KR101671883B1 - Method for Differentiating Pluripotent Stem Cell induced from adipose-derived Mesenchymal Stem Cell into Hematocyte - Google Patents

Abstract

The invention Ecklonia cava extract (Ecklonia The present invention relates to a culture medium for regenerating an induced pluripotency stem cell. The present invention also relates to a method for differentiating into pluripotent stem cells from induced pluripotent stem cells prepared using the culture medium composition. Using the medium composition according to the present invention, it is possible to efficiently produce induced pluripotent stem cells using mesenchymal stem cells, and the pluripotent pluripotent stem cells can be used as a patient-customized cell therapy agent because they can differentiate into adipocytes have.

Description

The present invention relates to a method for producing pluripotent pluripotent stem cells from adipose-derived mesenchymal stem cells,

TECHNICAL FIELD The present invention relates to a method for preparing patient-induced pluripotent stem cells using human pluripotent stem cell-derived medium composition of human fat-derived mesenchymal stem cells and differentiating them into hepatocytes.

Stem cells are collectively referred to as undifferentiated cells in the pre-differentiation stage that can be obtained from each tissue. It has the property of continuously producing the same cells for a certain period of undifferentiated state and the ability to differentiate into various cells constituting biological tissue under proper conditions.

Stem cells can be classified into embryonic stem cells and adult stem cells depending on the differentiation ability and generation time. Another classification is based on the ability of stem cells to differentiate, and can be divided into pluripotency, multipotency, and unipotency stem cells.

Adult stem cells can be divided into multipotent or monodisperse stem cells. Representative adult stem cells include mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). Mesenchymal stem cells differentiate into chondrocytes, osteoblasts, adipocytes, myocytes, and neurons. Hematopoietic stem cells differentiate into hematopoietic cells such as red blood cells, white blood cells, It is known to differentiate into cells.

On the other hand, pluripotent stem cells, which can differentiate into all three germ layers constituting the living body, are capable of differentiating into all cells or organ tissues of the human body. In general, embryonic stem cells . Although human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life forms, they are known to have superior cell proliferation and differentiation ability compared to adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., and have few ethical problems, but have limited differentiation ability compared to embryonic stem cells.

As an alternative to overcome these problems, various methods have been attempted to produce tailored pluripotent stem cells similar to embryonic stem cells by degenerating adult-derived cells. Representative methods include fusion with ES cell, somatic cell nuclear transfer, and reprogramming by gene factor. The cell fusion method has problems in terms of cell stability because the induced cells have two pairs of genes, and the somatic cell nuclear replacement method has a problem in that a large amount of eggs are required and the efficiency is also very low. In addition, the specific factor injection method uses a virus including a carcinogen to induce the differentiation by inserting a specific gene. Therefore, there is a high risk of cancer development. Due to the low efficiency and difficulty in the method, It is becoming a problem.

In order to obtain pluripotent stem cells successfully and in large quantities, a culture composition in the step of culturing the isolated adipose-derived mononuclear cells is very important, and studies for producing pluripotent stem cells with a higher amount and a higher efficiency induction method are needed State.

Meanwhile, a composition for treating or preventing atopic disease (Patent Publication No. 10-2009-0043115), a skin composition such as a cosmetic (Patent Document 10-2013-0017159) or a hair dye composition for oxidation dyeing Patent No. 10-2012-0126148), it has been used for the purpose of reverse-differentiating adipose-derived mesenchymal stem cells into induced pluripotency stem cells There is no such thing.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

1. Korean Patent Laid-Open No. 10-2009-0043115 2. Korean Patent Publication No. 10-2013-0017159 3. Korean Patent Publication No. 10-2012-0126148

The present inventors have sought to find a method for inducing pluripotent stem cells with high efficiency for commercialization of a cell therapy agent having high safety and high production efficiency. As a result, it has been confirmed that induction of pluripotent stem cells can be safely and efficiently performed using fat-derived mesenchymal stem cells when a gentle extract, which is a safe natural extract, is added to a cell culture medium, thereby completing the present invention .

Accordingly, it is an object of the present invention to provide a culture medium for the reverse-differentiation of fat-derived mesenchymal stem cells, including Ganoderma lucidum extract, into induced pluripotent stem cells.

