HK1220487B - Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method - Google Patents

Description

Method for inducing totipotent stem cells, and totipotent stem cells prepared by the method

Technical Field

The present invention relates to a method for inducing totipotent stem cells, totipotent stem cells prepared by the method, and a composition containing the totipotent stem cells, wherein the method is to induce totipotent stem cells by reprogramming and/or dedifferentiating differentiated adult cells.

The present invention also relates to a pharmaceutical composition or a cosmetic composition comprising totipotent stem cells.

The present invention also relates to a composition for activating stem cells, proliferating skin cells, regenerating skin or anti-aging.

Background Art

Stem cells are undifferentiated cells that can differentiate into various types of cells that make up biological tissues and can be obtained from embryos, fetuses, and adult tissues. Among the different cell types of stem cells, totipotent stem cells refer to those that can differentiate into any of the three germ layers, i.e., endoderm, mesoderm, and ectoderm.

The stem cells can be classified based on anatomical location, cellular function, antigens expressed on the cell surface, transcription factors, proteins produced by the cell, and the specific cell type from which they are derived.

As a relatively clear classification standard, the stem cells can be classified based on their origin. Embryonic stem cells (ES cells) are isolated from embryos, and adult stem cells are isolated from adult tissues.

Alternatively, the stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to their ability to differentiate into specialized cell types. Typically, embryonic stem cells (ES cells) can be divided into totipotent stem cells, and adult stem cells are divided into totipotent stem cells and unipotent stem cells.

Embryonic stem cells (ES cells) derived from the inner cell mass of the early embryo, the blastocyst, are totipotent stem cells, capable of differentiating into all tissues that make up the adult body. In other words, ES cells are undifferentiated cells that can proliferate without restriction and differentiate into all cell types. Unlike adult stem cells, ES cells can form germ cells and therefore be passed down to the next generation.

However, the production of totipotent stem cells raises serious religious and ethical concerns due to the destruction of embryos during their production. Furthermore, because they are derived from limited embryos and lack immune compatibility between individuals, immune rejection is unavoidable. To overcome these problems, various methods have been attempted to artificially produce totipotent stem cells, such as by using induced embryonic stem cells derived from adult cells or cells with totipotency similar to embryonic stem cells.

Representative examples include somatic cell nuclear transfer (SCNT), fusion with embryonic stem cells, and reprogramming by defined factors. SCNT has extremely low efficiency and raises ethical concerns due to the large number of eggs required. Fusion with embryonic stem cells raises serious concerns about cell stability due to the induced cells possessing two additional pairs of genes. Reprogramming by defined factors involves the serious risk of carcinogenesis due to the use of viruses containing oncogenes.

Therefore, in order to develop cell therapeutic agents, a method for preparing induced pluripotent stem cells that can ensure stability and safety and does not raise ethical issues is needed.

To meet these needs, researchers have studied a method for inducing pluripotent stem cells by dedifferentiating them by introducing four genes into somatic cells (Takahashi K, Yamanaka S (2006), Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676). This method is ethical because it uses adult cells and solves the problem of immune rejection because it uses autologous cells.

The inventors of the present invention obtained dedifferentiated stem cells from extracts of induced pluripotent stem cells (iPSCs) derived from animals, but there are several limitations.

First, this method requires large amounts (20 mg or more) of iPSC extract. Therefore, using extracts from dedifferentiated human stem cells to induce dedifferentiation has been costly and labor-intensive, and has not been successful. For example, to produce 20 mg of dedifferentiated human stem cell extract, a single expert would need to work diligently for at least three months, which is very costly.

Second, when somatic cells to be induced are treated with extracts of animal stem cells or dedifferentiated stem cells, if the cells are not completely destroyed in the extract and survive, it is not easy to distinguish the dedifferentiation-induced cells from the surviving dedifferentiated stem cells, and genomic DNA analysis is required, which is very costly and time-consuming.

Third, because the production of human dedifferentiated stem cells using proteins is almost impossible, extracts of human dedifferentiated stem cells produced using viruses are required. Because the resulting cells may contain carcinogens derived from these viruses, their clinical application is difficult.

Summary of the Invention

Technical issues

In one aspect, to address existing issues in the related art, the present invention provides a method for preparing totipotent stem cells, which ensures stability and safety and does not raise ethical concerns. The present invention also provides a method for inducing dedifferentiation of human stem cells, a method that has been largely unrealized.

The inventors of the present invention have developed a method for inducing totipotent stem cells using adult-derived cells, thereby generating totipotent stem cells with the same genetic background as the adult. According to the present invention, the same results can be achieved using adult-derived cells of various genetic backgrounds. Therefore, the method of the present invention is suitable for the production of totipotent stem cells.

Technical Solution

In one aspect, the present invention provides a method for inducing stem cells, comprising treating adult-derived cells with shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition containing the same.

Specifically, in one aspect, the present invention provides a method for preparing induced pluripotent stem cells, which comprises: extracting an extract containing active ingredients from plant stem cells or any kind of induced pluripotent plant stem cells induced by various methods; injecting the extract into adult-derived cells; and preparing pluripotent cells, such as embryonic stem cells, by culturing the cells injected with the extract.

In another aspect, the present invention provides a method for preparing stem cells, which further comprises: injecting shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition containing them into cells derived from an adult; and culturing the cells injected with the shikimic acid, the extract, or the composition.

The extract may be a callus tissue extract.

In an exemplary embodiment of the present invention, the method may further include: treating the adult-derived cells with a cell membrane permeabilizer before injecting the extract. The cell membrane permeabilizer may include streptolysin O and digitalis, and is not limited thereto as long as the cell membrane permeabilizer can easily inject the shikimic acid or extract of the present invention into the cell membrane.

In another exemplary embodiment of the present invention, the method further comprises: culturing the adult-derived cells injected with the extract after being transferred to a feeder cell layer. The feeder cell layer may include, but is not limited to, STO cells.

In another aspect, the present invention provides a method for preparing induced pluripotent stem cells, comprising: extracting an extract containing an active ingredient from plant stem cells, callus tissue, or any type of induced pluripotent plant stem cells induced by various methods; injecting the extract into adult-derived cells; culturing the cells injected with the extract using a conventional cell culture medium; and further culturing the cells, after being transferred to a feeder cell layer, using an embryonic stem cell culture medium.

In another aspect, the present invention provides a composition prepared by the above method.

In another aspect, the present invention provides a composition comprising the stem cells. In another aspect, the present invention provides a method for inducing induced pluripotent stem cells (iPSCs), pluripotent stem cells prepared using the method, and a cell therapy agent comprising the pluripotent stem cells, wherein the method is to induce reprogramming and/or dedifferentiation of dedifferentiated somatic cells using shikimic acid, or a plant extract or plant stem cell extract comprising shikimic acid.

The plant extract or plant stem cell extract containing shikimic acid used in the present invention may further contain a plant selected from the group consisting of redwood (Sequoia glauca), yellow iris, Jerusalem artichoke, blue fir (Picea pungens), white spruce (Picea glauca), silvertop eucalyptus (Eucalyptus sieberiana), king eucalyptus (Eucalyptus regnans), North American cypress (Thuja plicata), date palm (Phoenix dactylifera), Dahlia variabilis, chasteberry (Malus baccata), pear (Pyrus communis), wheat, red pine (Pinus densiflora), black pine (Pinus thunbergii), Japanese shikimic acid (Illicium anisatum), lotus magnolia (Magnolia grandiflora), Houttuynia cordata, saxifrage (Saxifraga stolonifera), Terminalia arjuna, Pistacia chinensis (Pistacia chinensis), and pineapple (Pinus chinensis). lentiscus), Ribes aureum, Symphytum officinalis, Actaea pachypoda, Alangium salvifollium, Gingkobiloba, Veratrum viride, Dipsacus laciniatus, Agastacheurticifolia, Inula helenium, Hypericum spp., Commelina bengalensis, Gymnema sylvestris, Terminalia chebula, Illicium floridanum, Illicium diffengri, Illicium henryi, Illicium verum, Illicium lancealatum, Illicium spp. pachyphyllum), Illicium anisatum, Illicium religiosum, Hemidesmus indicus, Cistus incanus, Sida acuta, Celastrus paniculata, Glycosmis uricata, Tanacetum parthenium, Triticum aestivum, Hypericum dolabriforme, Dipsacus pilosus, Triadenum walteri, Hypericum flondosum and Terminalia pallid (Denis V. Bochkov et.al., Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources, J Chem. Biol. (2012) 5; 5-17).

