WO2024195863A1 - Mesenchymal stem cell growth promoter, medium for mesenchymal stem cells, mesenchymal stem cells and culture supernatant - Google Patents

Abstract

The purpose of the present invention is to provide a medium for culture excellent in growth of mesenchymal stem cells.　The present invention is a mesenchymal stem cell growth promoter containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. The mesenchymal stem cell growth promoter of the present invention preferably also contains 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof. The present invention also includes a medium for mesenchymal stem cells that contains16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.

Description

Mesenchymal stem cell proliferation promoter, medium for mesenchymal stem cells, mesenchymal stem cells and culture supernatant

The present invention relates to a mesenchymal stem cell proliferation promoter, a medium for mesenchymal stem cells, mesenchymal stem cells, and a culture supernatant.

Regenerative medicine is a medical treatment that uses cells and tissues to support and regenerate dysfunctional tissues and organs. This regenerative medicine has the potential to become a new treatment for diseases that are difficult to treat with conventional treatments, and its practical application is an urgent issue. In particular, regenerative medicine that uses stem cells derived from the body has attracted attention and research is being conducted. Of the above-mentioned stem cells derived from the body, research is being conducted mainly on ES cells and iPS cells as tissue regeneration techniques. On the other hand, somatic stem cells such as mesenchymal stem cells are attracting attention as a more feasible cell therapy because they are expected to have various effects such as anti-inflammatory and immunosuppressive effects in addition to their function of filling in the gaps in tissue damage.

Mesenchymal stem cells are multipotent precursor cells that were first isolated from bone marrow by Friedenstein (1982) (see Non-Patent Document 1). It has been revealed that these mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Patent Documents 1 and 2). In addition, it has been reported that mesenchymal stem cells have an immunosuppressive effect and accumulate in tumors, and research is being conducted on using mesenchymal stem cells to prevent rejection after transplantation (Non-Patent Document 2; Stem Cell Res Ther. 2021; 12: 192.) and to deliver cancer therapeutic drugs (Non-Patent Document 3; Cancer Res (2013) 73 (1): 364-372). Recently, it has become known that cells with equivalent functions exist in the interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc., and mesenchymal stem cells are sometimes called stromal cells.

Various media have been developed for culturing mesenchymal stem cells (see, for example, Patent Documents 3 and 4), but there is a need for a medium that can efficiently grow large amounts of mesenchymal stem cells that are more suitable for disease treatment while retaining their undifferentiated state.

JP 2012-157263 A Special Publication No. 2012-508733 US Patent Publication No. 2010/0015710 JP 2010-094062 A

Pittenger F. M. etal. , Science 284, pp. 143-147, 1999 Stem Cell Res Ther. , 12, pp. 192-, 2021 Cancer Res, 73(1), pp. 364-372, 2013

The present invention aims to provide a method for efficiently proliferating mesenchymal stem cells while maintaining their undifferentiated state under the circumstances described above, that is, to provide an agent that is highly effective in promoting the proliferation of mesenchymal stem cells, and a medium for mesenchymal stem cells.

As a result of intensive research to solve the above problems, the present inventors discovered that a medium containing a specific lysophosphatidic acid (LPA) is excellent in promoting the proliferation of mesenchymal stem (stromal) cells (MSCs), and thus completed the present invention. According to the present invention, it is possible to promote the proliferation of mesenchymal stem cells while maintaining their undifferentiated state. In other words, the gist of the present invention is as follows.

[1] A mesenchymal stem cell proliferation promoter comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[2] The mesenchymal stem cell proliferation promoter according to [1], further comprising 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof.
[3] The mesenchymal stem cell proliferation promoter according to [2], wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof is 0.1 to 10.
[4] The mesenchymal stem cell proliferation promoter described in [1], wherein the mesenchymal stem cells are derived from adipose tissue.
[5] An additive for mesenchymal stem cell culture, comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[6] A medium for mesenchymal stem cells, comprising 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[7] The medium for mesenchymal stem cells described in [6], further containing 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof.
[8] The medium for mesenchymal stem cells according to [7], wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof is 0.1 to 10.
[9] The medium for mesenchymal stem cells according to any one of [6] to [8], which is used for promoting the proliferation of mesenchymal stem cells.
[10] Mesenchymal stem cells obtained by culturing using the mesenchymal stem cell proliferation promoter according to any one of [1] to [4], the additive for mesenchymal stem cell culture according to [5], or the medium for mesenchymal stem cells according to any one of [6] to [9].
[11] A culture supernatant containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, obtained by culturing mesenchymal stem cells in a medium for mesenchymal stem cells according to any one of [6] to [9].
[12] A method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in a medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[13] The culture method according to [12], wherein the culture is a suspension/agitation culture.
[14] Use of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof for promoting the proliferation of mesenchymal stem cells.
[15] Use of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof for promoting the proliferation of mesenchymal stem cells.
[16] Use of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof for the manufacture of an agent for promoting the proliferation of mesenchymal stem cells.
[17] Use of 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof for the manufacture of a mesenchymal stem cell proliferation promoter.
[18] A method for promoting the proliferation of mesenchymal stem cells, comprising using a medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[19] A method for promoting the proliferation of mesenchymal stem cells, characterized by using a medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof.
[20] A method for promoting the proliferation of mesenchymal stem cells, comprising using 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.
[21] A method for promoting the proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof.

The mesenchymal stem cell proliferation promoter and mesenchymal stem cell culture medium of the present invention allow mesenchymal stem cells to grow efficiently while retaining their undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells in the mesenchymal stem cell culture medium of the present invention contains a large amount of various humoral factors such as exosomes and HGF, and therefore can be suitably used for disease treatment, etc.

