WO2016111377A1

WO2016111377A1 - MEDICINAL COMPOSITION COMPRISING iPS CELL CULTURE SUPERNATANT AND METHOD FOR PRODUCING SAME, COSMETIC AND METHOD FOR PRODUCING SAME, ANTIAGING COMPOSITION, METHOD FOR INHIBITING DISEASE ONSET, METHOD FOR TREATING DISEASE, METHOD FOR TREATING TISSUE ABNORMALITY AND BEAUTIFICATION METHOD

Info

Publication number: WO2016111377A1
Application number: PCT/JP2016/050728
Authority: WO
Inventors: 上田　実
Original assignee: 上田　実
Priority date: 2015-01-09
Filing date: 2016-01-12
Publication date: 2016-07-14
Also published as: JP2016128396A

Abstract

The medicinal composition and cosmetic according to the present disclosure each comprise an iPS cell culture supernatant that is obtained by culturing iPS cells. The present disclosure also provides: a method for producing the aforesaid medicinal composition; a method for producing the aforesaid cosmetic; an antiaging composition; a method for inhibiting the onset of a disease; a method for treating a disease; a method for treating a tissue abnormality; and a beautification method.

Description

Pharmaceutical composition containing iPS cell culture supernatant and method for producing the same, cosmetic and method for producing the same, anti-aging composition, disease onset inhibiting method, disease treating method, tissue abnormality treating method and cosmetic method

The present invention relates to a pharmaceutical composition containing an iPS cell culture supernatant, a method for producing the same, a cosmetic and a method for producing the same, an anti-aging composition, a disease onset inhibiting method, a disease treating method, a tissue abnormality treating method and a cosmetic method. .

Regenerative medicine using stem cells has attracted attention as an alternative technique for diseases that are difficult to treat depending on conventional medicine.
Regenerative medicine using stem cells is a promising tool in a new clinical platform for all intractable diseases. Various stem cells including embryonic stem cells (ES cells) and somatic stem cells have been reported. Among somatic stem cells, mesenchymal stem cells (MSC) isolated from various tissues such as bone marrow, adipose tissue, skin, umbilical cord, and placenta have been particularly used for clinical application in skin regeneration.

In addition, induced pluripotent stem cells (iPS cells) are cells created by introducing several types of reprogramming factors into somatic cells, and can be differentiated into all three germ layers. And “self-replicating” capable of growing while maintaining an undifferentiated state (see, for example, Japanese Patent No. 5098028). Attempts have also been made to differentiate iPS cells and obtain transplant materials using this differentiation pluripotency (see, for example, International Publication No. WO2011-136378).

On the other hand, attempts have been made to use the stem cell culture supernatant obtained by culturing stem cells to repair the damaged part of the target tissue (see, for example, International Publication No. WO2011 / 118795).

Attempts to use iPS cells for regenerative medicine have made use of the pluripotency and self-replication of iPS cells themselves. In this case, for example, transplantation of a transplant material obtained by differentiating iPS cells is necessary, which places a burden on the patient. Furthermore, if it is intended to avoid rejection, it is necessary to prepare iPS cells from the patient's own somatic cells one by one, and the time and labor required for preparation for treatment become relatively large.
On the other hand, culture supernatants obtained by culturing somatic stem cells existing in the body, such as dental pulp stem cells, exhibit a certain effect in damage repair, but their effectiveness varies depending on the type of tissue to which they are applied. It was difficult to say that it has universal effectiveness for the body tissues in general.
The present invention has been made in view of the above situation, and a new pharmaceutical composition using the culture supernatant of iPS cells and a method for producing the same, a cosmetic and a method for producing the same, an anti-aging composition, and a disease onset suppression It is an object to provide a method, a disease treatment method, a tissue abnormality treatment method, and a beauty method.

The pharmaceutical composition according to the first aspect of the present invention is a pharmaceutical composition comprising an iPS cell culture supernatant obtained by culturing iPS cells.
The method for producing a pharmaceutical composition according to the second aspect of the present invention comprises:
(1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells;
Is a method for producing a pharmaceutical composition.
The use according to the third aspect of the present invention is the use of an iPS cell culture supernatant obtained by culturing iPS cells in the production of a pharmaceutical composition.

In the method according to the fourth aspect of the present invention, the pharmaceutical composition according to the first aspect is administered to a subject before the onset of a disease or tissue abnormality in an amount effective for suppressing the onset of the disease or tissue abnormality. A method of suppressing the onset of the disease or tissue abnormality before the onset of the disease or tissue abnormality in the subject.
The method according to the fifth aspect of the present invention comprises administering the pharmaceutical composition according to the first aspect to a subject having a disease in an amount effective to treat the disease. Is the method.
The method according to the sixth aspect of the present invention comprises administering the pharmaceutical composition according to the first aspect to a subject having a tissue abnormality in an amount effective for treating the tissue abnormality. This is a method for treating abnormalities.
The cosmetic according to the seventh aspect of the present invention is a cosmetic containing an iPS cell culture supernatant obtained by culturing iPS cells.

A beauty method according to an eighth aspect of the present invention is a beauty method for improving the cosmetic appearance of a subject by applying the cosmetic according to the seventh aspect to the subject.
The method for producing a cosmetic according to the ninth aspect of the present invention comprises:
(1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells;
A method for producing cosmetics.

According to the present invention, a new pharmaceutical composition using the culture supernatant of iPS cells and a production method thereof, a cosmetic and a production method thereof, an anti-aging composition, a disease onset suppression method, a disease treatment method, and a tissue abnormality treatment method As well as a cosmetic method.

It is a figure which shows the pulmonary fibrosis onset inhibitory effect by performing iPS cell culture supernatant mist process with a 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. It is a figure which shows the Langerhans island atrophy suppression effect by performing an iPS cell culture supernatant mist process with a 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. It is a figure which shows the hepatic fibrosis onset inhibitory effect by performing iPS cell culture supernatant mist process with a 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. It is a figure which shows the collagen decrease inhibitory effect in the dermis by performing iPS cell culture supernatant mist process in 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. . It is a figure which shows the inhibitory effect about the learning memory ability fall by performing iPS cell culture supernatant mist process in 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. . It is a figure which shows the cranial nerve degeneration inhibitory effect by performing the iPS cell culture supernatant mist process with a 12 hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging-promoting mouse. The measurement target part of the test for the suppression of the decrease in the number of brain neurons by performing the iPS cell culture supernatant mist treatment in a 12-hour cycle for SAMP8 (Japan SLC, 6 weeks old), which is an aging-promoting mouse. FIG. It is a figure which shows the brain nerve cell number reduction inhibitory effect by performing iPS cell culture supernatant mist process in 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging promotion mouse | mouth. It is a figure which shows the lifetime extension effect by performing the iPS cell culture supernatant mist process in a 12-hour cycle with respect to SAMP8 (Japan SLC Co., Ltd., 6 weeks old) which is an aging promotion mouse | mouth.

<Pharmaceutical composition>
The pharmaceutical composition according to the present disclosure includes an iPS cell culture supernatant obtained by culturing iPS cells.
iPS cells have pluripotency and self-renewal. As described above, there has been an attempt to differentiate iPS cells and use the obtained differentiated tissue as a graft. However, focusing on substances secreted from iPS cells, there has been no attempt to obtain, for example, medical or cosmetic effects using the culture supernatant of iPS cells. On the contrary, nothing was known about what biological effect the culture supernatant of iPS cells had.

The inventor of the present invention has made an unprecedented attempt to use the iPS cell culture supernatant to obtain medical and cosmetic effects. As a result, surprisingly, iPS cell culture supernatants have a wide repair ability for tissues in general, and the repair ability is shown by the somatic stem cell culture supernatants such as mesenchymal stem cells and hematopoietic stem cells. The inventor has discovered that it is outstanding compared to ability. This is because the secretory product secreted from somatic stem cells (including growth factors, cytokines, etc.) secreted from iPS cells with pluripotency has a greatly different composition. As a result, it is presumed that, regardless of the tissue, the repair ability is widely shown to general tissues, and the degree of the repair ability is high. Specifically, for many growth factors and cytokines, the content of iPS cells in the culture supernatant is higher than the content of somatic stem cells in the culture supernatant, or the specific growth factors and cytokines are iPS cells. It is considered to be contained in the cell culture supernatant but not substantially contained in the somatic stem cell culture supernatant. In addition, the culture supernatant of iPS cells contains an anti-aging substance (for example, a specific type of protein) that is not included in the culture supernatant of somatic stem cells. Kiyoshi is considered to have an excellent anti-aging effect not seen in the culture supernatant of somatic stem cells.
However, the present invention is not restricted by the estimation.
In the present disclosure, the term “repair” means that a part or all of the tissue function lost due to the tissue abnormality or disease is maintained or recovered as compared with the function of the tissue before the tissue abnormality or disease occurs. This means not only that the function of the tissue is restored, but also that it is regenerated as a functional tissue. The evaluation of whether the function is maintained or restored differs depending on the tissue in which the tissue abnormality or disease is occurring, but if it is performed based on the appearance, the assay usually used for evaluating the degree of the target function, etc. Good.