In another aspect, the present invention provides a composition for activating stem cells, regenerating skin or anti-aging, the composition comprising stem cells prepared by the above method, or provides a pharmaceutical composition or cosmetic composition comprising the stem cells.

Beneficial effects

According to the present invention, stem cells can be prepared using plant stem cell extracts, any type of induced pluripotent plant stem cell extract induced by various methods, shikimic acid, plant extracts containing shikimic acid, or compositions containing these. The methods of the present invention are applicable to cells from all species with various genetic backgrounds, including humans. Furthermore, the methods of the present invention do not raise ethical concerns because they use plant-derived stem cell extracts and can produce induced pluripotent stem cells with guaranteed safety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram illustrating the experimental process of induced pluripotent stem cells of the present invention.

FIG2 shows induced pluripotent stem cells observed on the fifth day after treatment with the plant stem cell extract.

FIG. 3 shows induced pluripotent stem cells observed on the eighth day after treatment with a plant stem cell extract and on the second day after transfer to a feeder cell layer.

Figure 4A shows induced pluripotent stem cells observed 32 days after treatment with plant stem cell extracts and after transfer to a feeder cell layer and subsequent subculture for four times. Figure 4B shows typical embryonic stem cells.

FIG5 shows the results of alkaline phosphatase staining of induced pluripotent stem cells observed on day 32 after treatment with the plant stem cell extract and after transfer to a feeder cell layer and four subcultures.

Figure 6A shows induced pluripotent stem cells observed 50 days after treatment with plant stem cell extracts and after transfer to a feeder cell layer and seven subcultures, as well as the results of alkaline phosphatase staining of the stem cells. Figure 6B shows human pluripotent stem cells induced using the four-factor viruses of Yamanaka and the results of alkaline phosphatase staining of the stem cells.

FIG. 7 shows gene expression in pluripotent stem cells induced according to the present invention.

8 shows the HPLC results of the redwood callus extract of the present invention. Shikimic acid has the structure of Chemical Formula 1.

FIG. 9 is a schematic diagram illustrating an experimental process of inducing totipotent stem cells by using shikimic acid or a plant stem cell extract containing shikimic acid according to the present invention.

FIG. 10 shows the expression of Oct3/4 genes in HDFs after treatment with shikimic acid or Sequoia callus extract.

FIG. 11 shows the expression of Oct3/4 genes in HDFs after treatment with different concentrations of shikimic acid.

FIG. 12 shows that ALP expression was increased after treatment with shikimic acid or Sequoia callus extract.

FIG13 shows that ALP expression was increased after treatment with different concentrations of shikimic acid.

FIG14 shows the colony-forming ability of dermal cells after treatment with shikimic acid or Sequoia callus extract.

FIG16 shows the colony-forming ability of dermal cells after treatment with shikimic acid or Sequoia callus extract.

FIG15 shows the colony-forming ability of dermal cells after treatment with different concentrations of shikimic acid.

FIG17 shows the colony-forming ability of dermal cells after treatment with different concentrations of shikimic acid.

FIG. 18 shows that the proliferation capacity of dermal cells was increased after treatment with shikimic acid or Sequoia callus extract.

DETAILED DESCRIPTION

The entire contents of Korean Patent Application No. 10-2013-0123860, filed on October 17, 2013, are incorporated herein for all purposes. Further, this application claims priority to Korean Patent Application No. 10-2013-0123860 and all rights arising therefrom, the entire contents of which are incorporated herein by reference.

In one aspect, the present invention provides a method for preparing induced pluripotent stem cells, the method comprising:

a) extracting an extract containing active ingredients from plant stem cells or induced pluripotent plant stem cells;

b) a step of injecting the extract into cells derived from an adult; and

c) a step of preparing totipotent stem cells such as embryonic stem cells by culturing the cells injected with the extract.

In another aspect, the present invention provides a method for preparing stem cells, comprising treating adult-derived cells by using shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition containing the same.

In another aspect, the present invention provides a method for preparing stem cells, comprising: injecting shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition containing them into cells derived from an adult; and culturing the cells into which the shikimic acid, the extract, or the composition is injected.

In the present invention, the extract may be a callus tissue extract.

In another aspect of the present invention, the plant extract or the plant stem cell extract may comprise shikimic acid, caffeic acid and ferulic acid.

In the present invention, "shikimic acid" can be represented by Chemical Formula 1 and is used in a broad sense, including precursors and derivatives of shikimic acid, etc. The shikimic acid may have a molecular weight of 174.15 g/mol.

【Chemical Formula 1】

In the present invention, "caffeic acid" can be represented by Chemical Formula 2, and is used in a broad sense to include precursors and derivatives of caffeic acid. The caffeic acid may have a molecular weight of 180.16 g/mol. Caffeic acid is a phenolic compound found in coffee beans, fruits including pears, and medicinal plants such as basil, thyme, bananas, tarragon, oregano, and dandelion.

【Chemical Formula 2】

In the present invention, "ferulic acid" can be represented by Chemical Formula 3 and is a broad concept that includes precursors and derivatives of ferulic acid. The caffeic acid may have a molecular weight of 191.18 g/mol. Ferulic acid is a precursor of lignin, which makes up plant cell walls and is abundant in plant cell walls. Ferulic acid can be found in seeds of plants such as wheat, oats, coffee, apples, oranges, and peanuts.

【Chemical Formula 3】

In the present invention, the term "stem cell" refers to a principal cell that can proliferate unrestricted to form cells specific to various tissues and organs. The stem cell is a totipotent or omnipotent cell that can develop. A stem cell can divide into two daughter stem cells, or a daughter stem cell and a transit cell.

In the present invention, the term "embryonic stem cells" refers to totipotent cells isolated from the inner cell mass of an early embryo, blastocyst, cultured after fertilization.

In the present invention, the term "dedifferentiated stem cell or induced pluripotent stem cell (iPS) extract" refers to a substance obtained by physically/chemically chopping dedifferentiated stem cells or induced pluripotent stem cells (iPS) induced and cultured using various methods in a test tube and separating them by centrifugation or the like.

In the present invention, the term "plant stem cell" refers to plant stem cells derived from the cambium. In particular, such plant stem cells include physically intact, pure cambial meristematic cells (CMCs) found in the cambium at the boundary between the xylem and phloem. In the present invention, the term "plant stem cell" is used in a broad sense to include any type of induced pluripotent stem cells induced by various methods.

In the present invention, the term "callus" refers to an unorganized mass of thin-walled cells, a representative example of which is tumor tissue formed by meristems surrounding plant wounds. Plant tissues are broadly divided into meristems that exhibit cell division and permanent tissues that fail to divide. When cells from meristems are cultured in a nutrient medium, callus initially forms. This then forms adventitious embryos, which then differentiate into plant organs. Callus is often referred to as "plant stem cells." The type of callus used in the present invention is not limited.

In the present invention, the term "totipotent stem cells" refers to stem cells that have the multipotency to differentiate into any of the three germ layers of an organism, such as the endoderm, mesoderm, and ectoderm. Traditionally, embryonic stem cells are stem cells that fall within this category.

In the present invention, the term "induced pluripotent stem cells" refers to pluripotent stem cells that are genetically identical to the donor cells used to prepare the induced pluripotent stem cells.

In the present invention, the terms "induced pluripotent stem cells", "dedifferentiated stem cells" and "inducible pluripotent stem cells" are used interchangeably.

In the present invention, the term "cells derived from an adult" refers to cells derived from a living adult, as opposed to embryonic cells.