Fig. 1 shows the proliferation promoting effect of LPA-containing medium on mesenchymal stem cells in planar adherent culture. AD: adipose-derived mesenchymal stem cells, UC: umbilical cord-derived mesenchymal stem cells. FIG. 2 shows the proliferation-promoting effect of an LPA-containing medium on adipose-derived mesenchymal stem cells in planar adherent culture. FIG. 3 shows the proliferation-promoting effect of an LPA-containing medium on umbilical cord-derived mesenchymal stem cells in planar adherent culture. FIG. 4 shows the results of investigating the concentration ratio of 16:0 LPA and 18:1 LPA in the culture of mesenchymal stem cells in an LPA-containing medium. FIG. 5 shows the results of investigating the LPA concentration when mesenchymal stem cells were cultured in an LPA-containing medium. FIG. 6 shows the proliferation-promoting effect of an LPA-containing medium on adipose-derived mesenchymal stem cells in suspension/agitated culture. FIG. 7 shows the exosome production promoting effect of an LPA-containing medium in planar adherent culture of mesenchymal stem cells. FIG. 8 shows the results of evaluating the adhesiveness of mesenchymal stem cells cultured in a medium containing LPA to various culture flasks. FIG. 9 shows the results of investigating the effect of LPA added to the medium on the number of cell divisions in planar adherent culture of adipose-derived mesenchymal stem cells. FIG. 10 shows the results of investigating the effect of LPA added to the medium on the variation in cell division number between cell lots in planar adherent culture of adipose-derived mesenchymal stem cells. FIG. 11 shows the results of counting the number of cell divisions during subculture of adipose-derived mesenchymal stem cells in planar adherent culture in an LPA-containing medium. FIG. 12 is a photograph showing the results of an angiogenesis test using HUVEC. FIG. 13 shows the effect of an LPA-containing medium on the number of cell divisions of umbilical cord-derived mesenchymal stem cells. FIG. 14 shows the effect of an LPA-containing medium on the number of divisions of dental pulp-derived mesenchymal stem cells.

The mesenchymal stem cell proliferation promoter, mesenchymal stem cell medium, mesenchymal stem cell culture method, mesenchymal stem cells, and culture supernatant according to the present invention will be described in detail below.

[Mesenchymal stem cell proliferation promoter]
The mesenchymal stem cell proliferation promoter of the present invention is characterized by containing a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof. By using the mesenchymal stem cell proliferation promoter of the present invention, mesenchymal stem cells can be efficiently proliferated while maintaining their undifferentiated state. In addition to the above-mentioned lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof, the mesenchymal stem cell proliferation promoter of the present invention may contain other components within a range that does not impair the effects of the present invention. The mesenchymal stem cell proliferation promoter of the present invention will be described in detail below.

In the present invention, the "undifferentiated" state of mesenchymal stem cells refers to a state in which mesenchymal stem cells maintain their ability to differentiate into bone cells, chondrocytes, and adipocytes.

In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (such as bone cells, cardiac muscle cells, chondrocytes, tendon cells, and adipocytes) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as interstitial cells and does not particularly distinguish between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Mesenchymal stem cells in the present invention include those derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc., among which adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are more preferred, and adipose tissue-derived mesenchymal stem cells are even more preferred.

In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically removing or aspirating subcutaneous fat from a mammal.

In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells.

In the present invention, bone marrow refers to the soft tissue that fills the inner cavity of bones, and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present therein are called bone marrow cells. Bone marrow cells include red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, and other cells, as well as mesenchymal stem cells, hematopoietic stem cells, and vascular endothelial precursor cells. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

In this invention, dental pulp is the connective tissue found in the core of the tooth, rich in blood vessels and nerves, and refers to the growth of the dental papilla in the tooth germ. It is the soft tissue that fills the pulp cavity inside the tooth.

In the present invention, the species of mesenchymal stem cells is preferably a mammal. Examples of mammals include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, rats, and monkeys. Of these, humans, horses, cows, and cats are preferred.

Mesenchymal stem cells may be cells provided by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, and Corning, or may be cells prepared by methods well known to those skilled in the art. In addition, mesenchymal stem cells may be primary cells isolated from donor tissue, or may be established cell lines.

In the present invention, lysophosphatidic acid (LPA) is a lysophospholipid having an unsubstituted phosphate group, and there are several types that have different combinations of alkyl chain length-number of double bonds (number of carbon atoms-degree of unsaturation). The above-mentioned carbon number-degree of unsaturation combinations (carbon number: degree of unsaturation) include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, 22:6, etc., and among these, from the viewpoint of excellent mesenchymal stem cell proliferation promoting effect, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3 are preferable, 16:0, 18:1 are more preferable, and 16:0 is even more preferable. In addition, the mesenchymal stem cell proliferation promoter of the present invention may contain multiple types of lysophosphatidic acid having different combinations of alkyl chain length-number of double bonds (carbon number-degree of unsaturation). The lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention may be a derivative of lysophosphatidic acid. Examples of derivatives of lysophosphatidic acid include lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol. The lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention may be in any form, such as a salt. When lysophosphatidic acid is in the form of a salt, examples of the salt include monosodium salt, disodium salt, monopotassium salt, dipotassium salt, magnesium salt, and calcium salt.

The mesenchymal stem cell proliferation promoter of the present invention preferably contains 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, as this more significantly exhibits the effects of the present invention, and more preferably contains 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof in addition to 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.

The concentration of each lysophosphatidic acid contained in the mesenchymal stem cell proliferation promoter of the present invention, when applied to cells, is from 0.0025 μg/mL to 1 mg/mL, and may be from 0.01 μg/mL to 1 mg/mL, preferably from 0.1 μg/mL to 100 μg/mL, more preferably from 1 μg/mL to 50 μg/mL, even more preferably from 1.5 μg/mL to 25 μg/mL, particularly preferably from 2 μg/mL to 20 μg/mL, even more preferably from 2 μg/mL to 10 μg/mL, and most preferably from 2.5 μg/mL to 5 μg/mL.

The mesenchymal stem cell proliferation promoter of the present invention may contain a mixture of multiple lysophosphatidic acids, in which case the total concentration of all the lysophosphatidic acids when applied to cells is from 0.05 μg/mL to 1.25 mg/mL, preferably from 0.1 μg/mL to 100 μg/mL, more preferably from 0.3 μg/mL to 50 μg/mL, even more preferably from 1 μg/mL to 40 μg/mL, particularly preferably from 2 μg/mL to 30 μg/mL, and even more preferably from 5 μg/mL to 20 μg/mL. In addition, the mixing ratio of multiple lysophosphatidic acids is not particularly limited, but when two types of lysophosphatidic acid, 16:0 lysophosphatidic acid and 18:1 lysophosphatidic acid, are included, from the viewpoint of excellent mesenchymal stem cell proliferation promoting effect, the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid and/or its salt to the content (μg/mL) of 18:1 lysophosphatidic acid and/or its salt is 0.001 to 1000, preferably 0.01 to 100, more preferably 0.1 to 10, even more preferably 0.1 to 5, particularly preferably 0.3 to 3, and even more preferably 0.5 to 1.5.

The form of the mesenchymal stem cell proliferation promoter of the present invention is not particularly limited, and may be the above-mentioned lysophosphatidic acid itself, or may be a composition in which the above-mentioned lysophosphatidic acid is combined with other components. Furthermore, the form of the above-mentioned composition is not particularly limited. The above-mentioned composition may be, for example, a liquid medium used for culturing mesenchymal stem cells, or may be an additive for mesenchymal stem cell culture that is added during the preparation of the liquid medium.

