JP2022087596A - Stem cell activator - Google Patents

Abstract

To provide a stem cell activator that can grow stem cells such as induced pluripotent stem cells (iPS cells) to increase cell survival rates.SOLUTION: A stem cell activator contains quercetin glycosides or derivatives thereof.SELECTED DRAWING: None

Description

The present invention relates to a stem cell activator.

In recent years, in the field of regenerative medicine, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), etc., which have proliferative ability and ability to differentiate into various cells for repairing damaged tissues, etc. The possibility of practical application of stem cells is increasing, and a method for proliferating these stem cells has been proposed.

For example, an embryonic or somatic stem cell growth-promoting agent containing an ethanol extract of jujube as an active ingredient has been proposed. Then, it is described that by using such a growth-promoting agent, stem cells can be efficiently proliferated while maintaining an undifferentiated state. (See, for example, Patent Document 1).

Japanese Patent No. 6587899

However, in the growth promoter described in Patent Document 1, the ethanol extract of jujube is merely used as an active ingredient, and a specific active ingredient has not been specified. There is a problem that the content and activity (inactivation) of the active ingredient cannot be controlled and evaluated, and the proliferative action of stem cells may not be stably obtained.

Therefore, the present invention has been made in view of these points, and an object of the present invention is to provide a stem cell activating agent capable of reliably proliferating stem cells such as iPS cells and improving the cell viability. do.

In order to achieve the above object, the stem cell activator of the present invention is characterized by containing a quercetin glycoside or a derivative thereof.

According to the present invention, it is possible to provide a stem cell activating agent capable of proliferating stem cells and improving cell viability.

It is a figure which shows the result of the gene expression level by RT-PCR (Reverse Translation Polymerase Chain Reaction, reverse transcription polymerase chain reaction) in an Example.

The stem cell activator of the present invention contains a quercetin glycoside or a derivative thereof, and activates stem cells such as iPS cells.

More specifically, when the stem cells were treated with the activator of the present invention, it was confirmed that the stem cells proliferated and the cell viability was improved.

This is because the activator of the present invention increases the expression of genes (FOSL1 gene and JUN gene), and thus the formation of transcription factor (AP-1) is promoted due to the increase in the expression of these genes. As a result, it is considered that the cell cycle was promoted and the cell proliferation was promoted.

In the present invention, improvement of cell viability can be confirmed by culturing stem cells in a medium containing the activator of the present invention by the method described in Examples described later.

The content of the quercetin glycoside or a derivative thereof with respect to the entire activator of the present invention is preferably 0.5 μM or more and 100 μM or less. If it is less than 0.5 μM, the above-mentioned effect of improving the cell viability may be reduced, and if it is larger than 100 μM, the cell viability is high, but the concentration-dependent increase is leveled off. This is because it may start to appear and it may be difficult to activate stem cells efficiently.

From the viewpoint of further improving the cell viability, the content of the quercetin glycoside or its derivative is preferably 25 μM or more and 100 μM or less.

(Quercetin glycoside or its derivative)
Examples of the quercetin glycoside in the activator of the present invention include rutin, quercitrin, hyperoside and the like. Examples of the derivative of the quercetin glycoside include α-glucosylrutin, troxerutin, disodium rutinyl disulfate and the like.

Among the quercetin glycosides or derivatives thereof, α-glucosyllutin can be used from the viewpoint of excellent solubility in water (in the following examples, a medium containing a large amount of water). preferable.

(Stem cells)
Examples of stem cells to which the activator of the present invention is applied include the above-mentioned iPS cells and ES cells. As these stem cells, those prepared by a known method may be used, or commercially available products may be used. Further, these stem cells may be any of primary cultured cells, subcultured cells, and frozen cells.

(culture)
The instruments and devices used for culturing are not particularly limited as long as they can cultivate stem cells and other cells, and for example, a dish (culture dish), a culture flask, a multi-well plate, a microtube, centrifuge tubes, and a measuring pipette. , Micropipets, Coulter counters, cell freezing ampoules, carbon dioxide incubators, safety cabinets, phase difference microscopes, autoclaves, cooling centrifuges, liquid nitrogen frozen cell storage vessels, etc. can be used.

(solvent)
The solvent used in the activator of the present invention is not particularly limited, and is, for example, purified water (distilled water, ion-exchanged water, sterile water, etc.), physiological saline, PBS (Propylene Buffered Saline; phosphate buffered saline, etc.). Water), ethanol, isopropanol, glycerin, 1,3-butylene glycol, propylene glycol, dipropylene glycol, polyethylene glycol, PEG-8 (capryl / capric acid) glyceryl, tri (capric acid / capric acid) glyceryl, isododecane, fluid Paraffin, light liquid isoparaffin, etc. can be used.