In the present invention, the term "differentiation" refers to the process by which cells specialize in morphology or function during cell division and proliferation. The morphology or function of the cells is altered to achieve the specific functions of the cells, tissues, etc. of an organism. Generally, differentiation refers to the phenomenon in which a relatively simple system is split into two or more qualitatively distinct subsystems. In other words, differentiation refers to the phenomenon in which essentially identical parts of an organism become differentiated or, as a result, are divided into qualitatively distinct systems, such as the formation of a head or body from an egg during ontogeny, or the differentiation of muscle cells into nerve cells.

In one aspect of the present invention, the plant may be redwood.

In one aspect of the present invention, the plant stem cells may be callus tissue.

In one aspect of the present invention, the plant stem cells may be redwood stem cells.

In one aspect of the present invention, the plant stem cell extract may be a callus tissue extract.

In one aspect of the present invention, the extract may be a Sequoia callus extract.

In one aspect of the present invention, the redwood may be Sequoiadendron giganteum.

In one aspect of the present invention, the extract or the composition may comprise shikimic acid.

In one aspect of the present invention, the composition may contain shikimic acid at a concentration of 10 μM to 30 mM based on the total volume of the composition. In another aspect of the present invention, the composition may contain shikimic acid, the plant extract, or the plant stem cell at a concentration of 10 μM or more, 20 μM or more, 30 μM or more, 50 μM or more, 100 μM or more, 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, or 30 mM or more, and 1 M or less, or 100 M or less, based on the total volume of the composition. When the content of shikimic acid in the composition is 10 μM or less, the effects of the present invention are difficult to achieve. Moreover, when the content of shikimic acid is 30 mM or more, skin irritation may occur. Specifically, the shikimic acid may be included at a concentration of 0.1 mM or more, 0.5 mM or more, 0.6 mM or more, 0.7 mM or more, 0.8 mM or more, or 0.9 mM or more, and 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1.5 mM or less, 1.4 mM or less, 1.3 mM or less, 1.2 mM or less, or 1.1 mM or less, based on the total volume of the composition. More specifically, the shikimic acid may be included at a concentration of 0.8-1.2 mM, based on the total volume of the composition. Most specifically, the shikimic acid may be included at a concentration of 1 mM, based on the total volume of the composition.

In another aspect of the present invention, the plant extract or plant stem cell extract may contain shikimic acid at a concentration of 0.0001-45% (w/v) based on the total volume of the extract. When shikimic acid within the above range is included, excellent Oct3/4 gene expression effect, ALP expression effect and fibroblast proliferation promoting effect can be obtained. In view of this, in the present invention, the plant extract or plant stem cell extract may contain shikimic acid at the following concentrations based on the total volume of the extract: 0.001% (w/v) or more, 0.01% (w/v or more, 0.1% (w/v) or more, 1% (w/v) or more, 5% (w/v) or more, 10% (w/v) or more, 15% (w/v) or more, 20% (w/v) or more, 25% (w/v) or more, or 30% (w/v) or more. v) or more, and 45% (w/v) or less, 40% (w/v) or less, 38% (w/v) or less, 36% (w/v) or less, 34% (w/v) or less, 33% (w/v) or less, or 32% (w/v) or less. Specifically, in the present invention, the plant extract or plant stem cell extract may contain shikimic acid at a concentration of 32-34% (w/v), more specifically 32.75% (w/v), based on the total volume of the extract.

In the present invention, the composition may comprise a plant extract or a plant stem cell extract having a concentration of shikimic acid as described above based on the total volume of the composition. For example, the composition can comprise the following concentrations of plant extract or plant stem cell extract, based on the total volume of the composition: 0.001 μg/mL or more, 0.01 μg/mL or more, 0.1 μg/mL or more, 1 μg/mL or more, 10 μg/mL or more, 50 μg/mL or more, 100 μg/mL or more, 0.3 mg/mL or more, 0.4 mg/mL or more, 0.5 mg/mL or more, 0.6 mg/mL or more, 0.7 mg/mL or more, 1 mg/mL or more, 1.5 mg/mL or more, 2 mg/mL or more, 5 mg/mL or more, 10 mg/mL or more, 20 mg/mL or more, or 50 mg/mL or more, and 1 g/mL or less, or 10 g/mL or less.

In the present invention, the concentration of shikimic acid contained in the plant extract or plant stem cell extract can be measured by using solvent A (water, containing 0.1% TFA) and solvent B (acetonitrile, containing 0.1% TFA) as mobile phases using a Waters 1525μ Binary HPLC pump and a Gemini 5u C18 110A column (5μm, 4.60mm x250nm, Phenomenex).

In the present invention, "sequoia" includes redwood (Sequoia sempervirens), giant redwood (Sequoiadendron giganteum) or metasequoia (Metasequoia glyptostroboides).

The redwood (Sequoia glauca) belongs to the Cupressaceae family and the Pinales order. It grows exclusively along the northwestern California coast and southwestern Oregon coast in the United States, as well as in New Zealand. It can live for approximately 2,500–3,000 years and is the tallest tree in the world, reaching heights of up to 112 meters. Redwoods have a diameter of 2.5–4.5 meters and a height of 50–100 meters, with thick bark reaching 20–30 cm. The leaves are similar to those of yew trees, measuring 1–3 cm long, with pointed tips and distinct main veins. The leaves are dark green above and whitish below.

The giant redwood (Sequoiadendron giganteum) is the only surviving species of the genus Sequoia. It grows on the western slopes of California's Sierra Nevada Mountains at elevations of 1,500 to 2,500 meters. It reaches a diameter of 3.5 to 6 meters and a height of 60 to 90 meters, with a diameter near the base of the tree reaching approximately 10 meters. Its leaves are similar to those of cedars, up to 1 cm long and arranged in spirals. However, on mature branches, the leaves have a scaly texture.

Metasequoia glyptostroboides is the only surviving species of the genus Metasequoia, Cupressaceae. It grows up to 35 meters tall. Its gray-brown bark fissures vertically. Its branches spread out laterally, but the leaves are opposite, measuring 10-23 mm long and 1.5-2 mm wide. In autumn, the tips of the leaves turn pale red. Hermaphroditic flowers bloom from February to March. Male flowers hang in racemes at the axils of the branches, with 20 stamens. Female flowers hang at the tips of the branches and bloom in March. The cones are spherical, measuring 18-25 mm long. They turn brown when mature and produce winged, oval seeds. This deciduous conifer is native to Sichuan and Hubei provinces in China and is also found in South Korea, China, and other regions. It is primarily cultivated as a garden tree.

Callus tissue refers to an undifferentiated, unorganized mass of thin-walled cells. A representative example is the tumor tissue formed by the meristem surrounding a plant wound. Plant tissues are broadly divided into meristems, which exhibit cell division, and permanent tissues, which fail to divide. When meristem cells are cultured in a nutrient medium, they initially form callus. This then forms adventitious embryos, which then differentiate into plant organs. Callus tissue is often referred to as "plant stem cells."

In the present invention, the "extract" includes any substance extracted from natural products, regardless of the extraction method, extraction solvent, extraction ingredients or extraction type. "Extract" uses a broad concept and includes substances obtained by processing or treating the extract in different ways.

In the present invention, the redwood can be used in the form of an extract, a crushed plant product, or a dried crushed plant product, but is not limited thereto. Furthermore, the method of obtaining the redwood used in the present invention is not limited. It can be cultivated or purchased, and can include the above-ground part, the underground part, or both of the redwood. The above-ground part can include the fruit, leaves, and stems of the redwood, and the underground part can include the roots, but is not limited thereto.

In the present invention, the term "plant extract" includes any substance extracted from plants such as redwood, regardless of the extraction method, extraction solvent, extraction ingredients, or extraction type. The term "plant extract" is used in a broad sense and includes substances extracted by treatment with heat, acid, alkali, enzymes, etc. Furthermore, the term "plant extract" also includes substances obtained by processing or treating the extract using various methods.

For the purposes of the present invention, the term "plant stem cell extract" encompasses any substance obtained by culturing redwood plant stem cells and extracting active ingredients, regardless of the extraction method, solvent, ingredients, or type of extraction. The term "plant stem cell extract" is used in a broad sense to encompass any substance extracted by treating redwood plant stem cells with heat, acid, alkali, enzymes, or the like to extract active ingredients. Furthermore, the term "plant stem cell extract" also encompasses substances obtained by processing or treating the extract using various methods.