In the pharmaceutical composition according to the present disclosure, an iPS cell culture supernatant obtained by culturing iPS cells is used as an active ingredient of the pharmaceutical composition. Since the iPS cell culture supernatant contains a cytokine mixture and the like, it exhibits repair ability in various tissues. In one form of the present invention, the mixture of cytokines in the iPS cell culture supernatant acts as an induction signal for the tissue's endogenous stem cells, so that the endogenous stem cells can differentiate and proliferate, which expresses the repair ability. It can be estimated that However, the present invention is not restricted by the estimation.

Since the iPS cell culture supernatant in the present disclosure is obtained by culturing iPS cells, the production method is also described in the following description.

Artificial pluripotent stem cells (iPS cells), which are produced by introducing reprogramming factors into somatic cells, have almost the same characteristics as ES cells, such as differentiation pluripotency and proliferation ability by self-replication. Cell-derived artificial stem cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666). The reprogramming factor can be introduced into somatic cells, for example, in the form of DNA or protein. The reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or noncoding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or noncoding RNA, or a small molecule It may be constituted by a compound. Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, as described in JP2013-247934A, for example. , L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1.
In addition to the above method, iPS cells can be prepared by various iPS cell preparation methods reported so far. It is naturally assumed that an iPS cell production method developed in the future will be applied.

The most basic method for producing iPS cells is to introduce four factors, transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). Three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), Oct3 / 4 and Klf4, two factors (Kim J B, et al: Nature 454 (7204 ), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136 (3), 411-419, 2009) has been reported to establish iPS cells. In addition, a method of introducing a gene expression product into a cell (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al : Cell Stem Cell 4, 472-476, 2009) has also been reported. On the other hand, by using the inhibitor BIX-01294 for histone methyltransferase G9a, the histone deacetylase inhibitor valproic acid (VPA) or BayK8644, production efficiency can be improved and factors to be introduced can be reduced. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J , Et al: PLoS. Biol. Studies on gene transfer methods are also underway. In addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008; Okita K. et al .: Nat Methods 8, 409-412), transposon vector (Woltjen K , Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009) or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) Technology has been developed.

The somatic cell into which the reprogramming factor is introduced in the preparation of the iPS cell may be any cell other than a germ cell derived from a mammal (eg, human, mouse, monkey, pig, rat, etc.), for example, As described in JP2013-247934A, keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface layer), exocrine gland epithelial cells (eg, mammary cells) , Hormone-secreting cells (eg, adrenal medullary cells), metabolism / storage cells (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), lumens of inner chain vessels Epithelial cells (eg, vascular endothelial cells), cilia cells with transport ability (eg, airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells) , Blood cells, immune cells (eg, T lymphocytes) Sensory organ cells (eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory organs and peripheral neuron support cells (eg, accessory cells), central nervous system neurons and glial cells ( Examples include astrocytes), pigment cells (eg, retinal pigment epithelial cells), and precursor cells thereof (tissue precursor cells). There is no particular limitation on the degree of cell differentiation and the age of the animal from which the cells are collected, whether it is undifferentiated progenitor cells (including somatic stem cells) or terminally differentiated mature cells, It can be used as a source of somatic cells in the present disclosure. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells. Among these, bone marrow-derived mesenchymal stem cells are preferable from the viewpoint of stability. From the viewpoint of repair ability, the somatic cell is preferably derived from a human. In addition, it may be advantageous for avoiding rejection to use somatic cells obtained from the subject to whom the pharmaceutical composition is administered as the somatic cells.

As a culture solution for iPS cell induction, as disclosed in JP2013-247934A, for example, DMEM, DMEM / F12 or DME culture solution containing 10 to 15% FBS (in these culture solutions) Further, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc. may be included as appropriate.) Or a commercially available culture solution [for example, a culture solution for mouse ES cell culture (TX- WES culture medium, Thrombo X), culture medium for primate ES cell culture (culture medium for primate ES / iPS cells, Reprocell), serum-free medium (mTeSR, Stemcell Technology)) and the like.

Examples of the culture method include somatic cells on DMEM or DMEM / F12 culture medium containing 10% FBS in the presence of 5% CO ₂ at 37 ° C., for example, as described in JP2013-247934A. Contact with reprogramming factor and culture for about 4-7 days, then re-seed cells on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) Ten days later, the cells can be cultured in a bFGF-containing primate ES cell culture medium, and iPS-like colonies can be generated about 30 to about 45 days or more after the contact.

Alternatively, 10% FBS-containing DMEM culture medium (including LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO ₂ at 37 ° C. And can be appropriately cultured with puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) to give ES-like colonies after about 25 to about 30 days or more. Desirably, instead of feeder cells, somatic cells to be initialized themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or an extracellular matrix (for example, Laminin ( WO2009 / 123349) and Matrigel (BD)) are exemplified.

In addition, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci USA 106.15720-15725). Furthermore, in order to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
During the culture, the culture medium is exchanged with a fresh culture medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

IPS cells can be selected according to the shape of the formed colonies as described in JP2013-247934A. In addition, expression of pluripotent stem cell markers (undifferentiation markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1 and Cript can be selected as an index. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing with the culture medium. When the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate. can do. Selected cells are collected as iPS cells.

In order to obtain a culture supernatant from the obtained iPS cells, conditions usually used as culture conditions for iPS cells can be applied as they are. For example, the culture medium and culture conditions for producing iPS cells include the culture medium and culture conditions described in the above description. The use of feeder cells is not essential for maintaining iPS cells. For example, feeder cells can be omitted by using laminin 511 (A novel efficient feeder-free culture system for the derivation of human induced). pluripotent stem cells
Masato Nakagawa, et al. Scientific Reports 4, Article number: 3594doi: 10.1038 / srep03594 Received 09 October 2013 Accepted 06 December 2013 Published 08 January 2014 (Nature)). Moreover, you may add a ROCK inhibitor in order to suppress a cell death in the case of culture | cultivation.

The pharmaceutical composition according to the present disclosure may not contain serum. Safety may be improved by not containing serum. For example, a culture supernatant not containing serum can be prepared by culturing iPS cells in a serum-free medium (serum-free medium). A serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.
In order to prepare “iPS cell culture supernatant” that does not contain serum, a serum-free medium may be used throughout the entire process or for the last or several subcultures from the end. In addition to DMEM, Iscov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of ingredients that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

The culture time for obtaining the culture supernatant is, for example, 5 hours to 7 days, and may be 1 day to 6 days. The culture temperature is, for example, 36 ° C. to 38 ° C., for example 37 ° C., and the CO ₂ concentration is 4 to 6%, for example 5%. The culture may be performed, for example, by three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregated suspension culture, etc.).

After culturing, the culture supernatant of iPS cells can be obtained by separating and removing cell components. In the present disclosure, the culture supernatant is not only the supernatant itself from which the cell components are separated and removed from the culture solution, but also various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution) , Desalted, preserved, etc.) are also included in the range. Details of the culture supernatant treatment method will be described later. In the present disclosure, the culture supernatant does not contain cell components. For this reason, the culture supernatant in this indication does not contain the iPS cell used for culture.

The pharmaceutical composition according to the present disclosure includes the culture supernatant of iPS cells obtained as described above as an active ingredient. In one embodiment, the pharmaceutical composition includes the iPS cells as a whole composition. Absent. In another embodiment, the pharmaceutical composition does not contain cells (regardless of cell type) as a whole composition. That is, it is cell-free. Due to this characteristic, the pharmaceutical composition of the embodiment is clearly distinguished from various compositions containing iPS cells as well as iPS cells themselves. A typical example of this embodiment is a pharmaceutical composition that does not contain iPS cells and is composed only of culture supernatants of iPS cells.

