TW201534724A - Low oxygen cultivation method for mesenchymal stem cells - Google Patents

Abstract

Provided is a novel cultivation method for mesenchymal stem cells that keeps the cells undifferentiated and healthy. A method for cultivating mesenchymal stem cells that keeps the cells undifferentiated and healthy, the method comprising the cultivation of the mesenchymal stem cells at an oxygen concentration suitable for keeping the mesenchymal stem cells undifferentiated and healthy.

Description

Hypoxic culture method of mesenchymal stem cells

The present invention relates to a method for cultivating mesenchymal stem cells between undifferentiated and healthy, and for cultivating the mesenchyme under an oxygen concentration suitable for maintaining undifferentiation and health of mesenchymal stem cells. The steps of stem cells are made.

Due to the amazing advances in medicine in recent years, the opportunity to save lives has increased with the removal of diseases, such as the treatment of symptomatic treatments such as cancer excision, or the transplantation techniques of tissues and organs. However, with the removal of the affected part, the patient sometimes suffers from a significant decrease in QOL (Quality Of Life) after treatment. In addition, there are limits to the treatment of living donors, such as lack of transplant donors, rejection reactions, and the like. If the tissue, organs, organs, etc. lost due to surgical treatment or accidents can be regenerated, the QOL of the patient can be greatly improved. Moreover, regenerative medicine can also eliminate the problems of living transplantation. From this point of view, the industry has a higher expectation of regenerative medicine.

In connection with this, mesenchymal stem cells have the ability to differentiate into cells belonging to the mesenchymal system (osteocytes, cardiomyocytes, chondrocytes, sputum cells, fat cells, etc.), and therefore, in recent years, the industry has extensively performed with the bone. Or research related to the application of regenerative medicine such as remodeling of blood vessels and myocardium. In order to realize regenerative medicine, it is necessary to obtain a large amount of leaf cell stem cells to maintain undifferentiation and health, but in subculture of mesenchymal stem cells, it is difficult to obtain a sufficient amount of active cells. It is known that, for example, in the case of subculture of dermal papilla cells as mesenchymal stem cells, it is confirmed that extreme decline in proliferative properties or loss of hair follicle inducing ability is difficult to maintain such properties (Jahoda CAB et al. 1984) Nature 311:560- 562).

Heretofore, a method for culturing frond stem cells between the ability to maintain proliferation and differentiation by introducing a specific gene such as Sox2 or Nanog into mesenchymal stem cells has been known (Japanese Patent Laid-Open No. 2009-11254) However, it is not known that the undifferentiation and health of mesenchymal stem cells can be maintained in vitro for a long period of time by culturing under a certain low oxygen concentration condition.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2009-11254

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2012-157263

[Patent Document 3] Japanese Patent Laid-Open No. Hei 7-274950

[Non-patent literature]

[Non-Patent Document 1] Jahoda CAB et al. (1984) Nature 311: 560-562

[Non-Patent Document 2] Noburo Sato et al., Nature Medicine Vol. 10, No. 1, Jan. 2004

An object of the present invention is to provide a novel culture method for maintaining leaf cell stem cells between undifferentiated and healthy.

The present inventors performed metabolome analysis and DNA (Deoxyribonucleic Acid) detection analysis, and conducted an effort to obtain a surprising result: culturing mesenchymal lobe by a certain low oxygen concentration condition Stem cells can maintain their undifferentiation and health.

Therefore, the present application includes the following inventions:

[1] A method for culturing a leaf cell stem cell that maintains undifferentiation and health, and is included in an oxygen concentration suitable for maintaining undifferentiation and health of mesenchymal stem cells. The steps of cultivating mesenchymal stem cells are obtained.

[2] The method according to the above [1], characterized in that the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is more than about 1% to less than about 21%.

[3] The method according to the above [1], characterized in that the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is from about 3% to about 7%.

[4] The method according to the above [1], characterized in that the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is more than about 1% to about 5% or less.

[5] The method according to [1] above, wherein the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is about 5%.

[6] The method according to any one of the above [1] to [5] wherein the mesenchymal stem cells are dermal root sheath (DS, Dermal Sheath) cells.

[7] The method according to any one of the above [1] to [5] wherein the mesenchymal stem cells are derived from adipose stem cells.

According to the present invention, leaf line stem cells which maintain undifferentiation and health can be obtained in large quantities.

Figure 1 shows NAD (Nicotinamide Adenine Dinucleotide) in adipose Derived Stem Cell (ADSC) cultured under various oxygen concentrations (1%, 5%, 21%). Comparison of the amount of amine adenine dinucleotide) ⁺ / NADH (Nicotinamide Adenine Dinucleotide Hydrogen, reduced nicotine indoleamine adenine dinucleotide).

Figure 2 shows reduced glutathione (GSH) in phylogenetic stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). Comparison of oxidized glutathione (GSSG) and cysteine (Cys, cysteine) and GSH/GSSG ratio.

