WO2016088932A1 - Method for preparing induced pluripotent stem cells from adipose-derived mesenchymal stem cells and differentiating same into adipocytes - Google Patents

Method for preparing induced pluripotent stem cells from adipose-derived mesenchymal stem cells and differentiating same into adipocytes Download PDF

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WO2016088932A1
WO2016088932A1 PCT/KR2014/012247 KR2014012247W WO2016088932A1 WO 2016088932 A1 WO2016088932 A1 WO 2016088932A1 KR 2014012247 W KR2014012247 W KR 2014012247W WO 2016088932 A1 WO2016088932 A1 WO 2016088932A1
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stem cells
medium
pluripotent stem
cells
present
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이상연
정원주
김호빈
오민선
이계호
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주식회사 비비에이치씨
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • the present invention relates to a method for producing patient-specific induced pluripotent stem cells using the pluripotent stem cell induction medium composition of human adipose derived mesenchymal stem cells and differentiating them into adipocytes.
  • Stem cells collectively refer to the undifferentiated cells in the pre-differentiated stage that can be obtained from each tissue. In the undifferentiated state, it is capable of continuously producing the same cells as itself for a certain period of time, and under proper conditions, it is capable of differentiating into various cells constituting biological tissues.
  • Stem cells can be largely divided into embryonic stem cells and adult stem cells according to their differentiation capacity and production time. Another classification is according to the differentiation capacity of stem cells, which can be divided into pluripotency, multipotency and unipotency stem cells.
  • Adult stem cells can be divided into multipotent or unipotent stem cells.
  • Representative adult stem cells include mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs).
  • MSCs mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • Mesenchymal stem cells differentiate into chondrocytes, osteoblasts, adipocytes, myocytes and neurons.
  • Hematopoietic stem cells mainly contain blood cells such as red blood cells, white blood cells, and platelets. It is known to differentiate into cells.
  • Pluripotent stem cells refer to stem cells that have the versatility to differentiate into all three germ layers that make up a living body and to differentiate into all cells or organ tissues of the human body, and generally embryonic stem cells. This is the case.
  • Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells.
  • Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited differentiation ability compared to embryonic stem cells.
  • various methods have been attempted to prepare customized pluripotent stem cells similar to embryonic stem cells by dedifferentiating adult-derived cells.
  • Representative methods include fusion with ES cells, somatic cell nuclear transfer, and reprogramming by gene factor.
  • the cell fusion method has a problem in that the induced cells have two more pairs of genes, and the somatic cell nuclear transfer method requires a large amount of eggs and a very low efficiency.
  • the specific factor injection method uses a virus containing a carcinogenic gene to induce differentiation by inserting a specific gene, which poses a high risk of cancer, and in view of the possibility of developing a cell therapy due to low efficiency and difficulty in terms of methodology. It is a problem.
  • the culture composition at the stage of culturing isolated adipose-derived mononuclear cells is very important. It is a state.
  • compositions for treating or preventing atopic diseases using Ecklonia cava Patent No. 10-2009-0043115
  • a skin composition such as cosmetics
  • a hair dye composition for oxidation dyeing publishing Patent No. 10-2012-0126148, but is used to dedifferentiate adipose-derived mesenchymal stem cells into induced pluripotency stem cells. There is nothing.
  • Patent Document 1 Korean Patent Publication No. 10-2009-0043115
  • Patent Document 2 Korea Patent Publication No. 10-2013-0017159
  • Patent Document 3 Korean Patent Publication No. 10-2012-0126148
  • the present inventors endeavored to find a method of inducing pluripotent stem cells with high efficiency for the practical use of cell therapy with high safety and production efficiency.
  • the Ecklonia cava extract which is a safe natural extract
  • the present invention was completed by confirming that induced pluripotent stem cells can be prepared at a safe and high efficiency using fat-derived mesenchymal stem cells. .
  • the present invention is to differentiate the adipose-derived mesenchymal stem cells containing Ecklonia cava extract into induced pluripotency stem cells It provides a medium composition for.
  • the invention provides a method of differentiating adipocytes from mesenchymal stem cells, comprising the following steps:
  • the present inventors have tried to find a method of inducing high efficiency pluripotent stem cells for the practical use of developing high-efficiency cell therapies without the risk of cancer cell formation without the ethical problem of destroying embryos and without the use of viruses.
  • the Ecklonia cava extract which is a safe natural extract, was added to the cell culture medium, it was confirmed that the induced pluripotent stem cells could be produced with surprisingly high efficiency.
  • Ecklonia cava an active ingredient included in the medium composition of the present invention, is a perennial seaweed of seaweed kelp, kelp, which is inhabited in the southern coast, Jeju island coast, and the Ulleungdo coastal area, and is mainly fed to abalone and conch It is also used as a major raw material for making alginic acid, iodine and potassium, and for food.
  • Ecklonia cava extract comprises water, (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol, etc.), (b) the Mixed solvent of lower alcohol and water, organic solvents such as (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethyl ether
  • the extraction may be performed using a solvent, and preferably, extraction may be performed using a mixed solvent of methanol or ethanol and water or each of them.
  • the content of methanol or ethanol is preferably 50-80 v / v%.
  • the term “embryonic stem cell” refers to a cell having pluripotency as a cell cultured by separating from an inner cell mass of a blastocyst that is early in development after fertilization.
  • pluripotency refers to a stem having pluripotency capable of differentiating into three germ layers constituting a living body, that is, endoderm, mesoderm, and ectoderm. Refers to a cell.
  • differentiation refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. It means to change.
  • cell therapeutic agent refers to a medicament used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared by isolation, culture, and special manipulation from humans, and is used to restore the function of cells or tissues. Or a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise modifying a cell's biological properties in vitro.
  • Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
  • “Mesenchymal Stem Cells” of the present invention are cells isolated from embryonic stem cells or adult stem cells derived from mammals, preferably fat-derived mesenchymal stem cells, and more preferably fat-derived mesenchymal stem cells of the human body. It is a cell.
  • the fat-derived stem cells can be obtained from adipose tissue of the human body. Sampling of mesenchymal stem cells from fat can be performed using a variety of methods. For example, in order to separate mononuclear cells from adipose tissue, adipose tissue is collected from the human body to DPBS (Dulbecco's Phosphate-Buffered Saline). Rinse until no blood comes out, and the washed umbilical cord is cut with a surgical blade and incubated at 37 ° C. to obtain a mononuclear cell-containing solution.
  • DPBS Dynamiconitride-Buffered Saline
  • the term “medium” refers to cells such as stem cells in vitro, including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals, and the like. Refers to a mixture for incubation or differentiation.
  • the medium of the present invention is a medium for culturing mesenchymal stem cells.
  • the mesenchymal stem cells are cells isolated from embryonic stem cells or adult stem cells derived from mammals, and have the ability to feed indefinitely and various cell types (eg, fat cells, chondrocytes, muscle cells, bones). Cells, etc.).
  • a multipotent mesenchymal stem cell having an immunophenotype that shows a positive response to antibodies against CD73, CD90 and CD105, and a negative response to antibodies to CD34 and CD45 is used.
  • Various media are commercially available in the art, and may be artificially prepared and used.
  • commercially available media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, DMEM F-12, and ⁇ -MEM ( ⁇ -Minimal Essential Medium, G-MEM (Glasgow's Minimal Essential Medium), IMPM (Iscove's Modified Dulbecco's Medium), AmnioMax, AminoMax II complete Medium (Gibco, Newyork, USA), Chang's Medium MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada) and the like, and can be used as a basal medium included in the medium composition of the present invention together with a medium that can be artificially produced.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basic Medium Eagle
  • Serum components for example, Fetal Bovine Serum (FBS)
  • antibiotics for example, penicillin, streptomycin
  • concentration of serum component or antibiotic component added to the basal medium may vary within a range capable of achieving the effect of the present invention, preferably 10% FBS, 100 unit / ml penicillin, 50 ⁇ g / ml streptomycin, or the like. Can be added.
  • the medium of the present invention may further comprise a nutrient mixture (Nutrient Mixture).
  • the nutrition mixture is a mixture containing various amino acids, vitamins, inorganic salts, and the like generally used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, and the like, or use a commercially prepared nutrition mixture.
  • Commercially prepared nutrient mixtures include, but are not limited to, M199, MCDB110, MCDB202, MCDB302, and the like.
  • 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 at 0.01 to 10 v / v%, more preferably at 0.05 to 0.5 v / v%.
  • the medium composition of the present invention is a medium specific for pluripotent stem cell induction, and may be achieved by adding Ecklonia cava extract to the basal medium, and may preferably comprise Ecklonia cava extract at a concentration of 10 to 400 ⁇ g / ml.
  • the present invention comprises the steps of adding the Ecklonia cava extract to the cell culture medium; And dedifferentiating adipose-derived mesenchymal stem cells into induced pluripotency stem cells in the medium.
  • the medium composition containing the Ecklonia cava extract of the present invention as the experimental group (medium extract containing Ecklonia cava extract, and energy water in DMEM F-12 medium) and only DMEM F-12 medium as a control group Unlike when used, it was confirmed that pluripotent stem cell colonies were formed on day 8-10 (FIG. 2 and FIG. 3).
  • the present invention provides an induced pluripotent stem cell prepared by the above method.
