of stem cell spheroids on culture days 1, 3, 5 and 7, we present a commercially available two-color, based on plasma membrane integrity and esterase activity. -color) assay kit (Live/Dead Kit assay, Molecular Probes, Eugene, OR, USA) was used. In the Live/Dead Kit assay, live cells show strong, single green fluorescence and dead cells show red fluorescence. In addition, a quantitative cell viability test was performed using an assay kit (Cell Counting Kit-8, Dojindo, Tokyo, Japan) based on a water-soluble tetrazolium salt.

3. Alkaline phosphatase activity level and calcium deposition

Bone differentiation capacity was evaluated by anthraquinone dye assay for evaluation of alkaline phosphatase activity level and calcium deposition. Cell spheroids grown on a culture plate together with an osteodifferentiation induction medium were obtained on day 14 of culture. A commercially available kit (K412-500, BioVision, Inc., Milpitas) was used for the evaluation of alkaline phosphatase activity levels. Anthraquinone dye assay for calcium deposition was used to evaluate osteogenic potential on culture days 7, 14 and 21. Stem cell spheroids were stained with Alizarin Red S for 30 min at room temperature after washing and fixation procedures. Quantification of bound dye was performed on day 21.

4. Evaluation of Runx2 and Col1 by Quantitative Polymerase Chain Reaction

Total RNA was isolated from the cells obtained on day 7 of culture using a purification kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA was used as a template for reverse transcription using SuperiorScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and the amount of RNA was measured by spectrophotometric (ND-2000, Thermo Fisher Scientific, Inc.) absorbance at 260 nm and 280 nm. determined to be within the ratio.

mRNA expression was detected by quantitative polymerase chain reaction. Sense and antisense primers were designed based on GenBank, and the base sequences of the primers are shown in Table 1 below. Normalization was performed by applying β-actin as a housekeeping gene.

primer
designation direction order Runx2 F 5'-AAT GAT GGT GTT GAC GCT GA-3' (SEQ ID NO: 1) R 5'-TTG ATA CGT GTG GGA TGT GG-3' (SEQ ID NO: 2) Col1 F 5'-CCA GAA GAA CTG GTA CAT CAG CAA-3' (SEQ ID NO: 3) R 5'-CGC CAT ACT CGA ACT GGA ATC-3' (SEQ ID NO: 4) β-actin F 5'-TGG CAC CCA GCA CAA TGA A-3' (SEQ ID NO: 5) R 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3' (SEQ ID NO: 6)

5. Statistical Analysis

Experimental data are presented as mean ± standard deviation. Tests for normality and variance were performed. Two-way ANOVA was performed to evaluate the effect of concentration and time point. One-way ANOVA with Tukey's post hoc test was applied to test for differences between groups (SPSS 12 for Windows, SPSS Inc., Chicago, IL, USA) (P < 0.05).

Experiment result

1. Formation of cell spheroids using human bone marrow-derived mesenchymal stem cells

Spherical stem cell aggregates were identified on day 1 of culture (see FIG. 2 ). By the addition of BMP-2, a significant morphological change of the cell spheroids cultured in the osteodifferentiation induction medium was not observed. The shape of the cell spheroids at 3, 7, 14 and 21 days of culture is shown in FIG. 2, and no significant change was observed at longer incubation times.

2. Cell viability

Qualitative results for the viability of cell spheroids were analyzed by Live/Dead Kit assay on day 1 of culture, as shown in FIG. 3a. In all cases, most cells within the cell spheroids displayed green fluorescence indicating that the cells were alive. No significant change was found at longer incubation times (see FIGS. 3b to 3d).

Quantitative values for cell viability on days 1, 3, 5 and 7 of culture are shown in FIG. 4 . When BMP-2 was treated at a concentration of 0, 10 and 100 ng/ml on day 1 of culture, the absorbance values at 450 nm were 0.247 ± 0.005, 0.241 ± 0.011 and 0.251 ± 0.006, respectively (P > 0.05).

3. Alkaline phosphatase activity level and calcium deposition

The results of analysis of alkaline phosphatase activity on day 14 of culture are shown in FIG. 5 . When BMP-2 was treated at a concentration of 0, 10 and 100 ng/ml on day 14 of culture, the absorbance values at 405 nm were 0.081 ± 0.001, 0.084 ± 0.007 and 0.103 ± 0.006, respectively. Compared with the control group (0 ng/ml), the absorbance value was significantly higher when BMP-2 was treated at a concentration of 100 ng/ml (P < 0.05).

The results of mineralization analysis on days 7, 14 and 21 of culture are shown in FIG. 6A . Mineralized extracellular deposits were observed evenly in each group. Quantitative analysis results on day 21 of culture showed that the absorbance value was significantly higher when BMP-2 was treated at a concentration of 100 ng/ml compared with the control group (P < 0.05) (see FIG. 6b ).

4. Runx2 by quantitative polymerase chain reaction and evaluation of Col1

As a result of analyzing the mRNA expression level using a quantitative polymerase chain reaction, it was observed that the mRNA expression level of Runx2 was significantly higher when BMP-2 was treated at a concentration of 100 ng/ml compared to the control group (P < 0.05) (see Figure 7a). When BMP-2 was treated at a concentration of 100 ng/ml, it was confirmed that there was no significant change in collagen I (Col1) mRNA expression compared to the control group (P > 0.05) (see FIG. 7b ).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Composition for Facilitating Osteogenic Differentiation of Stem Cell Spheroids Comprising Bone Morphogenetic Protein-2 As Active Ingredient <130> DPC193736 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for Runx2 gene <400> 1 aatgatggtg ttgacgctga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for Runx2 gene <400> 2 ttgatacgtg tgggatgtgg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for Col1 gene <400> 3 ccagaagaac tggtacatca gcaa 24 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for Col1 gene <400> 4 cgccatactc gaactggaat c 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for beta actin gene <400> 5 tggcacccag cacaatgaa 19 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for beta actin gene <400> 6 ctaagtcata gtccgcctag aagca 25

Claims (7)

delete delete delete In the method for promoting osteodifferentiation of stem cell spheroids,
Comprising the step of treating the bone morphogenetic protein-2 to the isolated stem cells,
The stem cells are human bone marrow-derived mesenchymal stem cells,
The stem cell spheroids are cultured on a concave surface,
A method for promoting osteodifferentiation of stem cell spheroids, characterized in that the mRNA expression of Runx2 is increased in the osteogenic protein-2 treated stem cells. 5. The method of claim 4,
The osteogenic protein-2 is a method for promoting osteodifferentiation of stem cell spheroids, characterized in that it is included in an amount of 0.0001 to 500 ng/ml. delete delete

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