A preferred embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid medium containing the above-mentioned lysophosphatidic acid at the above-mentioned concentration and/or ratio.

When the embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid medium, the mesenchymal stem cell proliferation promoter of the present invention is a conventionally known basal medium for animal cell culture containing the above-mentioned lysophosphatidic acid at the above-mentioned concentration and/or ratio.

The basal medium for animal cell culture in the present invention refers to a medium containing carbon sources, nitrogen sources, inorganic salts, etc., essential for culturing animal cells. Here, animal cells refer to mammalian cells, particularly human cells. The basal medium for animal cell culture in the present invention may contain biologically derived raw materials, but considering the possibility that the cells obtained by culturing and their culture supernatants may be used to treat diseases in animals (including humans), it is preferable that the medium contains as few biologically derived raw materials as possible from the standpoint of infectious risk.

As the basal medium for animal cell culture, a medium for animal cell culture known to those skilled in the art can be used. Specific examples include Minimum Essential Medium (MEM) such as Eagle's medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium α (MEM-α), Mesenchymal Cell Basal Medium (MSCBM), Ham's F-12 and F-10 medium, DMEM/F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove's Modified Dulbecco's Medium (IMDM), McCoy's modified medium, and mixtures thereof.

The basal medium for animal cell culture may be a known medium prepared for mesenchymal stem cells, such as Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use) manufactured by PromoCell, Mesenchymal Stem Cell Growth Medium XF (Ready-to-use) manufactured by PromoCell, MSCGM Bullet Kit, MSCGM Mesenchymal Stem Cell Growth Medium Bullet Kit. tm (Lonza), xeno-free medium for human mesenchymal stem cells (MSC NutriStem (registered trademark) XF, Biological Industries), MesenCult-ACF Plus (Veritas), StemXVivotm Serum-Free Human MSC Expansion Media (R&D Systems, Corning), serum-free medium for adipose-derived stem cells (KBM ADSC-4, Kohjin Bio), serum-free medium for mesenchymal stem cells (R:STEM Medium for hMSC High Growth, Rohto), etc.

When the embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid medium, the mesenchymal stem cell proliferation promoter of the present invention may contain, in addition to the above-mentioned basal medium for animal cell culture and the above-mentioned lysophosphatidic acid, as necessary, amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and magnesium sulfate, trace metals such as selenium, lipids (excluding the above-mentioned lysophosphatidic acid and its salts), vitamins such as pantothenic acid, albumin, insulin, transferrin, insulin, growth factors (e.g., epidermal growth factor, basic fibroblast growth factor), growth factors, proteins such as cytokines, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and other substances.

When the embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid medium, the mesenchymal stem cell proliferation promoter of the present invention may contain serum as necessary in addition to the basal animal cell culture medium and the lysophosphatidic acid. The serum is not particularly limited as long as it does not inhibit the proliferation of mesenchymal stem cells, but is an animal-derived serum, preferably a mammal-derived serum (e.g., fetal bovine serum, human serum, etc.), and more preferably a human serum. The concentration of the serum in the mesenchymal stem cell proliferation promoter of the present invention may be within a concentration range known to those skilled in the art. When the mesenchymal stem cells after culture are used for medical purposes, other animal-derived components may be a source of infection for blood-borne pathogens or xenoantigens, so it is preferable that the mesenchymal stem cell proliferation promoter of the present invention does not contain serum. If serum is not included, a serum substitute (e.g., Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid Concentrated (Gibco), etc.) may be used.

When the embodiment of the mesenchymal stem cell proliferation promoter of the present invention is a liquid medium, the osmotic pressure ratio is preferably 0.9 to 1.1. The pH is preferably 6.0 to 9.0, and more preferably 6.5 to 8.5. In addition, the liquid medium is preferably sterile, and the endotoxin amount is preferably 2.5 EU/mL or less.

The mesenchymal stem cell proliferation promoter of the present invention can be produced by mixing the above-mentioned components by a conventional method. The mesenchymal stem cell proliferation promoter of the present invention may also be provided in a concentrated state and diluted at the time of use.

As can be understood from the above explanation, the present invention relates to "use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof for promoting the proliferation of mesenchymal stem cells", "use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof, and use of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and/or salts thereof for promoting the proliferation of mesenchymal stem cells", Also included within the scope are "use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof to manufacture a mesenchymal stem cell proliferation promoter," and "use of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof, as well as 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and/or salts thereof to manufacture a mesenchymal stem cell proliferation promoter."

As can be understood from the above explanation, the present invention relates to "a method for promoting the proliferation of mesenchymal stem cells, characterized by using a medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof," and "a method for promoting the proliferation of mesenchymal stem cells, characterized by using a medium containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof." Also included within its scope are "a method for promoting cell proliferation," "a method for promoting proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof," and "a method for promoting proliferation of mesenchymal stem cells, characterized by using 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof."

[Medium for mesenchymal stem cells]
The present invention includes a medium for mesenchymal stem cells, which contains a basal medium for animal cell culture and a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof. The medium for stem cells has an excellent effect of promoting the proliferation of mesenchymal stem cells, and therefore can be suitably used for promoting the proliferation of stem cells. This corresponds to one embodiment of the stem cell proliferation promoting agent (when it is a liquid medium).

There are several types of lysophosphatidic acid with different combinations of alkyl chain length-number of double bonds (carbon number-degree of unsaturation), and specific examples of the carbon number-degree of unsaturation combinations (carbon number: degree of unsaturation) include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, 22:6, etc. Among these, from the viewpoint of excellent effect of promoting proliferation of mesenchymal stem cells, 16:0, 16:1, 18:0, 18:1, 18:2, and 18:3 are preferred, 16:0 and 18:1 are more preferred, and 16:0 is even more preferred. Furthermore, the mesenchymal stem cell medium of the present invention may contain multiple types of lysophosphatidic acid with different combinations of alkyl chain length-number of double bonds (number of carbon atoms-degree of unsaturation). Furthermore, the lysophosphatidic acid contained in the mesenchymal stem cell medium of the present invention may be a derivative of lysophosphatidic acid or may be in any form such as a salt. Specific examples of the above derivatives and salts are described in the section [Mesenchymal stem cell proliferation promoter].

The medium for mesenchymal stem cells of the present invention preferably contains 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, and more preferably contains 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof in addition to 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof.

The concentration of each lysophosphatidic acid contained in the mesenchymal stem cell medium of the present invention is 0.0025 μg/mL or more and 1 mg/mL or less, and may be 0.01 μg/mL or more and 1 mg/mL or less, preferably 0.1 μg/mL or more and 100 μg/mL or less, more preferably 1 μg/mL or more and 50 μg/mL or less, even more preferably 1.5 μg/mL or more and 25 μg/mL or less, particularly preferably 2 μg/mL or more and 20 μg/mL or less, even more preferably 2 μg/mL or more and 10 μg/mL or less, and most preferably 2.5 μg/mL or more and 5 μg/mL or less.