Another object of the present invention is to provide a method for producing inducible pluripotent stem cells comprising the step of defatting the fat-derived mesenchymal stem cells into inducible pluripotent stem cells in a medium containing the menthol extract.

Another object of the present invention is to provide an inducible pluripotent stem cell produced by the above-described method.

It is another object of the present invention to provide a method for differentiating mesenchymal stem cells into inducible pluripotent stem cells and further dividing the mesenchymal stem cells into hepatocytes in a culture medium containing the extracts of menthol.

It is still another object of the present invention to provide hepatocytes produced by the above method.

It is still another object of the present invention to provide a composition for patient-customized cell therapy comprising inducible pluripotent stem cells prepared by the above-described method by isolating stem cells from the fat of a patient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention there is provided Ecklonia cava (Ecklonia The present invention provides a medium composition for the de-differentiation of an adipose-derived mesenchymal stem cell containing an extract of a cava extract into an induced pluripotency stem cell.

According to another aspect of the present invention, the present invention provides a method of differentiating adipocytes from mesenchymal stem cells comprising the steps of:

(a) Ecklonia cava ) extract to a cell culture medium;

(b) de-differentiating adipose-derived mesenchymal stem cells into induced pluripotency stem cells in the medium; And

(c) differentiating the induced pluripotent stem cells into hepatocytes.

The present inventors have searched for a method for efficiently inducing pluripotent stem cells for the practical use of a cell therapy agent having high safety and production efficiency without the risk of cancer cell formation because there is no ethical problem of destroying the embryo and no virus is used. As a result, it was confirmed that induction of pluripotent stem cells can be achieved with surprisingly high efficiency when the gentian extract, which is a safe natural extract, is added to the cell culture medium.

Ecklonia cava , an active ingredient contained in the culture medium composition of the present invention, is a perennial sea anther alfalfa, which mainly occurs in the southern coast, Jeju island coast, and Ulleungdo coastal area. It is used as a main raw material for making alginic acid, iodine and potassium, and for food.

(A) anhydrous or low-boiling alcohol having 1-4 carbon atoms (methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol) (D) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, and (h) diethyl ether, which are mixtures of lower alcohol and water. And may be extracted using a solvent, preferably methanol or a mixed solvent of ethanol and water, or each of them. When extracted with a mixed solvent, the content of methanol or ethanol is preferably 50-80 v / v%.

As used herein, the term "embryonic stem cell" refers to a cell cultured in the inner cell mass of a blastocyst, which is an early stage of development, and has pluripotency. The term "pluripotent stem cell" used in the present invention refers to a stem having pluripotency that can differentiate into all three germ layers constituting the living body, that is, endoderm, mesoderm and ectoderm Cells.

The term " differentiation "as used herein refers to a phenomenon in which the structure or function of a cell is specialized during its growth by proliferation and proliferation, that is, the cell or tissue of a living organism has a form or function It means changing.

The term "cell therapeutic agent" used in the present invention is a drug used for the purpose of treatment, diagnosis and prevention with cells and tissues prepared by isolation, culture and special manipulation from a human. Diagnosis, and prevention through a series of actions such as, for example, proliferation, screening, or otherwise altering the biological characteristics of a cell, or a xenogeneic cell in vitro. The cell therapy agent is classified into a somatic cell therapy agent and a stem cell treatment agent according to the degree of cell differentiation, and the present invention particularly relates to a stem cell therapeutic agent.

The 'mesenchymal stem cell' of the present invention is a cell isolated from embryonic stem cells or adult stem cells derived from a mammal, preferably a fat-derived mesenchymal stem cell, more preferably a human mesenchymal stem cell, It is a stem cell. The fat-derived stem cells can be obtained from adipose tissue of the human body. For example, in order to isolate mononuclear cells from adipose tissue, adipose tissue is collected from the human body, and the blood is collected by DPBS (Dulbecco's Phosphate-Buffered Saline) , Wash the washed cord with a surgical blade, and incubate at 37 ° C to obtain a solution containing mononuclear cells.

