Method of making human cells expressing OCT4, SOX2, and Nanog using an Ecklonia cava extract

Abstract

The present disclosure relates to a medium composition for r25/eprogramming induced pluripotent stem cells, containing an Ecklonia cava extract. Also, the present disclosure relates to a method for manufacturing induced pluripotent stem cells by using the medium composition. When the medium composition according to the present disclosure is used, induced pluripotent stem cells can be efficiently produced using adipose-derived mesenchymal stem cells safely and easily. The manufactured pluripotent stem cells are differentiable into various cells, and thus can be favorably used as a cell therapeutic agent.

Claims

1. A method for manufacturing stem cells expressing Oct4, SOX2 and Nanog, comprising: (a) adding an Ecklonia cava extract which is extracted by water to a cell culture medium in an amount of 20-50 μg/ml relative to a volume of the cell culture medium, or by anhydrous or water-containing low alcohol having 1 to 4 carbon atoms to the cell culture medium in an amount of 10-400 μg/ml relative to the volume of the cell culture medium; and (b) culturing mammalian adipose-derived mesenchymal stem cells in the cell culture medium where the Ecklonia cava extract has been added to dedifferentiate into stem cells expressing Oct4, SOX2 and Nanog, wherein the stem cells are capable of differentiating into endoderm, ectoderm and mesodermal cells.

2. The method of claim 1, wherein the cell culture medium is selected from the group consisting of a Dulbecco's modified eagle's medium (DMEM), a minimal essential medium (MEM), a basal medium eagle (BME), RPMI 1640, F-10, F-12, DMEM F-12, a-minimal essential medium (a-MEM), a Glasgow's minimal essential medium (G-MEM), an Iscove's modified Dulbecco's medium (IMDM), and a MacCoy's 5A medium.

3. The method of claim 1, wherein the cell culture medium further comprises 0.01-10% (v/v) of 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram illustrating that substantially the same pluripotent stem cells as embryonic stem cells are induced in adipose-derived mesenchymal stem cells by injecting and culturing an Ecklonia cava extract medium.

(2) FIG. 2 illustrates formation of colonies of pluripotent stem cells induced according to a concentration of an Ecklonia cava extract (an ethanol extract) by a method (Example 1-1) of the present disclosure.

(3) FIG. 3 illustrates formation of colonies of pluripotent stem cells induced according to a concentration of an Ecklonia cava extract (a water extract) by a method (Example 1-2) of the present disclosure.

(4) FIG. 4 verifies that pluripotent stem cells induced by a method (Experimental Example 1-1) of the present disclosure are pluripotent stem cells by using specific-protein expression.

(5) FIG. 5 verifies that pluripotent stem cells induced by a method (Experimental Example 1-2) of the present disclosure are pluripotent stem cells by using specific-protein expression.

(6) FIG. 6 illustrates gene expression of pluripotent stem cells induced by the method (Experimental Example 1-1) of the present disclosure by a graph.

(7) FIG. 7 illustrates gene expression of pluripotent stem cells induced by the method (Experimental Example 1-2) of the present disclosure by a graph.

DETAILED DESCRIPTION

(8) In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

(9) Hereinafter, the present disclosure will be described in more detail through Examples. However, the present disclosure is not limited to the exemplary embodiments disclosed below, but can be implemented in various forms. The following exemplary embodiments are described in order to enable those of ordinary skill in the art to embody and practice the disclosure.

EXAMPLES

Example 1: Preparation of Ecklonia cava Extract

1.) Example 1-1: Preparation of Ecklonia cava Extract Using Ethanol Solvent

(10) Herb medicine samples used in an experiment were purchased in the Jeju island, exactly evaluated by the expert, and used in the experiment. 100 g of a dried herb medicine sample was added in 1 L of 70% ethanol, reflux-extracted for 16 hours with ethanol, and filtrated by using a filter. A filtrate was concentrated in a rotary decompression evaporator and immediately lyophilized.