The method of the present invention comprises the steps of culturing plant stem cells or induced pluripotent plant stem cells obtained by various methods, and then preparing an extract therefrom, using methods known in the art. For example, after culturing the plant stem cells or induced pluripotent plant stem cells obtained by various methods, they can be treated with a protease, and the supernatant can be collected. Alternatively, the plant stem cells can be incubated at 65°C for 2 hours and then filtered to extract proteins or shikimic acid derived from each cell. In one exemplary embodiment of the present invention, callus tissue powder can be used.

In the present invention, the redwood callus extract can be obtained by the following steps: i) a step of inducing callus from redwood; ii) a step of establishing a stem cell line by culturing the callus in a solid culture medium; iii) a step of generating a large amount of active ingredients by suspension culture of the cell line; and iv) a step of extracting the generated active ingredients.

In the present invention, the active ingredients from the redwood callus can be extracted by culturing a cell line derived from a tissue block of the plant as described in Korean Patent Publication No. 2007-0113193. Specifically, a stable plant cell line derived from redwood can be extracted using a mixture of _C5 or lower alcohols, but is not limited thereto.

Alternatively, the redwood callus extract or redwood callus powder used in the present invention can be purchased from the market.

In the present invention, the redwood callus extract can be prepared by dissolving redwood callus powder in a solvent. The solvent can include one or more substances selected from water, an organic solvent, and a mixture of water and an organic solvent. The water can include distilled water or purified water, and the organic solvent can include a mixed solvent of one or more selected from alcohols such as _C1 - _C5 lower alcohols, acetone, ether, ethyl acetate, diethyl ether, methyl ethyl ketone, and chloroform with n-hexane, dichloromethane, ethyl acetate, n-butanol, butanediol (BG), and ethanol (EtOH), dimethyl sulfoxide (DMSO), etc., but is not limited thereto.

Those skilled in the art will readily appreciate that existing protein extraction methods can be used to prepare high-concentration protein extracts. The concentration of the plant-derived shikimic acid or protein extract, based on the total volume of the composition containing the extract, can specifically be 10 μg/mL to 1 mg/mL, more specifically about 500 μg/mL. When the concentration of the protein extract exceeds this range, the induction efficiency of the induced pluripotent stem cells may decrease, or cells treated with the extract may die.

The method of the present invention comprises the following steps: injecting a protein extract isolated from plant stem cells or induced totipotent plant stem cells induced by various methods into cells derived from an adult.

The adult-derived cells may include human skin fibroblasts or human neonatal skin fibroblasts.

For the injection, the cells can be permeabilized by treatment with a cell membrane permeabilizing agent (e.g., streptolysin O or digitalis), thereby allowing the extract to be introduced into the cells. After the permeabilization, the shikimic acid, the plant extract, the plant stem cell extract, the induced pluripotent plant stem cells, or the composition can be emulsified and injected into the adult-derived cells.

In one aspect of the present invention, a method for preparing totipotent stem cells may comprise:

1. A step of preparing shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract, or a composition containing the same according to the present invention.

2. A step of injecting the shikimic acid, the extract or the composition into cells derived from an adult.

3. A step of culturing the adult-derived cells by using a common culture medium.

4. The step of transferring the cells to a feeder cell layer and further culturing them.

5. A step of recovering the cultured totipotent stem cells.

Specifically, the step of injecting the shikimic acid, the extract or the composition into the adult-derived cells may include:

1. A step of separating adult-derived cells into individual cells and transferring them into a tube after resuspending them;

2. separating the cells by centrifugation and resuspending the resulting cell pellet in a water bath;

3. Step of adding cell membrane permeabilization agent;

4. The step of mixing the sample up and down in a water bath to react;

5. a step of centrifuging the sample;

6. A step of resuspending the cell pellet by using shikimic acid, the extract or the composition;

7. A step of mixing the sample up and down in a water bath to react by an ATP regeneration system;

8. Adding ES culture medium and incubating in an incubator; and

9. A step of washing and resuspending the cell pellet in embryonic stem cell culture medium and seeding the cell pellet on a dish.

The method of the present invention includes the step of preparing totipotent cells such as embryonic stem cells by culturing cells injected with the shikimic acid, the extract or the composition.

The method of the present invention may further include the step of culturing the adult-derived cells injected with the shikimic acid, the extract, or the composition by using a common cell culture medium, then transferring the cells to a feeder cell layer and further culturing the cells.

More specifically, after the injection of the shikimic acid, the extract, or the composition, the adult-derived cells can be cultured in a standard cell culture medium (DMEM, 5-15% FBS, 10-100 U/mL penicillin, 20-80 mg/mL streptomycin) until colonies form. After the colonies form, the cells are transferred to a feeder cell layer and then subcultured every 5-8 days in embryonic stem cell culture medium, with the culture medium being replaced daily. The feeder cells used in the present invention may include, but are not limited to, STO cells.

The pluripotent stem cells induced by the extract can be cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 15-25% KSR (knockout serum replacement), 1-42 mM L-glutamine, 0.05-0.2 mM non-essential amino acids, 0.05-0.2 mM β-mercaptoethanol, 30-70 U/ml penicillin, 30-70 mg/mL streptomycin, and 1-30 μg/mL bFGF. Those skilled in the art will readily appreciate that the concentration of the compound added to the DMEM can be varied within a range that achieves the effects of the present invention.

The present invention also provides a method for inducing induced pluripotent stem cells, comprising:

a) preparing an extract by isolating proteins from plant stem cells or induced pluripotent plant stem cells induced by various methods;

b) a step of treating adult-derived cells with a cell membrane permeabilizing agent and then injecting the extract into the adult-derived cells; and

c) culturing the cells injected with the extract in a common cell culture medium, and then transferring them to a feeder cell layer and culturing them in an embryonic stem cell culture medium.

The method of the present invention is characterized in that totipotent stem cells that are almost indistinguishable from embryonic stem cells can be prepared from adult-derived cells by using shikimic acid, plant extracts, plant stem cells, extracts of induced totipotent plant stem cells induced by various methods, or compositions containing them.

The inventors of the present invention have confirmed that totipotent stem cells are induced by the method of the present invention.

Specifically, the totipotent stem cells induced by the method of the present invention are nearly identical in shape to embryonic stem cells (see Figure 4 ). Furthermore, by examining the expression of genes specific to embryonic stem cells (Nanog, Oct4), it was found that, as in embryonic stem cells, these genes are also expressed in the totipotent stem cells induced by the method of the present invention (see Figures 7 and 10 ).

In another aspect, the present invention provides induced pluripotent stem cells induced by the method of one aspect of the present invention.

The inventors of the present invention have confirmed self-renewal, which is a characteristic of stem cells, after 8-12 subcultures using the method of the present invention (see FIG. 6 ).

In another aspect, the present invention provides a composition comprising totipotent stem cells produced by a method according to one aspect of the present invention.

In one aspect, the present invention provides a composition comprising as an active ingredient one or more of shikimic acid, a plant extract containing shikimic acid, and a plant stem cell extract containing shikimic acid.

In one aspect of the present invention, the composition may be a pharmaceutical composition, a food composition or a cosmetic composition.

In one aspect of the present invention, the composition may be a composition for activating stem cells, regenerating skin, or anti-aging.

In one aspect, the present invention may relate to a method for activating stem cells, comprising administering shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances to an individual in need of stem cell activation. The administration may be performed according to the administration method or dosage described herein.

In one aspect, the present invention may relate to a method for regenerating skin, comprising the step of administering shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances to an individual in need of skin regeneration.

In one aspect, the present invention may relate to a method for anti-aging, comprising the step of administering shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances to an individual in need of anti-aging.

In one aspect, the present invention may relate to the use of shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances for activating stem cells, skin regeneration, or anti-aging.

In one aspect, the present invention may relate to a composition for activating stem cells, skin regeneration or anti-aging, comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances.

In one aspect of the invention, the composition may be a cell therapy agent.