Figure 3 shows glucose 6 phosphate (G6P, Glucose-6-phosphate) in phylloblastic stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). Comparison of the amount of lactic acid and ATP (Adenosine Triphosphate, adenosine triphosphate).

Figure 4 is a graph showing the comparison of the amount of glucose 6-phosphate and ribulose 5 phosphate in leaf cell stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). .

Figure 5 shows β-catenin (CTNNB1) in phylloblastic stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). Comparison of gene expression levels of casein kinase 1D (CSNK1D, Casein kinase 1D) and PPARγ (PPARG (Peroxisome Proliferator Activated Receptor Gamma)).

Figure 6 shows the Leukemia Inhibitory Factor Receptor (LIFR) in the leafy stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). And comparison of gene expression levels of T cell factor-4 (TCF-4, T Cell Factor-4).

Figure 7 shows the basic fibroblast growth factor (FGF2, Fibroblast Growth Factor) in phylogenetic stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). 2) Comparison of gene expression levels.

Figure 8 shows vascular endothelial cell proliferation factor (VEGFA, Vascular Endothelial Growth Factor A) in phylogenetic stem cells (ADSC) derived from subcutaneous fat cultured under various oxygen concentrations (1%, 5%, 21%). And comparison of gene expression levels of transforming growth factor 2 (TGFB2, Transforming growth factor 2).

Fig. 9 is a graph showing the comparison of alkaline phosphatase (AP) activity in dermal root sheath (DS) cells cultured under various oxygen concentrations (1%, 5%, 21%).

Figure 10 shows the dermal root sheath cultured under the conditions of oxygen concentration (5%, 21%). The amount of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the (DS) cells, and the GSH/GSSG ratio were compared.

Figure 11 is a graph showing the comparison of the amount of hypoxanthine in dermal root sheath (DS) cells cultured under various oxygen concentrations (5%, 21%).

Figure 12 shows the Glycolysis (Grucose-6-phosphate, F6-6) in dermal root sheath (DS) cells cultured under various oxygen concentrations (5%, 21%). Comparison of the amounts of -phosphoric acid), F1, 6P, 3PG (3-Phosphoglyceric acid, 3-phosphoglycerate), phosphoenolpyruvate, and lactic acid.

Fig. 13 is a graph showing the amount of NADH and the NADH/NAD ⁺ ratio in dermal root sheath (DS) cells cultured under the conditions of respective oxygen concentrations (5%, 21%).

Figure 14 shows the various factors (GSK-3β, OCT-4 (Octamer-Binding Transcription Factor-4) in dermal root sheath (DS) cells cultured under various oxygen concentrations (5%, 21%). The results of the Western blot method of the body-bound transcription factor-4)) (due to the inability to detect HIF-1α (Hypoxia Inducible Factor-1α, hypoxia-inducible factor-1α), no data).

Fig. 15 shows the results of analysis of a luciferase assay using Wnt3A in dermal root sheath (DS) cells cultured under various oxygen concentrations (5%, 21%).

The present invention provides a method for culturing a leaf cell stem cell that maintains undifferentiation and health, and includes culturing a mesenchymal line at an oxygen concentration suitable for maintaining undifferentiation and health of mesenchymal stem cells. The steps of stem cells are made.

The mesenchymal stem cell line is present in the body, such as fat, umbilical cord, epiphysis, synovial membrane and the like. The mesenchymal stem cells can differentiate into various mesodermal cells such as osteoblasts, chondrocytes, skeletal muscle cells, cardiomyocytes, and vascular endothelial cells. Mesenchymal stem cells are expected to be applied to bone, joint, muscle, liver, due to their multi-differentiation ability. Organisms such as kidney, heart, central nervous system, and pancreas, for example, the industry is undergoing myocardial infarction using transplants of mesenchymal stem cells, Chronic Obstructive Pulmonary Disease (COPD), autoimmune diseases, Treatment of chronic renal failure, osteoarthritis, cerebral infarction, and diseases of the cranial nervous system (eg, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease) (eg, refer to Japanese Patent Special Edition) Bulletin 2012-157263).

The phylogenetic stem cells in the present invention may be derived from all mammals such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, and the like. Experimental animals such as rats, mice, guinea pigs, more preferably nude mice, Severe Combined Immunodeficiency Disease mouse, fat of nude rats, umbilical cord, epiphysis, synovial membrane, From the viewpoint of the transplantation of humans or the manufacture of a three-dimensional model for research, it is preferably a cell derived from humans. The mesenchymal stem cells are preferably dermal root sheath (DS) cells or adipose derived mesenchymal stem cells (ADSC), and more preferably DS cells. The DS cell line is present in cells of the dermal root sheath and is known as one of the mesenchymal cells. The dermal sheath (DS, Dermal sheath), sometimes referred to as the connective tissue sheath or connective tissue sheath, is a dermal tissue surrounding the epithelial outer root sheath. Furthermore, DP (hair papilla) cells are considered to be derived from DS cells, wherein the proliferation of DP cells prior to the anagen phase is derived from the basal part of the dermal root sheath (DS) which is particularly close to the dermal papilla. The cells in the region of the Dermal Sheath Cup (DSC) are proliferated, and it is therefore considered that DS cells, especially cells derived from the DSC region (also referred to as DSC cells), are supplied to DP cells.