2.) Example 1-2: Preparation of Ecklonia cava Extract Using Water

(11) Herb medicine samples used in an experiment were purchased in the Jeju island, exactly evaluated by the expert, and used in the experiment. 100 g of a dried herb medicine sample was added in 1 L of water, extracted for 16 hours with water by applying an ultrasonic extractor, and filtrated by using a filter. A filtrate was concentrated in a rotary decompression evaporator and immediately lyophilized.

Example 2: Isolation and Incubation of Mesenchymal Stem Cells from Human Adipose Tissue

1). Example 2-1: Extraction of Human Adipose Tissue

(12) The adipose tissue was immediately collected after liposuction. Before the sample was transferred to a laboratory, the adipose tissue suctioned in a sterile glass bottle of 500 ml was collected. Thereafter, the sterile glass bottle was sealed and then transferred to the laboratory. In the laboratory, mesenchymal stem cells were extracted in a flow hood of class 100 under a sterile condition. The sample was first transferred to a sterile stainless steel container. The sample was washed with PBS several times and then the adipose tissue sample was cut with a length of 2 cm and transferred to a tube of 50 ml, and herein, additionally washed and treated with 70% ethanol for anti-infection, and then washed several times with PBS added with an antibiotic mixture (50 IU/ml of penicillin and 50 μg/ml of streptomycin (purchased from Invitrogen)) until the solution was cleaned.

2). Example 2-2: Isolation and Incubation of Mesenchymal Stem Cells from Human Adipose Tissue

(13) The isolated adipose tissue was washed with PBS and finely cut, shaken once per 10 min at 37° C. by using a DMEM medium added with collagenase type1 (1 mg/ml), and digested for 1 hr. Next, the adipose tissue was washed with PBS and centrifuged for 5 min at 1000 rpm. A supernatant was suctioned and a pellet remaining on the bottom was washed with PBS and centrifuged for 5 min at 1000 rpm. It was filtered by a filter having a mesh size of 100 μm and washed with PBS after removing debris.

(14) For isolation/incubation of the mesenchymal stem cells, the explanted tissue was immersed in 5 ml of a Dulbecco's modified eagle medium (DMEM) F-12 (Gibco) added with 10% fetal bovine serum (FBS, Hyclone), 10% FBS, 100 unit/ml of penicillin, and 50 μg/ml of streptomycin and maintained at 37° C. in a cell incubator of nitrogen 95% and carbon dioxide 5%, and cells except for the stem cells were killed while maintaining a hypoxic state to increase purity of the mesenchymal stem cells. The medium was replaced every 3 or 4 days. The outgrowth of the cells was monitored by an optical microscope. The outgrown cells were treated with Trypsin (0.125% Trypsin/0.05% EDTA) for additional expansion and refrigeration (using DMEM/10% FBS).

(15) For extraction of the mesenchymal stem cells, pellets of the cells were resuspended and counted in the medium DMEM F-12 (Gibco), 10% FBS, 100 unit/ml of penicillin, and 50 μg/ml of streptomycin and inoculated on a tissue culture dish of 10 cm at a density of 1×10.sup.6 cells/dish. The medium was replaced every 3 or 4 days. The growth and clone formation of the cells were monitored by an optical microscope. In approximately 90% cell number (confluence), the cells were sub-cultured as described above.

(i) Experimental Example 1: Induction of Pluripotent Stem Cells from Adipose-Derived Mesenchymal Stem Cells

Experimental Example 1-1: Manufacture of Pluripotent Stem Cells of Human Adipose-Derived Mesenchymal Stem Cells According to Concentration of Ecklonia cava Extract in Example 1-1

(16) As an experiment for inducing pluripotent stem cells from human adipose-derived stem cells according to a concentration of a Jeju Ecklonia cava extract, in a control group, DMEM F-12 (Gibco) as a dedicated medium of MSC, 10% FBS, 100 unit/ml of penicillin, and 50 μg/ml of streptomycin were used as a basic medium, and in an experimental group, human adipose-derived mesenchymal stem cells which was subjected to three sub-cultures were used, and in the medium, the Jeju Ecklonia cava extract having concentrations of normal, 1 μg/ml, 20 μg/ml, 50 μg/ml, 100 μg/ml, 400 μg/ml, 800μ/ml, and 1 mg/ml 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) were added (see FIG. 1). The human adipose-derived mesenchymal stem cells were isolated and washed and monocytes were inoculated in a 6-well plate (dish) with 1×10.sup.4 cells and maintained and incubated at 37° C. and 5% CO.sub.2.