  • Induced pluripotent stem cells of the present invention have the same differentiation capacity as embryonic stem cells, and are almost identical to embryonic stem cells in the shape of cells (FIGS. 2 and 3). According to an embodiment of the present invention, as a result of examining the expression of the genes (Oct4, Sox-2) and protein (SSEA-4) characteristic to the embryonic stem cells embryonic stem cells and pluripotent stem cells induced by the present invention It was confirmed that the same genes and proteins are expressed (Figs. 4 and 5).
  • mesenchymal stem cells that have not undergone the induction process have low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, whereas pluripotency induced by the method of the present invention In stem cells (Experimental Example 1-1: EtOH EPN, Experimental Example 1-2: Sonic EPN), these characteristic genes were significantly expressed (Figs. 6 and 7).
  • the induced pluripotent stem cells of the present invention were adipose Can effectively differentiate into cells.
  • the present invention provides a cell therapy composition comprising the differentiated adipocytes.
  • composition of the present invention can be administered by any route of administration, specifically by intraperitoneal or chest cavity administration, subcutaneous administration, intravenous or arterial vascular administration, intramuscular administration, topical administration by injection, or the like.
  • the composition can be administered in the form of injections, suspensions, emulsifiers and the like based on conventional methods, and if necessary, suspended in an adjuvant such as Freund's complete adjuvant or adjuvant activity such as BCG. It is also possible to administer with the substance having.
  • the composition may be sterile or contain adjuvants such as stabilizers, hydrating or emulsifying accelerators, salts or buffers for controlling osmotic pressure and other therapeutically valuable substances.
  • the cell therapy composition of the present invention can be applied to arthritis, nervous system diseases, endocrine diseases, liver diseases, and the like, and according to the results of clinical trials for humans in the future, there is also the possibility of treating allogeneic cells for humans.
  • the present invention provides a medium composition for induced pluripotent stem cell reverse differentiation comprising Ecklonia cava extract.
  • the present invention also provides a method for producing pluripotent stem cells using the medium composition.
  • the present invention eliminates ethical problems caused by embryo destruction by not using embryonic stem cells by producing pluripotent stem cells having the same differentiation capacity as embryonic stem cells, and thus does not use viruses that can cause cancer. Safe pluripotent stem cells can be produced without risk.
  • pluripotent stem cells can be produced very easily and significantly higher than conventional methods, and mesenchymal stem cells isolated from adipocytes of patients can be used for the practical application of stem cell therapeutics tailored to patients. Expect to be able to advance. It is believed that the present invention will greatly contribute to the treatment of various intractable diseases such as neurological diseases and immune diseases.
  • FIG. 1 is a diagram showing that the pluripotent stem cells almost the same as embryonic stem cells are induced when cultured by injection of Ecklonia cava extract medium from adipose-derived mesenchymal stem cells.
  • Figure 2 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (ethanol extract) by the method of the present invention (Example 1-1).
  • Figure 3 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (water extract) by the method of the present invention (Example 1-2).
  • Figure 4 confirms that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) by using specific protein expression.
  • Example 5 shows that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) are pluripotent stem cells using specific protein expression.
  • FIG. 6 is a graph showing the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) and graphs thereof.
  • Figure 7 shows the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) and graphically depicted them.
  • the herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of 70% ethanol, and ethanol was extracted under reflux for 16 hours and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and immediately freeze dried.
  • the herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of water, and 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 immediately freeze dried.
  • Adipose tissue is collected immediately after liposuction.
  • the sample collects aspirated adipose tissue in 500 ml sterile glass bottles before being transferred to the laboratory.
  • the sterile glass bottle is then sealed and transferred to the laboratory.
  • mesenchymal stem cells are extracted in a flow hood of class 100 under sterile conditions.
  • the sample is first transferred to a sterile stainless steel container.
  • the PBS was washed several times and then the adipose tissue sample was then cut into 2 cm lengths and transferred to a 50 ml tube where additional washing and anti-infection with 70% ethanol and antibiotic mixture (50 IU / ml penicillin, 50 ug / ml) Wash several times with PBS added with streptomycin (purchased from Invitrogen) until the solution is clear.
  • Example 2-2 Isolation and Culture of Mesenchymal Stem Cells from Human Adipose Tissue
  • the isolated adipose tissue was washed with PBS, the tissue was chopped, and digested for 1 hour while shaking once every 10 minutes at 37 ° C. using DMEM medium to which collagenase type 1 (1 mg / ml) was added. Next, washed with PBS and centrifuged for 5 minutes at 1000 rpm. The supernatant was aspirated and the pellet remaining on the bottom was washed with PBS and centrifuged at 1000 rpm for 5 minutes. Filtered with a 100 ⁇ m mesh size filler to remove debris and washed with PBS.
  • the explanted tissue was treated with 5 ml of DMEM (Dulbecco's modified eagle medium) F-12 (Gibco), 10% FBS, 100 units with 10% fetal calf serum (FBS, Hyclone). / Ml penicillin, 50 ⁇ g / ml streptomycin in a 95% nitrogen, 5% carbon dioxide cell culture incubator at 37 °C to maintain the hypoxic state to kill the cells other than stem cells, the purity of mesenchymal stem cells Raised. Medium was changed every 3 or 4 days. Outgrowth of cells was monitored by light microscopy. Elongating cells were trypsinized (0.125% trypsin / 0.05% EDTA) for further expansion and cryopreservation (using DMEM / 10% FBS).
  • DMEM Disbecco's modified eagle medium
  • F-12 Gibco
  • FBS fetal calf serum
  • FBS fetal calf serum
  • pellets of cells were resuspended and counted in medium DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, 50 ⁇ g / ml streptomycin, and 10 cm tissue culture dishes. Were inoculated at a density of 1 ⁇ 10 6 cells / dish. The medium was changed every 3 or 4 days. Cell growth and cloning were monitored by light microscopy. At about 90% confluence, the cells were sub-cultured as described above.
  • the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 100 to 400 ⁇ g / ml (Fig. 2), the microscope magnification was observed at a 200-fold ratio.
  • Example 1-2 Preparation of Pluripotent Stem Cells of Human Adipose-derived Mesenchymal Stem Cells According to Ecklonia cava Extract Concentrations of Example 1-2
  • Example 2 the Jeju Ecklonia cava extract was prepared in Example 1-2 was used.
  • the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 20 to 50 ⁇ g / ml (FIG. 3), and the microscope magnification was observed at a 200-fold ratio.
  • the staining process was first fixed with 4% paraformaldehyde (Paraformaldehyde), and then washed with PBS and blocked (blocking) with 1% BSA solution. After treatment with the primary antibody against OCT4, SOX3, SSEA-4 for 18 hours at 4 °C, washed with PBS and treated with a secondary antibody with a fluorescent pigment (FITC) for the primary antibody at room temperature The reaction was carried out for 1 hour.
  • FITC fluorescent pigment
  • FIGS. 4 and 5 are results for pluripotent stem cells induced by Ecklonia cava extract extracted with ethanol
  • Figure 5 is a result for pluripotent stem cells induced by Ecklonia cava extract extracted with water.
  • the mesenchymal stem cells that did not undergo the induction process had low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, while Pluripotent stem cells (pluripotent stem cells prepared by Experimental Example 1-1 (denoted by EtOH EPN) and Experimental Example 1-2 (denoted by Sonic EPN)) induced by the expression of these characteristic genes were significantly higher.
  • the expression levels of the stem cell genes OCT4, SOX2 and Nanog can be clearly seen through the graphs of FIGS. 6 and 7.
  • pluripotent stem cell cells were induced from mesenchymal stem cells by culturing in a culture medium of 95%, 37 ° C. and 5% CO 2 using a medium containing Ecklonia cava extract and energy water.
  • Adipocyte differentiation solution was incubated for 2 weeks in DMEM F-12, 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM hydrocortisone, 0.1 mM indomethacin 5% FBS.
  • Oil-Red-O histochemical staining to confirm intracellular fat accumulation for differentiation into adipocytes was shown to be positive for Oil-Red-O as shown in FIGS. 8A and 8B. It was confirmed that the expected cells could differentiate into adipocytes.

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Abstract

The present invention relates to a culture medium composition, for induced pluripotent stem cell dedifferentiation, comprising an Ecklonia cava extract. In addition, the present invention relates to a method for differentiating induced pluripotent stem cells, which are prepared by means of the culture medium composition, into adipocytes. By means of the culture medium composition, according to the present invention, induced pluripotent stem cells can be efficiently prepared by using mesenchymal stem cells, and the prepared pluripotent stem cells can be differentiated into adipocytes. Therefore, the present invention can be utilized for a patient-specific cell therapy product.

Description

지방-유래 중간엽 줄기세포로부터 유도만능 줄기세포를 제조하여 지방세포로 분화시키는 방법Method of producing induced pluripotent stem cells from adipose-derived mesenchymal stem cells to differentiate into adipocytes
본 발명은 인체 지방-유래 중간엽 줄기세포의 만능줄기세포 유도 배지 조성 물을 이용하여 환자 맞춤형 유도만능 줄기세포를 제조하고 이를 지방세포로 분화시키는 방법에 관한 것이다.The present invention relates to a method for producing patient-specific induced pluripotent stem cells using the pluripotent stem cell induction medium composition of human adipose derived mesenchymal stem cells and differentiating them into adipocytes.