Further, in the present invention, the phylogenetic stem cells can be obtained from the tissues of the mammals by primary culture, and can be cells obtained by subculture, or can be stem cells, iPS cells (induced pluripotent stem cells) and ES cells (Embryonic stem cells) Cells obtained by differentiation induction. From the viewpoint of transplantation, it is preferred to subculture cells in which cells obtained from a transplanted subject proliferate.

The basal medium which can be used in the present invention is not particularly limited as long as it can be used for the culture of cells of human or animal, and the commercially available nutrient medium can be cultured directly or by modifying it. As a representative medium which can be used to culture mesenchymal stem cells, for example, Dubeco's modified Eagle's medium (Gibco) or Chang Medium (Irvine Scientific) containing fetal bovine serum can be transplanted. From a viewpoint, a serum-free medium is preferred. A variety of serum-free media are commercially available, for example, HFDM-1 (+) (Cell Science Institute), HFDM-1 (-) (Cell Science Institute), StemPro MSC SFM CTS (sup+) (Lifetechnologies) StemPro MSC SFM CTS (sup-) (Lifetechnologies), Mosaic hMSC SF Culture Medium (Sup+) (BD), and the like. As a component of these commercially available media, HFDM-1(-) is known to contain a basal medium RITC80-7, insulin 5 μg/ml, dexamethasone 10 ^-7 M, and HFDM-1 (+) in addition to these, Contains EGF 10ng/ml.

The basal medium may contain a Platelet Derived Growth Factor (PDGF). The PDGF is a proliferative factor mainly involved in the movement or proliferation of mesenchymal stem cells (fibroblasts, smooth muscle cells, glial cells, etc.), and belongs to the family of PDGF/VEGF (Vascular Endothelial Growth Factor). In addition to being mainly produced by megakaryocytes, it is also known to be contained in alpha particles of platelets, which are produced by various cells such as epithelial cells or endothelial cells. PDGF has at least four kinds of PDGF-A, B, C, and D depending on the A chain, the B chain, the C chain, and the D chain. These all form dimers or heterodimers, and it is known that there are five isoforms (Isoform) of PDGF-AA, -AB, -BB, -CC, -DD. Among these, it is especially preferred to be PDGF-BB. The amount of PDGF added to the basal medium is not particularly limited, and is, for example, 0.01 ng/ml to 10 μg/ml, preferably 0.1 ng/ml to 100 ng/ml, more preferably 1 to 10 ng/ml.

Further, a Wnt signal activator may be added to the basal medium. The so-called Wnt signal refers to a series of functions that promote the nuclear translocation of β-catenin and play a role as a transcription factor. This signal is caused by intercellular interactions, for example, including a series of processes in which a protein of Wnt3A secreted by a cell acts on other cells, and the β-catenin in the cell undergoes nuclear translocation and functions as a transcription factor. effect. A series of processes have led to the initial phenomenon of organ construction as an example of the above-mentioned cutaneous mesenchymal interaction. The Wnt signal is known to control cell proliferation or differentiation, organ formation, or initial production by activating the β-catenin pathway, the PCP (Planar Cell Polarity) pathway, and the Ca ²⁺ pathway. Various cellular functions such as cell movement. The purpose of inhibiting differentiation by maintaining the function of the undifferentiated state of the Wnt signal is to utilize it when culturing ES cells (for example, Noburo Sato et al., Nature Medicine Vol. 10, No. 1, Jan. 2004). ).

The Wnt signal activator is not particularly limited, and may be any one as long as it exhibits an inhibitory activity of glycogen synthase kinase-3 (GSK-3, Glycogen Synthase Kinase-3), and examples thereof include:哚 (靛红) compound (BIO) ((2'Z, 3'E)-6-bromo ruthenium-3'-肟), its acetone oxime analog BIO-acetone oxime (2'Z, 3'E) -6-bromoindole-3'-acetone oxime), thiadiazolidine (TDZD) analogue (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-di Ketone), oxodithiazolidine-3-thione analog (2,4-dibenzyl-5-oxothiadiazolidine-3-thione), thienyl α-chloromethylketone compound ( 2-Chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone), phenyl alpha bromomethylketone compound (α-4-dibromoacetophenone), thiazole-containing urea compound (N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea) or a GSK-3β peptide inhibitor such as H-KEAPPAPPQSpP-NH ₂ , List lithium chloride and the like. Further, CT99021 (molecular weight: 465.34, purity: 99.1%, CAS No: 252917-06-9, alias: CHIR99021) is known as a very potent selective GSK-3 inhibitory substance, which is cultured in mouse ES cells. It can be used together with PD184352 (MEK/MAPK (Mitogen Activated Protein Kinase) inhibitor or SU5402 (FGFR (Fibroblast Growth Factor Receptor) inhibitor) for long-term culture. .