Specifically, the cell therapy agent can be used for the formation of hepatocytes, adipocytes, bone cells, cartilage cells, muscle cells, neurons, cardiomyocytes, vascular endothelial cells, and the like.

In one aspect, the present invention may relate to a method for cell therapy, comprising the step of administering shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances to an individual in need of cell therapy.

In one aspect, the present invention may relate to the use of shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances for cell therapy.

In one aspect, the present invention relates to a composition for cell therapy, comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more of these substances.

As used in the present invention, the term "cell therapeutic agent" refers to cells or tissues that are isolated from the human body and made into medicines through culture and specific operations for the purpose of treatment, diagnosis and prevention (USFDA regulations). Biological therapeutic agents are used for the purpose of treatment, diagnosis and prevention by proliferating and screening living autologous homologous or heterologous cells in vitro, or by changing the biological properties of the cells to restore the function of cells or tissues. The cell therapeutic agents can be roughly divided into somatic cell therapeutic agents and stem cell therapeutic agents according to the degree of differentiation of the cells. Specifically, the present invention relates to the stem cell therapeutic agent.

In one aspect, the present invention provides a food composition comprising shikimic acid, a plant extract containing shikimic acid, or a plant stem cell extract containing shikimic acid according to one aspect of the present invention. In one aspect of the present invention, the composition may contain other ingredients that do not negatively affect the intended primary effects of the present invention. For example, to improve physical properties, additives such as spices, colorants, bactericides, antioxidants, preservatives, humectants, thickeners, minerals, emulsifiers, synthetic polymers, etc. may be further included. In addition, other auxiliary ingredients such as water-soluble vitamins, oil-soluble vitamins, polypeptides, polysaccharides, and seaweed extracts may be further included. Depending on the dosage form type or intended use, those skilled in the art may appropriately select these ingredients, and the amounts added may be determined within a range that does not violate the purposes and effects of the present invention. For example, the amounts added may be 0.01-5wt%, more specifically 0.01-3wt%, of the total weight of the composition. In one aspect of the present invention, the food composition may include, but is not limited to, a health food composition, a functional food composition, a nutritional supplement composition, a processed food composition, a food additive composition, and the like.

In one aspect, the present invention provides a cosmetic composition comprising shikimic acid, a plant extract containing shikimic acid, or a plant stem cell extract containing shikimic acid according to one aspect of the present invention. The cosmetic composition comprises a cosmetically or dermatologically acceptable medium or matrix. The cosmetic composition can be in any form suitable for topical application, such as an emulsion, a gel, a solid, an anhydrous cream, an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a suspension, a microemulsion, a microcapsule, an ionic (liposome) or non-ionic vesicle dispersion, a foam, or a compressed propellant aerosol or patch. These compositions can be prepared according to methods commonly used in the art.

In addition to the above substances, the cosmetic composition may further include other ingredients that provide a synergistic effect to the main effect within the range that the main effect is not negatively affected. Those skilled in the art can easily select the other ingredients according to the dosage form type or the purpose of use. For example, in addition to the above-mentioned active ingredients, the cosmetic composition of the present invention may include other ingredients that are usually mixed in cosmetic compositions. Specific examples include fats, oils, moisturizers, emollients, surfactants, organic or inorganic colorants, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, stabilizers, thickeners, glycerin, pH regulators, alcohols, colorants, fragrances, blood circulation promoters, coolants, antiperspirants, purified water, etc. However, the other ingredients contained in the cosmetic composition may not be limited thereto, and their content is determined within the scope of not violating the purpose and effect of the present invention.

The dosage form of the cosmetic composition is not particularly limited and can be appropriately selected according to the purpose. For example, the cosmetic composition can be prepared into a formulation selected from toner, moisturizing lotion, essence, nourishing cream, massage cream, facial mask, gel, base makeup, foundation, powder, lipstick, patch, spray, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, cleanser, shampoo, conditioner, hair care product, hair essence, shampoo, scalp and hair nourisher, scalp essence, hair gel, hair spray, hair mask, body wash, body cream, body oil and body essence, but is not limited thereto.

In one aspect, the present invention provides a pharmaceutical composition comprising shikimic acid, a plant extract containing shikimic acid, or a plant stem cell extract containing shikimic acid according to one aspect of the present invention. In addition to the active ingredient, the pharmaceutical composition may further include a pharmaceutical adjuvant or other substance that contributes to treatment, such as a preservative, a stabilizer, a wetting agent, an emulsifier, a salt and/or buffer for controlling osmotic pressure, a diluent (e.g., lactose, glucose, sucrose, mannitol, sorbitol, cellulose, or glycine), a lubricant (e.g., silicon dioxide, talc, stearic acid, and its magnesium salt or calcium salt, or polyethylene glycol), a binder (e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, or polyvinyl pyrrolidone), etc. In some cases, the pharmaceutical composition may further include other pharmaceutical additives such as a disintegrant, for example, starch, agar, alginic acid, or its sodium salt, an absorbent, a colorant, a flavoring, a sweetener, etc.

The pharmaceutical composition can be prepared into dosage forms for oral or parenteral administration according to common methods.

The dosage form for oral administration can include, for example, tablets, fillers, hard/soft capsules, liquids, suspensions, emulsions, syrups, powders, fine powders, microgranules, granules, pills, etc. In addition to the above-mentioned active ingredients, these dosage forms can include surfactants, diluents (for example, lactose, glucose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (for example, silicon dioxide, talc, stearic acid and its magnesium salt or calcium salt, and polyethylene glycol). Tablets can include binding agents such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, and in some cases can further include pharmaceutical additives such as disintegrants such as starch, agar, alginic acid or its salts, absorbents, colorants, flavorings, sweeteners, etc. The tablets can be made by methods such as conventional mixing, granulation or coating.

Dosage forms for parenteral administration may include, for example, injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches and the like, but are not limited thereto.

The pharmaceutical compositions of one aspect of the present invention can be administered parenterally, for example, rectally, topically, transdermally, subcutaneously, etc. The compositions are formulated into dosage forms suitable for various methods of administration. In particular, compositions for intravenous injection are formulated to a very high purity by eliminating unsuitable additives.

The dosage of the active ingredient will vary depending on the age, sex, weight of the subject being treated, the specific disease or pathological condition being treated, the route of administration, or the diagnosis. Determination of dosage based on these factors is within the skill of those skilled in the art. Specifically, a typical dosage range is 30 μg/mL to 1 mg/mL, but is not limited thereto.

Specifically, in one aspect of the present invention, a pharmaceutical composition comprising totipotent stem cells produced by the methods of the present invention can be used as an injectable. Similar to Botox, totipotent stem cells produced by the methods of the present invention can be injected into the skin to remove wrinkles, activate skin stem cells, and promote skin cell proliferation, thereby achieving skin regeneration, anti-aging, improved skin elasticity, and wrinkle reduction effects.

In another aspect, the present invention provides a reagent or culture medium for use in an experiment, comprising the shikimic acid, the plant extract containing shikimic acid, or the plant stem cell extract containing shikimic acid according to one aspect of the present invention.

The present invention will be described in detail below by way of examples. However, the following examples are only used for illustrative purposes and it will be apparent to those skilled in the art that the examples do not limit the scope of the present invention.

[Example 1] Injection of plant cell extract into adult-derived cells

Callus tissue powder is used as a plant-derived stem cell extract.The callus tissue powder that can be used in the present invention is not particularly limited, and commercially available callus tissue powder can also be used.

Callus tissue is induced from leaves of redwood, cultured in a solid medium to establish a stem cell line, and then suspended to produce a large amount of active ingredients. Extraction is then performed to obtain a redwood callus extract. Extraction of active ingredients from the redwood callus can be performed by culturing a cell line derived from tissue blocks of the plant, as described in Korean Patent Publication No. 2007-0113193. Specifically, extraction can be performed on a stable plant cell line derived from redwood by dissolving it in a _C5 or lower alcohol, but is not limited thereto.

