Method for differentiating pluripotent stem cell induced from mesenchymal stem cell into chondrocyte

Abstract

The present invention relates to a medium composition containing an Ecklonia cava extract for dedifferentiating an induced pluripotent stem cell. Also, the present invention relates to a method for differentiating an induced pluripotent stem cell, produced by using the medium composition into a chondrocyte. When using the medium composition according to the present invention, induced pluripotent stem cells using mesenchymal stem cells can be produced efficiently, and the pluripotent stem cells which have been produced can be useful as a cell treatment agent by being capable of being differentiated into chondrocytes.

Claims

1. A method for differentiating mesenchymal stem cells into chondrocytes, comprising the steps of: (a) adding 100-400 g/ml of an Ecklonia cava extract and purified deionized water containing SiO.sub.2, Al.sub.2O.sub.3, TiO.sub.3, Fe.sub.2O.sub.3, CaO, Na.sub.2O, K.sub.2O, and LiO to a culture medium; (b) dedifferentiating mesenchymal stem cells into induced pluripotent stem cells in the medium; and (c) differentiating the induced pluripotent stem cells into chondrocytes in a chondrocyte differentiation medium.

2. The method of claim 1, wherein the cell culture medium is selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM-F12, -MEM (-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), and MacCoy's 5A medium.

3. The method of claim 1, wherein the purified deionized water is 0.01-10% v/v.

4. The method of claim 1, wherein the chondrocyte differentiation medium comprises dexamethasone, Acetylsalicylic acid (AsA), sodium pyruvate, proline, Insulin-Transferrin-Selenium (ITS) and TGF.

5. The method of claim 4, wherein the cell culture medium is selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM-F12, -MEM (-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), and MacCoy's 5A medium.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a view illustrating that pluripotent stem cells that are substantially the same as embryonic stem cells are induced when mesenchymal stem cells are cultured using a medium containing an Ecklonia cava extract medium.

(2) FIG. 2 shows the expression of specific proteins, which confirms that pluripotent stem cells induced by the method of the present invention are pluripotent stem cells.

(3) FIG. 3 illustrates the formation of pluripotent stem cell colonies induced using various concentrations of an Ecklonia cava extract according to the method of the present invention.

(4) FIG. 4 shows gene expression in pluripotent stem cells induced by the method of the present invention.

(5) FIG. 5 shows the results of testing the in vivo differentiation potential of pluripotent stem cells induced by the method of the present invention.

(6) FIG. 6 shows the results of differentiating pluripotent stem cells, induced by the method of the present invention, into chondrocytes by use of chondrocyte differentiation mediums.

BEST MODE

(7) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only intended to describe the present invention in further detail, and it will be apparent to a person having ordinary knowledge in the art that the scope of the present invention is not limited to these examples.

EXAMPLES

Example 1: Preparation of Ecklonia cava Extract

(8) An herbal sample used in the experiment was purchased from Jeju-do, and used in the experiment after precise accurate judgment. 100 g of a dried herbal sample was added to 1 L of 70% methanol, extracted under reflux for 16 hours, and filtered through a filter paper. The filtrate was concentrated in a rotary evaporator under reduced pressure, and then immediately, freeze-dried.

Example 2: Isolation and Culture of Mesenchymal Stem Cells from Human Umbilical Cord

Example 2-1: Collection of Human Umbilical Cord

(9) Umbilical cord tissue was collected immediately after delivery. Before the tissue sample was transferred to the laboratory, it was rinsed clean, and then immediately, transferred into a 500 mL sterilized glass bottle containing F-12 medium and a transport medium (50 IU/mL of penicillin, 50 g/mL of streptomycin (purchased from Invitrogen)). In the laboratory, extraction of stem cells was performed in a flow hood (Class 100) under sterile conditions. The sample was first transferred to a sterile stainless steel container. The umbilical cord tissue sample was washed several times with PBS, and then cut into 2 cm long pieces and transferred to a cell culture dish having a diameter of 10 cm. Herein, the sample was additionally washed and subjected to anti-infective treatment with 70% ethanol, and it was washed several times with PBS containing an antibiotic mixture (50 IU/mL of penicillin, 50 g/mL of streptomycin (purchased from Invitrogen)) until the solution was clean.