Therefore, for example, the above-mentioned quercetin glycoside or a derivative thereof dissolved in a solvent such as purified water can be used as the activator of the present invention.

(Form of activator)
The activator of the present invention may be in the form of liquid, milky liquid, milky, creamy, gelled, pasty, water-oil two-layered, foamy (shape when used), atomized (shape when used), etc. It can also be in the form of an aerosol.

(Other ingredients)
The activator of the present invention may contain other components as long as the effects of the present invention are not impaired. For example, pH adjusting components (eg, citric acid, lactic acid, phosphoric acid, etc.), oily components (eg, sphingolipids, ceramides, cholesterol derivatives, phytosterol derivatives, phospholipids, lanolin, lanolin fatty acid derivatives, perfluoropolyether, etc.) , Vegetable oils (eg olive oil, shea butter, macadamia nut oil, etc.), waxes (eg, jojoba seed oil, carnauba wax, candelilla wax, beeswax, etc.), hydrocarbons (eg, paraffin, microcrystallin wax, squalane, vaseline, iso). Hexadecane, etc.), higher fatty acids (eg, myristic acid, oleic acid, stearic acid, isostearic acid, branched fatty acids (C (carbon number) 14-28), hydroxystearic acid, etc.), alcohols (eg, cetearyl alcohol, cetanol). , Isostearyl alcohol, oleyl alcohol, hydrogenated rapeseed oil alcohol, cholesterol, citosterol, diglycerin, 1,2-hexanediol, etc.), sugars and their derivatives (eg, glucose, sucrose, D-sorbitol, maltose, trehalose) , Polyoxypropylene methyl glucoside, glyceryl glucoside, etc.), esters (eg, isopropyl myristate, cetyl palmitate, octyldodecyl oleate, glyceryl triisostearate, diethoxyethyl succinate, cetyl lactate, lactyl dodecyl, etc.) , Silicones (eg, dimethyl silicone, methylphenyl silicone, cyclic dimethyl silicone, polyether-modified silicone, alkyl-modified silicone, amino-modified silicone, dimethiconol, PCA dimethicone, etc.), amino acids and derivatives thereof (eg, glutamate, aspartic acid, etc.) Glycin, serine, methionine, trimethylglycine, sodium polyaspartate, sodium dilauroyl glutamate lysine, N-lauroyl-L-lysine, etc., polypeptides and proteins (eg, hydrolyzed silk, hydrolyzed wheat, hydrolyzed soybean, etc.) Hydrolyzed collagen, hydrolyzed keratin, silylated hydrolyzed silk, silylated hydrolyzed wheat, cationized hydrolyzed silk, cationized hydrolyzed collagen, keratin, etc.), natural polymers (eg, alginate, mannan, gum arabic) , Tamarind gum, chitosan, carrageenan, mutin, cellac, hyaluronate, cationized hyaluronic acid, xanthan gum, Dextrin, hydroxyethyl cellulose, cationized cellulose, guar gum, cationized guar gum, honey, etc.), synthetic polymers (eg, anionic polymers, cationic polymers, nonionic polymers, amphoteric polymers, polypropylene glycol, polyoxy) Propropylene diglyceryl ether, etc.), anionic surfactants (eg, alkyl ether carboxylate, N-acylglutamic acid, N-acylmethyl taurine salt, alkyl sulfosuccinate, alkyl ether sulfate, polyoxyethylene alkyl ether phosphate, etc.) , Disetyl phosphate, etc.), Cationic surfactants (eg, alkylamine salts, fatty acid amidamine salts, etc.), Amphoteric surfactants (eg, glycine-type amphoteric surfactants, aminopropionic acid-type amphoteric surfactants, aminoacetic acid Betain-type amphoteric surfactants, sulfobetaine-type amphoteric surfactants, etc.), nonionic surfactants (eg, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, alkyl glucoside, parent Oil-type glyceryl monostearate, etc.), dyes (eg, tar pigments, natural pigments, etc.), plant extracts (eg, chamomile extract, confree extract, sage extract, rosemary extract, oyster tannin, cha dry distillate, copper chlorophyllin) Sodium, etc.), vitamins (eg, L-ascorbic acid, DL-α-tocopherol, D-pantenol, natural vitamin E, etc.), UV absorbers (eg, paraaminobenzoic acid, octyl salicylate, paramethoxysilicate skin acid 2) -Ethylhexyl, ferulic acid, etc.), preservatives (eg, benzoic acid, sodium benzoate, salicylic acid, dehydroacetic acid, sodium dehydroacetate, paraben, phenoxyethanol, etc.), antioxidants (eg, sodium hydrogen sulfite, dibutylhydroxytoluene, etc.) , Metal sequestering agents (eg, edetate, sodium polyphosphate, hydroxyethandiphosphonic acid, phytic acid, etc.), other inorganic compounds (eg, titanium oxide, silver, platinum, iron chloride, iron oxide, etc.), other organic compounds (For example, urea, hydroxyethyl urea, dl-sodium pyrrolidone carboxylate, copper gluconate, etc.), solvent (for example, benzyl alcohol, etc.), propellant (for example, LPG (liquefied petroleum gas), DME (dimethyl ether), nitrogen gas). , Carbon acid gas, etc.), known cosmetic ingredients such as fragrances can be blended.