Depending on the condition of the subject to be applied, the pharmaceutical composition according to the present disclosure may additionally contain other components, provided that the expected therapeutic effect is maintained. An example of components that can be additionally included is as follows.
(i) Bioabsorbable material As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) and the like can be used.

(ii) Gelling material As the gelling material, a material having high biocompatibility is preferably used, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (application tissue) of the pharmaceutical composition according to the present disclosure. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(iii) Others Pharmaceutically acceptable other components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc. ) Can also be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used. Antibiotics, pH adjusting agents, growth factors (for example, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)) and the like may be included.

The final form of the pharmaceutical composition according to the present disclosure is not particularly limited. Examples of forms are liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.). The pharmaceutical composition according to the present disclosure may have a form suitable for inhalation, and for example, may have a liquid form that can be sprayed in a mist form with a nebulizer or a diffuser. The culture supernatant of iPS cells is more advantageous than the case of using iPS cells themselves in terms of prior preparation and storage, and can be said to be particularly suitable for the treatment of the acute phase and subacute phase of diseases. Moreover, the usefulness of the culture supernatant of iPS cells is extremely high in that it does not contain cellular components and can overcome the problem of immune rejection.

In recent years, research for the realization of regenerative medicine using cells has been promoted by many research groups. When cells are used, the cells collected from the living body are subjected to culture, selection, treatment, etc., and then collected and used as a component of the transplant. In such conventional research, the culture supernatant is usually discarded or replaced with a physiological buffer in the course of a series of operations. Therefore, the final transplant does not actively contain the culture supernatant. In view of this, in terms of including the culture supernatant of iPS cells, the pharmaceutical composition according to the present disclosure is a composition using iPS cells as an active ingredient, focusing on the usefulness of iPS cells themselves. Of course, the wording is also distinct.

However, in some embodiments, the pharmaceutical composition according to the present disclosure may include iPS cells in addition to the culture supernatant of iPS cells. In such a case, it is preferable to use iPS cells that have not been induced to differentiate after production (in other words, maintained in an undifferentiated state). The therapeutic effect may be improved by additionally using iPS cells.

The pharmaceutical composition according to the present disclosure has an effect of repairing a tissue against tissue abnormality or disease by containing a culture supernatant of iPS cells. The degree of this effect far surpasses the effect obtained by using the culture supernatant of conventional somatic stem cells. In addition, the pharmaceutical composition according to the present disclosure surprisingly exhibits a wide and general repair effect on general tissues in the body. For this reason, the pharmaceutical composition according to the present disclosure can be used as a universal pharmaceutical composition not limited to a specific disease or tissue abnormality. In other words, it can be used as a kind of panacea. For this reason, since the pharmaceutical composition of the present invention is before the onset of a disease or tissue abnormality, even if the disease or tissue abnormality is unknown, the disease or The progression of tissue abnormality can be effectively suppressed. For this reason, by using the pharmaceutical composition according to the present disclosure habitually, it is possible to treat various diseases and abnormal conditions before onset.

In this disclosure,
(1) culturing iPS cells;
(2) A step of recovering the culture supernatant obtained by culturing the iPS cells.
A method for producing a pharmaceutical composition is also provided. In this production method, the collected culture supernatant is subjected to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting. May further be included. By including such a step, handling, storage and transportation of the pharmaceutical composition become easier. The production method may further include a step of adding an additional component to the collected culture supernatant. By adding such an additional component, it is possible to change the physical properties of the entire pharmaceutical composition and to improve its properties. The production method may further include a step of creating the iPS cell by introducing an reprogramming factor into a somatic cell. For each step and additional components, the matters described in the explanation of the pharmaceutical composition according to the present disclosure are applied as they are. Subjecting the collected culture supernatant to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting; When the culture supernatant includes both steps of adding additional components, both steps may be performed first, and if possible, may be performed simultaneously.

In step (2) above, the culture supernatant of iPS cells is collected. For example, the culture solution can be collected by suction with a dropper or pipette. The collected culture supernatant is used as an active ingredient of the pharmaceutical composition according to the present disclosure as it is or after one or more treatments. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., −80 ° C.). It should be noted that the culture supernatant of iPS cells exhibits the desired action without complicated and sophisticated purification. For this reason, the pharmaceutical composition according to the present disclosure can be produced by a simple process. The fact that a complicated purification step is not required is also advantageous in that a decrease in activity associated with purification can be avoided.

<Concentration method of iPS cell culture supernatant>
The pharmaceutical composition according to the present disclosure may be formulated. As a method for concentrating the iPS cell culture supernatant for formulation, a method usually used for concentrating the culture supernatant can be applied. Examples of the concentration method include the following two methods.
1. Spin column concentration method The culture supernatant is concentrated using Amicon Ultra Centrifugal Filter Units-10K (Millipore) (maximum 75-fold concentration). The specific operation procedure is as follows.
(i) The culture supernatant (up to 15 ml) is put into Amicon Ultra Centrifugal Filter Units-10K, centrifuged at 4000 g for about 60 minutes, and concentrated to 200 μl.
(ii) Put the same amount of sterile PBS as the culture supernatant into the above tube, and centrifuge again at × 4000g for about 60 minutes to replace the base solution with PBS.
(iii) Collect 200 μl of the obtained solution into a micro test tube to obtain a concentrated iPS cell culture supernatant.

2. Ethanol precipitation concentration method Concentrate the culture supernatant using the ethanol precipitation method (concentration up to 10 times). The specific operation procedure is as follows.
(i) Add 45 ml of 100% ethanol to 5 ml of the culture supernatant, mix and leave at -20 ° C for 60 minutes.
(ii) Centrifuge for 15 minutes at 4 ° C and 15000g.
(iii) Remove the supernatant, add 10 ml of 90% ethanol and stir well.
(iv) Centrifuge at 4 ° C and 15000g for 5 minutes.
(v) The supernatant is removed, and the resulting pellet is dissolved in 500 μl of sterilized water, collected in a micro test tube, and used as a concentrated iPS cell culture supernatant.

<Method of freeze-drying iPS cell culture supernatant>
The iPS cell culture supernatant in the pharmaceutical composition according to the present disclosure may be lyophilized. Thereby, good storage stability is obtained. As a lyophilization method of the iPS cell culture supernatant, a method generally used for lyophilization of the culture supernatant can be applied. Examples of the lyophilization method include the following methods.
(i) The iPS cell culture supernatant or concentrated iPS cell culture supernatant obtained by the above method is frozen at −80 ° C. for 2 hours to half a day.
(ii) After freezing, open the lid of the sample tube and set it in the freeze dryer.
(iii) Freeze-dry for 1-2 days.
(iv) The obtained sample is used as a lyophilized iPS cell culture supernatant (can be stored at -80 ° C).

In the present disclosure, the pharmaceutical composition according to the present disclosure is administered to a subject prior to the onset of a disease or tissue abnormality in an amount effective for suppressing the onset of the disease or tissue abnormality. A method for suppressing the onset of the disease or tissue abnormality before the onset of the tissue abnormality is also provided. The subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ). The subject may be a subject determined to have a risk of developing a disease or tissue abnormality. Such a risk can be determined by genetic diagnosis, family analysis or the like. For example, in some cases, it has been statistically revealed that the presence of a specific allele in a specific gene correlates with the probability of suffering a specific disease.

In general, as disease and tissue abnormality progress, the treatment becomes more difficult. For this reason, early detection of such diseases and tissue abnormalities is important, but early detection is not always reliable. In consideration of this point, it is desirable that treatment can be started in advance before the onset of a disease or tissue abnormality, but since the drug used for such treatment is continuously used, the burden on the administration subject is small. In view of the necessity of suppressing the progression of a disease or tissue abnormality that is not yet clear at that stage, it is desirable that the disease or tissue abnormality generally has wide efficacy. Satisfying these requirements simultaneously has been difficult with conventional knowledge. However, if the pharmaceutical composition according to the present disclosure is used, it can be administered without invasive treatment such as transplantation or injection, and the pharmaceutical composition has wide efficacy in general for diseases or tissue abnormalities. From this, the said pharmaceutical composition has high effectiveness in the early treatment (progression suppression) of the disease and tissue abnormality before onset as mentioned above. Such early treatment before onset is referred to as preemptive medicine in the present disclosure.