The amount of the Wnt signal activator to be added is not particularly limited as long as the Wnt signal activation, in other words, the inhibition of GSK-3 is achieved, and the amount of cell proliferation is not stopped, depending on the type of the agent to be used and the proliferation of the drug to be used. The type of cells is determined by the industry. For example, when BIO is used as the Wnt signal activator, the amount thereof is, for example, 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably about 10 μM. Further, when CT99021 is used as the Wnt signal activating agent, the amount thereof is, for example, 0.1 to 10 μM, preferably about 1 μM.

Further, in the serum-free medium, other cell growth factors, hormones or other micronutrients may be added as needed. As such a specific one, for example, an epidermal growth factor (EGF), a tumor necrosis factor alpha (TNFα, Tumor Necrosis Factor α), a hepatocyte growth factor (HGF), a fibroblast Proliferation factor 7 (FGF7), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β1, 2, 3 (TGFβ1, 2, 3, Transforming Growth Factor β1 , 2, 3), bone formation factor (BMP (Bone Morphogenetic Protein)) or insulin-like growth factor-1, 2 (IGF-1, 2, Insulin-like Growth Factor-1, 2) In the case, the amount is, for example, about 0.1 μg/ml to 100 μg/ml. When a phospholipid (for example, phosphatidic acid, phospholipid osmolar, ethanolamine) is used, the amount thereof is, for example, about 0.1 μg/ml to 100 μg/ml. When a fatty acid (for example, linoleic acid, oleic acid or arachidonic acid) is used, the amount thereof is, for example, about 0.001 μg/ml to 100 μg/ml. In the case of using prostaglandins, the amount is from about 0.1 ng/ml to about 100 ng/ml. When a reducing agent (for example, ascorbic acid or reduced glutathione) is used, the amount thereof is, for example, about 1 μg/ml to 100 μg/ml. In the case of using mercaptoethanol, the amount is, for example, about 0.1 μg/ml to 100 μg/ml. When using transferrin or insulin, the amount is 0.01μg/ml~100. Μg/ml or so. In the case of using dexamethasone, the amount is about 0.000001 μM to 0.1 μM. In the case of using triiodothyronine, the amount is from about 0.1 pM to about 100 pM. In the case of using glycoside, the amount is about 0.0001 μM to 0.1 μM. In the case of using cholesterol, the amount is, for example, about 0.1 μg/ml to 100 μg/ml. In the case of using hydrocortisone, the amount is, for example, about 0.01 μg/ml to 10 μg/ml. In the case of using testosterone, the amount is, for example, about 0.1 μM to 100 μM. In the case of using estradiol or progesterone, the amount is, for example, about 0.01 ng/ml to 100 ng/ml. When a trace element (for example, copper, zinc, cobalt, manganese, molybdenum or selenium) is used, the amount thereof is, for example, about 0.000001 mg/ml to 0.1 mg/ml. In the case of using albumin, fibronectin or vitronectin, the amount is, for example, about 0.1 μg/ml to 1000 μg/ml.

In such media, the culture of mesenchymal stem cells is usually carried out by using a culture dish at 5% CO under "suitable for maintaining the undifferentiated and healthy oxygen concentration of mesenchymal stem cells". _{In the} atmosphere of ₂ , the mixture was allowed to stand in an incubator at 37 ° C, and after the growth was confirmed, the medium was replaced and the culture was continued (subculture). The cultured cells obtained in the above manner are subcultured through the desired subcultures. It can be subcultured until the amount of mesenchymal stem cells is reached, for example, it can be carried out 10 times or more. In the case where the required amount is large, it is preferable to carry out subculture for 15 times or more, and further preferably It is a generation of more than 20 times.

In the present invention, the mesenchymal stem cells are cultured under the "oxygen concentration suitable for maintaining undifferentiation and health". Here, "undifferentiating" means that the mesenchymal stem cells are not directed to bone cells or cardiomyocytes. Cells such as chondrocytes, sputum cells, and fat cells are differentiated and still have a multi-differentiation state. The term "health" means that the amount of reactive oxygen species or inflammatory cytokines that cause cytotoxicity is small and normal. The state of survival and proliferation.

The "undifferentiation" or "health" of such mesenchymal stem cells can be as follows Method determination: Metabolites and/or expression genes present in mesenchymal stem cells are comprehensively analyzed by metabolome analysis or DNA array analysis.