줄기세포는 각 조직에서 얻을 수 있는 분화되기 전 단계의 미분화 세포들을 총칭한다. 미분화 상태에서 일정기간 동안 자신과 동일한 세포를 지속적으로 만들어 낼 수 있는 성질과 적당한 조건하에서는 생물 조직을 구성하는 다양한 세포들로 분화될 수 있는 성질을 가지고 있다. Stem cells collectively refer to the undifferentiated cells in the pre-differentiated stage that can be obtained from each tissue. In the undifferentiated state, it is capable of continuously producing the same cells as itself for a certain period of time, and under proper conditions, it is capable of differentiating into various cells constituting biological tissues.
줄기세포는 분화능과 생성시기에 따라 크게 배아줄기세포(embryonic stem cell)와 성체 줄기세포(adult stem cell)로 구분될 수 있다. 또 다른 분류는 줄기 세포의 분화능에 따른 것으로, 만능(pluripotency), 다분화능(multipotency) 및 단분화능(unipotency) 줄기세포로 나눌 수 있다. Stem cells can be largely divided into embryonic stem cells and adult stem cells according to their differentiation capacity and production time. Another classification is according to the differentiation capacity of stem cells, which can be divided into pluripotency, multipotency and unipotency stem cells.
성체줄기세포는 다분화능 또는 단분화능 줄기세포로 구분할 수 있다. 대표적 인 성체줄기세포에는 중간엽 줄기세포(mesenchymal stem cells; MSCs)와 조혈모세포(hematopoietic stem cells; HSCs)가 있다. 중간엽 줄기세포는 연골세포 (chondrocyte), 골아세포(osteoblast), 지방세포(adipocyte), 근육세포(myocyte), 신경세포(neuron)로 분화하며 조혈모세포에는 적혈구, 백혈구, 혈소판 등 주로 혈액내의 혈구세포로 분화하는 것으로 알려져 있다. Adult stem cells can be divided into multipotent or unipotent 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 mainly contain blood cells such as red blood cells, white blood cells, and platelets. It is known to differentiate into cells.
반면에 만능 줄기세포는 생체를 구성하는 3가지 배엽(germ layer) 모두로 분화될 수 있어 인체의 모든 세포나 장기 조직으로 분화할 수 있는 다기능성을 지닌 줄기세포를 지칭하며, 일반적으로 배아줄기세포가 이에 해당된다. 인간 배아 줄기 세포는 인간 생명체로 발생할 수 있는 배아로부터 만들어지기 때문에 많은 윤리적 인 문제점을 가지고 있으나, 성체 줄기세포에 비하여 세포증식 및 분화 능력이 우수한 것으로 알려져 있다. 성체 줄기세포는 골수, 혈액, 뇌, 피부 등에서 얻을 수 있어 윤리적인 문제가 적으나, 배아 줄기세포에 비하여 한정된 분화능력을 가지고 있다. Pluripotent stem cells, on the other hand, refer to stem cells that have the versatility to differentiate into all three germ layers that make up a living body and to differentiate into all cells or organ tissues of the human body, and generally embryonic stem cells. This is the case. Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited differentiation ability compared to embryonic stem cells.
이러한 문제점을 극복하기 위한 대안으로, 성체에서 유래한 세포를 역분화시켜 배아줄기세포와 유사한 맞춤형 만능 줄기세포를 제조하기 위한 여러 가지 방법들이 시도되어 왔다. 대표적인 방법으로 세포 융합법(fusion with ES cell), 체세포 핵치환법(somatic cell nuclear transfer), 특정 인자 주입법(reprogramming by gene factor) 등이 있다. 세포융합법은 유도된 세포가 2쌍의 유전자를 더 가지고 있어 세포의 안정성 측면에서 문제점이 있고 체세포 핵치환법은 난자가 대량으로 필요하며 효율 또한 매우 낮다는 점에서 문제가 있다. 그리고 특정 인자 주입법은 특정 유전자를 삽입하여 역분화를 유도하기 위하여 발암유전자를 포함하는 바이러스를 이용하는 방법으로 암발생의 위험이 높으며, 낮은 효율과 방법적인 측면에서 의 난이도로 인해 세포 치료제 개발 가능성 면에서 문제시 되고 있다. As an alternative to overcome this problem, various methods have been attempted to prepare customized pluripotent stem cells similar to embryonic stem cells by dedifferentiating adult-derived cells. Representative methods include fusion with ES cells, somatic cell nuclear transfer, and reprogramming by gene factor. The cell fusion method has a problem in that the induced cells have two more pairs of genes, and the somatic cell nuclear transfer method requires a large amount of eggs and a very low efficiency. In addition, the specific factor injection method uses a virus containing a carcinogenic gene to induce differentiation by inserting a specific gene, which poses a high risk of cancer, and in view of the possibility of developing a cell therapy due to low efficiency and difficulty in terms of methodology. It is a problem.
만능 줄기세포를 성공적으로, 그리고 다량으로 얻기 위해서는 분리된 지방 유래 단핵세포를 배양하는 단계에서의 배양 조성물이 매우 중요한 바, 보다 많은 양, 고효율의 유도방법으로 만능 줄기세포를 제조하기 위한 연구가 필요한 상태이다. In order to obtain pluripotent stem cells successfully and in large quantities, the culture composition at the stage of culturing isolated adipose-derived mononuclear cells is very important. It is a state.
한편 감태를 이용하여 아토피 질환의 치료 또는 예방을 위한 조성물(공개특허 제10-2009-0043115호), 화장료 등의 피부 조성물(공개특허 제10-2013-0017159호) 또는 산화염색용 염모제 조성물(공개특허 제10-2012-0126148호)에 사용한 경우 는 있으나, 지방-유래 중간엽 줄기세포(adipose-derived mesenchymal stem cell)를 유도만능 줄기세포(induced pluripotency stem cell)로 역분화하기 위한 용도로 사용된 것은 전무한 상태이다. Meanwhile, a composition for treating or preventing atopic diseases using Ecklonia cava (Patent No. 10-2009-0043115), a skin composition such as cosmetics (Patent No. 10-2013-0017159), or a hair dye composition for oxidation dyeing (publishing Patent No. 10-2012-0126148, but is used to dedifferentiate adipose-derived mesenchymal stem cells into induced pluripotency stem cells. There is nothing.
상기한 배경기술로서 설명된 사항들은 본 발명의 배경에 대한 이해 증진을 위한 것일 뿐, 이 기술분야에서 통상의 지식을 가진 자에게 이미 알려진 종래기술에 해당함을 인정하는 것으로 받아들여져서는 안 될 것이다. The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.
[선행기술문헌][Preceding technical literature]
특허문헌 1. 한국공개특허 제10-2009-0043115호 Patent Document 1. Korean Patent Publication No. 10-2009-0043115
특허문헌 2. 한국공개특허 제10-2013-0017159호 Patent Document 2. Korea Patent Publication No. 10-2013-0017159
특허문헌 3. 한국공개특허 제10-2012-0126148호 Patent Document 3. Korean Patent Publication No. 10-2012-0126148
본 발명자들은 안전성와 생산효율이 높은 세포 치료제 개발의 실용화를 위한 만능 줄기세포를 고효율로 유도하는 방법을 찾고자 노력하였다. 그 결과, 안전한 천연 추출물인 감태 추출물을 세포 배양 배지에 첨가할 경우 지방-유래 중간엽 줄기세포를 이용하여 안전하고도 높은 효율로 유도만능 줄기세포를 제조할 수 있다는 것을 확인함으로써 본 발명을 완성하였다. The present inventors endeavored to find a method of inducing pluripotent stem cells with high efficiency for the practical use of cell therapy with high safety and production efficiency. As a result, when the Ecklonia cava extract, which is a safe natural extract, was added to the cell culture medium, the present invention was completed by confirming that induced pluripotent stem cells can be prepared at a safe and high efficiency using fat-derived mesenchymal stem cells. .
따라서, 본 발명의 목적은 감태 추출물을 포함하는 지방-유래 중간엽 줄기세포를 유도만능 줄기세포로 역분화시키기 위한 배지 조성물을 제공하는데 있다. Accordingly, it is an object of the present invention to provide a medium composition for dedifferentiating adipose derived mesenchymal stem cells into induced pluripotent stem cells comprising Ecklonia cava extract.
본 발명의 다른 목적은 감태 추출물이 포함된 배지에서 지방-유래 중간엽 줄기세포를 유도만능 줄기세포로 역분화시키는 단계를 포함하는 유도만능 줄기세포의 제조 방법을 제공하는데 있다. 본 발명의 또 다른 목적은 상기 제조 방법으로 제조된 유도만능 줄기세포를 제공하는데 있다. 본 발명의 다른 목적은 감태 추출물이 포함된 배지에서 중간엽 줄기세포를 유도만능 줄기세포로 역분화시키고 이를 다시 지방세포로 분화시키는 방법을 제공하는데 있다. 본 발명의 또 다른 목적은 상기 제조 방법으로 제조된 지방세포를 제공하는 데 있다. 본 발명의 또 다른 목적은 환자의 지방에서 줄기세포를 분리하여 상기 제조 방법으로 제조된 유도만능 줄기세포를 포함하는 환자 맞춤형 세포 치료용 조성물을 제공하는데 있다. Another object of the present invention to provide a method for producing induced pluripotent stem cells comprising the step of dedifferentiating adipose-derived mesenchymal stem cells into induced pluripotent stem cells in a medium containing Ecklonia cava extract. Another object of the present invention to provide an induced pluripotent stem cell prepared by the above method. Another object of the present invention is to provide a method for dedifferentiating mesenchymal stem cells into induced pluripotent stem cells in the medium containing the Ecklonia cava extract and then re-differentiating them into adipocytes. Another object of the present invention to provide an adipocyte prepared by the above production method. Still another object of the present invention is to provide a composition for treating a patient customized cell comprising induced pluripotent stem cells prepared by the above method by separating stem cells from the fat of the patient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 감태(Ecklonia cava) 추출물을 포함하는 지방-유래 중간엽 줄기세포(adipose-derived mesenchymal stem cell)를 유도 만능 줄기세포(induced pluripotency stem cell)로 역분화하기 위한 배지 조성물을 제공한다. According to one aspect of the present invention, the present invention is to differentiate the adipose-derived mesenchymal stem cells containing Ecklonia cava extract into induced pluripotency stem cells It provides a medium composition for.