The dosage of the pharmaceutical composition is, for example, 0.1 mg / kg / day to 1000 mg / kg / day in terms of the amount of untreated culture supernatant, and 1 mg / kg / day to 100 mg / kg. / Day may be sufficient. Moreover, the administration method is not particularly limited. For example, the pharmaceutical composition is preferably administered parenterally, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying. Examples of methods for administering the pharmaceutical composition include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary absorption), and nasal cavity. An internal administration etc. can be mentioned. Of these, intranasal administration, transpulmonary administration and the like are preferable because they are minimally invasive. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.

The administration method can be selected by those skilled in the art based on the type of tissue, the type of disease, and the like, which are targets for suppression of onset. For example, when the tissue is in the head, it is not necessary to consider the passage through the blood brain barrier, and it is particularly preferable to apply intranasal administration or the like because it is minimally invasive. For example, when the tissue is the brain, intranasal administration can be preferably applied. In addition, intranasal administration can be preferably applied in preemptive medical treatment for cerebral infarction. Transpulmonary administration is less burdensome on the subject of administration because it can be administered for a long time in the living environment, and can be carried out by spraying the pharmaceutical composition in a mist form using a diffuser or the like.

Depending on the embodiment, iPS cells can also be administered in addition to the pharmaceutical composition according to the present disclosure. For example, the pharmaceutical composition and iPS cells can be administered to a subject simultaneously or at different times. In such a case, it is preferable to use iPS cells that have not been differentiated after collection (in other words, maintained in an undifferentiated state). When using the said pharmaceutical composition and iPS together, you may administer using the kit which consists of the 1st component containing the said pharmaceutical composition, and the 2nd component containing an iPS cell. Each component may be a separate capsule, for example, or a separate ampoule or vial. In this case, you may administer a 2nd component with respect to the treatment target which administered the 1st component simultaneously with the administration of a 1st component, or after administration. Here, “simultaneous” does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without substantial time difference is also included in the concept of “simultaneous” herein.

The disease is not particularly limited. For example, gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular heart disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, pulmonary fibrosis, (I Langerhans island atrophy (as seen in type II diabetes), liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases (amyotrophic lateral sclerosis, Alzheimer's disease) Parkinson's disease, progressive supranuclear palsy, Huntington's disease, multiple system atrophy, spinocerebellar degeneration etc.), neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. , Retinal disease with neuronal damage (traumatic retinal detachment, retinal tear, retinal shaking, optic nerve tube fracture, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, colloidemia, label congenital blindness, cone Body Rofi, familial drusen, central areolar choroidal dystrophy, autosomal dominant optic atrophy etc.), intractable neurological diseases, periodontal disease, skin ulcers, osteoporosis, and the like. The disease may be a disease that develops or progresses with aging. Moreover, administration of the pharmaceutical composition may suppress the occurrence or progression of the disease associated with aging.

Said tissue abnormality refers to tissue degeneration as compared to normal tissue in younger age groups. So-called aging is also included in tissue abnormalities. Representative symptoms of aging include, for example, decreased visual acuity, decreased hearing, decreased muscle strength, decreased bone density, decreased renal function, decreased liver function, decreased pancreatic function, decreased number of neurons, decreased memory ability, and the like. The aging symptoms may be tissue atrophy, fibrosis, a decrease in the number of cells, pigmentation, a change in the tissue or the whole body that shortens the life span, and the like. In addition, tissue abnormalities include injured or damaged parts caused by physical or physiological defects in the tissue. The sites where the diseases or tissue abnormalities occur include the esophagus, stomach, pancreas, liver, spleen, kidney. , Small intestine, duodenum, large intestine, skin, eye, ear, nasal cavity, trachea, lung, blood vessel, brain, bone, muscle, pharynx, nerve (spinal cord, peripheral nerve, etc.).
The tissue abnormality may be a tissue abnormality that occurs or progresses with aging. Moreover, administration of the pharmaceutical composition may suppress the occurrence or progression of the tissue abnormality associated with aging.

In the above method, suppression of the onset of a disease or tissue abnormality may be achieved based on the ability of endogenous stem cells. The pharmaceutical composition according to the present disclosure includes various components including cytokines, and these components can stimulate the ability of endogenous stem cells to suppress the onset of diseases or tissue abnormalities. It is.

Of course, the use of the pharmaceutical composition according to the present disclosure is not limited to preemptive medicine. For this reason, according to the present disclosure, there is also provided a method for treating a disease, comprising administering the pharmaceutical composition according to the present disclosure to a subject having the disease in an amount effective for treating the disease.
The subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ).

In the method for treating a disease, the dose of the pharmaceutical composition is, for example, 0.1 mg / kg / day to 1000 mg / kg / day in terms of the amount of untreated culture supernatant, and 1 mg / kg It may be from kg / day to 100 mg / kg / day. Moreover, the administration method is not particularly limited. For example, the pharmaceutical composition is preferably administered parenterally, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying. Examples of methods for administering the pharmaceutical composition include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary absorption), and nasal cavity. An internal administration etc. can be mentioned. Of these, intranasal administration, transpulmonary administration and the like are preferable because they are minimally invasive. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.

In the present disclosure, the range of “treatment” includes not only a treatment to cure a disease or tissue abnormality, but also stops the progression of the disease or tissue abnormality until it is not completely cured, or delays compared to a case in which no treatment is performed. Treatment is also included. For example, it is also within the scope of treatment to stop or delay the progression of diseases and tissue abnormalities due to aging. Further, in the present disclosure, the start time of treatment may be after onset is observed, or may be performed before onset as described above.

In the disease treatment method, the administration method can be selected by those skilled in the art based on the type of disease tissue, the type of disease, and the like. For example, when the tissue is in the head, it is not necessary to consider the passage through the blood brain barrier, and it is particularly preferable to apply intranasal administration or the like because it is minimally invasive. For example, when the tissue is the brain, intranasal administration can be preferably applied. In addition, intranasal administration can be preferably applied to the treatment of cerebral infarction. Transpulmonary administration is less burdensome on the subject of administration because it can be administered for a long time in the living environment, and can be carried out by spraying the pharmaceutical composition in a mist form using a diffuser or the like.

Depending on the embodiment, iPS cells can also be administered in addition to the pharmaceutical composition according to the present disclosure. For example, the pharmaceutical composition and iPS cells can be administered to a subject simultaneously or at different times. In such a case, it is preferable to use iPS cells that have not been differentiated after collection (in other words, maintained in an undifferentiated state). When using the said pharmaceutical composition and iPS together, you may administer using the kit which consists of the 1st component containing the said pharmaceutical composition, and the 2nd component containing an iPS cell. Each component may be a separate capsule, for example, or a separate ampoule or vial.

In this case, the second component may be administered to the treatment target administered with the first component simultaneously with or after the administration of the first component. The use of administering the first component and the second component simultaneously is particularly suitable for application to acute and subacute disease. Here, “simultaneous” does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without substantial time difference is also included in the concept of “simultaneous” herein.

The disease treated by the disease treatment method is not particularly limited. For example, gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular heart disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, pulmonary fibrosis, (I Langerhans island atrophy (as seen in type II diabetes), liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases (amyotrophic lateral sclerosis, Alzheimer's disease) Parkinson's disease, progressive supranuclear palsy, Huntington's disease, multiple system atrophy, spinocerebellar degeneration etc.), neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. , Retinal disease with neuronal damage (traumatic retinal detachment, retinal tear, retinal shaking, optic nerve tube fracture, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, colloidemia, label congenital blindness, cone Body Rofi, familial drusen, central areolar choroidal dystrophy, autosomal dominant optic atrophy etc.), intractable neurological diseases, periodontal disease, skin ulcers, osteoporosis, and the like.

The site of the disease includes the esophagus, stomach, pancreas, liver, spleen, kidney, small intestine, duodenum, large intestine, skin, eye, ear, nasal cavity, trachea, lung, blood vessel, brain, bone, muscle, pharynx, nerve (spinal cord) Peripheral nerve, etc.). The disease may be a disease that develops or progresses with aging. Moreover, administration of the pharmaceutical composition may suppress the occurrence or progression of the disease associated with aging.

In the above method, treatment of a disease may be achieved based on the ability of endogenous stem cells. This is because the pharmaceutical composition according to the present disclosure includes various components including cytokines, and these components can stimulate the ability of endogenous stem cells to achieve treatment of a disease.