Metabolome analysis is carried out by collectively separating low molecular weight metabolites in cells containing amino acids, amines, nucleotides, sugars, lipids, etc., and identifying and quantifying them. As a separation method which can be used in the metabolome analysis, gas chromatography (GC, Gas Chromatography), liquid chromatography (LC, Liquid Chromatography), and capillary electrophoresis (CE, Capillary Electrophoresis) can be used. These separation methods are usually used as separation-detection devices by combining with mass spectrometry (MS, Mass Spectrometry). Furthermore, metabolome analysis can entrust companies and institutions that provide analytical services, such as Human Metabolome Technologies, Inc.

Among the metabolites thus identified and quantified in general, factors relating to the undifferentiation or health of the mesenchymal stem cells are evaluated. As such a factor, an in vivo low-molecular substance such as nicotine indoleamine adenine dinucleotide-related (NAD ⁺ /NADH), glutathione-related, related to energy production such as oxidation, reduction reaction or ATP in vivo may be mentioned. (GSH/GSSG), glycolytic system related (glucose 6 phosphate/lactic acid/ATP), and pentose phosphate cycle (glucose 6 phosphate/ribulose 5 phosphate).

In addition to the metabolome analysis described above, the gene expression of mesenchymal stem cells is comprehensively determined by microarray analysis, and the gene of the undifferentiated or healthy mesenchymal stem cells is used as an index, thereby further Sexual evaluation. Examples of such an indicator include a gene specifically expressed in stem cells or an inflammatory cytokine gene such as β-catenin (CTNNB1), casein kinase 1D (CSNK1D), PPARγ (PPARG), and leukemia. Inhibitory factor receptor (LIFR), T cell factor-4 (TCF-4), basic fibroblast growth factor (FGF2), vascular endothelial cell proliferation factor (VEGFA), transforming growth factor 2 (TGFB2), frizzy protein homologue (Frizzled homologue) 1 (Drosophila) (FRZ-1), Frizzled protein homolog 4 (Drosophila) (FRZ-4) and other genes.

Thus, by comprehensively analyzing the metabolites and expression genes present in the mesenchymal stem cells, it is possible to determine "the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells." Regarding such an oxygen concentration, specifically, it is a low oxygen concentration of more than 1% to less than 21% (normal oxygen concentration). Further, it is typically from 2% to 16%, preferably from 3% to 12%, more preferably from 3% to 7%, still more preferably from 4% to 6%, most preferably 5%.

Preferably, the leaf system stem cells are formed into a sphere between the cultures as described above (Japanese Patent Laid-Open No. Hei 7-274950). The formation of the spheres can be carried out by proliferating the cells until they are saturated, and after separating the cells, suspending the cells in the medium, and winding the suspension of the cells on a medium in a petri dish which is not subjected to the subsequent treatment, and placing Over the course of several days, a cell assembly of rounded cell masses (Spheroids) is formed. Preferably, the formation of the spheres is carried out in the absence of bFGF, but the formation of spheres in the presence of bFGF is also sufficiently completed. Further, the period in which the sphere is formed is not particularly limited, and it can be carried out on the cultured cells which are finished in the last subculture. As a culture method for forming a spheroid, a roller bottle culture, a spin bottle culture, a hanging drop culture, and the like are known, and a culture vessel having a depressed portion or a cell adherence is low. The coated culture container is commercially available. Further, the cell adhesion region and the cell non-adhesive region are coexisted, and the culture container coated with a phosphoric acid choline (PC) is used as a cell non-adhesive region to culture. This can form a large number of spheroids of uniform size.

The spheroidized stem cells prepared in the above manner maintain the multi-differentiation ability of the phylogenetic stem cells, and therefore, can be used for in vitro experiments, regenerative medicine, and the like for clarifying the mechanism of differentiation of mesenchymal stem cells.

The present invention will now be described in detail by way of examples.

[Example 1] 1-1) Culture of leaf line stem cells (ADSC) derived from subcutaneous fat

At the Shiseido Research Center (Xin Yokohama), prepare to fill with 1%, 5% or 21% oxygen, Each standard 5% carbon dioxide and the remaining standard nitrogen were used to culture ADSC (purchased from Invitrogenn) in a closed container. The culture was carried out using StemPro SFM MSC (Gibco) as a medium, and the container was placed in an incubator at 37 °C.

1-2) Metabolome analysis of ADSC (mass spectrometry) Pretreatment

The culture of ADSC was carried out in accordance with the above 1-1). Each of the oxygen conditions (1%, 5%, and 21%) was set to N=3, and the culture was used for 2 days as a sample. The cultured cell population was present in about 1 million per 10 cm dish. In order to obtain the living substance in the cell, it is sent to Human Metabolome Technologies Co., Ltd. (HMT) (specifically, it is washed twice with an isotonic mannitol aqueous solution, and extracted with 1 mL of a 50% aqueous methanol solution, and then borrowed. Deproteinization by centrifugation filtration. After the sample was taken up by HMT, the filtrate was dried and dissolved again in 50 μL of Milli-Q water (ultra-pure water) for measurement.

Determination

In this test, the measurement of the cation mode and the anion mode was carried out under the conditions shown below.