본 발명의 다른 양태에 따르면, 본 발명은 하기의 단계를 포함하는 중간엽 줄기세포(mesenchymal stem cell)로부터 지방세포(adipocyte)를 분화시키는 방법을 제공한다: According to another aspect of the invention, the invention provides a method of differentiating adipocytes from mesenchymal stem cells, comprising the following steps:
(a) 감태(Ecklonia cava) 추출물을 세포 배양 배지에 첨가하는 단계; (a) adding Ecklonia cava extract to the cell culture medium;
(b) 상기 배지에서 지방유래 중간엽 줄기세포를 유도만능 줄기세포(induced pluripotency stem cell)로 역분화시키는 단계; 및 (b) dedifferentiating the adipose derived mesenchymal stem cells into induced pluripotency stem cells in the medium; And
(c) 상기 유도만능 줄기세포를 지방세포로 분화시키는 단계. (c) differentiating the induced pluripotent stem cells into adipocytes.
본 발명자들은 배아를 파괴하는 윤리적 문제를 없고 바이러스를 사용하지 않으므로서 암세포 형성 위험이 없는 안전성와 생산효율이 높은 세포치료제 개발의 실용화를 위한 만능 줄기세포를 고효율로 유도하는 방법을 찾고자 노력하였다. 그 결과, 안전한 천연 추출물인 감태 추출물을 세포 배양 배지에 첨가할 경우 놀랍게도 높은 효율로 유도만능 줄기세포를 제조할 수 있다는 것을 확인하였다. The present inventors have tried to find a method of inducing high efficiency pluripotent stem cells for the practical use of developing high-efficiency cell therapies without the risk of cancer cell formation without the ethical problem of destroying embryos and without the use of viruses. As a result, when the Ecklonia cava extract, which is a safe natural extract, was added to the cell culture medium, it was confirmed that the induced pluripotent stem cells could be produced with surprisingly high efficiency.
본 발명의 배지 조성물에 포함되는 유효성분인 감태(Ecklonia cava)는 주로 남해안, 제주도 해안 일대 및 울릉도 해안 일대에서 서식하는 갈조식물 다시마목 다시마과의 여러해살이 해조류로서, 주로 전복과 소라 등의 먹이가 되며, 알긴산이나 요오드·칼륨을 만드는 주요 원료나, 식용으로 이용하기도 한다. Ecklonia cava ( Ecklonia cava ), an active ingredient included in the medium composition of the present invention, is a perennial seaweed of seaweed kelp, kelp, which is inhabited in the southern coast, Jeju island coast, and the Ulleungdo coastal area, and is mainly fed to abalone and conch It is also used as a major raw material for making alginic acid, iodine and potassium, and for food.
본 발명이 포함하는 감태 추출물은 물, (a) 탄소수 1-4의 무수 또는 함수 저급 알코올(메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올 및 노말-부탄올 등), (b) 상기 저급 알코올과 물과의 혼합용매, (c) 아세톤, (d) 에틸 아세테이트, (e) 클로로포름, (f) 1,3-부틸렌글리콜, (g) 헥산, (h) 디에틸에테르 등의 유기용매를 이용하여 추출할 수 있으며, 바람직하게는 메탄올 또는 에탄올과 물과의 혼합용매 또는 이들 각각을 이용하여 추출할 수 있다. 혼합용매를 이용하여 추출할 경우 메탄올 또는 에탄올의 함량은 50-80 v/v%가 바람직하다. Ecklonia cava extract according to the present invention comprises water, (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol, etc.), (b) the Mixed solvent of lower alcohol and water, organic solvents such as (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethyl ether The extraction may be performed using a solvent, and preferably, extraction may be performed using a mixed solvent of methanol or ethanol and water or each of them. When extracted using a mixed solvent, the content of methanol or ethanol is preferably 50-80 v / v%.
본 발명에서 사용된 용어 "배아줄기세포"는 수정 후 발생 초기인 배반포 기(blastocyst)의 내부세포덩어리(inner cell mass)에서 분리하여 배양한 세포로 만능성(pluripotency)을 지니는 세포를 지칭한다. 본 발명에서 사용된 용어 "만능 줄기세포"는 생체를 구성하는 3가지 배엽(germ layer), 즉 내배엽(endoderm), 중배엽(mesoderm), 외배엽(ectoderm) 모두로 분화할 수 있는 만능성을 지닌 줄기세포를 지칭한다. As used herein, the term "embryonic stem cell" refers to a cell having pluripotency as a cell cultured by separating from an inner cell mass of a blastocyst that is early in development after fertilization. As used herein, the term "pluripotent stem cell" refers to a stem having pluripotency capable of differentiating into three germ layers constituting a living body, that is, endoderm, mesoderm, and ectoderm. Refers to a cell.
본 발명에서 사용된 용어 "분화(differentiation)"는 세포가 분열 증식하여 성장하는 동안에 서로 구조나 기능이 특수화하는 현상, 즉 생물의 세포, 조직 등이 각각에게 주어진 일을 수행하기 위하여 형태나 기능이 변해가는 것을 말한다. As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. It means to change.
본 발명에서 사용된 용어 "세포 치료제"는 사람으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 동종, 또는 이종세포를 체외에서 증식, 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다. 세포 치료제는 세포의 분화정도에 따라 크게 체세포 치료제, 줄기세포 치료제로 분류되며 본 발명은 특히 줄기세포 치료제에 관한 것이다. As used herein, the term "cell therapeutic agent" refers to a medicament used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared by isolation, culture, and special manipulation from humans, and is used to restore the function of cells or tissues. Or a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise modifying a cell's biological properties in vitro. Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
본 발명의 "중간엽 줄기세포" 포유동물 유래의 배아 줄기세포 또는 성체 줄기세포에서 분리한 세포로서, 바람직하게는 지방-유래 중간엽 줄기세포이며, 보다 바람직하게는 인체의 지방-유래 중간엽 줄기세포이다. 상기 지방-유래 줄기세포 는 인체의 지방조직에서 채취하여 얻을 수 있다. 지방으로부터 중간엽 줄기세포의 채취는 다양한 방법을 이용하여 이를 수행할 수 있으며, 예를 들어, 지방조직으로 부터 단핵세포를 분리하기 위하여 인체에서 지방 조직을 채취하여 DPBS(Dulbecco's Phosphate-Buffered Saline)로 혈액이 나오지 않을 때까지 씻어주고, 씻은 제대를 수술용 칼날로 다지고 37℃에서 인큐베이션(incubation) 시켜서 단핵세포가 함유된 용액을 얻을 수 있다. "Mesenchymal Stem Cells" of the present invention are cells isolated from embryonic stem cells or adult stem cells derived from mammals, preferably fat-derived mesenchymal stem cells, and more preferably fat-derived mesenchymal stem cells of the human body. It is a cell. The fat-derived stem cells can be obtained from adipose tissue of the human body. Sampling of mesenchymal stem cells from fat can be performed using a variety of methods. For example, in order to separate mononuclear cells from adipose tissue, adipose tissue is collected from the human body to DPBS (Dulbecco's Phosphate-Buffered Saline). Rinse until no blood comes out, and the washed umbilical cord is cut with a surgical blade and incubated at 37 ° C. to obtain a mononuclear cell-containing solution.
본 발명에서 사용된 용어 "배지"는 당, 아미노산, 각종 영양물질, 혈청, 성장인자, 무기질 등의 세포의 성장 및 증식 등에 필수적인 요소를 포함하는 생체 외(in vitro)에서 줄기세포 등의 세포의 배양 또는 분화를 위한 혼합물을 말한다. As used herein, the term "medium" refers to cells such as stem cells in vitro, including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals, and the like. Refers to a mixture for incubation or differentiation.
특히 본 발명의 배지는 중간엽 줄기세포의 배양을 위한 배지이다. 이때 중간 엽 줄기세포는 포유동물 유래의 배아 줄기세포 또는 성체 줄기세포에서 분리한 세포로서, 무한정으로 중식할 수 있는 능력 및 여러 가지 세포형태(예를 들면, 지방 세포, 연골세포, 근육세포, 뼈세포 등)로 분화가 가능한 세포이다. 또한 본 발명에 서는 CD73, CD90, CD105에 대한 항체에는 양성반응으로, CD34, CD45에 대한 항체에 는 음성반응을 나타내는 면역표현형을 갖는 다분화능 중간엽 줄기세포를 사용한다. 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 embryonic stem cells or adult stem cells derived from mammals, and have the ability to feed indefinitely and various cell types (eg, fat cells, chondrocytes, muscle cells, bones). Cells, etc.). In the present invention, a multipotent mesenchymal stem cell having an immunophenotype that shows a positive response to antibodies against CD73, CD90 and CD105, and a negative response to antibodies to CD34 and CD45 is used.