In addition, according to the present disclosure, there is also provided a method for treating a tissue abnormality, comprising administering the pharmaceutical composition according to the present disclosure to a subject having a tissue abnormality in an amount effective for treating the tissue abnormality. Is done.
The subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ).
In the method for treating tissue abnormality, the dosage of the pharmaceutical composition is, for example, 0.1 mg / kg / day to 1000 mg / kg / day in terms of the amount of untreated culture supernatant, and 1 mg / Kg / day to 100 mg / kg / day. Moreover, the administration method is not particularly limited. For example, the pharmaceutical composition is preferably administered parenterally, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying. Examples of methods for administering the pharmaceutical composition include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary absorption), and nasal cavity. An internal administration etc. can be mentioned. Of these, intranasal administration, transpulmonary administration and the like are preferable because they are minimally invasive. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the sex, age, weight, disease state, etc. of the subject can be considered.

In the method for treating a tissue abnormality, the administration method can be selected by those skilled in the art based on the type of tissue causing the tissue abnormality, the type of disease, and the like. For example, when the tissue is in the head, it is not necessary to consider the passage through the blood brain barrier, and it is particularly preferable to apply intranasal administration or the like because it is minimally invasive. For example, when the tissue is the brain, intranasal administration can be preferably applied. Transpulmonary administration is less burdensome on the subject of administration because it can be administered for a long time in the living environment, and can be carried out by spraying the pharmaceutical composition in a mist form using a diffuser or the like.

Depending on the embodiment, iPS cells can also be administered in addition to the pharmaceutical composition according to the present disclosure. For example, the pharmaceutical composition and iPS cells can be administered to a subject simultaneously or at different times. In such a case, it is preferable to use iPS cells that have not been differentiated after collection (in other words, maintained in an undifferentiated state).

In this case, the second component may be administered to the treatment target administered with the first component simultaneously with or after the administration of the first component. The use of administering the first component and the second component simultaneously is particularly suitable for application to acute and subacute tissue abnormalities. Here, “simultaneous” does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein.

Examples of the tissue abnormalities include aging represented by decreased visual acuity, decreased hearing, decreased muscle strength, decreased bone density, decreased renal function, decreased liver function, decreased pancreatic function, decreased number of neurons, decreased memory ability, etc. Examples include trauma and chemical trauma caused by exposure of tissues to chemical substances. Further, an injured part or a damaged part due to a physical or physiological defect of the tissue is also included. The aging symptom may be tissue atrophy, fibrosis, a decrease in the number of cells, pigmentation, a tissue change or systemic change that shortens the life span, and the like.
Examples of the tissue abnormalities include esophagus, stomach, pancreas, liver, spleen, kidney, small intestine, duodenum, large intestine, skin, eye, ear, nasal cavity, trachea, lung, blood vessel, brain, bone, muscle, pharynx, nerve ( Spinal cord, peripheral nerve, etc.).
The tissue abnormality may be a tissue abnormality that occurs or progresses with aging. Moreover, administration of the pharmaceutical composition may suppress the occurrence or progression of the tissue abnormality associated with aging.

In the above method, treatment of tissue abnormality may be achieved based on the ability of endogenous stem cells. This is because the pharmaceutical composition according to the present disclosure includes various components including cytokines, and these components can stimulate the ability of endogenous stem cells to achieve treatment of tissue abnormalities. .

According to the present disclosure, use of the iPS cell culture supernatant obtained by culturing iPS cells in the production of a pharmaceutical composition is also provided. For details of the iPS cell culture supernatant, the pharmaceutical composition and the method of use, refer to the above description (see the description of the pharmaceutical composition according to the present disclosure and the production method thereof, and various treatment methods according to the present disclosure). It is as follows.

According to the present disclosure, a cosmetic product containing an iPS cell culture supernatant obtained by culturing iPS cells can also be provided. The details of the iPS cell culture supernatant and the production method thereof are the same as the details of the pharmaceutical composition and the production method.

Also in the production of the cosmetic product, as described above, the culture supernatant of iPS cells can be obtained by separating and removing the cell components after culturing iPS cells. It is not limited to the separated supernatant itself, but on cultures that have been appropriately subjected to various treatments (eg, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) It may be Qing. The details of the culture supernatant treatment method are as described above as the culture supernatant treatment method in the pharmaceutical composition.

The cosmetic according to the present disclosure contains the culture supernatant of iPS cells obtained as described above as an active ingredient, and in one embodiment, the cosmetic does not contain the iPS cells as a whole composition. Moreover, in another embodiment, the said cosmetics do not contain a cell (the kind of cell is not ask | required) as the whole composition. That is, it is cell-free.

Depending on the condition of the subject to be applied, the pharmaceutical composition according to the present disclosure may additionally contain other components, provided that the expected therapeutic effect is maintained. Examples of ingredients that can be additionally included include emollients, surfactants, external analgesics, keratin softeners, plasticizers, lubricants, solubilizers, reducing agents, buffers, repellents, insecticides , Foaming agent, adsorbent, chelating agent, binder, thinning agent polishing / scrub agent, anti-acne agent, anti-cariogenic agent, antidepressant agent, antibacterial agent, oral hygiene agent, oral care agent, anti-caking agent, enzyme Agent, anti-dandruff agent, flavoring agent, fragrance, bactericidal agent, oxidizing agent, antioxidant, ultraviolet absorber / scattering agent, astringent, deodorant, antifoaming agent, artificial nail agent, hydrophilic thickener, antibacterial agent Sweat agent, cleaning agent, extender, anti-fading agent, antistatic agent, hair remover, colorant, nail conditioning agent, emulsifying stabilizer, emulsifier, adhesive, permanent wave reducing agent, release agent, inactive agent system Dispersant, non-aqueous thickener, skin conditioning agent, skin bleach, skin protectant, film form Agent, surface modifier, corrosion inhibitor, physical hair remover, opacifier, dispersant, propellant, hair conditioning agent, hair styling agent, occlusive agent, pH adjuster, denaturant, antiseptic, moisturizing / wetting Agents, water retention agents, hair coloring agents, oral health care drugs, and skin protection solvents.

The final form of the cosmetic according to the present disclosure is not particularly limited. Examples of forms are liquid, emulsion, cream, ointment, spray, gel and the like.

In this disclosure,
(1) culturing iPS cells;
(2) A step of recovering the culture supernatant obtained by culturing the iPS cells.
A method for producing a cosmetic product is also provided. In this production method, the collected culture supernatant is subjected to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting. May further be included. The production method may further include a step of adding an additional component to the collected culture supernatant. The production method may further include a step of creating the iPS cell by introducing an reprogramming factor into a somatic cell. About each step, the matter described in description of the pharmaceutical composition based on this indication is applied as it is. For the additional components, the matters described in the description of the cosmetic product according to the present disclosure are applied as they are. Subjecting the collected culture supernatant to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting; When the culture supernatant includes both steps of adding additional components, both steps may be performed first, and if possible, may be performed simultaneously.

According to the present disclosure, a cosmetic method for improving the cosmetic appearance of a subject by applying the cosmetic according to the present disclosure to the subject is also provided.
The target is, for example, a human, and the target tissue is, for example, skin or hair. The cosmetic can be applied to parts such as the face, neck, arms, and feet, for example. It can also be applied to these areas to eliminate burns and trauma marks.
The application may be performed once to three times a day, or may be continuously applied by spraying in a mist using a diffuser or the like.

Since the cosmetic product according to the present disclosure includes the iPS cell culture supernatant, the cosmetic product exhibits an effect of repairing changes that impair the cosmetic appearance, such as injury to collagen and elastin tissue that cause skin wrinkles. Thus, the cosmetic product according to the present disclosure is considered to exhibit an effect of improving the cosmetic appearance of the target by being applied to a part related to the cosmetic appearance of the target.
The cosmetic product according to the present disclosure may be one that suppresses the occurrence or deterioration of cosmetic defects, such as wrinkles, sagging, dullness, and spots, that occur or progress with aging.

As described above, the iPS cell culture supernatant obtained by culturing iPS cells can widely suppress the occurrence or progression of aging-related diseases, tissue abnormalities, cosmetic defects, and the like. Therefore, according to the present disclosure, an anti-aging composition containing an iPS culture supernatant obtained by culturing iPS cells can also be provided. About the iPS culture supernatant contained in the anti-aging composition, the details of the production method, origin, preliminary treatment, form, content and the like are the same as the contents explained in the explanation of the pharmaceutical composition above. . The expression of aging may be tissue atrophy, fibrosis, cell number decrease, pigmentation, tissue or systemic changes that shorten life span, and the like. Also provided is a method for inhibiting aging in a subject, comprising administering to the subject an amount of the anti-aging composition effective to exert an anti-aging effect. Administration of the anti-aging composition to a subject may extend the life of the subject.