The mesenchymal stem cell culture medium of the present invention may contain a mixture of multiple lysophosphatidic acids, in which case the total concentration of all lysophosphatidic acids is preferably 0.1 μg/mL or more and 100 μg/mL or less, more preferably 0.3 μg/mL or more and 50 μg/mL or less, even more preferably 1 μg/mL or more and 40 μg/mL or less, particularly preferably 2 μg/mL or more and 30 μg/mL or less, and even more preferably 5 μg/mL or more and 20 μg/mL or less. In addition, the mixing ratio of multiple lysophosphatidic acids is not particularly limited, but when two types of lysophosphatidic acid, 16:0 lysophosphatidic acid and 18:1 lysophosphatidic acid, are included, from the viewpoint of excellent mesenchymal stem cell proliferation promoting effect, the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and/or salts thereof is 0.001 to 1000, preferably 0.01 to 100, more preferably 0.1 to 10, even more preferably 0.1 to 5, particularly preferably 0.3 to 3, and even more preferably 0.5 to 1.5.

The medium for mesenchymal stem cells of the present invention corresponds to one embodiment of the above-mentioned mesenchymal stem cell proliferation promoter of the present invention (when it is a liquid medium), so the descriptions in the explanation of the liquid medium in the section on the mesenchymal stem cell proliferation promoter can be applied as is with regard to the above-mentioned basal medium for animal cell culture, other ingredients, characteristics, etc.

The medium for mesenchymal stem cells of the present invention also includes the above-mentioned liquid medium to which a gelling material has been added to make it into a gel. The above-mentioned gelling material may be any material that can make the medium into a gel and that can be used for cell culture, such as agar, agarose, alginic acid, collagen, gelatin, cellulose, etc. The concentration of these gelling materials added may be any material that can make the medium into a gel, and may be appropriately set by those skilled in the art as needed. It is sufficient that the medium is in a gel state under cell culture or storage conditions, and it is preferable that the medium is in a gel state at any temperature within the temperature range of, for example, -80°C to 100°C.

[Additive for mesenchymal stem cell culture]
The present invention also includes an additive for mesenchymal stem cell culture containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. The additive for mesenchymal stem cell culture of the present invention contains the above-mentioned specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof, and when added to a medium or the like for culturing mesenchymal stem cells, it exhibits an excellent effect of promoting the proliferation of mesenchymal stem cells, and therefore can be suitably used for mesenchymal stem cell culture and/or for promoting the proliferation of mesenchymal stem cells. The additive for mesenchymal stem cell culture of the present invention corresponds to one embodiment of the above-mentioned mesenchymal stem cell proliferation promoter of the present invention. Therefore, the detailed description of the additive for mesenchymal stem cell culture of the present invention can be applied to the description of the mesenchymal stem cell proliferation promoter of the present invention.

[Method of culturing mesenchymal stem cells]
The present invention also includes a method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in a medium containing a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof. According to the culture method of the present invention, it is possible to efficiently proliferate mesenchymal stem cells while maintaining their undifferentiated state.

There are several types of lysophosphatidic acid with different combinations of alkyl chain length-number of double bonds (carbon number-degree of unsaturation), and specific examples of the carbon number-degree of unsaturation combinations (carbon number: degree of unsaturation) include 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:3, 22:4, 22:5, 22:6, etc. Among these, from the viewpoint of excellent effect of promoting proliferation of mesenchymal stem cells, 16:0, 16:1, 18:0, 18:1, 18:2, and 18:3 are preferred, 16:0 and 18:1 are more preferred, and 16:0 is even more preferred. The medium may contain multiple types of lysophosphatidic acid with different combinations of alkyl chain length-number of double bonds (number of carbon atoms-degree of unsaturation). The lysophosphatidic acid contained in the medium may be a derivative of lysophosphatidic acid or may be in any form, such as a salt thereof. Specific examples of the derivatives and salts are described in the section [Mesenchymal stem cell proliferation promoter].

The medium containing a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof in the culture method of the present invention corresponds to one embodiment of the mesenchymal stem cell proliferation promoter of the present invention described above (when it is a liquid medium), so the description of the liquid medium in the section on mesenchymal stem cell proliferation promoter can be applied as is to the details of the medium. In addition, the description of mesenchymal stem cells in the section on mesenchymal stem cell proliferation promoter can be applied as is to the explanation of mesenchymal stem cells.

The culture method of the present invention is not particularly limited as long as it includes a step of culturing mesenchymal stem cells in a medium containing a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof, and other than that, a method similar to that of the past can be used. Usually, the culture temperature is 30°C to 40°C, preferably 30 to 37°C. The _CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The _O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5 to 21%. In addition, during culture, mesenchymal stem cells may be subcultured or medium exchanged as necessary, and the timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and can be performed in the same manner as in the past while observing the morphology of the mesenchymal stem cells. The number of passages is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. The seeding density can be appropriately determined depending on the purpose of the culture, etc., and may be low density for long-term culture or high density for short-term culture. Generally, 2500 cells/ ^cm2 to 10000 cells/ ^cm2 is preferable, and 5000 cells/ ^cm2 to 7500 cells/ ^cm2 is more preferable.

In the method of culturing mesenchymal stem cells of the present invention, the mesenchymal stem cells may be cultured by general planar adhesion culture in which the mesenchymal stem cells are adhered to a culture vessel such as a flask, dish, or plate for cell culture, or by suspension culture (suspension culture) in which the mesenchymal stem cells are adhered to microbeads, microcarriers, etc. and the microbeads, microcarriers, etc. are suspended in a medium and cultured, or by suspension/agitation culture, or by adhering to a fibrous scaffold such as microfiber and culturing, or spheroid culture may be used.

The culture vessel used for culturing the mesenchymal stem cells of the present invention is not particularly limited as long as it is capable of culturing mesenchymal stem cells, and examples include flasks, flasks for tissue culture, dishes, Petri dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc.

[Mesenchymal stem cells]
The present invention also includes mesenchymal stem cells obtained by culturing using the above-mentioned mesenchymal stem cell proliferation promoter, mesenchymal stem cell culture additive, or mesenchymal stem cell medium of the present invention. The mesenchymal stem cells of the present invention are cultured in a medium containing a specific lysophosphatidic acid, a derivative of lysophosphatidic acid, and/or a salt thereof, and thus not only are their proliferation promoted, their viability high, and their condition good, but they are also able to release a large amount of various humoral factors such as exosomes and HGF compared to conventional mesenchymal stem cells, and therefore can be suitably used for the treatment of various diseases.