Cationic metabolite (cationic mode): device

Agilent CE-TOFMS system (Agilent Technologies) No. 3

Capillary: Fused silica capillary i.d.50μm×80cm

Measuring condition

Run buffer: Cation Buffer Solution (p/n: H3301-1001)

Rinse buffer: Cation Buffer Solution (p/n: H3301-1001)

Sample injection: Pressure injection 50 Barr, 10sec

CE voltage: Positive, 27kV

MS ionization: ESI (Electronic Spray Ion)

MS capillary voltage: 4,000V

MS scan range: m/z 50-1,000

Sheath liquid: HMT Sheath Liquid (p/n: H3301-1020)

Anionic metabolite (anion mode): device

Agilent CE-TOFMS system (Agilent Technologies) No. 1

Capillary: Fused silica capillary i.d.50μm×80cm

Measuring condition

Run buffer: Anion Buffer Solution (p/n: I3302-1023)

Rinse buffer: Anion Buffer Solution(p/n:I3302-1023)

Sample injection: Pressure injection 50mbar, 25sec

CE voltage: Positive, 30kV

MS ionization: ESI Negative

MS capillary voltage: 3,500V

MS scan range:m/z 50-1,000

Sheath liquid: HMT Sheath Liquid (p/n: H3301-1020)

Data processing and analysis

The peak detected by CE-TOFMS (Capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry) using the automatic integration software MasterHands ver.2.13.0.8.h (Qing Ying Developed by the University of the Private School, the automatic sampling is performed as the peak information, and the mass-to-charge ratio (m/z), the electrophoresis time (MT, migration time), and the peak area value are obtained. The peak area value obtained was converted into a relative area value using the following formula: relative area value = area value of the target peak / area value of the internal standard substance × sample amount.

Further, in these materials, since the adduct ions such as Na ⁺ or K ⁺ and the fragment ions such as dehydration or deammonium are contained, the molecular weight-related ions are deleted. However, since there are also substance-specific adducts or fragments, it is impossible to examine them in detail. Regarding the peak value of the detailed investigation, the comparison and alignment of the peaks between the samples were performed based on the values of m/z and MT.

The analysis results of the undifferentiated and healthy metabolites of the relevant ADSCs are shown below.

Nicotine indoleamine adenine dinucleotide correlation (NAD ⁺ /NADH)

NADH is a reducing agent in vivo, which is an energy currency. NADH is converted to NAD ⁺ by oxidation, and energy is supplied to other molecules (redox reaction) in exchange. NADH is mainly produced by the glycolysis system, so it can be seen that NADH is at a high level at an oxygen concentration of 21%. On the other hand, the used NADH, that is, NAD ⁺ is highest at an oxygen concentration of 1%, followed by a sequence of 21% and 5% (Fig. 1). When the oxygen concentration is 1%, the NADH is small and the NAD ^{+ is} large. Therefore, it is considered that there is a tendency to generate energy due to mitochondria other than the glycolysis system, and it is possible to estimate the generation of free radicals. The pressure on the cells becomes too large. Further, it is considered that when the oxygen concentration is 21%, the NADH is generated by excessive glycolysis, but the oxide NAD ^{+ is} also increased, and excess energy is consumed (wasted) by the entire cell. On the other hand, it has been found that the accumulation of oxides is less in the middle of the oxygen concentration of 5%, especially in the case of less NAD ⁺ . Regarding the case where NAD ^{+ is} small, it can be considered that the oxidative pressure of the corresponding cells is not applied.

Glutosin related (GSH/GSSG)

Reduced glutathione (GSH) decreased at 5% oxygen concentration and no change in oxidized form (GSSG) (Fig. 2). It is considered that if the amount of GSH is increased in the cells, the active oxygen removal is exerted. Role and have a good impact on the activity. On the other hand, it is understood that the amount of cysteine (Cys) which is a starting material of GSH is small. That is, it is considered that in the case of the stem cells, the case where the GSH is not required, that is, the case where the detoxification or the reduction reaction is not performed is caused by the oxygen concentration of 5%.

Glycolysis related (glucose 6 phosphate / lactic acid / ATP)

The lactic acid is increased as a hypoxic pressure marker. It is known that in an environment where lactic acid is increased, energy acquisition by the glycolysis system becomes unfavorable, and active oxygen increases. On the other hand, it is understood that the amount of lactic acid increases at an oxygen concentration of 1% (Fig. 3). Further, glucose 6-phosphate (G6P), which is an intermediate metabolite of the glycolysis system, increases at an oxygen concentration of 1%. These data show that the rate of reaction of the glycolysis pathway is reduced by insufficient oxygen, resulting in retention of intermediates. In either case, the hypoxic pressure at 1% oxygen concentration was significant, whereas at 5% and 21% oxygen concentration, no major obstacles were observed in the glycolysis system. Further, regarding ATP, an increase was also observed at an oxygen concentration of 1%. It was observed that at 1% oxygen concentration, ATP acquisition by mitochondria is superior to other conditions. The use of energy of mitochondria produces cytotoxicity at an oxygen concentration of 1% due to the production of peroxide in the form of by-products.