당업계에는 다양한 배지가 시판되고 있으며, 인위적으로 제조하여 사용할 수 도 있다. 일예로서, 시판 중인 배지로는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM F-12, α-MEM(α-Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMPM(Iscove's Modified Dulbecco's Medium), AmnioMax, AminoMaxⅡ complete Medium(Gibco, Newyork, USA), Chang's Medium MesemCult-XF Medium(STEMCELL Technologies, Vancouver, Canada) 등이 있으며, 인위적으로 제조할 수 있는 배지와 더불어 본 발명의 배지 조성물에 포함되는 기본 배지로 사용할 수 있다. Various media are commercially available in the art, and may be artificially prepared and used. For example, commercially available media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, DMEM F-12, and α-MEM ( α-Minimal Essential Medium, G-MEM (Glasgow's Minimal Essential Medium), IMPM (Iscove's Modified Dulbecco's Medium), AmnioMax, AminoMax II complete Medium (Gibco, Newyork, USA), Chang's Medium MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada) and the like, and can be used as a basal medium included in the medium composition of the present invention together with a medium that can be artificially produced.
상기 기본 배지에는 통상적으로 첨가되는 혈청 성분(예를 들어, FBS(Fetal Bovine Serum)) 및 항생제(예를 들어, 페니실린, 스트렙토마이신) 등을 첨가할 수 있다. 상기 기본 배지에 첨가되는 혈청 성분 또는 항생제 성분의 농도는 본 발명의 효과를 달성할 수 있는 범위 내에서 변할 수 있으며, 바람직하게는 10% FBS, 100 unit/㎖ 페니실린, 50 ㎍/㎖ 스트렙토마이신 등을 첨가할 수 있다. Serum components (for example, Fetal Bovine Serum (FBS)) and antibiotics (for example, penicillin, streptomycin), which are commonly added, may be added to the basal medium. The concentration of serum component or antibiotic component added to the basal medium may vary within a range capable of achieving the effect of the present invention, preferably 10% FBS, 100 unit / ml penicillin, 50 μg / ml streptomycin, or the like. Can be added.
또한, 본 발명의 배지는 영양혼합물(Nutrient Mixture)을 추가로 포함할 수 있다. 상기 영양 혼합물은 세포배양에 일반적으로 사용되는 각종 아미노산, 비타민, 무기염 등을 포함하는 혼합물로서, 상기 아미노산, 비타민, 무기염 등을 혼합하여 제조하거나 상업적으로 제조된 영양 혼합물을 사용할 수 있다. 상업적으로 제조된 영양혼합물은 M199, MCDB110, MCDB202, MCDB302 등을 예로 들 수 있으나, 이에 제한되는 것은 아니다. In addition, the medium of the present invention may further comprise a nutrient mixture (Nutrient Mixture). The nutrition mixture is a mixture containing various amino acids, vitamins, inorganic salts, and the like generally used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, and the like, or use a commercially prepared nutrition mixture. Commercially prepared nutrient mixtures include, but are not limited to, M199, MCDB110, MCDB202, MCDB302, and the like.
또한, 본 발명의 배지는 만능 줄기 세포의 유도와 안정화를 위해 에너지워터 를 추가적으로 포함할 수 있다. 상기 에너지워터는 0.01 내지 10 v/v%로 추가하는 것이 바람직하며, 보다 바람직하게는 0.05 내지 0.5 v/v%로 추가한다. 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 at 0.01 to 10 v / v%, more preferably at 0.05 to 0.5 v / v%.
본 발명의 배지 조성물은 만능 줄기세포 유도에 특이적인 배지로서, 상기 기본 배지에 감태 추출물을 첨가함으로써 달성될 수 있으며, 바람직하게는 10 내지 400 ㎍/㎖ 농도로 감태 추출물을 포함할 수 있다. The medium composition of the present invention is a medium specific for pluripotent stem cell induction, and may be achieved by adding Ecklonia cava extract to the basal medium, and may preferably comprise Ecklonia cava extract at a concentration of 10 to 400 μg / ml.
본 발명의 다른 양태에 따르면, 본 발명은 감태 추출물을 세포 배양 배지에 첨가하는 단계; 및 상기 배지에서 지방-유래 중간엽 줄기세포(adipose-derived mesenchymal stem cell)를 유도만능 줄기세포(induced pluripotency stem cell)로 역분화시키는 단계를 포함하는 유도만능 줄기세포의 제조 방법을 제공한다. According to another aspect of the invention, the present invention comprises the steps of adding the Ecklonia cava extract to the cell culture medium; And dedifferentiating adipose-derived mesenchymal stem cells into induced pluripotency stem cells in the medium.
본 발명의 일 실시예에 따르면, 실험군으로서 본 발명의 감태 추출물이 포함된 배지 조성물을 이용한 경우(DMEM F-12 배지에 감태 추출물, 및 에너지 워터를 포함한 배지)와 대조군으로서 DMEM F-12 배지만을 이용한 경우와 달리 8-10일째 되는 날 만능 줄기세포 콜로니들이 형성되었음을 확인할 수 있었다(도 2 및 도 3). According to one embodiment of the present invention, when using the medium composition containing the Ecklonia cava extract of the present invention as the experimental group (medium extract containing Ecklonia cava extract, and energy water in DMEM F-12 medium) and only DMEM F-12 medium as a control group Unlike when used, it was confirmed that pluripotent stem cell colonies were formed on day 8-10 (FIG. 2 and FIG. 3).
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 제조 방법으로 제조된 유도만능 줄기세포를 제공한다. According to another aspect of the present invention, the present invention provides an induced pluripotent stem cell prepared by the above method.
본 발명의 유도만능 줄기세포는 배아 줄기세포와 동일한 분화능을 가지며, 세포의 모양에 있어서도 배아 줄기세포와 거의 동일하다(도 2 및 도 3). 본 발명의 일 실시예에 따르면, 배아줄기세포에 특징적인 유전자(Oct4, Sox-2) 및 단백질(SSEA-4)의 발현여부를 조사한 결과 본 발명에 의해 유도된 만능 줄기세포에서 배아줄기세포와 동일하게 상기 유전자 및 단백질이 발현됨을 확인하였다(도 4 및 도 5). Induced pluripotent stem cells of the present invention have the same differentiation capacity as embryonic stem cells, and are almost identical to embryonic stem cells in the shape of cells (FIGS. 2 and 3). According to an embodiment of the present invention, as a result of examining the expression of the genes (Oct4, Sox-2) and protein (SSEA-4) characteristic to the embryonic stem cells embryonic stem cells and pluripotent stem cells induced by the present invention It was confirmed that the same genes and proteins are expressed (Figs. 4 and 5).
다른 실시예에 따르면, 유도 과정을 거치치 않은 중간엽 줄기세포(MSC)에서 는 만능 줄기세포의 특징적인 유전자인 OCT4, SOX2와 Nanog의 발현도가 낮은 반면 에, 본 발명의 방법에 의해 유도된 만능 줄기세포(실험예 1-1: EtOH EPN, 실험예 1-2: Sonic EPN)에서는 이들 특징적인 유전자들이 현저히 높게 발현되었다(도 6 및 도 7).따라서, 본 발명의 유도만능 줄기세포는 지방세포로 효과적으로 분화할 수 있다. According to another embodiment, mesenchymal stem cells (MSC) that have not undergone the induction process have low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, whereas pluripotency induced by the method of the present invention In stem cells (Experimental Example 1-1: EtOH EPN, Experimental Example 1-2: Sonic EPN), these characteristic genes were significantly expressed (Figs. 6 and 7). Thus, the induced pluripotent stem cells of the present invention were adipose Can effectively differentiate into cells.
본 발명의 일 실시예에 따르면, 만능 줄기세포로 예상되었던 세포들이 지방 세포로 분화될 수 있음을 확인할 수 있었다(도 8). According to one embodiment of the present invention, it was confirmed that cells expected to be pluripotent stem cells can be differentiated into adipocytes (FIG. 8).
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 분화된 지방세포를 포함하는 세포 치료용 조성물을 제공한다. According to another aspect of the present invention, the present invention provides a cell therapy composition comprising the differentiated adipocytes.
본 발명의 조성물은 임의의 투여경로에 의해서, 구체적으로는 복강 또는 흉강 투여, 피하 투여, 정맥 또는 동맥 혈관내 투여, 근육내 투여, 주사에 의한 국소 투여 등의 방법에 의해서 투여 가능하다. The composition of the present invention can be administered by any route of administration, specifically by intraperitoneal or chest cavity administration, subcutaneous administration, intravenous or arterial vascular administration, intramuscular administration, topical administration by injection, or the like.
본 발명에 있어서, 상기 조성물은 통상의 방법에 기초하여 주사제, 현탁제, 유화제 등의 형태로 투여할 수 있고, 필요에 따라서 프로인트 완전 보조제 등의 보조제에 현탁되거나, 또는 BCG와 같은 보조제 활성을 갖는 물질과 함께 투여하는 것도 가능하다. 상기 조성물은 멸균되거나 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 또는 완충제 등의 보조제 및 기타 치료적으로 유용한 물질을 함유할 수 있다. In the present invention, the composition can be administered in the form of injections, suspensions, emulsifiers and the like based on conventional methods, and if necessary, suspended in an adjuvant such as Freund's complete adjuvant or adjuvant activity such as BCG. It is also possible to administer with the substance having. The composition may be sterile or contain adjuvants such as stabilizers, hydrating or emulsifying accelerators, salts or buffers for controlling osmotic pressure and other therapeutically valuable substances.