The mesenchymal stem cells of the present invention can be obtained by culturing conventional mesenchymal stem cells using the above-mentioned mesenchymal stem cell proliferation promoter of the present invention or the medium for mesenchymal stem cells of the present invention. Usually, the culture temperature is 30°C to 40°C, preferably 30 to 37°C. The _CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The _O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5% to 21%. In addition, during culture, mesenchymal stem cells may be subcultured or the medium may be replaced as necessary, and the timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and can be performed in the same manner as conventional methods while observing the morphology of the mesenchymal stem cells. The number of passages is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. The seeding density is usually preferably 2,500 cells/cm ² to 10,000 cells/cm ² , and more preferably 5,000 cells/cm ² to 7,500 cells/cm ² .

The mesenchymal stem cells of the present invention can increase the amount of exosomes and various humoral factors such as HGF released into the culture supernatant by increasing the seeding density and cell density. Such a seeding density is preferably 5000 cells/cm ² to 40000 cells/cm ² , more preferably 7500 cells/cm ² to 30000 cells/cm ² , and even more preferably 15000 cells/cm ² to 20000 cells/cm ^2. In addition, it is also possible to increase the concentration of exosomes and various humoral factors such as HGF in the culture supernatant by lowering the seeding density and culturing for a long period of time.

The mesenchymal stem cells of the present invention are positive for CD73, CD90, and CD105, and retain their undifferentiated state. Furthermore, the mesenchymal stem cells of the present invention release more exosomes that are positive for CD9 and CD63 than conventional mesenchymal stem cells. Furthermore, the mesenchymal stem cells of the present invention release more humoral factors, such as hepatocyte growth factor (HGF), insulin-like growth factor (IGF-related factor), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), than conventional mesenchymal stem cells.

Diseases for which the mesenchymal stem cells of the present invention can be used as a medicine include, for example, cartilage degradation, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout, psoriasis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic valve stenosis, renal failure, lupus, pancreatitis, allergies, fibrosis, anemia, atherosclerosis, restenosis, chemotherapy/radiation-related complications, type I diabetes, type II diabetes, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease, and nonspecific colonic necrosis factor (NCT). inflammation, allergic conjunctivitis, diabetic retinopathy, Sjogren's syndrome, uveitis-allergic rhinitis, asthma, asbestosis, silicosis, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, sarcoidosis, glomerulonephritis, vasculitis, dermatitis, HIV-associated cachexia, cerebral malaria, ankylosing spondylitis, leprosy, pulmonary fibrosis, esophageal cancer, gastroesophageal reflux disease, Barrett's esophagus, stomach cancer, duodenal cancer, small intestine cancer, appendix cancer, large intestine cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, liver cancer, gallbladder cancer, spleen cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, breast cancer, lung cancer, thyroid cancer, fibromyalgia, etc.

When the mesenchymal stem cells of the present invention are used as a pharmaceutical, the method of administration is not particularly limited, but is preferably intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc., and among these, intravascular administration is more preferable.

The dosage of the mesenchymal stem cells of the present invention when used as a pharmaceutical product may vary depending on the type of disease, the severity of symptoms, the dosage form, the body weight of the subject, etc., but when administered to humans, mesenchymal stem cells can be administered in the range of 1X10 ⁵ to 1X10 ⁹ cells per day. When administered to small animals such as mice, the range of 1X10 ⁴ to 1X10 ⁹ cells per day can be administered, and the range of 1X10 ⁵ to 1X10 ⁹ cells is more preferable. When the mesenchymal stem cells of the present invention are used as a pharmaceutical product, they may be administered once or multiple times per day. The administration may be a single administration or may be continuously administered. When administered continuously, they may be administered, for example, at a frequency of at least once every three days, two or more times continuously.

When the mesenchymal stem cells of the present invention are used as a pharmaceutical, the mammal to which they are administered is not particularly limited, but is preferably a human, monkey, mouse, rat, hamster, guinea pig, cow, pig, horse, rabbit, sheep, goat, cat, dog, etc., and among these, human is more preferable. Furthermore, when the mesenchymal stem cells of the present invention are used as a pharmaceutical, it is preferable that the type of mammal to which they are administered matches the type of mammal to which they are administered, from the viewpoint of obtaining a more stable and excellent preventive and/or therapeutic effect against a disease.

[Culture Supernatant]
The present invention also includes a culture supernatant containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, obtained by culturing mesenchymal stem cells in the above-mentioned medium for mesenchymal stem cells of the present invention. The culture supernatant of the present invention contains a larger amount of CD9- and CD63-positive exosomes than culture supernatants obtained by culturing mesenchymal stem cells in conventional media. Furthermore, the culture supernatant of the present invention contains a large amount of various humoral factors such as Hepatocyte Growth Factor (HGF), Insulin-like Growth Factors (IGF-related factors), Platelet-Derived Growth Factor (PDGF), and Vascular Endothelial Growth Factor (VEGF). Therefore, the culture supernatant of the present invention can be suitably used for the treatment of various diseases. It can also be applied to cosmetics and foods.

(Preparation of culture supernatant)
The culture supernatant of mesenchymal stem cells obtained by the following method can be used as the culture supernatant of the present invention. The culture supernatant of the present invention may also be a product obtained by removing unnecessary components from the supernatant by means of dialysis, ultrafiltration, or the like, a fraction obtained by fractionating the supernatant using a column or the like, a fraction selected using an antibody against a specific molecule, or a fraction obtained by centrifugation.

The medium used to obtain the culture supernatant is the medium for mesenchymal stem cells of the present invention described above. The method for obtaining the culture supernatant is not particularly limited as long as it is a method suitable for the culture of each mesenchymal stem cell, but the culture temperature is usually 30°C to 40°C, preferably 30°C to 37°C. The _CO2 concentration during culture is 2% to 10%, preferably 2% to 7%, more preferably 5%. The _O2 concentration during culture is 0% to 100%, preferably 0.5% to 22%, more preferably 5% to 21%. In addition, during culture, mesenchymal stem cells may be subcultured or the medium may be replaced as necessary, and the timing and method are not particularly limited as long as they are suitable for each mesenchymal stem cell, and can be performed in the same manner as in the past while observing the morphology of the mesenchymal stem cells. The number of passages of the mesenchymal stem cells is preferably 0 to 8, more preferably 1 to 6. The culture period is preferably 3 to 50 days, more preferably 3 to 40 days, and even more preferably 3 to 30 days. In general, the seeding density is preferably 2,500 cells/cm ² to 10,000 cells/cm ² , and more preferably 5,000 cells/cm ² to 7,500 cells/cm ² .