(Cosmetics)
Further, the activator of the present invention can be blended and used in cosmetics, and examples of cosmetics include hair shampoo, hair conditioner, hair treatment, scalp lotion, scalp mist, hair restorer, body soap, and lotion. , Milky lotion, beauty liquid, moisturizing cream, hand cream, body cream, body lotion, body mist, hair styling agent, hair coloring agent, hair curling agent, hair straightening agent, sunscreen cream, pack, mask, lipstick, lipstick, Examples include lip creams, washing pigments, cosmetic soaps, foundations, massage creams, bathing agents and the like.

Hereinafter, the present invention will be described based on examples. The present invention is not limited to these examples, and these examples can be modified or modified based on the gist of the present invention, and these examples are excluded from the scope of the present invention. is not.

<Preparation of activator>
α-Glucosylrutin (manufactured by Toyo Sugar Refining Co., Ltd., trade name: αG rutin PS-C) in water (purified water) as a solvent, 50 μM, 0.5 mM, 1.25 mM, 2.5 mM, 5 mM, and 10 mM. To prepare an activator containing α-glucosylrutin by dissolving at each concentration of.

The activator containing α-glucosyllutin was used after filtration sterilization using a filter having a pore size of 0.2 μm, if necessary.

<Preparation of iPS cells>
NB1RGB, which is a human skin-derived fibroblast, was reprogrammed by an RNA reprogramming method to prepare iPS cells. For RNA reprogramming, Stemgent (registered trademark) StemRNA-NM Reprogramming Kit (manufactured by Stemgent) was used.

In addition, the prepared iPS cells have been confirmed to express the stem cell markers NANOG, Oct4, SSEA4, KLF4, differentiate into three germ layers, and differentiate into neural stem cells, and comprehensively analyze the expression of mRNA by RNA sequence. (M. Shimada, K. Tsukada, N. Kagawa, Y, Matsumoto, Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells, Journal of Radiation Research, Vol. . 60, No. 6, 2019).

<Evaluation of the effect of the activator containing α-glucosyllutin on the proliferation (survival rate%) of iPS cells, fibroblasts, and epidermal keratinocytes>
Next, an activator containing α-glucosyllutin prepared using iPS cells, NB1RGB cells (fibroblasts in the dermis), and epidermal keratinocytes prepared by the above method is used for each of these cells. The effect on the proliferation of the dermis was investigated.

As the epidermal keratinocytes, in addition to the epidermal keratinocytes induced to differentiate from iPS cells, HEKn (Human Epidermal Keratinocytes, neonatal; primary cultured epidermal keratinocytes, Thermo Fisher Scientific), which is a cultured epidermal keratinocyte, is used. ] And HaCaT (immortalized epidermal keratinocytes, manufactured by Cosmo Bio Co., Ltd.) were used.

iPS cells were cultured in a dish using Nutristem (Nutristem XF / FF Culture Medium, manufactured by Reprocell). Subculture is performed by washing with DPBS, treating with TrypLE Select (manufactured by Thermo Fisher Scientific), exfoliating and suspending the cells with Nutristem, centrifuging, centrifuging, removing the supernatant, and performing ROCK inhibitor (Rho-inhibitor) in Nutristem. ) Y27632 (manufactured by WAKO) was added to suspend and dilute the precipitated cells, and iMatrix-511 silk was further added, and then in a wet state (37 ° C.) in the presence of 5% CO ₂ . It was cultured. Then, 24 hours after the subculture, the medium was exchanged with Nutristem containing no Y27632, and then the medium was exchanged once every 24 hours, and the medium was subcultured at about 60% confluence.