본 발명의 세포 치료용 조성물은 관절염, 신경계질환, 내분비질환, 간질환 등에 적용이 가능하며, 추후 사람에 대한 임상시험 결과에 따라서는 사람에 대한 동종세포 치료제로의 가능성도 있다. The cell therapy composition of the present invention can be applied to arthritis, nervous system diseases, endocrine diseases, liver diseases, and the like, and according to the results of clinical trials for humans in the future, there is also the possibility of treating allogeneic cells for humans.
본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:
(i) 본 발명은 감태 추출물을 포함하는 유도만능 줄기세포 역분화용 배지 조성물을 제공한다. (i) The present invention provides a medium composition for induced pluripotent stem cell reverse differentiation comprising Ecklonia cava extract.
(ii) 또한, 본 발명은 상기 배지 조성물을 이용한 유도만능 줄기세포 제조 방법을 제공한다. (ii) The present invention also provides a method for producing pluripotent stem cells using the medium composition.
(iii) 본 발명에 따른 배지 조성물을 이용하면 지방-유래 중간엽 줄기세포를 이용하여 유도만능 줄기세포를 효율적으로 제조할 수 있으며, 제조된 만능 줄기세포는 다양한 세포로의 분화가 가능하므로 세포 치료제로서 유용하게 사용될 수 있다. (iii) By using the medium composition according to the present invention, it is possible to efficiently produce induced pluripotent stem cells using adipose-derived mesenchymal stem cells, and the prepared pluripotent stem cells are capable of differentiating into various cells, thereby treating cells. It can be usefully used as.
(iv) 본 발명은 배아줄기세포와 동일한 분화능을 가지는 만능줄기세포를 제조하므로써, 배아줄기세포를 사용하지 아니하므로 배아의 파괴로 인한 윤리적 문제 를 없애고 암을 유발할 수 있는 바이러스를 사용하지 않으므로 암세포 형성 위험이 없는 안전한 만능줄기세포를 제조할 수 있다. (iv) The present invention eliminates ethical problems caused by embryo destruction by not using embryonic stem cells by producing pluripotent stem cells having the same differentiation capacity as embryonic stem cells, and thus does not use viruses that can cause cancer. Safe pluripotent stem cells can be produced without risk.
(v) 나아가 천연추출물을 사용하므로 종래 방법에 비하여 매우 용이하게 현저히 높은 효율로 만능 줄기세포를 제조할 수 있고 환자의 지방세포에 분리한 중간 엽 줄기세포를 이용하므로 환자 맞춤형 줄기세포 치료제의 실용화를 앞당길 수 있을 것으로 기대가 된다. 본 발명은 신경계 질환, 면역 질환 등 다양한 난치병 질환 을 치료하는데 크게 기여할 것으로 여겨진다.(v) Furthermore, since natural extracts are used, pluripotent stem cells can be produced very easily and significantly higher than conventional methods, and mesenchymal stem cells isolated from adipocytes of patients can be used for the practical application of stem cell therapeutics tailored to patients. Expect to be able to advance. It is believed that the present invention will greatly contribute to the treatment of various intractable diseases such as neurological diseases and immune diseases.
도 1은 지방-유래 중간엽 줄기세포에서 감태추출물 배지를 주입하여 배양 시, 배아 줄기세포와 거의 동일한 만능 줄기세포가 유도되는 것을 보여주는 그림이 다. 1 is a diagram showing that the pluripotent stem cells almost the same as embryonic stem cells are induced when cultured by injection of Ecklonia cava extract medium from adipose-derived mesenchymal stem cells.
도 2는 본 발명의 방법(실시예 1-1)으로 감태 추출물(에탄올 추출물)의 농도 에 따라 유도된 만능 줄기세포 콜로니 형성을 나타낸 것이다. Figure 2 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (ethanol extract) by the method of the present invention (Example 1-1).
도 3은 본 발명의 방법(실시예 1-2)으로 감태 추출물(물 추출물)의 농도에 따라 유도된 만능 줄기세포 콜로니 형성을 나타낸 것이다. Figure 3 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (water extract) by the method of the present invention (Example 1-2).
도 4는 본 발명의 방법(실험예 1-1)으로 유도된 만능 줄기세포를 특이적 단백질 발현을 이용하여 만능 줄기세포임을 확인한 것이다. Figure 4 confirms that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) by using specific protein expression.
도 5는 본 발명의 방법(실험예 1-2)으로 유도된 만능 줄기세포를 특이적 단백질 발현을 이용하여 만능 줄기세포임을 확인한 것이다. 5 shows that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) are pluripotent stem cells using specific protein expression.
도 6은 본 발명의 방법(실험예 1-1)으로 유도된 만능 줄기세포의 유전자 발현 및 이를 그래프로 도식화하여 나타낸 것이다. 6 is a graph showing the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) and graphs thereof.
도 7은 본 발명의 방법(실험예 1-2)으로 유도된 만능 줄기세포의 유전자 발현 및 이를 그래프로 도식화하여 나타낸 것이다. Figure 7 shows the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) and graphically depicted them.
도 8은 본 발명의 방법으로 유도된 만능줄기세포로 지방세포 분화용 배지를 이용하여 지방세포로 분화한 결과이다. 8 is a result of differentiation into adipocytes using adipose cell differentiation medium into pluripotent stem cells induced by the method of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예 1: 감태 추출물의 제조 Example 1: Preparation of Ecklonia cava Extract
실시예 1-1: 에탄올 용매를 이용한 감태 추출물의 제조Example 1-1 Preparation of Ecklonia cava Extract Using Ethanol Solvent
실험에 사용된 생약 시료들은 제주도에서 구입하여 전문가의 정확한 감정을 거친 후 실험에 사용하였다. 건조된 생약 시료 100 g을 70% 에탄올 1 ℓ에 넣고 에탄올은 16시간 동안 환류 추출하고 여과지를 사용하여 여과하였다. 여액을 회전감압증발기에서 농축시키고 즉시 동결 건조하였다. The herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of 70% ethanol, and ethanol was extracted under reflux for 16 hours and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and immediately freeze dried.
실시예 1-2: 물을 이용한 감태 추출물의 제조Example 1-2 Preparation of Ecklonia cava Extract Using Water
실험에 사용된 생약 시료들은 제주도에서 구입하여 전문가의 정확한 감정을 거친 후 실험에 사용하였다. 건조된 생약 시료 100 g을 물 1 ℓ에 넣고 물은 16시간 동안 초음파추출기를 적용하여 추출하고 여과지를 사용하여 여과하였다. 여액을 회전감압증발기에서 농축시키고 즉시 동결 건조하였다. The herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of water, and 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 immediately freeze dried.
실시예 2: 인체 지방조직에서 중간엽 줄기세포의 분리 및 배양 Example 2: Isolation and Culture of Mesenchymal Stem Cells from Human Adipose Tissue
실시예 2-1: 인체 지방조직 채취Example 2-1: Collection of Human Adipose Tissue
지방조직은 지방 흡입한 후 바로 수집된다. 시료는 실험실로 옮겨지기 전에 500 ㎖의 멸균 유리병에 흡입된 지방조직을 모은다. 이후 살균유리병을 밀봉한 후 실험실로 옮겨진다. 실험실에서는 멸균 상태 하에서 class 100의 플로우 후드에서 중간엽 줄기세포의 추출이 수행된다. 시료는 우선 멸균 스테인레스스틸의 용기로 옮겨진다. PBS는 수회 세정한 후 지방 조직 시료는 이후 2 cm 길이로 잘라져 50 ㎖ 튜브로 옮겨지며, 여기서 추가적인 세정 및 70% 에탄올로 항감염처리하고, 항생제 혼합물(50 IU/㎖의 페니실린, 50 ug/㎖의 스트렙토마이신(Invitrogen으로부터 구매))이 첨가된 PBS로 상기 용액이 깨끗해질 때까지 수차례 세정한다. Adipose tissue is collected immediately after liposuction. The sample collects aspirated adipose tissue in 500 ml sterile glass bottles before being transferred to the laboratory. The sterile glass bottle is then sealed and transferred to the laboratory. In the laboratory, mesenchymal stem cells are extracted in a flow hood of class 100 under sterile conditions. The sample is first transferred to a sterile stainless steel container. The PBS was washed several times and then the adipose tissue sample was then cut into 2 cm lengths and transferred to a 50 ml tube where additional washing and anti-infection with 70% ethanol and antibiotic mixture (50 IU / ml penicillin, 50 ug / ml) Wash several times with PBS added with streptomycin (purchased from Invitrogen) until the solution is clear.
실시예 2-2: 인체 지방조직에서 중간엽 줄기세포 분리 및 배양Example 2-2: Isolation and Culture of Mesenchymal Stem Cells from Human Adipose Tissue
분리된 지방조직을 PBS로 세척한 후 조직을 잘게 자르고, collagenase type 1(1mg/㎖)을 첨가한 DMEM 배지를 이용해 37℃에서 10분에 1번씩 흔들어 주면서 1시간 동안 절단(digestion) 하였다. 다음으로, PBS로 세척 후 1000 rpm에서 5분간 원심분리 하였다. 상층액은 흡입(suction)하고 바닥에 남은 펠렛은 PBS로 세척한 후 1000 rpm으로 5분간 원심분리하였다. 100 ㎛ 메쉬(mesh) 크기의 필러로 필터링하여 잔해(debris)를 제거한 후 PBS로 세척하였다. The isolated adipose tissue was washed with PBS, the tissue was chopped, and digested for 1 hour while shaking once every 10 minutes at 37 ° C. using DMEM medium to which collagenase type 1 (1 mg / ml) was added. Next, washed with PBS and centrifuged for 5 minutes at 1000 rpm. The supernatant was aspirated and the pellet remaining on the bottom was washed with PBS and centrifuged at 1000 rpm for 5 minutes. Filtered with a 100 μm mesh size filler to remove debris and washed with PBS.