When obtaining the culture supernatant, the seeding density and cell density of the mesenchymal stem cells are increased, thereby increasing the concentration of various humoral factors such as exosomes and HGF in the culture supernatant. Such a seeding density is preferably 5000 cells/cm ² to 40000 cells/cm ² , more preferably 7500 cells/cm ² to 30000 cells/cm ² , and even more preferably 15000 cells/cm ² to 20000 cells/cm ^2. In addition, by lowering the seeding density of the mesenchymal stem cells and culturing them for a long period of time, it is also possible to increase the concentration of various humoral factors such as exosomes and HGF in the culture supernatant.

After obtaining the culture supernatant, the cells may be passaged and cultured multiple times in the mesenchymal stem cell medium of the present invention, and the culture supernatant may be obtained multiple times.

The culture for obtaining the culture supernatant of the present invention may be planar adhesion culture in which the cells are attached to a flask, dish, plate, etc. for cell culture, or may be suspension or agitation culture in which the cells are attached to a microcarrier, microbeads, etc.

When provided, the culture supernatant of the present invention is preferably sterile, having been subjected to a sterilization process, and the endotoxin content is preferably 2.5 EU/mL or less.

The culture supernatant of the present invention can be used for the same diseases as those for which the mesenchymal stem cells of the present invention can be used as a medicine. The same applies to the species to which it is administered. The culture supernatant of the present invention can also be used in quasi-drugs, cosmetics, and foods. When the culture supernatant of the present invention is used as a medicine, the preferred administration methods include intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc., and among these, intravascular administration is more preferred.

The dosage of the culture supernatant of the present invention when used as a pharmaceutical product may vary depending on the type of disease, the severity of symptoms, the dosage form, the body weight of the subject, and the like. When the culture supernatant of the present invention is used as a pharmaceutical product, it may be administered once or multiple times a day. The administration may be a single dose or may be administered continuously. When administered continuously, it may be administered, for example, twice or more continuously at a frequency of once or more every three days.

The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[Example 1: Examination of the effect of LPA-containing medium on promoting proliferation of mesenchymal stem cells-1]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) and human umbilical cord-derived mesenchymal stem cells (UC-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/cm ² in a T25 flask for adherent cells (manufactured by Corning), and cultured in R:STEM Medium for hMSC High Growth medium (manufactured by Rohto Pharmaceutical Co., Ltd., serum-free medium, hereinafter referred to as RS medium, RS, or RSTEM), or RS medium supplemented with 18:1 LPA and/or 16:0 LPA. When 18:1 LPA alone or 16:0 LPA alone was added, it was added so that the final concentration was 5.0 μg/mL, and when both 18:1 LPA and 16:0 LPA were added, each was added so that the final concentration was 2.5 μg/mL. After culturing in each medium for 3 days under 37°C and 5% _CO2 conditions, the cells were detached with trypsin and the number of cells in each flask was counted. The control (RS) was cultured in RS medium without the addition of either 18:1 LPA or 16:0 LPA, and the relative value (%) of the number of recovered cells in each medium condition was calculated based on the number of recovered cells in the control as 100%. The results are shown in Figures 1 and 2. Figure 1 shows the effect of adding 16:0 LPA (AD-MSC, UC-MSC), and Figure 2 shows the effect of adding either 18:1 LPA or 16:0 LPA, or both (AD-MSC).

When cultured in a medium supplemented with 16:0 LPA, the proliferation of both AD-MSCs and UC-MSCs was significantly promoted (Figure 1). Furthermore, a medium supplemented with both 18:1 LPA and 16:0 LPA showed a synergistic proliferation-promoting effect on AD-MSCs compared to a medium supplemented with either 18:1 LPA or 16:0 LPA alone (Figure 2).

[Example 2: Examination of the mesenchymal stem cell proliferation-promoting effect of planar adhesion culture/LPA-containing medium-2]
Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (Corning) and cultured in a basal medium (MEM, bFGF, albumin, insulin, transferrin, 3% FBS) supplemented with 18:1 LPA and/or 16:0 LPA. When 18:1 LPA alone or 16:0 LPA alone was added, it was added so that the final concentration was _2.5 μg/mL, and when both 18:1 LPA and 16:0 LPA were added, each was added so that the final concentration was 2.5 μg/mL. After culturing in each medium at 37°C and 5% CO2 for 3 days, the cells were detached with trypsin and the number of cells in each flask was counted. The number of cells recovered in the control culture medium without the addition of either 18:1 LPA or 16:0 LPA was set as 100%, and the relative value (%) of the number of cells recovered in each culture medium condition was calculated. The results are shown in Figure 3.

As shown in Figure 3, for UC-MSCs, both the medium containing 18:1 LPA alone and the medium containing 16:0 LPA alone showed superior proliferation-promoting effects compared to the basal medium. Furthermore, the medium containing both 18:1 LPA and 16:0 LPA showed a significant proliferation-promoting effect compared to the control. Although data is not shown, the proliferation-promoting effect of adding 18:1 LPA and/or 16:0 LPA was also confirmed for AD-MSCs, as in Example 1.

[Example 3: Planar adhesion culture/examination of concentration ratio of 16:0 LPA and 18:1 LPA]
Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (Corning) and cultured in R:STEM Medium for hMSC High Growth medium (Rohto Pharmaceutical, serum-free medium, hereinafter referred to as RS medium), or in a medium containing 18:1 LPA and 16:0 LPA added at a total concentration of 5.0 μg/ml at various concentration ratios. After culturing for 3 days in each of the _LPA media at 37°C and 5% CO2, the cells were detached with trypsin and the number of cells in each flask was counted. The control was cultured in RS medium without the addition of either 18:1 LPA or 16:0 LPA, and the number of recovered cells in the control was set as 100%, and the relative value (%) of the number of recovered cells under each medium condition was calculated. The results are shown in Figure 4.

As shown in Figure 4, a more significant proliferation-promoting effect was obtained for UC-MSCs when the ratio of 16:0 LPA to 18:1 LPA was between 10:90 and 75:25. Although the data is not shown, the proliferation-promoting effect was also confirmed for AD-MSCs by adding various concentrations of 18:1 LPA and 16:0 LPA, as in Example 3.