Pentose phosphate cycle related (glucose 6 phosphate / ribulose 5 phosphate)

The pentose phosphate cycle for supplying nicotine decylamine was activated at an oxygen concentration of 1%, and specifically, an increase in glucose 6-phosphate and ribulose 5-phosphate was confirmed (Fig. 4). This cycle is a bypass of the glycolysis system that is stagnant under hypoxia and supports the production of nicotine guanamine required for the elimination of hypoxic ROS (Reactive Oxygen Species).

1-3) Microarray analysis of ADSC

Microarray analysis was performed using an Agilent human 4x44K microarray (Agilent Technologies).

Preparation and hybridization of the samples are based on the operating instructions recommended by Agilent Technologies. Further, the cells used in the sample were each set to N=4 under each oxygen condition (1%, 5%, 21%) according to the above 1-1), and the leaf system derived from subcutaneous fat was cultured for 3 days. stem cell. For the total amount of RNA (Ribonucleic Acid, ribonucleic acid) 1~2μg, use A cRNA (Complementary Ribonucleic Acid) labeling kit (Agilent Technologies) was used for labeling. After purification by Qiaquick (purification kit) (Qiagen, manufacturer's recommended operating instructions), the solvent was dried using a SpeedVac (centrifugal concentrator). The slides were then washed after hybridization using an Agilent human 4x44K microarray. Data was read by a scanner (Agilent Technologies).

The analysis results of the microarray of the undifferentiated and healthy ADSC can be shown below.

CTNNB1/CSNK1D/PPARG

As an advantage of 5% oxygen concentration, the expression of self-replicating β-catenin (CTNNB1) of related stem cells was specifically increased (Fig. 5). At the same time, the performance of casein kinase 1D (CSNK1D) located upstream of the signal was also improved. Further, PPARγ (PPARG), which determines the properties of ADSC under the same conditions, is also specifically hyperactive.

LIFR/TCF-4

It is known that LIFR is a leukemia inhibitory factor (LIF) receptor, and by combining LIFR with LIF, the undifferentiated maintenance mechanism of stem cells is advanced. Further, TCF-4 is a transcription factor that functions by specifically conjugating stem cells with β-catenin, and is associated with self-replication ability and production induction. Significant performance of LIFR and TCF-4 was confirmed in an environment with an oxygen concentration of 5% (Fig. 6).

FGF2

The basic fibroblast growth factor (FGF2) showed a decrease in the case of a higher oxygen concentration (21%) (Fig. 7). The factor required for the culture of FGF2 lineage stem cells.

VEGFA/TGFB2

As a factor causing excessive angiogenesis, vascular endothelial cell proliferation factor (VEGFA) is exemplified, and this gene is also shown to be hyperactive at an oxygen concentration of 1% (Fig. 8).

In addition, a transforming growth factor that is a factor associated with cell death has also been identified. 2 (TGFB2) performance is improved.

[Embodiment 2] 2-1) Culture of dermal root sheath (DS) cells

At the Shiseido Research Center (Xin Yokohama), prepare a standard gas filled with 1%, 5% or 21% oxygen, 5% carbon dioxide, and the remaining amount of nitrogen. Use this standard gas to culture DS in a closed container. Cells (purchased from Invitrogenn). The culture was carried out using StemPro SFM MSC (Gibco) as a medium, and the container was placed in an incubator at 37 °C.

2-2) Alkaline phosphatase (AP) detection of DS cells

The AP activity as a stem cell and a hair follicle-inducing marker was measured for each DS cell cultured according to the above 2-1).

The DS cells cultured in a 6-well cell culture plate were washed once with PBS (Phosphate Buffer Solution, phosphate buffer) (-), and 50 μl of the fixing solution (4% paraformaldehyde-phosphate buffer) was added. To each hole. After 5 minutes, the mixture was washed twice with 1 ml of PBS(-), and then washed once with TBS (Tris buffered saline, trishydroxymethylaminomethane buffer) (pH 9.5), using alkaline phosphatase ( The matrix BM-purple (Roche) of AP) was subjected to a color reaction at 37 ° C for 1 hour. Four washes were performed using ultrapure water, followed by replacement with PBS(-).

As shown in Fig. 9, it is understood that the AP activity in DS cells is inversely proportional to the oxygen concentration. It is believed that those with lower oxygen concentrations have a tendency to enhance stem cell identity.

2-3) Metabolome analysis of DS cells (mass spectrometry)

In the same manner as in the above 1-2), intracellular metabolite changes in DS cells cultured at 5% oxygen concentration and 21% oxygen concentration were quantitatively analyzed by a mass spectrometer (registered analysis: HMT Company).

The analysis results of the undifferentiated and healthy metabolites of the relevant DS cells are shown below.