Of such as the term "medium" is a sugar, amino acids, and various nutrients, serum, growth factors in vitro that includes essential elements such as growth and proliferation of cells, such as minerals (in vitro) from the stem cells used in the present invention, cells Refers to a mixture for cultivation or differentiation.

In particular, the medium of the present invention is a medium for culturing mesenchymal stem cells. Herein, the mesenchymal stem cells are cells isolated from mammalian embryonic stem cells or adult stem cells, and have an ability to indefinitely pause and have various cell types (for example, adipocytes, cartilage cells, muscle cells, bone Cells, etc.). In addition, in the present invention, multipotent mesenchymal stem cells having an immunophenotype showing a negative response to antibodies against CD34, CD45 and positive for antibodies against CD73, CD90 and CD105 are used.

Various media are commercially available in the art and can be manufactured and used artificially. For example, commercially available media include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM F-12, Minimal Essential Medium, G-MEM, Iscove's Modified Dulbecco's Medium, AmnioMax, AminoMaxII complete Medium (Gibco, New York, USA), Chang's Medium MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada) and the like can be used as a basic medium to be included in the culture medium of the present invention together with a culture medium which can be produced artificially.

(For example, FBS (Fetal Bovine Serum)) and antibiotics (for example, penicillin, streptomycin) may be added to the above-mentioned basal medium. The concentration of the serum component or the antibiotic component added to the basic medium may vary within a range that can achieve the effects of the present invention and preferably 10% FBS, 100 unit / ml penicillin, 50 μg / ml streptomycin Can be added.

In addition, the medium of the present invention may further comprise a nutrient mixture. The nutritional mixture is a mixture containing various amino acids, vitamins, inorganic salts and the like generally used for cell culture, and may be prepared by mixing the above amino acids, vitamins, inorganic salts or the like or a commercially prepared nutritional mixture. Commercially produced nutritional mixtures include, but are not limited to, M199, MCDB110, MCDB202, MCDB302, and the like.

In addition, the medium of the present invention may further include energy water for induction and stabilization of pluripotent stem cells. The energy water is preferably added in an amount of 0.01 to 10 v / v%, more preferably 0.05 to 0.5 v / v%.

The medium composition of the present invention is a medium specific for induction of pluripotent stem cells, and can be obtained by adding a gangue extract to the basic medium, preferably 10 to 400 g / ml.

According to another aspect of the present invention, the present invention provides a method for producing a cell culture comprising the steps of: adding a gangue extract to a cell culture medium; And a step of infertilizing adipose-derived mesenchymal stem cells into induced pluripotency stem cells in the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

According to one embodiment of the present invention, DMEM F-12 medium (DMEM F-12 medium, Ganoderma lucidum extract, and energy water medium) as a control group and DMEM F-12 medium (Fig. 2 and Fig. 3). On the 8th to 10th day, colonies of pluripotent stem cells were formed.

According to still another aspect of the present invention, there is provided an inducible pluripotent stem cell prepared by the above method.

The inducible pluripotent stem cells of the present invention have the same pluripotency as the embryonic stem cells and are almost identical to the embryonic stem cells in the shape of the cells (FIGS. 2 and 3). According to one embodiment of the present invention, the expression of genes (Oct4, Sox-2) and proteins (SSEA-4) characteristic of embryonic stem cells was examined. As a result, embryonic stem cells It was confirmed that the gene and protein were expressed in the same manner (FIGS. 4 and 5).

According to another embodiment, the expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, are low in mesenchymal stem cells (MSCs) that do not undergo induction, whereas the pluripotent stem These characteristic genes were remarkably expressed in the cells (Experimental Example 1-1: EtOH EPN, Experimental Example 1-2: Sonic EPN) (FIGS. 6 and 7) It can be effectively differentiated.

According to one embodiment of the present invention, it was confirmed that cells that were expected to be pluripotent stem cells could be differentiated into hepatocytes (Fig. 8)

According to still another aspect of the present invention, there is provided a composition for treating cells comprising the differentiated hepatocytes.

The composition of the present invention can be administered by any route of administration, specifically, intraperitoneal or thoracic administration, subcutaneous administration, intravenous or intraarterial administration, intramuscular administration, topical administration by injection, and the like.