[Example 4: Examination of flat adhesion culture/LPA concentration]
Human umbilical cord-derived mesenchymal stem cells (UC-MSC, Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (Corning), and cultured in R:STEM Medium for hMSC High Growth medium (Rohto Pharmaceutical, serum-free medium, hereinafter referred to as RS medium), or in a medium in which 18:1 LPA and 16:0 LPA were added in equal amounts at various concentrations to RS medium. After culturing in each medium for 3 days under conditions of ₃₇ °C and 5% CO2, the cells were detached with trypsin and the number of cells in each flask was counted. The control was cultured only in RS medium without the addition of either 18:1 LPA or 16:0 LPA, and the relative value (%) of the number of cells recovered in each medium condition was calculated, assuming the number of cells recovered in the control to be 100%. The results are shown in FIG. 5.

As shown in Figure 5, a superior proliferation-promoting effect was observed when the combined concentration of 18:1 LPA and 16:0 LPA was 0.3125 μg/mL or higher, but a particularly notable proliferation-promoting effect was observed at concentrations between 5 μg/mL and 20 μg/mL.

[Example 5: Suspension culture]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 7,500 cells/cm ² on Corning® low-concentration Synthemax® II microcarriers (manufactured by Corning) in a single-use bioreactor (manufactured by Able), and suspension culture was performed in RS medium or a medium in which 18:1 LPA and 16:0 LPA were added at 2.5 μg/mL each to RS medium. After 3 days of culture under 37°C and 5% CO ₂ conditions, the cells were detached with trypsin and the number of cells in each flask was counted. The control (RS) was cultured only in RS medium without the addition of either 18:1 LPA or 16:0 LPA, and the relative value (%) of the number of recovered cells under each medium condition was calculated, with the number of recovered cells in the control being 100%. The results are shown in FIG. 6.

As shown in Figure 6, the medium containing both 18:1 LPA and 16:0 LPA exhibited a significant proliferation-promoting effect on AD-MSCs compared to the control. This effect obtained in suspension culture was more significant than that in planar adherent culture (Example 1).

[Example 6: Evaluation of exosome production from adipose-derived mesenchymal stem cells]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/cm ² in a T25 flask for adherent cells (manufactured by Corning), and cultured in RS medium (RSTEM) and medium (LPA-added) in which 2.5 μg/mL of 18:1 LPA and 16:0 LPA were added to RS medium. After culturing in each medium for 4 days under conditions of 37 ° C. and 5% CO ₂ , the culture supernatant was collected and the amount of exosomes was quantified using CD9, CD63 ELISA kit (manufactured by Cosmo Bio Co., Ltd.). The results are shown in FIG. 7.

As shown in Figure 7, the amount of exosomes in the culture supernatant obtained by culturing AD-MSCs in the medium supplemented with 18:1 LPA and 16:0 LPA was approximately four times higher than in the serum medium and RS medium (RSTEM), demonstrating that the addition of 18:1 LPA and 16:0 LPA significantly promoted exosome production from AD-MSCs.

[Example 7: Evaluation of humoral factor production from adipose-derived mesenchymal stem cells]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in a T25 flask for adherent cells (manufactured by Corning), and cultured in RS medium and medium containing 2.5 μg/mL each of 18:1 LPA and 16:0 LPA added to RS medium. After culturing in each medium for 4 days under conditions of 37°C and 5% _CO2 , the culture supernatant was collected, and each humoral factor in the culture supernatant was analyzed using RayBio Label-Based Antibody Array.

The results of the above analysis showed that compared to RS medium, RS medium supplemented with LPA (a mixture of 16:0 and 18:1) had more than five times the amount of hepatocyte growth factor (HGF), more than twice the amount of insulin-like growth factor (IGF-related factor), more than three times the amount of platelet-derived growth factor (PDGF), and more than twice the amount of vascular endothelial growth factor (VEGF).

[Example 8: Evaluation of adhesiveness of mesenchymal stem cells]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded at 5,000 cells/ ^cm2 in a commercially available flask for adherent cells, and cultured for 3 days at 37°C and 5% _CO2 in RS medium or RS medium supplemented with 18:1 LPA and 16:0 LPA at 2.5 μg/mL each (RS+LPA). After 3 days of culture, the cells were detached with trypsin, and the number of cells in each flask was counted. The results are shown in FIG. 8. Details of the flasks used are as follows:
- The polystyrene surface of the Corning CellBind/Corning (registered trademark) CellBIND surface culture vessel is negatively charged by incorporating oxygen-containing functional groups.
・Nunc/Nunc EasYFlask Cell Culture Flasks
The culture surface is given a unique surface treatment.
- The culture surface of the Falcon/Falcon (registered trademark) cell culture flask is vacuum gas plasma treated.
・Sumitomo Bakelite/Adherent cell culture flask with filter cap (green cap)
The culture surface has been subjected to a physical hydrophilic treatment.

As shown in Figure 8, when cultured using RS medium supplemented with 18:1 LPA and 16:0 LPA, the culture performance was better due to adhesion than RS in both flasks.

[Example 9: Evaluation of proliferation promoting effect and culture variation of mesenchymal stem cells]
Three different lots of human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were used, each seeded at 5,000 cells/ ^cm2 , and cultured in RS medium or RS medium supplemented with 18:1 LPA and 16:0 LPA at 2.5 μg/mL each (RS+LPA) for a total of 9 days under conditions of 37°C and 5% _CO2 . The number of cell divisions over 9 days was measured and shown in Figure 9. In addition, the variation (coefficient of variation) in the number of divisions between lots was compared, and the results are shown in Figure 10.

As shown in Figure 9, when cultured using RS + LPA, the number of cell divisions over 9 days was approximately twice as high as when cultured with RS, demonstrating good culture performance. In addition, the variation in the number of divisions between cell lots was smaller when cultured using RS + LPA compared to cultured with RS. It was suggested that the improvement in the adhesive performance of mesenchymal stem cells in culture using a medium containing LPA contributed to improved proliferation and reduced differences between lots (donors).

[Example 10: Over-passage culture of mesenchymal stem cells]
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded in a flask for adherent cells at 5,000 cells/ ^cm2 , and _{an over-} subculture test was carried out in RS medium or RS medium supplemented with 18:1 LPA and 16:0 LPA at 2.5 μg/mL each (RS+LPA) at 37°C and 5% CO2, with subculture repeated every 3 to 4 days. The results are shown in FIG. 11.

As shown in Figure 11, when RS+LPA was used, AD-MSCs could be cultured up to passage number 13 (P13) while maintaining their proliferation rate.