Embodiments of the present invention include the following.
<1> A pharmaceutical composition comprising an iPS cell culture supernatant obtained by culturing iPS cells.
<2> The pharmaceutical composition according to <1>, which does not include the iPS cell.
<3> The pharmaceutical composition according to <1> or <2>, which is cell-free.
<4> The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. The pharmaceutical composition according to any one of <1> to <3>.
<5> The pharmaceutical composition according to any one of <1> to <4>, wherein the iPS cell is derived from a bone marrow-derived mesenchymal stem cell.
<6> The pharmaceutical composition according to any one of <1> to <5>, which does not contain serum.

<7> The pharmaceutical composition according to any one of <1> to <6>, which is used for suppressing the onset of the disease or tissue abnormality before the onset of the disease or tissue abnormality in the subject.
<8> The pharmaceutical composition according to <7>, wherein the subject is a subject determined to have a risk of developing the disease or tissue abnormality.
<9> The above diseases are gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. <7> or <8>, wherein the pharmaceutical composition is selected from the group consisting of dropout, periventricular leukomalacia, retinal disease with neuronal damage, intractable neuropathy, periodontal disease, skin ulcer, and osteoporosis object.
<10> The pharmaceutical composition according to any one of <7> to <9>, wherein the tissue abnormality is aging.
<11> The pharmaceutical composition according to <7>, which is a universal pharmaceutical composition in which the disease or tissue abnormality is not limited to a specific disease or tissue abnormality.
<12> The pharmaceutical composition according to any one of <1> to <6>, which is used for treating a disease.
<13> The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. <12> The pharmaceutical composition according to <12>, selected from the group consisting of dropout, periventricular leukomalacia, retinal disease associated with nerve cell damage, intractable nerve disease, periodontal disease, skin ulcer, and osteoporosis.

<14> The pharmaceutical composition according to any one of <1> to <6>, which is used for treating a tissue abnormality.
<15> The pharmaceutical composition according to <14>, wherein the tissue abnormality is aging.
<16> (1) a step of culturing iPS cells; and (2) a step of collecting a culture supernatant obtained by culturing the iPS cells,
A method for producing a pharmaceutical composition, comprising:
<17> The method according to <16>, further comprising a step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.
<18> The collected culture supernatant is subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The method according to <16> or <17>, further comprising a step.
<19> The method according to any one of <16> to <18>, further comprising a step of adding an additional component to the collected culture supernatant.
<20> Use of an iPS cell culture supernatant obtained by culturing iPS cells in the production of a pharmaceutical composition.

<21> The use according to <20>, wherein the pharmaceutical composition does not contain the iPS cell.
<22> The use according to <20> or <21>, wherein the pharmaceutical composition is cell-free.
<23> The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The use according to any one of <20> to <22>.
<24> The use according to any one of <20> to <23>, wherein the iPS cell is derived from a bone marrow-derived mesenchymal stem cell.
<25> The use according to any one of <20> to <24>, wherein the pharmaceutical composition does not contain serum.
<26> The use according to any one of <20> to <25>, wherein the pharmaceutical composition is used to suppress the onset of the disease or tissue abnormality before the onset of the disease or tissue abnormality in the subject.
<27> The use according to <26>, wherein the subject is a subject determined to have a risk of developing the disease or tissue abnormality.

<28> The above diseases are gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, aging change of heart, hypertension, ischemic heart disease, valvular heart disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. The use according to <26> or <27>, which is selected from the group consisting of dropout, periventricular leukomalacia, retinal disease with neuronal damage, intractable neuropathy, periodontal disease, skin ulcer, and osteoporosis.
<29> The use according to any one of <26> to <28>, wherein the tissue abnormality is aging.
<30> The use according to <26>, wherein the disease or tissue abnormality is not limited to a specific disease or tissue abnormality, and the pharmaceutical composition is a universal pharmaceutical composition.
<31> The use according to any one of <20> to <25>, wherein the pharmaceutical composition is used for treating a disease.
<32> The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. The use according to <31>, which is selected from the group consisting of dropout, periventricular leukomalacia, retinal disease with neuronal damage, intractable neuropathy, periodontal disease, skin ulcer, and osteoporosis.
<33> The use according to any one of <20> to <25>, wherein the pharmaceutical composition is used for treating a tissue abnormality.
<34> The use according to <33>, wherein the tissue abnormality is aging.

<35> The pharmaceutical composition according to any one of <1> to <6> is administered to a subject before the onset of the disease or tissue abnormality in an amount effective for suppressing the onset of the disease or tissue abnormality. A method of suppressing the onset of the disease or tissue abnormality before the onset of the disease or tissue abnormality in the subject.
<36> The method according to <35>, wherein the subject is a subject determined to have a risk of developing the disease or tissue abnormality.
<37> The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. <35> or <36>, wherein the method is selected from the group consisting of dropout, periventricular leukomalacia, retinal disease with neuronal damage, intractable nerve disease, periodontal disease, skin ulcer, and osteoporosis.
<38> The method according to any one of <35> to <37>, wherein the tissue abnormality is aging.
<39> The method according to <35>, wherein the disease or tissue abnormality is not limited to a specific disease or tissue abnormality, and the pharmaceutical composition is a universal pharmaceutical composition.
<40> The method according to any one of <35> to <39>, wherein the suppression of the onset of the disease or tissue abnormality is achieved based on the ability of endogenous stem cells.

<41> A method for treating a disease, comprising administering the pharmaceutical composition according to any one of <1> to <6> to a subject having a disease in an amount effective for treating the disease. .
<42> The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related change of heart, hypertension, ischemic heart disease, valvular disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, lung Fibrosis, Langerhans Island atrophy, liver fibrosis, chronic bronchitis, stroke, cerebral ischemia, cerebral infarction, dementia, epilepsy, neurodegenerative diseases, cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, etc. <41> The method according to <41>, which is selected from the group consisting of dropout, periventricular leukomalacia, retinal disease associated with nerve cell damage, intractable nerve disease, periodontal disease, skin ulcer, and osteoporosis.
<43> The method according to <41> or <42>, wherein the treatment of the disease is achieved based on the ability of endogenous stem cells.
<44> A tissue abnormality comprising administering the pharmaceutical composition according to any one of <1> to <6> to a subject having a tissue abnormality in an amount effective for treating the tissue abnormality. Treatment methods.
<45> The method according to <44>, wherein the tissue abnormality is aging.
<46> The method according to <45>, wherein the treatment of the tissue abnormality is achieved based on the ability of endogenous stem cells.
<47>
The administration of the pharmaceutical composition was selected from the group consisting of intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration, and intranasal administration. The method according to any one of <41> to <46>, which is performed by a method.

<48> The method according to any one of <41> to <47>, wherein the pharmaceutical composition is administered by pulmonary administration or intranasal administration.
<49> A cosmetic comprising an iPS cell culture supernatant obtained by culturing iPS cells.
<50> The cosmetic according to <49>, which does not include the iPS cell.
<51>
The cosmetic according to <49> or <50>, which is cell-free.
<52>
The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The cosmetic product according to any one of 49> to <51>.
<53>
The cosmetic according to any one of <49> to <52>, wherein the iPS cells are derived from bone marrow-derived mesenchymal stem cells.
<54>
The cosmetic according to any one of <49> to <53>, which does not contain serum.
<55>
The cosmetic according to any one of <49> to <54>, which is used for improving a skin condition selected from the group consisting of stains, wrinkles, sagging and dullness.

<56>
A cosmetic method for improving the cosmetic appearance of a subject by applying the cosmetic according to any one of <49> to <54> to the subject.
<57>
The cosmetic method according to <56>, wherein the improvement of the cosmetic appearance is achieved based on the ability of endogenous stem cells.
<58>
The cosmetic method according to <56> or <57>, wherein the cosmetic is applied to the subject's skin to improve the skin condition selected from the group consisting of spots, wrinkles, sagging and dullness.
<59>
(1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells;
A method for producing cosmetics, comprising:
<60>
The method according to <59>, further comprising the step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.

