Method for producing brown adipose tissue from stem cells, hydrogel, and differential induction agent for stem cells into brown adipose tissue.

Abstract

To provide a novel method for obtaining brown adipose tissue by differentiating stem cells. [Solution] A method for producing brown adipose tissue, comprising a culture step of culturing a hydrogel containing stem cells and a cationic polymer in a medium containing 3,3',5-triiodo-L-thyronine and insulin, thereby inducing differentiation of the stem cells into brown adipose tissue.

Description

[Technical Field]

[0001] This invention relates to a method for producing brown adipose tissue from stem cells, a hydrogel, and an agent for promoting differentiation of stem cells into brown adipose tissue. [Background technology]

[0002] Brown adipose tissue (BAT) promotes heat dissipation by detaching proton transport from ATP synthesis. BAT, thanks to its abundant mitochondria and uncoupling protein 1 (UCP1), can convert the electrochemical energy generated during respiration into heat by detaching mitochondrial lipid oxidation from ATP production (Non-Patent Literature 1-3). This metabolic process contributes to oxygen intake, calorie expenditure, and thermoregulation (Non-Patent Literature 3). Because BAT strongly influences calorie expenditure, it is considered a potential therapeutic target for obesity. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] DF Pisani, V. Barquissau,JC Chambard, D. Beuzelin, RA Ghandour, M. Giroud, A. Mairal, S. Pagnotta,S. Cinti, D. Langin, EZ Amri, Mitochondrial fission is associated with UCP1activity in human brite / beige adipocytes, Mol. Metabol. 7 (2018), https: / / doi.org / 10.1016 / j.molmet.2017.11.007. [Non-Patent Document 2] ET Chouchani, L. Kazak, BMSpiegelman, New advances in adaptive thermogenesis: UCP1 and beyond, CellMetabol. 29 (2019), https: / / doi.org / 10.1016 / j.cmet.2018.11.002. [Non-Patent Document 3] M. Christian, In vitro models for study of brown adipocyte biology, Handb. Exp. Pharmacol. 251 (2019) 85-96, https: / / doi.org / 10.1007 / 164_2018_122. [Overview of the project] [Problems that the invention aims to solve]

[0004] There is no established method for inducing differentiation from stem cells into brown adipose tissue, and new methods are needed.

[0005] The present invention aims to provide a novel method for obtaining brown adipose tissue by differentiating stem cells. The present invention also aims to provide a hydrogel containing brown adipose tissue obtained by this method. The present invention also aims to provide an agent that promotes differentiation of stem cells into brown adipose tissue. [Means for solving the problem]

[0006] This invention encompasses the following inventions. [1] A method for producing brown adipose cells from stem cells, comprising a culture step of culturing a hydrogel containing stem cells and a cationic polymer in a medium containing 3,3',5-triiodo-L-thyronine and insulin to induce differentiation of the stem cells into brown adipose cells. [2] The method according to [1], wherein the cationic polymer is polylysine. [3] The method according to [1] or [2], wherein the hydrogel is a fibrin gel. [4] The method according to any one of [1] to [3], wherein the stem cells are dedifferentiated adipocytes. [5] The method according to any one of [1] to [4], wherein the concentration of the cationic polymer in the hydrogel is 5 μg / mL or more and 20 μg / mL or less. [6] The method according to any one of [1] to [5], further comprising a gelation step of mixing stem cells, a cationic polymer, and a hydrogel precursor before the culture step, to gel a mixture obtained therefrom, thereby forming a hydrogel containing stem cells and a cationic polymer. [7] The method according to [6], wherein the hydrogel precursor comprises fibrinogen and thrombin. [8] A hydrogel containing brown adipose tissue and a cationic polymer. [9] The hydrogel according to [8], wherein the cationic polymer is polylysine.

[10] A fibrin gel, the hydrogel described in [8] or [9].

[11] The hydrogel according to any one of [8] to

[10] , wherein the concentration of the cationic polymer in the hydrogel is 5 μg / mL or more and 20 μg / mL or less.

[12] A differentiation inducer that promotes the differentiation of stem cells into brown adipose tissue, comprising a cationic polymer as an active ingredient.

[13] The differentiation-inducing agent according to

[12] , wherein the cationic polymer is polylysine. [Effects of the Invention]

[0007] According to the present invention, a new method for inducing the differentiation of stem cells to obtain brown adipocytes can be provided. According to the present invention, a hydrogel containing brown adipocytes obtained by the method can be provided. According to the present invention, an agent for promoting the induction of differentiation from stem cells to brown adipocytes can be provided.