In this example, redwood callus powder purchased from BIO-FD&C was used. Specifically, human skin fibroblasts were separated into individual cells using trypsin-EDTA and then washed with cold PBS (phosphate buffered saline). The resulting cell pellet was resuspended in cold HBSS (Hank's balanced salt solution) free of Ca ²⁺ - and Mg ²⁺ - to a concentration of 100,000 cells/100 μL and then transferred to a 1.5 mL tube. After centrifugation at 120 g using a horizontal swing rotor at 4°C for 5 minutes, the supernatant was discarded and the remaining cell pellet was resuspended in 97.7 μL of cold HBSS and then incubated in a 37°C water bath for 2 minutes. Then, 2.3 μL of streptolysin O (SLO, 100 g/mL stock solution diluted 1:10 in cold HBSS) was added (final SLO concentration was 230 ng/mL). Alternatively, digitonin (20 μg/mL) and 200 μL carrier solution (110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, protease inhibitor cocktail, 20 mM HEPES, pH 7.3) can be added instead of the SLO. The sample was incubated in a 37°C water bath for 50 minutes while mixing up and down every 10 minutes. After incubation, the sample was placed on ice and, after adding 200 μL of cold HBSS, centrifuged at 120 g using a horizontal rotor at 4°C for 5 minutes. After this permeabilization process, the resulting cell pellet was resuspended in 200 μL of plant stem cell extract to form 1000 cells/1 μL. Callus tissue was used as the plant stem cell extract (500 μg/mL). Then, 1 mM nucleoside triphosphate and an ATP regeneration system (10 mM creatine phosphate and 25 g/mL creatine kinase) were added to the suspended cell pellet and incubated in a 37°C water bath for 1 hour, while mixing up and down every 10 minutes. After incubation, ES medium containing ₂ mM CaCl2 was added and incubated in a 37°C water bath for 2 hours to reconstitute the plasma membrane. After washing with PBS, the cell pellet was resuspended in embryonic stem cell culture medium and then plated on a dish coated with 0.1% gelatin.

[Example 2] Preparation of totipotent cells such as embryonic stem cells by culturing cells injected with extracts

In an incubator maintained at 37°C and 5% _CO2 , the cells injected with the extract were cultured in a common cell culture medium supplemented with 10% FBS, 50U/mL penicillin and 50mg/mL streptomycin in DMEM. Adult-derived cells (human dermal fibroblasts) were cultured on a dish coated with 0.1% gelatin, and the adult-derived cells were injected with the plant stem cell extract (callus powder). After the first two days, the culture medium was replaced. After 10 days of culture and daily culture medium replacement, the cells were transferred to a feeder cell (STO cell) layer treated with mitomycin (MMC) at a ratio of 1:2. The cells were then transferred to a new feeder cell layer every 7 days, while daily replacement of DMEM (Dulbecco's modified Eagle's medium)/F1 supplemented with 20% KSR (knockout serum replacement), 2 mM L-glutamine, 0.01 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, 50 U/ml penicillin, 50 mg/mL streptomycin, and 10 μg/mL bFGF was performed. Cultivation of the induced stem cells required for analysis took an average of approximately 21 days.

Figure 1 is a schematic diagram illustrating the experimental process for inducing totipotent stem cells according to the methods of the present invention. Figures 2-4 show the induced totipotent stem cells observed on days 5, 10, and 32, respectively, after treatment with a plant stem cell extract. Furthermore, Figures 5 and 6 show the results of alkaline phosphatase staining on days 32 and 50, respectively, after culture. Alkaline phosphatase staining was performed using a commonly used staining kit.

As shown in FIG. 6 , the totipotent stem cells induced according to the method of the present invention showed a positive result (violet) for alkaline phosphatase staining, which is a characteristic of embryonic stem cells.

[Example 3] Characteristics of induced pluripotent stem cells (gene expression analysis)

The cultured cells were recovered and all RNA was isolated using TRIzol reagent (Invitrogen). After cDNA was synthesized by reverse transcription polymerase chain reaction (RT-PCR), PCR was performed using specific primers for the Nanog gene, Oct3/4 gene, and the GAPDH gene as a control gene. The expression of these genes was analyzed by electrophoresis of the PCR products on agarose gel. The results are shown in Figure 7.

As shown in FIG. 7 , the pluripotent stem cells (hiPS) induced according to the method of the present invention exhibited the expression of Nanog and Oct3/4 genes, which are characteristics of embryonic stem cells (hES).

[Example 4] Preparation of Sequoiadendron giganteum callus extract containing shikimic acid

20 mg of the redwood callus powder of Example 1 was dissolved in 1 mL of DMSO. Similarly, 1 g of the redwood callus powder was dissolved in 10 mL of a mixed solvent of BG and EtOH to prepare a redwood callus extract containing shikimic acid. The obtained extract was used as a sample in the following test examples.

[Test Example 1] Composition Analysis of Sequoia Callus Extract

Among the Sequoia extracts prepared in Example 4, the Sequoia callus extract prepared by using a mixed solvent of BG and EtOH was subjected to composition analysis using HPLC.

The composition analysis of the extract was performed using a Waters 1525μ Binary HPLC pump and a Gemini 5u C18 110A column (5μm, 4.6mm x 250nm, Phenomenex). Solvent A (water, containing 0.1% TFA) and solvent B (acetonitrile, containing 0.1% TFA) were used as mobile phases, and a 230-nm UV lamp was used. The measurement was performed at a flow rate of 1 mL/min, the run time was 46 minutes, and the extract injection volume was 20 μL.

Figure 8 shows the LC chromatogram of the redwood extract, and the results are shown in Table 1. In Table 1, area % represents the percentage (w/v) of a substance contained in the redwood extract. This indicates that the redwood callus extract, which contains shikimic acid, contains a greater amount of shikimic acid than any other substance. This indicates that, in addition to shikimic acid, the redwood callus extract also contains caffeic acid and ferulic acid.

【Table 1】

RT area area% high 1 3.589 2413779 7.91 122548 2(Shikimic acid) 4.248 9990137 32.75 633894 3 17.115 2660895 8.72 146243 4 (caffeic acid) 18.666 7252283 23.78 363051 5(Ferulic acid) 21.672 5225473 17.13 320196 6 22.411 2775302 9.10 159198 7 28.044 183049 0.60 15075

[Test Example 2-1] Increase in Oct 3/4 expression

5x10 ⁵ human neonatal dermal fibroblasts (NHDF-Neonatal) (CC-2509, Lonza, USA) were treated with permeabilization buffer and 10 μg/mL digitonin, and then treated with 20 μg/mL redwood callus extract (a mixed solvent of BG and EtOH) or 5 μg/mL shikimic acid of Example 4. Untreated NHDF-Neonatal was used as a negative control group. On the third day after treatment, the cells (5,000 cells) were affixed to 4 chamber slides. The next day, i.e., the fourth day, the cells were fixed with 3.8% formaldehyde (diluted with 38% paraformaldehyde) in PBS and then allowed to stand at room temperature for 10 minutes. Immunohistochemistry was then performed using Oct3/4 antibody (Genetex, GTX100622, x200) and Alexa488 goat anti-rabbit IgG, and 400 times the image was then obtained using Carl Zeiss confocal microscope LSM510. The resulting image is shown in Figure 10. The proportion of Oct3/4-positive cells was determined by analyzing Carl Zeiss confocal microscopy images obtained from the Oct3/4 ICC experiment, with four or more images analyzed per sample, and an average of five images analyzed. The results are shown in Table 2.

【Table 2】

As shown in FIG10 , HDFs treated with the redwood callus extract of Example 4 showed increased expression of the Oct3/4 gene compared to normal HDFs (negative control group), and the expression was higher than that of HDFs treated with shikimic acid.

Furthermore, as can be seen from Table 2, the percentage of cells positive for the Oct3/4 gene was the highest, reaching 33%, when the redwood callus extract was extracted with a mixed solvent of BG (butylene glycol) and EtOH, which was higher than that with shikimic acid.