Example 11: Angiogenesis test
Human adipose-derived mesenchymal stem cells (AD-MSC, manufactured by Promo Cell) were seeded in each medium (negative control, positive control, RSTEM, RSTEM + LPA (16:0 LPA and 18:1 LPA at 2.5 ug/mL each)) at 5,000 cells/cm2 in a ^flask for adherent cells (manufactured by Corning), and cultured for 3 days under conditions of ₃₇ °C and 5% CO2. Then, each medium was removed, the cells were detached with trypsin, and then seeded in new media of the same type, and cultured for 3 days under the same conditions. Then, the cells were detached with trypsin and seeded in 2% FBS-HuMedia at 120,000 cells/ ^cm2 , and further cultured for 3 days under conditions of ₃₇ °C and 5% CO2, and the supernatant was collected. As a negative control, 2% FBS-HuMedia was used. As a positive control, a medium containing 2% FBS-HuMedia supplemented with hEGF (final concentration: 10 ng/mL) and hFGF-b (final concentration: 5 ng/mL) was used.

Matrigel basement membrane matrix (Corning) was added to a black 96-well plate on ice at 40 ul/well and incubated at 37°C and 5% CO2 for 1 hour or more. Human umbilical vein endothelial cells (HUVEC) dispersed in each of the above-prepared supernatants were seeded on a Matrigel-coated 96- _well plate at 7,500 cells/well. Incubated for 5 hours at 37°C and 5% _CO2 . After culturing, HUVEC was stained with calcein. Furthermore, the percentage (%) of the stained area of HUVEC in the entire field of view was calculated using ImageJ to evaluate the proliferation rate of HUVEC (angiogenic potential of each supernatant). Comparison of the photographs at the time of staining and the stained area is shown in Figure 12 and Table 1, respectively.

As shown in Figure 12 and Table 1, the angiogenic area was larger in the culture supernatant of RSTEM + LPA, confirming significantly superior angiogenic ability. In addition, as a result of proteomic analysis of each supernatant, factors known to be involved in angiogenesis were detected at higher levels in the culture supernatant of RSTEM + LPA compared to the culture supernatant of RSTEM (data not shown).

[Example 12: Evaluation of proliferation-promoting effect of umbilical cord-derived mesenchymal stem cells]
One type of human umbilical cord-derived mesenchymal stem cells (UC-MSC, manufactured by LIFELINE) was used, and seeded at 4000 cells/ ^cm2 , and cultured in RS medium (RSTEM) and medium containing 2.5 μg/mL each of 18:1 LPA and 16:0 LPA (LPA-containing medium) at 37°C and 5% CO2 for _a total of 10 days. The number of cell divisions (PDL) over 10 days was measured and shown in FIG. 13.

As shown in Figure 13, when cultured using LPA-containing medium, the number of cell divisions increased over 10 days compared to culture in RS medium, demonstrating good culture performance. It was suggested that culture using a medium containing LPA contributed to improved proliferation ability even when using umbilical cord-derived mesenchymal stem cells.

[Example 13: Evaluation of cell surface antigen markers]
UC-MSCs cultured for 10 days under the conditions of Example 12 were frozen and thawed using a thermostatic bath set at 37 ° C. 3.4 × 10^6 cells were collected from the thawed cell suspension and suspended in medium. After centrifugation, the supernatant was removed and resuspended in 1.7 mL of 1% BSA-PBS. 100 μL of the cell suspension and each antibody reagent were stirred with a vortex mixer and reacted for 30 minutes in the dark and on ice. After the reaction, the cells were washed twice with 1% BSA-PBS, and after removing the supernatant, 500 μL of 1% BSA-PBS was added to make the cell suspension concentration uniform. The cell suspension was added to a tube through a cell strainer and reacted with 7-AAD for 10 minutes in the dark and at room temperature, and each cell surface marker was measured by a flow cytometer. The results are shown in Table 2.

As shown in Table 2, it was confirmed that UC-MSCs cultured using LPA-containing medium meet the definition of mesenchymal stem cells set forth by the International Society for Cellular Therapy (ISCT).

[Example 14: Evaluation of proliferation-promoting effect of dental pulp-derived mesenchymal stem cells]
Human dental pulp-derived mesenchymal stem cells (manufactured by Lonza) were used, seeded at 3000 cells/ ^cm2 , and cultured in RS medium (RSTEM) and medium containing 2.5 μg/mL each of 18:1 LPA and 16:0 LPA (LPA-containing medium) at 37°C and 5% CO2 for _a total of 10 days. The number of cell divisions (PDL) measured over 4 days is shown in Figure 14.

As shown in Figure 14, when cultured using LPA-containing medium, the number of cell divisions increased over four days compared to culture in conventional medium, demonstrating good culture performance. Furthermore, LPA-containing medium showed a good number of cell divisions even when harvested after seven and ten days compared to culture in conventional medium. These results suggest that medium supplemented with LPA contributes to improved proliferation ability even when used for dental pulp-derived mesenchymal stem cells.

The mesenchymal stem cell proliferation promoter and mesenchymal stem cell culture medium of the present invention allow mesenchymal stem cells to grow efficiently while retaining their undifferentiated state. Furthermore, the culture supernatant obtained by culturing mesenchymal stem cells in the mesenchymal stem cell culture medium of the present invention contains a large amount of various humoral factors such as exosomes and HGF, and therefore can be suitably used for disease treatment, etc.

Claims (11)

A mesenchymal stem cell proliferation promoter containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. The mesenchymal stem cell proliferation promoter according to claim 1, further comprising 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof. The mesenchymal stem cell proliferation promoter according to claim 2, wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and/or salts thereof is 0.1 to 10. The mesenchymal stem cell proliferation promoter according to claim 1, wherein the mesenchymal stem cells are derived from adipose tissue. An additive for mesenchymal stem cell culture containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. A medium for mesenchymal stem cells containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof. The medium for mesenchymal stem cells according to claim 6, further comprising 18:1 lysophosphatidic acid, a derivative of 18:1 lysophosphatidic acid, and/or a salt thereof. The medium for mesenchymal stem cells according to claim 7, wherein the ratio of the content (μg/mL) of 16:0 lysophosphatidic acid, derivatives of 16:0 lysophosphatidic acid, and/or salts thereof to the content (μg/mL) of 18:1 lysophosphatidic acid, derivatives of 18:1 lysophosphatidic acid, and/or salts thereof is 0.1 to 10. The medium for mesenchymal stem cells according to claim 6, which is for promoting the proliferation of mesenchymal stem cells. Mesenchymal stem cells obtained by culturing using the mesenchymal stem cell proliferation promoter according to any one of claims 1 to 4, the additive for mesenchymal stem cell culture according to claim 5, or the medium for mesenchymal stem cells according to any one of claims 6 to 9. A culture supernatant containing 16:0 lysophosphatidic acid, a derivative of 16:0 lysophosphatidic acid, and/or a salt thereof, obtained by culturing mesenchymal stem cells in a medium for mesenchymal stem cells according to any one of claims 6 to 9.

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