In the present invention, the composition can be administered in the form of injections, suspensions, emulsifiers and the like based on conventional methods, suspended in adjuvants such as Freund's complete adjuvant or, if necessary, It is also possible to administer it together with the substance having. The composition may contain sterilized or stabilizers, wettable or emulsifying accelerators, adjuvants such as salts or buffers for controlling osmotic pressure, and other therapeutically useful substances.

The composition for cell therapy of the present invention can be applied to arthritis, neurological diseases, endocrine diseases, liver diseases and the like.

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

(i) The present invention provides a medium composition for the induction of pluripotent stem cells containing a gangrene extract.

(Ii) In addition, the present invention provides a method for producing induced pluripotent stem cells using the above-mentioned culture medium composition.

(Iii) Using the medium composition according to the present invention, inducible pluripotent stem cells can be efficiently produced using the fat-derived mesenchymal stem cells, and since the pluripotent pluripotent stem cells can be differentiated into various cells, And can be usefully used as a cell therapy agent.

(Iv) Since the present invention does not use embryonic stem cells by producing pluripotent pluripotent stem cells having the same differentiation potential as embryonic stem cells, it eliminates ethical problems caused by destruction of embryos and does not use a virus that can cause cancer, Safe and safe all-round stem cells can be produced.

(V) Further, since natural extracts are used, it is possible to produce pluripotent stem cells with remarkably high efficiency compared with the conventional methods, and since the mesenchymal stem cells isolated from the adipocytes of the patient are used, practical use of the patient- I expect to be able to move forward. The present invention is believed to contribute greatly to the treatment of various intractable diseases such as neurological diseases and immune diseases.

FIG. 1 is a graph showing the induction of pluripotent stem cells, which are almost the same as embryonic stem cells, when cultured by injection of a menthol extract medium in fat-derived mesenchymal stem cells.
Fig. 2 shows the formation of pluripotent stem cell colonies induced by the concentration of the extract of menthol (ethanol extract) in the method of the present invention (Example 1-1).
Fig. 3 shows the formation of pluripotent stem cell colonies induced by the concentration of the gentian extract (water extract) in the method of the present invention (Example 1-2).
FIG. 4 shows that pluripotent stem cells derived from the method of the present invention (Experimental Example 1-1) were pluripotent stem cells using specific protein expression.
FIG. 5 shows that pluripotent stem cells derived from the method of the present invention (Experimental Example 1-2) were pluripotent stem cells using specific protein expression.
FIG. 6 is a graphical representation of gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1).
FIG. 7 is a graphical representation of gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2).
FIG. 8 shows the results of differentiation into hepatocytes using the medium for differentiation of hepatocytes into pluripotent stem cells induced by the method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One: Moth  Preparation of extract

Example  1-1: Using an ethanol solvent Moth  Preparation of extract

The herbal medicine samples used in the experiment were purchased from Jeju Island and used for the experiment after having passed the experts' feelings. 100 g of the dried herbal medicine sample was placed in 1 liter of 70% ethanol, and the ethanol was refluxed for 16 hours and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and lyophilized immediately.

Example  1-2: Using water Moth  Preparation of extract

The herbal medicine samples used in the experiment were purchased from Jeju Island and used for the experiment after having passed the experts' feelings. 100 g of the dried herbal medicine sample was added to 1 liter of water, and the water was extracted using an ultrasonic extractor for 16 hours and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and lyophilized immediately.

Example  2: In human fatty tissue Intermediate lobe  Isolation and culture of stem cells

Example  2-1: Collection of human fat tissue

Fat tissue is collected immediately after liposuction. Samples are collected in 500 ml sterile glass bottles before they are transferred to the laboratory. After sealing the sterilized glass bottle, it is transferred to the laboratory. In the laboratory, the extraction of mesenchymal stem cells from a class 100 flow hood is performed under sterile conditions. The sample is first transferred to a sterile stainless steel container. After several washings of PBS, the fat tissue samples were then cut into 2-cm lengths and transferred to a 50-ml tube where they were further rinsed and treated with 70% ethanol, and the antibiotic mixture (50 IU / ml penicillin, 50 μg / Of streptomycin (purchased from Invitrogen)) is added several times until the solution is cleaned.