[Test Example 2-2] Increase in Oct 3/4 expression

1x10 ⁶ human neonatal dermal fibroblasts (NHDF-Neonatal) (CC-2509, Lonza, USA) were treated with 10 μM or 10 mM shikimic acid, respectively. Untreated NHDF-Neonatal was used as a negative control group. On the 5th day after treatment, the cells (5,000 cells) were affixed to a 4-chamber slide. After three days, i.e., on the 8th day, the cells were fixed with 3.8% formaldehyde (diluted with 38% paraformaldehyde) in PBS and then allowed to stand at room temperature for 10 minutes. Immunohistochemistry was then performed using Oct3/4 antibody (Genetex, GTX100622, x200) and Alexa488 goat anti-rabbit IgG, and 400 times the image was obtained using a Carl Zeiss confocal microscope LSM510. The resulting image is shown in Figure 11. The proportion of Oct3/4-positive cells was determined by analyzing Carl Zeiss confocal microscopy images obtained from the Oct3/4 ICC experiment, with four or more images analyzed per sample, and an average of five images analyzed. The results are shown in Table 3.

【Table 3】

As shown in Figure 11, HDFs treated with shikimic acid showed increased expression of the Oct3/4 gene compared to untreated normal HDFs (negative control group). The expression of the Oct3/4 gene increased with the concentration of shikimic acid used.

Furthermore, as shown in Table 3, the percentage of cells positive for the Oct3/4 gene when treated with shikimic acid in water was 27-29% on average, which was significantly higher than that of the untreated group.

The above results confirm that shikimic acid, a major component of the redwood callus extract, effectively increases expression of the Oct3/4 genes, which play a key role in the induction of induced pluripotent stem cells. Therefore, by injecting shikimic acid or an extract containing shikimic acid of the present invention into somatic cells to express the Oct3/4 genes, induced pluripotent stem cells can be induced.

[Test Example 3-1] Increased expression of stem cell marker alkaline phosphatase (ALP)

5x10 ⁵ NHDF-Neonatal (CC-2509, Lonza, USA) were treated with permeabilization buffer and 10 μg/mL digitonin, and then treated with 20 μg/mL of the redwood callus extract of Example 4 (a mixed solvent of BG and EtOH) or 5 μg/mL shikimic acid. Untreated NHDF-Neonatal was used as a negative control group. On the 6th day after treatment, the cells (5,000 cells) were affixed to a 12-well plate. Five days later, on the 11th day, the cells were fixed with 3.8% formaldehyde in PBS and then allowed to stand at room temperature for 15 minutes. The cells were then treated with 200 μL ALP matrix solution diluted in 10 mL ALP buffer, 0.5 mL at a time. After 20 hours, the cells were observed under an Olympus CKX41 optical microscope at a magnification of 40 times. The results are shown in Figure 12.

As shown in Figure 12 , HDFs treated with the redwood callus extract exhibited larger staining areas than normal HDFs, indicating a significant increase in ALP expression. Furthermore, it was found that cells treated with the redwood callus extract exhibited higher ALP expression than cells treated with shikimic acid.

During the process of converting fibroblasts into induced pluripotent stem cells, ALP begins to express three days after the Oct4 gene is expressed in the cells. ALP is known to play an important role in the early stages of dedifferentiated stem cell formation. Therefore, when the redwood callus extract of the present invention is injected into somatic cells, ALP expression significantly increases and the Oct4 gene is expressed. Thus, induced pluripotent stem cells can be induced.

From the experimental results, it was confirmed that the redwood callus extract of the present invention exhibited a significant effect of activating stem cells by promoting the expression of the stem cell marker ALP.

[Test Example 3-2] Increased expression of stem cell marker alkaline phosphatase (ALP)

1x10 ⁶ NHDF-Neonatal (CC-2509, Lonza, USA) were treated with 10 μM or 10 mM shikimic acid, respectively. Untreated NHDF-Neonatal was used as a negative control group. On the 5th day after treatment, the cells (100,000 cells) were affixed to a 6-well plate. After 7 days, i.e., on the 12th day, the cells were fixed with 3.8% formaldehyde in PBS and then allowed to stand at room temperature for 15 minutes. Then, the cells were treated with 200 μL ALP matrix solution diluted in 10 mL ALP buffer, 0.5 mL per treatment. After 20 hours, the cells were observed under an Olympus CKX41 optical microscope at 40 times magnification. The results are shown in Figure 13.

As shown in Figure 13, HDFs treated with shikimic acid showed a larger dark blue staining area than normal HDFs, indicating a significant increase in ALP expression. Furthermore, it can be seen that ALP expression increases with the concentration of shikimic acid.

During the process of converting fibroblasts into induced pluripotent stem cells, ALP begins to express three days after the Oct4 gene is expressed in the cells. ALP is known to be a marker that plays an important role in the early stages of dedifferentiated stem cell formation. Therefore, when shikimic acid and plant extracts containing shikimic acid of the present invention are injected into somatic cells, ALP expression significantly increases and the Oct4 gene is expressed. Thus, induced pluripotent stem cells can be induced.

From the experimental results, it was confirmed that shikimic acid and the plant extract containing shikimic acid of the present invention exhibited a significant effect of activating stem cells by promoting the expression of the stem cell marker ALP.

[Test Example 4-1] Increase in colony-forming ability of dermal cells

5x10 ⁵ NHDF-Neonatal (CC-2509, Lonza, USA) were treated with permeabilization buffer and 10 μg/mL digitonin, and then treated with 100ppm or 200ppm of the redwood callus extract of Example 4 (a mixed solvent of BG and EtOH) filtered with a 0.4 μm filter and 20ppm DMSO solution. Untreated NHDF-Neonatal was used as a negative control group. On the 10th day after treatment, the cells (200 cells) were attached to a 60-mm plate. On the 23rd day, the cells were washed with ice-cold PBS and then fixed with ice-cold methanol stored at -20°C for 10 minutes. The cells were stained with a working solution for 5-10 minutes. The working solution was prepared by diluting crystal violet in an ethanol stock solution with PBS to 1/10, washed 4 times with PBS, and then photographed. For quantitative analysis, the cells were eluted for 5 minutes using an elution buffer consisting of 50% ethanol, 40% DW, and 10% acetic acid. 200 μL of the cells were then transferred to a 96-well plate, and the absorbance was measured at 580 nm. The resulting photograph is shown in Figure 14 . Furthermore, the absorbance measurement results relative to the negative control group are shown in Figure 16 .

As shown in FIG14 , the cells treated with the Sequoia callus extract showed larger staining areas than the negative control group NHDF-Neonatal, indicating that epidermal cells were actively differentiating and forming large colonies.

16 , the colony-forming ability of cells treated with the redwood callus extract increased by about 2.6 times compared to the negative control group, which was statistically significant. The colony-forming ability of cells treated with shikimic acid increased by about 3.8 times compared to the negative control group.

This indicates that the redwood callus extract of the present invention promotes skin regeneration by significantly promoting the proliferation of fibroblasts.

Furthermore, it was found that the redwood callus extract of the present invention exhibits the effect of promoting skin regeneration by significantly promoting the proliferation of fibroblasts.

[Test Example 4-2] Increase in colony-forming ability of dermal cells

1x10 ⁶ NHDF-Neonatal (CC-2509, Lonza, USA) were treated with 10μM, 50μM, 100μM, 1mM or 10mM shikimic acid, respectively. Untreated NHDF-Neonatal was used as a negative control group. On the 5th day after secondary culture, the cells (200 cells) were attached to a 60-mm plate. On the 17th day, the cells were washed with ice-cold PBS and then fixed with ice-cold methanol stored at -20°C for 10 minutes. The cells were stained by treating with an experimental solution (working solution) for 5-10 minutes. The experimental solution was prepared by diluting the crystal violet in the ethanol stock solution with PBS to 1/10, washing with PBS 4 times, and then taking pictures. For quantitative analysis, the cells were eluted for 5 minutes by treating with an elution buffer consisting of 50% ethanol, 40% DW and 10% acetic acid. Then, 200 μL of the cells were transferred to a 96-well plate, and the absorbance was measured at 580 nm. The obtained photograph is shown in Figure 15. In addition, the absorbance measurement results relative to the negative control group are shown in Figure 17.

As shown in FIG15 , the cells treated with shikimic acid showed larger staining areas than the negative control group NHDF-Neonatal, indicating that epidermal cells were actively differentiated and formed large colonies.