<61>
The recovered culture supernatant is further subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. The method according to <59> or <60>.
<62>
The method according to any one of <59> to <61>, further comprising a step of adding an additional component to the collected culture supernatant.
<63> An anti-aging composition comprising an iPS cell culture supernatant obtained by culturing iPS cells.
<64> The anti-aging composition according to <63>, which does not include the iPS cell.
<65> The anti-aging composition according to <63> or <64>, which is cell-free.
<66> The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The anti-aging composition according to any one of <63> to <65>.
<67> The anti-aging composition according to any one of <63> to <66>, wherein the iPS cells are derived from bone marrow-derived mesenchymal stem cells.
<68> The anti-aging composition according to any one of <63> to <67>, which does not contain serum.
<69> (1) a step of culturing iPS cells; and (2) a step of collecting a culture supernatant obtained by culturing the iPS cells.
The manufacturing method of an anti-aging composition containing this.
<70> The method according to <69>, further comprising a step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.
<71> The collected culture supernatant is subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The method according to <69> or <70>, further comprising a step.
<72> The method according to any one of <69> to <71>, further comprising a step of adding an additional component to the collected culture supernatant.
<73> Aging in a subject comprising administering an anti-aging composition according to any one of <63> to <68> to the subject in an amount effective to exert an anti-aging effect. How to suppress.

Hereinafter, examples of the present invention will be described, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

Example 1: Preemptive medicine in various tissues in senescence-accelerated model mice Diseases with and without administration of the iPS cell culture supernatant according to the present disclosure using senescence-accelerated mouse (SAM) Differences in tissue degeneration were examined.
Specifically, a culture supernatant of human iPS cells was obtained as follows. In the following, culture was performed at 37 ° C.
(IPS cells)
Except for using human bone marrow-derived mesenchymal cells as a source of iPS cells, Takahashi K, et al. "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors" Cell. 131 (5): 861 -PS (2007), iPS cells prepared according to the method described in the above were obtained from Kyoto University. Specifically, a retrovirus (pMX vector) containing human Oct3 / 4, Sox2, Klf4 and c-Myc was introduced into human bone marrow-derived mesenchymal cells, 6 days after the introduction, the cells were trypsinized, and mitomycin C Were plated on SNL feeder cells treated with (McMahon and Bradley, 1990). On the next day, the medium was changed from 10% FBS-containing DMEM to a primate ES cell culture medium (primate ES cell culture, manufactured by Reprocell) supplemented with 4 ng / ml bFGF.
After about 2 weeks, spherical colonies having human ES cell-like morphology appeared. On day 25, colonies that were flat and similar to human ES cells were observed. The morphology of each cell was similar to that of human ES cells, and spontaneous differentiation was sometimes seen in the center of the colony. In addition, although it has feeder cell dependency or remains undifferentiated in the mouse embryonic fibroblast (MEF) primate ES cell culture medium on the matrigel-coated plate, it remains undifferentiated in the unconditioned medium. The obtained cells showed similar properties to human ES cells in that they were not retained.
The iPS cell culture supernatant was prepared using the human iPS cells thus obtained.

(Preparation of culture supernatant)
The human iPS cells were maintained by adhesion culture in a petri dish for adhesion culture. In culture, primate ES cell medium supplemented with 4 ng / ml human basic FGF (bFGF) (serum-free; manufactured by Reprocell Corporation; hereinafter, this bFGF-added primate ES cell medium is referred to as iPS cell medium. Used). During culture, passage is required every week, which involves treating human iPS cells with a solution containing 0.25% trypsin, 0.1 mg / mL collagenase IV, 1 mM CaCl ₂ and 20% KSR. It was done by doing.
The human iPS cells maintained as described above were detached from the adhesion culture petri dish using an ES cell dissociation solution (manufactured by Reprocell Co., Ltd.), and suspended in the iPS cell culture medium placed in the non-adhesion culture petri dish for 1 week. . As a result, an embryoid body (EB) was formed.
The formed embryoid bodies (EBs) were seeded on adherent culture dishes and grown for 1 week in DMEM containing 10% FBS and 1% anti-anti (registered trademark, antifungal agent) ( For cell outgrowth, see Stem Cells Dev. 22, 102-113, 2013).
Next, the human iPS cells were detached from the adhesion culture dish using a 0.05% trypsin-EDTA solution, and seeded in a new adhesion culture dish. As a result, human iPS cells became single cells. Using DMEM containing 10% FBS and 1% anti-anti (registered trademark, antifungal agent) as a medium, the cells were cultured for one week.
After confirming that human iPS cells became 70-80% or more confluent, the medium was replaced with serum-free medium (DMEM containing 1% anti-anti (registered trademark, antifungal agent) without FBS). After culturing for one day (48 hours), the supernatant was collected. The collected supernatant was centrifuged at 1500 rpm for 5 minutes, and the supernatant was collected again and then centrifuged at 3000 rpm for 3 minutes. The supernatant was collected again and used as a human iPS cell culture supernatant in subsequent experiments. The centrifugation was performed using a tabletop multi-centrifuge LC-120 manufactured by Tommy Seiko.

SAMP8 (6 weeks old, Nippon SLC Co., Ltd.) was prepared. Further, the iPS cell culture supernatant obtained above was diluted with purified water to a concentration of 1% and atomized with ultrasound to fill the animal cage. Further, without using the iPS cell culture supernatant, only purified water was atomized to fill the animal cage. For the experimental group (n = 5 for each male and female), a cycle of 12 hours in an animal cage filled with the mist containing the above iPS cell culture supernatant and then 12 hours in an animal cage not containing the mist. Went. For the control group (n = 5 for each male and female), a cycle of 12 hours in an animal cage filled with the purified water mist was followed by 12 hours in an animal cage not containing the mist.

On the 7th, 40th and 60th weeks after the start of the experiment, the aging degree criterion proposed by Toshio Takeda (grading score system; “Grading score system: a method for evaluation of the degree of senescence in senescence accelerated mouse ( SAM) "authored by Hosokawa M, Kasai R, Higuchi K, Takeshita S, Shimizu K, Hamamoto H, Honma A, Irino M, Toda K, Matsumura A, et al., Mech Ageing Dev. : 91-102) and the survival time during the observation period was measured.

In an environment of 100% humidity, the mouse absorbs approximately 2% of water every breathing cycle through the alveoli. By absorption of the mist containing the above iPS cell culture supernatant, about 0.01% of the culture supernatant is absorbed for each respiratory cycle. Since the respiratory rate of the mouse is about 50 times per minute, it is considered that about 0.5% of the iPS cell culture supernatant was absorbed into the blood per minute.

FIG. 1 shows lung conditions at 6 weeks of age (at the start of the experiment) and lung conditions of the experimental group (iPS-CM) and control group at 30 weeks of age. As can be seen from FIG. 1, pulmonary fibrosis is progressing in the control group, whereas the progression of pulmonary fibrosis is significantly suppressed in the experimental group. From this, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of pulmonary diseases such as pulmonary fibrosis and abnormal tissue of the lung.

FIG. 2 shows the state of pancreatic islets at 6 weeks of age (at the start of the experiment) and the state of pancreatic islets in the experimental group (iPS-CM) and the control group at 30 weeks of age. As can be seen from FIG. 2, the Langerhans island atrophy is progressing in the control group, while the Langerhans island atrophy is remarkably suppressed in the experimental group. Therefore, the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of pancreatic diseases and pancreatic tissue abnormalities such as Langerhans island atrophy (as seen in type I and type II diabetes). I understand.

FIG. 3 shows the state of the liver at 6 weeks of age (at the start of the experiment) and the state of the livers of the experimental group (iPS-CM) and the control group at 30 weeks of age. As can be seen from FIG. 3, liver fibrosis is progressing in the control group, whereas the progression of liver fibrosis is significantly suppressed in the experimental group. From this, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of liver diseases such as liver fibrosis and liver tissue abnormalities.

FIG. 4 shows the state of collagen accumulation in the rat dermis at 7 weeks, 30 weeks and 70 weeks of age. As can be seen from FIG. 4, in the control group, the decrease in collagen content has already occurred at 7 weeks of age, whereas in the experimental group, the decrease in collagen content is significantly suppressed even at 40 and 70 weeks of age. ing. From this, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of skin diseases and skin tissue abnormalities including a decrease in collagen content.