Example  2-2: In human adipose tissue Intermediate lobe  Isolation and culture of stem cells

The isolated adipose tissue was washed with PBS, and the tissue was chopped and digested with DMEM medium supplemented with collagenase type 1 (1 mg / ml) for 1 hour at 37 ° C with shaking every 10 minutes. Next, the cells were washed with PBS and centrifuged at 1000 rpm for 5 minutes. The supernatant was suctioned and the remaining pellet on the bottom was washed with PBS and centrifuged at 1000 rpm for 5 minutes. The debris was removed by filtering with a 100 탆 mesh filler and then washed with PBS.

For the isolation / culture of mesenchymal cells, the above-mentioned explanted tissues were cultured in 5 ml of Dulbecco's modified eagle medium F-12 (Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), 10% FBS, 100 units / Ml penicillin, 50 μg / ml streptomycin, maintained in a nitrogen incubator at 37 ° C in a 95% CO2 5% cell incubator to maintain hypoxic conditions to kill cells other than stem cells, . The medium was replaced every 3 or 4 days. Cell growth (outgrowth) was monitored by light microscopy. Growing cells were trypsinized (0.125% trypsin / 0.05% EDTA) for further expansion and cryopreservation (using DMEM / 10% FBS).

For extraction of mesenchymal stem cells, the cell pellet was resuspended and counted in medium DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, 50 ug / ml streptomycin, At a density of 1x10 ⁶ cells / dish. The medium was changed every 3 or 4 days. Cell growth and clonal formation were monitored by light microscopy. At about 90% confluence, the cells were sub-cultured as described above.

Experimental Example  1: province-derived Intermediate lobe  Induction of pluripotent stem cells from stem cells

Experimental Example  1-1: Example  1-1 of Moth  Human fat-derived by concentration of extract Intermediate lobe  Production of pluripotent stem cells from stem cells

As a test for inducing pluripotent stem cells from human fat-derived stem cells according to the concentration of Jeju gut extract, the control group was DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, / Ml streptomycin was used as a basic medium. In the experimental group, the human fat-derived mesenchymal stem cells, which were the third subculture, were used to culture the Jeju ginseng extract prepared in Example 1-1 in Normal, 1 / / ml (SiO ₂ , Al ₂ O ₃ , TiO ₃ , and Fe ₂ O) at a concentration of 1 μg / ml, 20 μg / ml, 50 μg / ml, 100 μg / ₃ , CaO, Na ₂ O, K ₂ O, purified deionized water containing LiO, Est.) Was added (FIG. 1). The human fat-derived mesenchymal stem cells were separated and the washed mononuclear cells were inoculated in a 6-well plate (1 x 10 ⁴ cells) and cultured at 37 ° C and 5% CO ₂ .

As a result, it was observed that colonies were clearly formed after 10 days when the concentration of Jeju ganoderma extract was 100 to 400 / / ㎖ in the experimental group (FIG. 2), and the microscope magnification was observed at a ratio of 200 times.

Experimental Example  1-2: Example  1-2 Moth  Human fat-derived by concentration of extract Intermediate lobe  Production of pluripotent stem cells from stem cells

Experiments were conducted in the same manner as in Experimental Example 1-1, except that the Jeju ginseng extract prepared in Example 1-2 was used. As a result, in the experimental group, colonies were apparently formed after 10 days when the concentration of the extract was 20 to 50 占 퐂 / ml (FIG. 3), and the microscopic magnification was observed at a ratio of 200 times.

Experimental Example  1-3: Immunochemical staining of pluripotent stem cells induced by the method of the present invention

Expression of OCT4, SOX2, and SSEA-4 (stage-specific embryonic antigen-4), specific genes of embryonic stem cells, on pluripotent stem cells induced by the methods of Experimental Examples 1-1 and 1-2 Immunohistochemistry was used to analyze the expression of protein vesicles.

The cells were fixed with 4% paraformaldehyde, washed with PBS, and blocked with 1% BSA solution. OCT4, SOX3, and SSEA-4, and incubated at 4 ° C for 18 hours. Then, the cells were washed with PBS, treated with a secondary antibody with fluorescent dye (FITC) for the primary antibody, And reacted for 1 hour.