Furthermore, as shown in Figure 17, the colony-forming ability of cells treated with shikimic acid increased by approximately 21.27-5.06 times compared to the negative control group, which was statistically significant. In particular, cells treated with 1 mM shikimic acid exhibited the highest colony-forming ability. Therefore, it can be seen that treatment with 1 mM shikimic acid can maximize the promotion of cell proliferation.

This indicates that the shikimic acid of the present invention can significantly promote the proliferation of fibroblasts.

Furthermore, it was found that the shikimic acid of the present invention can promote skin regeneration by significantly promoting the proliferation of fibroblasts.

[Test Example 5] Increase in the proliferation ability of dermal cells

2,000 NHDF-Neonatal (CC-2509, Lonza, USA) were placed on a 96-well plate. The next day, the cells were treated with 20 μg/mL of the redwood callus extract of Example 4 (a mixed solvent of BG and EtOH) and 5 μg/mL of shikimic acid. Untreated NHDF-Neonatal was used as a negative control group. Then, secondary culture was performed for 7 days (on the 7th day, confluence became 90% or less). After 6 days, 10 μL of WST-1 was treated, and the absorbance was measured at 450 nm. The results relative to the negative control group are shown in Figure 18.

As shown in Figure 18, the treatment with the redwood callus extract of the present invention increased cell division by 1.5 times or more compared to the negative control group, which is statistically significant. Moreover, the shikimic acid increased cell division by 2 times compared to the ordinary control group.

This shows that the redwood callus extract of the present invention can significantly promote the cell division of fibroblasts.

Furthermore, it was found that the redwood callus extract of the present invention can promote skin regeneration by significantly promoting cell division of fibroblasts.

The above description of the present invention is used as an illustration only, and those skilled in the art will appreciate that changes can be easily made thereto without departing from the technical spirit and scope of the present invention.

Hereinafter, dosage form examples of the composition will be described. However, the following dosage form examples are for illustrative purposes only.

[Dosage Form Example 1] Soft Capsule

According to a conventional method, 40 μg of shikimic acid or the redwood callus extract of Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 2 mg of palm oil, 8 mg of hydrogenated vegetable oil, 4 mg of yellow beeswax, and 9 mg of lecithin were mixed. In addition, 66 parts by weight of gelatin, 24 parts by weight of glycerin, and 10 parts by weight of sorbitol solution were used to prepare soft capsule tablets, which were filled with the above mixture to prepare soft capsules containing 400 mg of the composition of the present invention.

[Dosage Form Example 2] Tablet

40 μg of shikimic acid or the redwood callus extract of Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 200 mg of galacto-oligosaccharides, 60 mg of lactose, and 140 mg of maltose were mixed and granulated using a fluidized bed dryer. After adding 6 mg of sugar ester, 500 mg of the resulting composition was formed into tablets using conventional methods.

[Dosage Form Example 3] Drink

40 μg of shikimic acid or the redwood callus extract of Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 10 g of glucose, 0.6 g of citric acid and 25 g of oligosaccharide syrup were mixed, and after adding 300 mL of purified water, 200 mL of the resulting mixture was filled into each bottle and sterilized at 130° C. for 4-5 seconds.

[Dosage Form Example 4] Granules

40 μg of shikimic acid or the sequoia callus extract of Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed and granulated by using a fluidized bed dryer, and then bagged.

[Dosage Form Example 5] Injection

As described in Table 4, injections (2 mL ampoules) were prepared by conventional methods.

【Table 4】

Element content Shikimic acid or redwood callus extract of Example 4 40 μg Sterile distilled water for injection appropriate amount pH adjusters appropriate amount

[Dosage Form Example 6] Softening Water (Skin Toner)

As described in Table 5, toner was prepared by conventional method.

【Table 5】

Element Content (wt%) Shikimic acid or redwood callus extract of Example 4 0.2 glycerin 3.0 Butanediol 2.0 Propylene glycol 2.0 Carbopol 0.1 PEG-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 ethanol 10.0 triethanolamine 0.1 Preservatives, colorants, and flavors appropriate amount purified water margin

[Dosage Form Example 7] Nourishing Lotion

As described in Table 6, a nourishing lotion was prepared by a common method.

【Table 6】

Element Content (wt%) Shikimic acid or redwood callus extract of Example 4 1.0 glycerin 3.0 Butanediol 3.0 Propylene glycol 3.0 Carbopol 0.1 beeswax 4.0 Polysorbate 60 1.5 Caprylic/capric triglyceride 5.0 Squalane 5.0 Sorbitan sesquioleate 1.5 liquid paraffin 0.5 Cetearyl Alcohol 1.0 triethanolamine 0.2 Preservatives, colorants, and flavors appropriate amount purified water margin

[Dosage Form Example 8] Nutritional Cream

As described in Table 7, a nourishing cream was prepared by a conventional method.

【Table 7】

Element Content (wt%) Shikimic acid or redwood callus extract of Example 4 2.0 glycerin 3.0 Butanediol 3.0 liquid paraffin 7.0 β-glucan 7.0 Carbomer 0.1 Caprylic/capric triglyceride 3.0 Squalane 5.0 Cetearyl Glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 triethanolamine 0.1 Preservatives, colorants, and flavors appropriate amount purified water margin

[Dosage Form Example 9] Massage Cream

As described in Table 8, massage cream was prepared by conventional methods.

【Table 8】

Element Content (wt%) Shikimic acid or redwood callus extract of Example 4 2.0 glycerin 8.0 Butanediol 4.0 liquid paraffin 45.0 β-glucan 7.0 Carbomer 0.1 Caprylic/capric triglyceride 3.0 beeswax 4.0 Cetearyl Glucoside 1.5 Sorbitan sesquioleate 0.9 Petrolatum 3.0 paraffin 1.5 Preservatives, colorants, and flavors appropriate amount purified water margin

[Dosage Form Example 10] Facial Mask

As described in Table 9, facial masks were prepared by conventional methods.

【Table 9】

Element Content (wt%) Shikimic acid or redwood callus extract of Example 4 0.2 glycerin 4.0 polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 β-glucan 7.0 Allantoin 0.1 Nonylphenyl ether 0.4 Polysorbate 60 1.2 ethanol 6.0 Preservatives, colorants, and flavors appropriate amount purified water margin

[Dosage Form Example 11] Health Food

As described in Table 10, health foods were prepared by conventional methods.

【Table 10】

Element content Shikimic acid or redwood callus extract of Example 4 20 μg Vitamin A acetate 70 μg Vitamin E 1.0mg 0.13mg 0.15mg 0.5mg 0.2 μg Vitamin C 10mg Biotin 10 μg Niacinamide 1.7mg folic acid 50 μg Calcium pantothenate 0.5mg Ferrous sulfate 1.75mg zinc oxide 0.82mg magnesium carbonate 25.3mg Potassium dihydrogen phosphate 15mg Calcium hydrogen phosphate 55mg Potassium citrate 90mg calcium carbonate 100mg magnesium chloride 24.8mg

The composition of the above-mentioned vitamin and mineral mixture is shown only as an example and can be changed as needed.

[Dosage Form Example 12] Health Drink

As described in Table 11, health drinks were prepared by conventional methods.

【Table 11】

Element content Shikimic acid or redwood callus extract of Example 4 20 μg Citric acid 1000mg Oligosaccharides 100g Taurine 1g purified water margin

According to a common health drink preparation method, the above ingredients were mixed and heated with stirring at 85° C. for about 1 hour. The resulting solution was filtered and sterilized.

[Dosage Form Example 13] Injection containing totipotent stem cells

As described in Table 12, injections containing totipotent stem cells were prepared by conventional methods.

【Table 12】

Element content Totipotent stem cells of Example 2 40 μg Sterile distilled water for injection appropriate amount pH adjusters appropriate amount

While exemplary embodiments have been shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined in the following claims.

Claims (2)

1. Use of shikimic acid in the preparation of compositions for promoting the induction of human skin fibroblasts into induced pluripotent stem cells. 2. The use according to claim 1, wherein the composition comprises shikimic acid at a concentration of 10 μM-10 mM based on the total volume of the composition.