중간엽 세포의 분리/배양을 위해 상기의 외식된 조직은 10% 우태혈청(FBS, Hyclone)이 첨가된 5 ㎖의 DMEM (Dulbecco's modified eagle medium) F-12 (Gibco), 10% FBS, 100 unit/㎖ 페니실린, 50 ㎍/㎖ 스트렙토마이신에 담가져 질소 95%, 이산화탄소 5% 세포배양기에서 37℃로 유지하여 저산소증(Hypoxic) 상태를 유지하여 줄기세포 이외의 세포는 죽게 만들어 중간엽 줄기세포의 순도를 높였다. 배지는 매 3일 또는 4일 마다 교체되었다. 세포의 성장(outgrowth)은 광학현미경으로 모니터링되었다. 신장하는 세포들은 추가적인 확장 및 냉동보관(DMEM/10% FBS 이용)을 위해 트립신처리(0.125% 트립신/0.05% EDTA)하였다. For the isolation / culture of mesenchymal cells, the explanted tissue was treated with 5 ml of DMEM (Dulbecco's modified eagle medium) F-12 (Gibco), 10% FBS, 100 units with 10% fetal calf serum (FBS, Hyclone). / Ml penicillin, 50 ㎍ / ㎖ streptomycin in a 95% nitrogen, 5% carbon dioxide cell culture incubator at 37 ℃ to maintain the hypoxic state to kill the cells other than stem cells, the purity of mesenchymal stem cells Raised. Medium was changed every 3 or 4 days. Outgrowth of cells was monitored by light microscopy. Elongating cells were trypsinized (0.125% trypsin / 0.05% EDTA) for further expansion and cryopreservation (using DMEM / 10% FBS).
중간엽 줄기세포의 추출을 위해, 세포의 펠렛은 배지 DMEM F-12(Gibco), 10% FBS, 100 unit/㎖ 페니실린, 50 ㎍/㎖ 스트렙토마이신에 재현탁 및 카운트되었으며, 10 cm 조직배양 접시에 1x106 세포/접시의 밀도로 접종되었다. 상기 배지는 매 3일 또는 4일 마다 교환되었다. 세포의 성장(growth) 및 클론형성은 광학현미경으로 모니터링되었다. 약 90%의 세포수(confluence)에서, 세포들은 상기에 설명된 바와 같이 서브-배양(sub-culture) 되었다. For extraction of mesenchymal stem cells, pellets of cells were resuspended and counted in medium DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, 50 μg / ml streptomycin, and 10 cm tissue culture dishes. Were inoculated at a density of 1 × 10 6 cells / dish. The medium was changed every 3 or 4 days. Cell growth and cloning were monitored by light microscopy. At about 90% confluence, the cells were sub-cultured as described above.
실험예 1: 지방-유래 중간엽 줄기세포로부터 만능 줄기세포 유도 Experimental Example 1 Induction of Pluripotent Stem Cells from Adipose-Derived Mesenchymal Stem Cells
실험예 1-1: 실시예 1-1의 감태 추출물 농도에 따른 인간 지방-유래 중간엽Experimental Example 1-1 Human fat-derived mesenchyme according to Ecklonia cava extract concentration of Example 1-1 줄기세포의 만능 줄기세포 제조Pluripotent stem cell production of stem cells
제주 감태 추출물의 농도에 따라 인간 지방-유래 줄기세포로부터 만능 줄기 세포를 유도하기 위한 실험으로 대조군은 MSC의 전용 배지로 DMEM F-12(Gibco), 10% FBS, 100 unit/㎖ 페니실린, 50 ㎍/㎖ 스트렙토마이신을 기본배지로 사용하였으며, 실험군은 계대배양을 세 번째 한 인간 지방-유래 중간엽 줄기세포를 사용하여 배지에 실시예 1-1에서 제조한 제주 감태 추출물을 Normal, 1 ㎍/㎖, 20 ㎍/㎖, 50 ㎍/㎖, 100 ㎍/㎖, 400 ㎍/㎖, 800 ㎍/㎖, 1 ㎎/㎖의 농도와 에너지 워터(SiO2, Al2O3, TiO3, Fe2O3, CaO, Na2O, K2O, LiO를 함유하는 정제 탈이온수, 에스티씨나라) 0.1 v/v%를 첨가하였다(도 1). 인간 지방-유래 중간엽 줄기세포들을 분리하여 세척된 단핵구 세포를 6-웰 플레이트(dish)에 1 x 104 개의 세포를 접종하여 37℃와 5% CO2를 유지하여 배양하였다. Experiments to induce pluripotent stem cells from human adipose-derived stem cells according to the concentration of Jeju Ecklonia cava extract. The control group was a medium of MSC. DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, 50 ㎍ / Ml streptomycin was used as the primary medium, and the experimental group used the Jeju Ecklonia cava extract prepared in Example 1-1 in medium using human adipose-derived mesenchymal stem cells subjected to subculture 3, Normal, 1 ㎍ / ml Concentrations of 20 μg / ml, 50 μg / ml, 100 μg / ml, 400 μg / ml, 800 μg / ml, 1 mg / ml and energy water (SiO 2 , Al 2 O 3 , TiO 3 , Fe 2 O 3 , CaO, Na 2 O, K 2 O, purified de-ionized water containing LiO, Estinicin) 0.1 v / v% was added (FIG. 1). Monocytes isolated from human adipose derived mesenchymal stem cells were inoculated with 1 × 10 4 cells in 6-well plates and cultured at 37 ° C. and 5% CO 2 .
그 결과, 실험군에서는 제주감태 추출물의 농도가 100 내지 400 ㎍/㎖일 때 10일 후 콜로니가 분명하게 형성하는 것이 관찰 되었으며(도 2), 이때 현미경 배율 은 200배 비율로 관찰한 것이다. As a result, in the experimental group, the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 100 to 400 ㎍ / ㎖ (Fig. 2), the microscope magnification was observed at a 200-fold ratio.
실험예 1-2: 실시예 1-2의 감태 추출물 농도에 따른 인간 지방-유래 중간엽 줄기세포의 만능 줄기세포 제조Experimental Example 1-2: Preparation of Pluripotent Stem Cells of Human Adipose-derived Mesenchymal Stem Cells According to Ecklonia cava Extract Concentrations of Example 1-2
실험예 1-1과 동일한 방법으로 실험하되, 제주 감태 추출물을 실시예 1-2에 서 제조한 것을 사용하였다. 그 결과, 실험군에서는 제주감태 추출물의 농도가 20 내지 50 ㎍/㎖일 때 10일 후 콜로니가 분명하게 형성하는 것이 관찰 되었으며(도 3), 이때 현미경 배율은 200배 비율로 관찰한 것이다. Experimental in the same manner as in Experimental Example 1-1, the Jeju Ecklonia cava extract was prepared in Example 1-2 was used. As a result, in the experimental group, the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 20 to 50 μg / ml (FIG. 3), and the microscope magnification was observed at a 200-fold ratio.
실험예 1-3: 본 발명의 방법에 의해 유도된 만능 줄기세포의 면역화학적 염색 분석Experimental Example 1-3 Analysis of Immunochemical Staining of Pluripotent Stem Cells Induced by the Method of the Present Invention
상기 실험예 1-1 및 1-2의 방법에 의해 유도된 만능 줄기세포에 대하여 배아 줄기세포의 특이 유전자인 OCT4, SOX2, 단백질인 SSEA-4(stage-specific embryonic antigen-4)의 발현 여부를 이에 대한 항체를 사용하여 면역화학적 염색법을 사용하여 단백빌 발현 여부를 분석하였다. Whether the expression of OCT4, SOX2, or the protein SSEA-4 (stage-specific embryonic antigen-4), which is a specific gene of embryonic stem cells, was expressed in the pluripotent stem cells induced by the methods of Experimental Examples 1-1 and 1-2 above Antibodies were used to analyze protein expression using immunochemical staining.
염색 과정은 우선 4% 파라포르말데하이드(Paraformaldehyde)를 이용하여 세포를 고정한 후 PBS로 세정하고 1% BSA 용액으로 블로킹(blocking)을 하였다. OCT4, SOX3, SSEA-4에 대한 1차 항체를 처리하여 4℃에서 18 시간 동안 반응시킨 후, PBS로 세정을 하고 1차 항체에 대한 형광색소(FITC)가 붙은 2차 항체를 처리하여 실온에서 1 시간 동안 반응시켰다. The staining process was first fixed with 4% paraformaldehyde (Paraformaldehyde), and then washed with PBS and blocked (blocking) with 1% BSA solution. After treatment with the primary antibody against OCT4, SOX3, SSEA-4 for 18 hours at 4 ℃, washed with PBS and treated with a secondary antibody with a fluorescent pigment (FITC) for the primary antibody at room temperature The reaction was carried out for 1 hour.