Glutosin related (GSH/GSSG)

As shown in Figure 10, both oxidized glutathione and reduced glutathione were increased under 21% oxygen conditions. Further, when the reduction ratio of glutathione was observed, it was found that the ratio of the reduced form was higher under 5% oxygen conditions than 21%.

It is believed that under hyperoxic conditions (21%), the activity of the cells increases, and the overall capacity of the oxidized reduction glutathione is increased. On the other hand, it is considered that the glutathione has a reduced capacity and a reduced oxidative pressure at a pH of 5% close to the concentration in the living body.

Hypoxanthine

Analysis of hypoxanthine associated with reactive oxygen species pressure during DNA replication in related cells.

Regarding the amount of hypoxanthine, it was reduced to 1/4 under 5% oxygen conditions compared to 21% (Fig. 11). It is believed that under hyperoxic conditions (21%), cell activity increases and DNA metabolism becomes active. On the other hand, it is considered that the oxidative pressure generated by the active oxygen caused by the decrease in hypoxanthine is lowered under the condition of 5% oxygen concentration close to the living body concentration.

Glycolysis related (glucose 6 phosphate / lactic acid / ATP)

As shown in Fig. 12, the 21% oxygen condition was more than twice as high as the initial metabolite glucose 6 phosphate (G6P) and the late metabolite phosphoenolpyruvate compared to the 5% condition. It is considered that under high oxygen conditions (21%), the activity of the glycolysis of the cells is increased. It is considered that high oxygen conditions (21%) obtain more energy, and on the other hand, the above high oxidation pressure is also induced. It is considered that under 5% oxygen conditions, energy generation is reduced, and on the other hand, as described above, oxidation pressure can be avoided.

Nicotine indoleamine adenine dinucleotide correlation (NAD ⁺ /NADH)

The central carbon metabolism (NADH/NAD ⁺ ratio), which is an indicator of energy metabolism in cells, is twice as high as that under 5% oxygen conditions under high oxygen conditions (21%). Further, the amount of NADH exhibiting the activity of the TCA cycle (Tricarboxylic acid cycle) was also doubled under high oxygen conditions (Fig. 13).

It is believed that under high oxygen conditions (21%), not only the glycolysis system, but also more energy is obtained in the mitochondrial TCA cycle.

2-4) Western blot analysis of DS cells

Intracellular proteins were extracted from DS cells cultured for 2 days at 5% oxygen concentration and 21% oxygen concentration and analyzed by Western blot method (primary antibody was taken from Cell Signaling).

The antibody corresponds to GSK-3β (stem cell identity), OCT-4 (stem cell identity), and HIF-1α (hypoxia transcription factor). As shown in Figure 14, the stem cell identity was controlled to be negative for GSK-3β to increase under hyperoxia, the stem cell identity was controlled to be positive for OCT-4 to decrease under high oxygen, and the induced transcription factor (HIF) under hypoxia. -1α) is neutral (undetectable).

Based on these results, it can be considered that the oxygen condition (5%) in the living state is in a state in which the stem cell identity is increased as compared with the hyperoxic condition (21%).

2-5) Detection of luciferase in DS cells

For the DS cells cultured for 2 days in MesenPro RS medium (Gibco) at 5% oxygen concentration and 21% oxygen concentration, in order to evaluate stem cell identity, the transcription factor of the Wnt signal pathway was detected by double luciferase assay ( TCF-Lef (Tricresyl phosphate-Lef, glycerol phosphate-Lef) activity reporter assay (Promega). Recombinant human Wnt-3a protein was used as a stimulating substance.

Depending on the Wnt-3A concentration, an increase in TCF-Lef activity was observed at either oxygen concentration (Figure 15). In the comparison of 21% and 5% oxygen conditions in the presence of 50 ng/ml of Wnt3a, TCF-Lef activity was more than 2-fold.

It is believed that the Wnt signal is also aggressive in the state of the characteristic signal path when stem cells and hair hair are induced. It is considered that in this experiment, the in vivo oxygen condition (5%) is superior to the hyperoxic condition (21%) in maintaining the stem cell identity of the DS cells and the Wnt signaling system of the hair follicle inducing ability.

Claims (7)

A method for cultivating mesenchymal stem cells between undifferentiated and healthy, and comprising culturing mesenchymal stem cells under an oxygen concentration suitable for maintaining undifferentiation and health of mesenchymal stem cells Made. The method of claim 1, wherein the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is from more than about 1% to less than about 21%. The method of claim 1, wherein the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is from about 3% to about 7%. The method of claim 1, wherein the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is more than about 1% to about 5% or less. The method of claim 1, wherein the oxygen concentration suitable for maintaining the undifferentiation and health of the mesenchymal stem cells is about 5%. The method of any one of claims 1 to 5, wherein the mesenchymal stem cells are dermal root sheath (DS) cells. The method of any one of claims 1 to 5, wherein the mesenchymal stem cells are derived from adipose mesenchymal stem cells.