Example 2-2: Isolation of Stem Cells from Human Umbilical Cord and Culture of thereof

(10) In order to isolate Wharton's jelly (base of umbilical cord) from the blood vessels and other internal elements of umbilical cord, the umbilical cord was first incised. After removing blood vessels, the isolated Wharton's jelly was cut into small pieces (0.5 cm0.5 cm) in order to extract cells.

(11) Explant was performed by placing the umbilical cord Wharton's jelly pieces in different tissue culture dishes having cell culture conditions suitable for extraction of epithelial stem cells or mesenchymal stem cells. In order to isolate and culture mesenchymal stem cells, the explanted tissue was added to 5 mL of Dulbecco's modified eagle medium (DMEM) F-12 (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), 10% FBS, 100 unit/mL of penicillin and 50 g/mL of streptomycin, and was maintained in a CO.sub.2 incubator at 37 C. The medium was replaced every three or four days. The outgrowth of cells was monitored with an optical microscope. The outgrowing cells were treated with trypsin (0.125% trypsin/0.05% EDTA) for further expansion and frozen storage (using DMEM/10% FBS).

(12) The medium was replaced every three or four days. The outgrowth of cells from the explanted tissue is monitored with an optical microscope.

(13) In order to extract mesenchymal stem cells, the pellets of the cells were resuspended in medium DMEM F-12 (Gibco), 10% FBS, 100 unit/mL of penicillin, and 50 g/mL of streptomycin and counted. Then, the cells were inoculated into 10-cm tissue culture dishes at a density of 110.sup.6 cells/dish. The medium was replaced every three or four days. The growth of cells and formation of clones were monitored with an optical microscope. At a confluence of about 90%, the cells were sub-cultured as described above.

Experimental Example 1: Induction of Pluripotent Stem Cells from Mesenchymal Stem Cells

Experimental Example 1-1: Production of Pluripotent Stem Cells of Human Mesenchymal Stem Cells Using Various Concentrations of Ecklonia cava Extract

(14) An experiment for inducing pluripotent stem cells from human umbilical cord stem cells using various concentrations of the Jeju Ecklonia cava extract was performed. For a control group, the MSC culture medium DMEM F-12 (Gibco) (containing 10% FBS, 100 unit/mL of penicillin, and 50 g/mL of streptomycin) was used as a basal medium. For an experimental group, 1 g/mL, 10 g/mL, 100 g/mL, 200 g/mL, 400 g/mL, 800 g/mL and 1000 g/mL of the Jeju Ecklonia cava extract, and 0.1 v/v % of energy water (purified deionized water containing SiO.sub.2, Al.sub.2O.sub.3, TiO.sub.3, Fe.sub.2O.sub.3, CaO, Na.sub.2O, K.sub.2O, and LiO; STC Nara Co., Ltd., Korea), were added to media with human umbilical cord mesenchymal stem cells subcultured three times (FIG. 1). Human umbilical cord mesenchymal stem cells were isolated and washed, and the resulting mononuclear cells were inoculated into 6-well plates (dishes) at a density of 110.sup.4 cells and cultured under the conditions of 37 C. and 5% CO.sub.2.

(15) For the pluripotent stem cells induced by the method of the present invention, the expressions of OCT4, SOX2 and stage-specific embryonic antigen4 (SSEA4), which are proteins specific for embryonic stem cells, were analyzed by immunochemical staining using antibodies against the proteins. For straining, the cells were fixed with 4% paraformaldehyde, washed with PBS, and blocked with 1% BSA solution. Then, the cells were treated with primary antibodies against OCT4, SOX3 and SSEA4 and incubated at 4 C. for 18 hours. Then, the cells were washed with PBS, and treated with fluorescence (FITC)-labeled secondary antibodies against the primary antibodies and incubated at room temperature for 1 hour. After washing with PBS, expression of the proteins was analyzed using a confocal microscope, and the results of the analysis are shown in FIG. 2. In FIG. 2, BF indicates bright field, the second figure indicates the staining results for expression of each protein, and the third figure indicates the merge of the two figures (FIG. 2).

(16) As a result, in the experimental group, it was observed that only when the concentration of the Jeju Ecklonia cava extract was between 100 and 400 g/mL, colonies were formed after 10 days (FIG. 3). Further, OCT4, SOX2 and SSEA4, which are markers specific for pluripotent stem cells, were stained only in colonies, suggesting that the cells induced by the method of the present invention are pluripotent stem cells.