PBS로 세정을 한 후 형광현미경(fluorescence microscope)을 사용하여 발현 여부를 분석하여 그 결과를 도 4 및 도 5에 나타내었다. 도 4는 에탄올을 이용하여 추출한 감태 추출물에 의해 유도된 만능 줄기세포에 대한 결과이고, 도 5는 물을 이용하여 추출한 감태 추출물에 의해 유도된 만능 줄기세포에 대한 결과이다. After washing with PBS, the expression was analyzed using a fluorescence microscope, and the results are shown in FIGS. 4 and 5. Figure 4 is a result for pluripotent stem cells induced by Ecklonia cava extract extracted with ethanol, Figure 5 is a result for pluripotent stem cells induced by Ecklonia cava extract extracted with water.
도 4 (a) 및 도 5 (b)의 Bright field 결과에 의해 줄기세포 콜로니의 형태 를 알 수 있다. The shape of stem cell colonies can be seen by the bright field results of FIGS. 4 (a) and 5 (b).
또한, 도 4 (b) 및 도 5 (b)의 만능 줄기세포 특이적 마커인 OCT4, SOX2, SSEA-4로부터 발현된 단백질에 대한 염색 결과에 있어서, 모두 양성 반응이 나타나 만능 줄기세포가 제조되었음을 확인할 수 있었다. In addition, in the staining results for proteins expressed from the pluripotent stem cell specific markers OCT4, SOX2, and SSEA-4 of FIGS. 4 (b) and 5 (b), all positive reactions occurred, indicating that pluripotent stem cells were prepared. I could confirm it.
도 4 (c) 및 도 5 (c)의 DAPI에 의한 세포핵의 염색 결과를 통해 줄기세포들 의 존재를 다시 한 번 확인할 수 있었다. The presence of stem cells was once again confirmed through staining of the cell nuclei by DAPI of FIGS. 4 (c) and 5 (c).
실험예 1-4: 만능 줄기세포 유전자 분석 비교Experimental Example 1-4: Pluripotent stem cell gene analysis comparison
상기 실험예 1-1과 1-2에서 제조된 만능 줄기세포를 현미경으로 보면서 200 ㎕ 파이펫을 사용하여 콜로니만 떼어낸 후, TRIzol 시약(Invitrogen사 제조)을 사용하여 전체 RNA를 분리하였다. 역전사-중합효소연쇄반응(RT-PCR)을 이용하여 cDNA를 합성한 후 OCT4, Sox-2, Nanog 및 대조유전자인 GAPDH(glyceraldehyde 3-phosphate dehydrogenase) 유전자에 특이적인 프라이머를 이용하여 PCR을 진행하였다. Nanog, OCT4, Sox-2는 배아줄기세포에서 보이는 특징적 유전자이다. PCR 산물을 아가로스 겔 전기영동으로 분석하여, 이들 유전자의 발현을 확인한 결과를 도 6과 7에 나타내었다. After observing the pluripotent stem cells prepared in Experimental Examples 1-1 and 1-2 under a microscope, only colonies were removed using a 200 μl pipette, and total RNA was isolated using TRIzol reagent (manufactured by Invitrogen). CDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR), and then PCR was performed using primers specific for OCT4, Sox-2, Nanog and the control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. . Nanog, OCT4 and Sox-2 are characteristic genes seen in embryonic stem cells. PCR products were analyzed by agarose gel electrophoresis, and the results of confirming the expression of these genes are shown in FIGS. 6 and 7.
그 결과 도 6과 7에 따르면, 유도 과정을 거치치 않은 중간엽 줄기세포(대조군, MSC)에서는 만능 줄기세포의 특징적인 유전자인 OCT4, SOX2와 Nanog의 발현도 가 낮은 반면에 본 발명의 방법에 의해 유도된 만능 줄기세포(실험예 1-1(EtOH EPN 으로 나타냄) 및 실험예 1-2(Sonic EPN으로 나타냄)에 의해 제조된 만능 줄기세포)에서는 이들 특징적인 유전자들이 현저히 높게 발현되었다. 줄기세포 유전자인 OCT4, SOX2와 Nanog의 발현 정도는 도 6 및 도 7의 그래프를 통해 명확하게 확인할 수 있다. As a result, according to FIGS. 6 and 7, the mesenchymal stem cells (control group, MSC) that did not undergo the induction process had low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, while Pluripotent stem cells (pluripotent stem cells prepared by Experimental Example 1-1 (denoted by EtOH EPN) and Experimental Example 1-2 (denoted by Sonic EPN)) induced by the expression of these characteristic genes were significantly higher. The expression levels of the stem cell genes OCT4, SOX2 and Nanog can be clearly seen through the graphs of FIGS. 6 and 7.
실험예 2: 지방세포로의 분화Experimental Example 2: Differentiation into Adipocytes
지방세포로의 분화를 유도하기 위하여 감태 추출물과 에너지워터를 혼합한 배지를 사용하여 습도 95%, 37℃, 5% CO2 조건의 배양기에 배양하여 중간엽 줄기세포로부터 만능 줄기세포세포를 유도한 다음 지방세포 분화용액 DMEM F-12, 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM hydrocortisone, 0.1 mM indomethacin 5% FBS 에서 2주 동안 배양하였다. 지방세포로의 분화 검증을 위해 세포내 지방 축적을 확인하기 위한 Oil-Red-O 조직화학 염색을 통하여 확인한 결과 도 8 A 및 B에서 나타난 바와 같이 Oil-Red-O에 양성반응을 보여 만능 줄기세포로 예상되었던 세포들이 지방세포로 분화될 수 있음을 확인할 수 있었다. In order to induce differentiation into adipocytes, pluripotent stem cell cells were induced from mesenchymal stem cells by culturing in a culture medium of 95%, 37 ° C. and 5% CO 2 using a medium containing Ecklonia cava extract and energy water. Adipocyte differentiation solution was incubated for 2 weeks in DMEM F-12, 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM hydrocortisone, 0.1 mM indomethacin 5% FBS. Oil-Red-O histochemical staining to confirm intracellular fat accumulation for differentiation into adipocytes was shown to be positive for Oil-Red-O as shown in FIGS. 8A and 8B. It was confirmed that the expected cells could differentiate into adipocytes.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (9)

  1. 하기의 단계를 포함하는 중간엽 줄기세포(mesenchymal stem cell)로부터 지방세포(adipocyte)를 분화시키는 방법: A method of differentiating adipocytes from mesenchymal stem cells, comprising the following steps:
    (a) 감태(Ecklonia cava) 추출물을 포함하는 배지에서 인간의 지방으로부터 수득한 지방-유래 중간엽 줄기세포(mesenchymal stem cell)를 유도만능 줄기세포(induced pluripotency stem cell)로 역분화시키는 단계; 및 (a) dedifferentiating adipose-derived mesenchymal stem cells obtained from human fat in a medium comprising Ecklonia cava extract into induced pluripotency stem cells; And
    (b) 상기 유도만능 줄기세포를 지방세포로 분화시키는 단계.(b) differentiating the induced pluripotent stem cells into adipocytes.
  2. 제 1 항에 있어서, 상기 (a) 단계의 배지는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), BME(Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM-F12, α-MEM(α-Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), MacCoy's 5A 배지, AmnioMax, AminoMaxⅡ complete Medium 또는 Chang's Medium MesemCult-XF Medium인 것을 특징으로 하는 방법. According to claim 1, wherein the medium of step (a) Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, DMEM-F12 , 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, AminoMaxII complete Medium or Chang's Medium MesemCult-XF Medium How to.
  3. 제 1 항에 있어서, 상기 감태 추출물은 상기 배지 중 10 내지 400 ㎍/㎖의 농도로 포함되는 것을 특징으로 방법. According to claim 1, wherein the Ecklonia cava extract is characterized in that it comprises a concentration of 10 to 400 ㎍ / ㎖ in the medium.
  4. 제 1 항에 있어서, 상기 (a) 단계의 상기 감태 추출물은 에탄올 용매로 추출 한 것으로 상기 배지 중 100 내지 400 ㎍/㎖의 농도로 포함되는 것을 특징으로 방법. The method of claim 1, wherein the Ecklonia cava extract of step (a) is extracted with an ethanol solvent, characterized in that it comprises a concentration of 100 to 400 ㎍ / ㎖ in the medium.
  5. 제 1 항에 있어서, 상기 (a) 단계의 상기 감태 추출물은 물로 추출한 것으로 상기 배지 중 20 내지 50 ㎍/㎖의 농도로 포함되는 것을 특징으로 방법. The method according to claim 1, wherein the Ecklonia cava extract of step (a) is extracted with water and is contained at a concentration of 20 to 50 μg / ml in the medium.
  6. 제 1 항에 있어서, 상기 (a) 단계의 배지는 에너지워터 0.01 내지 10 v/v%를 더 포함하는 것을 특징으로 하는 방법. The method of claim 1, wherein the medium of step (a) further comprises an energy water of 0.01 to 10 v / v%.
  7. 제 1 항에 있어서, 상기 (b) 단계는 3-이소부틸-1-메틸잔틴(3-isobutyl-1-methylxanthine), 하이드로코르티손(hydrocortisone) 및 인도메타신(indomethacin)을 포함하는 배지를 이용하는 것을 특징으로 하는 방법.The method of claim 1, wherein step (b) comprises using a medium containing 3-isobutyl-1-methylxanthine, hydrocortisone and indomethacin. How to feature.
  8. 제 1 항 내지 제 7 항 중에서 선택된 어느 한 항의 방법에 의해 분화된 지방 세포. Fat cells differentiated by the method of any one of claims 1 to 7.
  9. 제 8 항의 지방세포를 포함하는 세포 치료용 조성물. Cell therapy composition comprising the fat cells of claim 8.
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