After washing with PBS, expression was analyzed using a fluorescence microscope. The results are shown in FIGS. 4 and 5. FIG. 4 shows the results of the pluripotent stem cells induced by the ethanol extract, and FIG. 5 shows the results of the pluripotent stem cells induced by the water extract.

The bright field results in FIGS. 4 (a) and 5 (b) show the colony morphology of the stem cells.

In addition, all of the staining results for the proteins expressed from OCT4, SOX2 and SSEA-4, which are the pluripotent stem cell-specific markers of FIGS. 4 (b) and 5 (b) I could confirm.

The presence of stem cells was confirmed once again by the result of DAPI staining of the nuclei of FIG. 4 (c) and FIG. 5 (c).

Experimental Example  1-4: Comparisons of Genetic Stem Cell Genetic Analysis

The pluripotent stem cells prepared in Experimental Examples 1-1 and 1-2 were observed under a microscope, and only the colonies were removed using a 200-μl pipet, and total RNA was isolated using TRIzol reagent (Invitrogen). CDNA was synthesized using reverse transcription-polymerase chain reaction (RT-PCR), and PCR was performed using primers specific for OCT4, Sox-2, Nanog and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) . Nanog, OCT4, and Sox-2 are characteristic genes found in embryonic stem cells. PCR products were analyzed by agarose gel electrophoresis and the expression of these genes was confirmed. The results are shown in FIGS. 6 and 7. FIG.

As a result, according to FIGS. 6 and 7, the expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, are low in mesenchymal stem cells (control group, MSC) These characteristic genes were significantly higher in induced pluripotent stem cells (allele stem cells prepared by Experimental Example 1-1 (represented by EtOH EPN) and Experimental Example 1-2 (represented by Sonic EPN)). The degree of expression of the stem cell genes OCT4, SOX2 and Nanog can be clearly confirmed through the graphs of FIG. 6 and FIG.

Experimental Example  2: Differentiation into hepatocytes

In order to induce differentiation into hepatocytes, pluripotent stem cell cells were induced from mesenchymal stem cells by culturing in a culture medium containing 95%, 37%, and 5% CO ₂ at a humidity of 95% Ml Hepatocyte Differentiation Solution DMEM F-12, 10 nM dexamethason, 5.5 占 퐂 / ml ITS (Insulin-Transferrin-Selenium; 6.25 占 퐂 / ml transferring, 6.25 ng / ml selenius scid), 1.25 mg / ml bovine serum albumin, 5.35 mg / ml lioleic acid, 100 ng / ml HGF, and 50 ng / ml FGF for 2 weeks. As shown in Fig. 8, the cells were stained with green fluorescence after being treated with differentiation medium as shown in Fig. 8, and the cells, which were expected to be pluripotent stem cells, were differentiated into hepatocytes It can be confirmed. The immunochemical tissue staining method is the same as the above immunohistochemical method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A method of differentiating hepatocytes from mesenchymal stem cells comprising the steps of:
(a) Ecklonia deficient mesenchymal stem cells obtained from human fats in a medium containing a cava extract of the present invention into an induced pluripotency stem cell; And
(b) differentiating the induced pluripotent stem cells into hepatocytes.
The method of claim 1, wherein the medium of step (a) 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- , α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, AminoMaxII complete medium, Chang's medium and MesenCult- ≪ / RTI >
The method according to claim 1, wherein the phlegm extract is contained at a concentration of 10 to 400 μg / ml in the medium.
[3] The method according to claim 1, wherein the ghatti extract of step (a) is extracted with an ethanol solvent at a concentration of 100 to 400 [mu] g / ml in the medium.
[3] The method according to claim 1, wherein the ghatti extract of step (a) is extracted with water at a concentration of 20 to 50 [mu] g / ml in the medium.
According to claim 1, wherein the medium of step (a) _{SiO 2, Al 2 O 3,} TiO 3, Fe 2 O 3, CaO, Na 2 O, K 2 O, and deionized water of 0.01 to 10 containing LiO v / v%. < / RTI >
The method according to claim 1, wherein the step (b) is performed using a medium containing dexamethasone, transferrin, sodium selenite, insulin, and growth factor How to. delete delete

Images