In addition, NB1RGB was cultured in a dish using DMEM (Dulvecco's Modified Eagle Medium, manufactured by Thermo Fisher Scientific). Subculture is performed by washing with DPBS, treating with Trypin / EDTA (manufactured by Thermo Fisher Scientific), then exfoliating and suspending the cells with DMEM, centrifuging, removing the supernatant after centrifugation, and suspending the cells precipitated with DMEM. It was diluted and cultured in a wet state (37 ° C.) in the presence of 5% CO ₂ . After that, the medium was changed twice a week, and the cells were subcultured at about 80% confluent.

In addition, HEKn was prepared by adding HKGS (Human Keratinocyte Growth Supplement, Thermo Fisher Scientific) and calcium chloride to EpiLife (manufactured by Thermo Fisher Scientific) and calcium chloride. Subculture is performed by washing with DPBS, treating with TrypLE Select, then exfoliating and suspending the cells with a Definated Trypsin Inhibitor & Trypsin Neutralizer (manufactured by Invitrogen), centrifuging, centrifuging, removing the supernatant, and HKGS and calcium chloride in EpiLife. The cells precipitated in the added medium were suspended and diluted, and cultured in a wet state (37 ° C.) in the presence of 5% CO ₂ . Then, the medium was changed every 2 to 3 days, and the cells were subcultured at about 80% confluent.

HaCaT was also cultured in a dish using DMEM. Subculture is performed by washing with DPBS, treating with Trypin / EDTA, then exfoliating / suspending the cells with DMEM and centrifuging, removing the supernatant after centrifugation, suspending / diluting the cells precipitated with DMEM, and 5%. The cells were cultured in a wet state (37 ° C.) in the presence of CO ₂ . After that, the medium was changed twice a week, and the cells were subcultured at about 80% confluent.

The differentiation of iPS cells into epidermal keratinocytes was induced by the following method. First, iPS cells were cultured in a dish for 24 hours using a medium in which Y27632 and iMatrix-511 silk were added to Nutristem in the same manner as in passage. Next, RA (Retinoic acid, manufactured by Sigma) and BMP4 (bone morphogenetic) were added to DKSFM (Defined keratinocyte serum-free medium, Thermo Fisher Scientific), and BMP4 (bone morphogenetic) was added to the medium. Further, on the 4th day, the medium was replaced with a medium in which EGF (Epidermal Growth Factor, manufactured by R & D systems) was added to DKSFM, and the cells were cultured until the 14th day. In the first passage, after washing with DPBS and TripLE Select treatment, cells are detached / suspended and centrifuged with DKSFM, and after centrifugation, the supernatant is removed, and the cells precipitated with DKSFM are suspended / diluted and dished. Was sown in. The dish used was previously coated with Type I collagen (manufactured by Advanced BioMatrix) and Fibronectin (manufactured by Sigma). Then, after culturing for 1 to 2 hours, the medium was replaced with a medium containing Y27632 and EGF added to DKSFM. The second passage was carried out on the 24th day, the third passage was carried out on the 34th day in the same manner as the first passage, and all the cultures were carried out in a wet state (37) in the presence of 5% CO ₂ . It was carried out under the condition of ℃).

The viability of each cell was then evaluated by the WST-8 assay. The WST-8 assay is a method of measuring the metabolic activity associated with electron transfer of nicotinamide adenine dinucleotide (NADH) in cells to calculate cell viability.

More specifically, first, each cell is seeded and cultured on a 96-well plate by the same method as the above-mentioned passage, and then an activator containing each concentration of α-glucosyllutin described above is added to the medium. did. It should be noted that the activated agent containing each concentration of α-glucosyllutin prepared and the medium were mixed at a ratio of activator: medium = 1:99, and the activated agent containing each concentration of α-glucosyllutin produced was activated. The agent was diluted 100-fold with medium before use. Therefore, for example, an activator containing 5 mM α-glucosyllutin was diluted 100-fold with a medium and used as an activator containing 50 μM α-glucosyllutin in the medium.

Then, after culturing each cell in the medium to which the activator was added by the above method, WST-8 reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well of the plate, and 5% CO was added. In the presence of ₂ , the cells were allowed to stand in a wet state (37 ° C.) for 2 hours. After that, the absorbance at 450 nm in each well was measured with a plate reader (Bio-Rad Laboratories, Inc., trade name: iMark microplate reader), and no activator was added (that is, α-glucosyllutin was added). Cell viability was calculated using the absorbance of the (not) well as a comparative control (ie, viability 100%).