Fig. 5 shows Morris, R (May 1984). "Developments of a water-maze procedure for studying spatial learning in the rat."", Journal of neuroscience methods 11 (1): 47-60). Specifically, water was spread to a depth of 30 cm in a cylindrical container having a diameter of 150 cm, and an invisible platform rising from the water surface to 1 cm to 2 cm was provided therein. Using this platform, we measured the time to escape from the water maze. Compared to the control group, the experimental group showed a significant decrease in the required time with an increase in the number of experiments, and high learning memory ability. It can be seen that it is maintained.
From this result, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of brain function deterioration and brain diseases including learning memory ability decline.

FIG. 6 shows the state in the brain at 30 weeks of age. As can be seen from FIG. 6, in the control group, nerve degeneration and nerve cell decrease occurred, whereas in the experimental group, nerve tissue was kept normal and the decrease in nerve cells was remarkably suppressed. From this, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the onset of brain diseases and brain tissue abnormalities including prevention of neurodegeneration and prevention of neuronal cell loss.

FIG. 7 shows the target site for measuring the number of nerve cells in the brain at 30 weeks of age. The results of measuring the number of nerve cells are shown in FIG. In FIG. 8, “Pre” represents the control group, “Post” represents the experimental group, and the vertical axis represents the decrease in the number of cells. As can be seen from FIG. 8, in the control group, a decrease in the number of neurons occurs, whereas in the experimental group, the decrease in the number of neurons is remarkably suppressed. From this, it can be seen that the pharmaceutical composition according to the present disclosure has the effect of suppressing the onset of brain diseases such as prevention of neuronal cell loss and brain tissue abnormalities.

FIG. 9 shows survival curves for the experimental group (iPS-CM group) and the control group. As can be seen from FIG. 9, in the control group, the survival rate began to decrease at an early stage after the start of the experiment in both the male and female cases, and the life expectancy was 63 weeks. On the other hand, in the experimental group, the lifespan was significantly extended in both the male and female cases, with an average life of 120 weeks. From this, it can be seen that the pharmaceutical composition according to the present disclosure has an effect of suppressing the progress of aging.

(Human iPS cells prepared using plasmid)
In addition, human iPS cells prepared according to the method described in Okita K. et al .: Nat Methods 8, 409-412, except that human bone marrow-derived mesenchymal cells were used as the source cells of iPS cells. But I did an experiment. Specifically, the p53 shRNA expression cassette driven by the mouse U6 promoter was inserted into the BamHI site of plasmid pCXLE-hOCT3 / 4-shp53 (pCXLE-hOCT3 / 4 with OCT3 / 4 (Addgene accession code 27076)) ), PCXLE-hSK (with Addgene accession code 27078, SOX2 and KLF4) and pCXLE-hUL (with Addgene accession code 27080, L-MYC and LIN28), and a microporator (Invitrogen) ) Was introduced into 6 × 10 ⁵ human bone marrow-derived mesenchymal cells by electroporation. The introduction conditions were 1800 V, 20 ms, and one pulse. Cells were trypsinized 7 days after transfection and 1 × 10 ⁵ cells were re-plated into 100 mm dishes covered with SNL or MEF feeder layers. On the next day, the medium was replaced with the medium for iPS cells. Colonies were counted 26-32 days after plating. Colonies similar to human ES cells were selected. Using the human iPS cells thus obtained, the culture supernatant was prepared in the same manner as described above. And the effect at the time of making it inhale to a SAMP8 mouse | mouth similarly to the above using this culture supernatant was investigated.
As a result, the same results as in the case of human iPS cells prepared using a retrovirus were obtained. Thus, regardless of the iPS cell production method, the iPS cell culture supernatant had excellent properties in terms of preemptive medicine and anti-aging.

As described above, due to the universal and powerful action of the iPS cell culture supernatant according to the present disclosure, if the pharmaceutical composition or cosmetic according to the present disclosure is used, preemptive medical treatment or treatment of disease or tissue abnormality (including progression inhibition) ) Or maintaining a cosmetic appearance.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually described to be incorporated by reference, Incorporated herein by reference.
The entire disclosure of Japanese Patent Application No. 2015-003493 filed on January 9, 2015 is incorporated herein by reference.

Claims

A pharmaceutical composition comprising an iPS cell culture supernatant obtained by culturing iPS cells.
The pharmaceutical composition according to claim 1, which does not contain the iPS cell.
The pharmaceutical composition according to claim 1 or 2, which is cell-free.
The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. The pharmaceutical composition according to any one of claims 1 to 3.
The pharmaceutical composition according to any one of claims 1 to 4, wherein the iPS cells are derived from bone marrow-derived mesenchymal stem cells.
6. The pharmaceutical composition according to any one of claims 1 to 5, which does not contain serum.
The pharmaceutical composition according to any one of claims 1 to 6, which is used for suppressing the onset of the disease or tissue abnormality before the onset of the disease or tissue abnormality in the subject.
The pharmaceutical composition according to claim 7, wherein the subject is determined to have a risk of developing the disease or tissue abnormality.
The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular heart disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, pulmonary fibrosis, Langerhans Island Atrophy, Liver Fibrosis, Chronic Bronchitis, Stroke, Cerebral Ischemia, Cerebral Infarction, Dementia, Epilepsy, Neurodegenerative Diseases, Neuronal Degeneration / Dropping due to Cerebral Infarction due to Cerebral Ischemia or Intracerebral Hemorrhage, Brain The pharmaceutical composition according to claim 7 or 8, selected from the group consisting of periventricular leukomalacia, retinal disease with nerve cell damage, intractable nerve disease, periodontal disease, skin ulcer, and osteoporosis.
The pharmaceutical composition according to any one of claims 7 to 9, wherein the tissue abnormality is aging.
The pharmaceutical composition according to claim 7, which is a universal pharmaceutical composition in which the disease or tissue abnormality is not limited to a specific disease or tissue abnormality.
The pharmaceutical composition according to any one of claims 1 to 6, which is used for treating a disease.
The disease is gastric ulcer, cirrhosis, liver failure, gallstone, cholangitis, jaundice, age-related changes in heart, hypertension, ischemic heart disease, valvular heart disease, arrhythmia, heart failure, arteriosclerosis, pneumonia, emphysema, pulmonary fibrosis, Langerhans Island Atrophy, Liver Fibrosis, Chronic Bronchitis, Stroke, Cerebral Ischemia, Cerebral Infarction, Dementia, Epilepsy, Neurodegenerative Diseases, Neuronal Degeneration / Dropping due to Cerebral Infarction due to Cerebral Ischemia or Intracerebral Hemorrhage, Brain 13. The pharmaceutical composition according to claim 12, selected from the group consisting of periventricular leukomalacia, retinal disease with neuronal damage, intractable nerve disease, periodontal disease, skin ulcer, and osteoporosis.
The pharmaceutical composition according to any one of claims 1 to 6, which is used for treating a tissue abnormality.
The pharmaceutical composition according to claim 14, wherein the tissue abnormality is aging.
(1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells;
A method for producing a pharmaceutical composition, comprising:
The method according to claim 16, further comprising the step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.
The recovered culture supernatant is further subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. 18. A method according to claim 16 or claim 17 comprising.
The method according to any one of claims 16 to 18, further comprising a step of adding an additional component to the collected culture supernatant.
Cosmetics containing iPS cell culture supernatant obtained by culturing iPS cells.
The cosmetic according to claim 20, which does not contain the iPS cells.
The cosmetic according to claim 20 or 21, which is cell-free.
The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. The cosmetic according to any one of Items 20 to 22.
The cosmetic according to any one of claims 20 to 23, wherein the iPS cells are derived from bone marrow-derived mesenchymal stem cells.
The cosmetic according to any one of claims 20 to 24, which does not contain serum.
The cosmetic according to any one of claims 20 to 25, which is used for improving a skin condition selected from the group consisting of spots, wrinkles, sagging and dullness.
A cosmetic method for improving the cosmetic appearance of a subject by applying the cosmetic according to any one of claims 20 to 25 to the subject.
The cosmetic method according to claim 27, wherein the improvement of the cosmetic appearance is achieved based on the ability of endogenous stem cells.
The cosmetic method according to claim 27 or 28, wherein the cosmetic is applied to a target skin to improve a skin condition selected from the group consisting of spots, wrinkles, sagging and dullness.
(1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells;
A method for producing cosmetics, comprising:
The method according to claim 30, further comprising the step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.
The recovered culture supernatant is further subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. 32. A method according to claim 30 or claim 31 comprising.
The method according to any one of claims 30 to 32, further comprising a step of adding an additional component to the collected culture supernatant.
An anti-aging composition comprising an iPS cell culture supernatant obtained by culturing iPS cells.