Experimental Example 1-2: Analysis and Comparison of Pluripotent Stem Cell Genes

(17) Colonies were detached from the pluripotent stem cells (produced in Example 2-1 above) using a 200 l pipette while observing the pluripotent stem cells with a microscope, and then total RNA was isolated using TRIzol reagent (manufactured by Invitrogen). cDNA was synthesized using reverse transcription-polymerase chain reaction (RT-PCR), and then PCR was performed using primers specific for OCT4, Sox-2, Nanog, c-Myc, and the control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Nanog, OCT4 and Sox-2 are characteristic genes appearing in embryonic stem cells, and c-Myc gene is a non-specific gene that may be positive in both embryonic stem cells and adult stem cells. The PCR products were analyzed by agarose gel electrophoresis to confirm the expression of these genes, and the results of the analysis are shown in FIG. 4. Referring to FIG. 4, the expression level of OCT4, which is a characteristic gene of pluripotent stem cells, was low in mesenchymal stem cells which did not undergo the induction process, whereas the expression levels of the characteristic genes were significantly high in the pluripotent stem cells (STC2013-F002) induced by the method of the present invention. SOX2 and Nanog, which are stem cell genes, were expressed at similar levels, and the expression level of c-Myc, a non-specific gene, was lower in cells (STC2013-F002) which underwent the induction process, than in the cells which did not undergo the induction process.

Experimental Example 2: Identification of Pluripotent Stem Cells by Teratoma Test

(18) In order to analyze the in vivo differentiation potential of the pluripotent stem cells induced by the method of the present invention, undifferentiated pluripotent stem cell colonies cultured on support cells were detached by treatment with trypsin-EDTA at day 5 of culture, and then added to collagenase and maintained in an incubator for 30 minutes. The undifferentiated pluripotent stem cells were recovered, and 110.sup.6 cells were injected subcutaneously into mice with severe combined immune deficiency (SCID). After 4 weeks, formed teratomas were harvested, fixed with 4% paraformaldehyde, and embedded in paraffin according to a conventional method. The tissue was sectioned to a thickness of 10 m and stained with hematoxylin and eosin.

(19) Referring to FIG. 5, it can be seen that teratoma was visibly formed in the area into which the induced pluripotent stem cells produced by the method of the present invention were injected. Specifically, it was histologically shown that teratomas were formed which are capable of differentiating into ectooderm-derived nervous tissue (FIG. 5a), mesoderm-derived muscle tissue (FIG. 5b), and endoderm-derived stomach tissue (columnar epithelium, FIG. 5c). Through the experiment, it can be confirmed that the cells induced by the method of the present invention have substantially the same in vivo differentiation potential as embryonic stem cells, i.e., pluripotency to differentiate into ectoderm, mesoderm, and endoderm.

Experimental Example 3: Dedifferentiation into Chondrocytes

(20) In order to induce differentiation into chondrocytes, mesenchymal stem cells were cultured in an incubator under the conditions of 95% humidity, 37 C. and 5% CO.sub.2 by use of a medium comprising a mixture of the Ecklonia cava extract and energy water, thereby inducing pluripotent stem cells from the mesenchymal stem cells. Then, the cells were cultured in the chondrocyte differentiation medium DMEM F-12 (containing 0.1 uM of dexamethasone, 50 g/ml of Acetylsalicylic acid (AsA), 100 g/ml of sodium pyruvate, 40 g/ml of proline, 10 ng/ml of TGF-1, 5% of ITS (Insulin-Transferrin-Selenium; 6.25 g/ml of insulin, 6.25 g/ml of transferring and 6.25 ng/ml of selenium), 1.25 mg/ml of bovine serum albumin, 5.35 mg.Math.ml of lioleic acid for 2 weeks. In order to verify differentiation into chondrocytes, Alcian blue histochemical staining was performed. As a result, as shown in FIG. 6, the cells were negative for Alcian blue before treatment with the differentiation medium (FIG. 6A) and positive to Alcian blue after treatment with the differentiation medium (FIGS. 6B and 6C), suggesting that the cells considered pluripotent stem cells could be differentiated into chondrocyte. The immunohistochemical staining method is same as the above immunohistochemical staining method.

(21) Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.