Α at 50 μM, 0.5 mM, 1.25 mM, 2.5 mM, 5 mM, and 10 mM concentrations (ie, concentrations after 100-fold dilution are 0.5 μM, 5 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM). -Table 1 shows the results on iPS cell proliferation (% survival rate) when an activator containing glucosyllutin was used, and the concentrations of 0.5 mM, 2.5 mM, and 5 mM (that is, 100-fold). Results on proliferation (% survival rate) of iPS cells, fibroblasts, and epidermal keratinized cells when an activator containing α-glucosyllutin with diluted concentrations of 5 μM, 25 μM, and 50 μM) were used. It is shown in Table 2.

As shown in Table 1, when the content of α-glucosyllutin was 0.5 μM or more and 100 μM or less, the iPS cells were proliferating and the iPS cells were proliferated as compared with the comparison target to which the activator was not added. It can be seen that the survival rate is improved.

Further, as shown in Table 2, when the same concentration of α-glucosyllutin was added to iPS cells, fibroblasts, and epidermal keratinized cells, the survival rate of iPS cells was most improved, and the present invention. It can be seen that the activator containing α-glucosyllutin of the above has an effect of proliferating iPS cells in particular.

<Measurement of gene expression level by RT-PCR>
After culturing iPS cells for 4 hours, 8 hours, and 24 hours in a medium containing an activator (α-glucosyllutin concentration: 50 μM), the cells were washed twice with DPBS, and FastGene RNA Basic kit (Nippon Genetic). Total RNA was extracted using (Su). Then, RT-PCR (reverse transcription polymerase chain reaction) was performed on all RNA using PrimeScript High Fidelity RT-PCR Kit and TakaRa PCR Thermal Cycler Dice TP 650 (manufactured by Takara Bio Co., Ltd.), and the FOSL1 gene and JUN gene were subjected to RT-PCR (reverse transcription polymerase chain reaction). Gene expression was confirmed.

Both the FOSL1 gene and the JUN gene are genes encoding the subunit proteins constituting the transcription factor AP-1 (Activator Protein 1), and AP-1 responds to various stimuli such as cytokines and growth factors. , Regulates the expression of genes involved in cell differentiation, proliferation, apoptosis, etc. (J. Hess, P. Angel, M. Schorpp-Kistner, AP-1 subunits: quarrel and harmony among siblings, J. Cell. Sci . 117 (2004)).

Annealing was performed at 65 ° C. for 5 minutes, and reverse transcription to cDNA was performed at 42 ° C. for 30 minutes. Then, the treatment was carried out at 95 ° C. for 5 minutes to stop the enzymatic reaction. RT-PCR was performed for 30 cycles at 98 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute using the primers shown below.

In addition, gene expression of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was also confirmed as an internal standard for RT-PCR. The above results are shown in FIG.

Primer sequence used for RT-PCR FOSL1 forward: 5'-ACCGGAGGAAGGAACTGAC-3'(SEQ ID NO: 1)
FOSL1 reverse 5'-CTGCAGCCCCAGATTTCCAT-3'(SEQ ID NO: 2)
JUN forward 5'-CCCCAAGATCCTGAAACAGA-3' (SEQ ID NO: 3)
JUN reverse 5'-CCGTTGCTGGACTGGATTAT-3'(SEQ ID NO: 4)
GAPDH forward 5'-TGCACCACCACACTGCTTAGC-3'(SEQ ID NO: 5)
GAPDH reverse 5'-GGCATGGACTGTGGTCATTGAG-3'(SEQ ID NO: 6)

As shown in FIG. 1, when iPS cells were treated with α-glucosyllutin (concentration: 50 μM), increased expression of the FOSL1 gene and the JUN gene was observed after 4 hours of treatment. Therefore, treatment of iPS cells with α-glucosyllutin (concentration: 50 μM) promotes the formation of transcription factor (AP-1) due to the increased expression of these genes, resulting in a cell cycle. It is considered that cell proliferation was promoted as well as being promoted.

Examples of utilization of the present invention include stem cell activators that activate stem cells such as iPS cells.

Claims (4)

A stem cell activator characterized by containing a quercetin glycoside or a derivative thereof. The stem cell activator according to claim 1, wherein the stem cell is an iPS cell. The stem cell activator according to claim 1 or 2, wherein the content of the quercetin glycoside or a derivative thereof is 0.5 μM or more and 100 μM or less. The stem cell according to any one of claims 1 to 3, wherein the derivative is at least one selected from the group consisting of α-glucosylrutin, troxerutin, and disulfuric acid disulfate disodium. Activator.

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