(17) As a result, in the experimental group, it was observed that only when the concentration of the Jeju Ecklonia cava extract was 100 to 400 μg/ml, colonies were formed after 10 days (see FIG. 2), and in this case, the microscope magnification was observed at 200× magnification.

(ii) Experimental Example 1-2: Manufacture of Pluripotent Stem Cells of Human Adipose-Derived Mesenchymal Stem Cells According to Concentration of Ecklonia cava Extract in Example 1-2

(18) Experimental Example 1-2 was experimented by the same method as Experimental Example 1-1 and a Jeju Ecklonia cava extract which was prepared in Example 1-2 was used. As a result, in the experimental group, it was observed that only when the concentration of the Jeju Ecklonia cava extract was 20 to 50 μg/ml, colonies were formed after 10 days (see FIG. 3), and in this case, the microscope magnification was observed at 200× magnification.

(iii) Experimental Example 1-3: Immunochemical Staining Analysis of Pluripotent Stem Cells Induced by Method of the Present Disclosure

(19) With respect to the pluripotent stem cells induced by the methods of Experimental Examples 1 and 2, whether to express specific genes OCT4 and SOX2 and protein of stage-specific embryonic antigen-4 (SSEA-4) to the embryonic stem cells was analyzed by using antibodies thereto and whether to express the protein was analyzed by using an immunochemical staining method.

(20) In the staining process, cells were first fixed by using 4% paraformaldehyde and washed with PBS, and blocked with a 1% BSA solution. The cells were treated with primary antibodies for OCT4, SOX3, and SSEA-4 and reacted at 4° C. for 18 hours, and then washed with PBS, treated with secondary antibodies with fluorescence pigment (fluorescein isothiocyanate, FITC) to the primary antibodies, and reacted at room temperature for 1 hour.

(21) The cells were washed with PBS and then the expression was analyzed by using a fluorescence microscope, and the result thereof was illustrated in FIGS. 4 and 5. The BF means a bright field and the second diagram means a staining result for each protein expression, and the third diagram illustrates a cell nucleus strained with DAPI.

(22) As a result, in both the Ecklonia cava extract extracted by using ethanol (Experimental Example 1-1) and the Ecklonia cava extract extracted by using water (Experimental Example 1-2), the pluripotent stem cells having a positive reaction only in the colony of OCT4, SOX2, and SSEA-4 as pluripotent stem cell-specific markers were verified (see FIGS. 4 and 5).

(iv) Experimental Example 1-4: Comparison of Gene Analysis of Pluripotent Stem Cells

(23) While the pluripotent stem cells manufactured in Experimental Examples 1-1 and 1-2 was observed by a microscope, only the colony was picked by using a pipette of 200 μl, and then the total RNA was isolated by using a TRIzol reagent (manufactured by Invitrogen Corporation). cDNA was synthesized by using reverse transcription-polymerase chain reaction (RT-PCR) and the PCR was performed by using a specific primer to OCT4, Sox-2, and Nanog genes and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a control gene. The Nanog, OCT4, and Sox-2 are specific genes in the embryonic stem cells. The PCR products were analyzed by agarose gel electrophoresis and results of verifying expression of these genes were illustrated in FIGS. 6 and 7.

(24) As a result, as illustrated in FIGS. 6 and 7, in mesenchymal stem cells (MSC, control group) without an induction process, an expression level of OCT4, SOX2 and Nanog as specific genes of the pluripotent stem cells is low, whereas in the pluripotent stem cells manufactured by the pluripotent stem cells (Experimental Example 1-1 (illustrated by EtOH EPN) and Experimental Example 1-2 (illustrated by Sonic EPN) induced by the method of the present disclosure, these specific genes were significantly highly expressed. The expression level of OCT4, SOX2 and Nanog as the stem cell genes can be clearly verified through graphs of FIGS. 6 and 7.

(25) From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.