Brief Description of Drawings

[0008] [Figure 1] Immunofluorescence imaging images of nuclei, lipids, and UCP1 of the hydrogel of the example under the condition of using PLL for differentiation induction culture, merged images thereof, and high-magnification images of the merged images. [Figure 2] A graph showing the ratio of the amount of uncoupling protein 1 (UCP1) to the DNA fluorescence intensity and the ratio of the amount of lipid to the DNA fluorescence intensity of the hydrogel of the example under differentiation induction culture. [Figure 3] A graph showing the ELISA results of UCP1 of the hydrogel of the example under differentiation induction culture. [Figure 4] A graph showing the results of the relative gene expression level of UCP1 of the hydrogel of the example under differentiation induction culture. [Figure 5] A graph showing the results of the relative gene expression level of Cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) of the hydrogel of the example under differentiation induction culture. [Figure 6] A graph showing the results of the relative gene expression level of PR domain containing 16 (PRDM16) of the hydrogel of the example under differentiation induction culture. [Figure 7] A representative MitoTracker staining image of mitochondria (red) and nuclei (blue) by Hoechst counterstaining of the hydrogel of the example under the condition of using PLL for differentiation induction culture. [Figure 8] A graph showing the results of the ratio of the amount of mitochondria to the DNA fluorescence intensity of the hydrogel of the example under differentiation induction culture. [Figure 9] This graph shows the 24-hour oxygen consumption rate of hydrogels from the differentiated and induced cultured examples. [Modes for carrying out the invention]

[0009] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments. In the numerical ranges described step by step in this specification, the upper or lower limit of a numerical range in one step can be arbitrarily combined with the upper or lower limit of a numerical range in another step. In the numerical ranges described in this specification, the upper or lower limit of a numerical range may be replaced with the values ​​shown in the examples.

[0010] [Method for producing brown adipose tissue] The method according to this embodiment is a method for producing brown adipose cells from stem cells, comprising a culture step of culturing stem cells and a hydrogel containing a cationic polymer in a culture medium containing 3,3',5-triiodo-L-thyronine (hereinafter also referred to as "T3") and insulin to induce differentiation of stem cells into brown adipose cells.

[0011] The method according to this embodiment may further include a gelation step, prior to the culture step, in which a mixture obtained by mixing stem cells, a cationic polymer, and a hydrogel precursor is gelled to form a hydrogel containing stem cells and a cationic polymer.

[0012] The method according to this embodiment, including the gelation step and the culture step, will be described below.

[0013] <Gelation Process> In the gelation process, a mixture obtained by mixing stem cells, a cationic polymer, and a hydrogel precursor is gelled to form a hydrogel containing stem cells and a cationic polymer.

[0014] In this specification, "stem cells" means cells that have the ability to self-renew and multipotency. Stem cells include pluripotent stem cells, which have the ability to differentiate into any cell tumor, and tissue stem cells (also called somatic stem cells), which have the ability to differentiate into specific cell tumors. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells, hematopoietic stem cells, and neural stem cells.

[0015] Stem cells may be adipose-derived stem cells. In this specification, "adipose-derived stem cells" include adipose-derived mesenchymal stem cells and dedifferentiated adipocytes. Adipose-derived mesenchymal stem cells are mesenchymal stem cells harvested from adipose tissue. Dedifferentiated adipocytes are stem cells that can be obtained by dedifferentiating mature adipocytes through ceiling culture. Mature adipocytes can be obtained by isolation from adipose tissue.

[0016] The origin of stem cells is not particularly limited, but may be human, for example, or other mammals. Examples of non-human mammals include non-human primates (e.g., monkeys), dogs, cats, rabbits, pigs, cows, mice, rats, etc.

[0017] In this specification, "cationic polymer" means a polymer having a cationic group in its molecule. Examples of cationic groups include primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium groups.

[0018] The molecular weight of the cationic polymer may be 10 kDa or more, 30 kDa or more, or 50 kDa or more, and may be 300 kDa or less, 200 kDa or less, or 150 kDa or less. The molecular weight of the cationic polymer can be measured by, for example, GPC method, light scattering method, etc.

[0019] The cationic polymer may be, for example, a cationic polyamino acid. Examples of cationic polymers include polylysine (poly-L-lysine or poly-D-lysine) and its salts, polydiallyldimethylammonium salts (e.g., polydiallyldimethylammonium chloride), and polyallylamine and its salts (e.g., polyallylamine hydrochloride).

[0020] The cationic polymer is preferably a polymer with a lower risk of causing cytotoxicity, and more preferably at least one selected from the group consisting of polylysine and polydiallyldimethylammonium chloride, and even more preferably polylysine, as this reduces the risk of causing cytotoxicity. The polylysine may be ε-polylysine or α-polylysine.

[0021] ε-Polylysine is a cationic polyamino acid containing a structure in which the amino group at the ε position of lysine (ε-amino group) is peptide-bonded to a carboxyl group. ε-Polylysine may be a polyamino acid in which 25 to 35 units of the L-lysine unit, in which the ε-amino group is peptide-bonded to a carboxyl group, are linked in a linear chain. α-Polylysine is a cationic polyamino acid containing a structure in which the amino group at the α position of lysine (α-amino group) is peptide-bonded to a carboxyl group.

[0022] In this specification, "hydrogel" means a polymer that has been crosslinked by hydrogen bonds, ionic bonds, coordination bonds, covalent bonds, etc., to form a three-dimensional network structure, and which contains a liquid such as water inside the three-dimensional network structure. In this specification, "hydrogel precursor" means a substance that forms a hydrogel upon some external stimulus such as chemical or physical stimulation, and which is in a state in which a hydrogel has not yet formed.

[0023] The hydrogel may be a gel composed of biocompatible molecules such as fibrin, collagen, gelatin, hyaluronic acid, alginic acid, pectin, and chitosan. The hydrogel may also be a gel composed of crosslinked biocompatible molecules, such as those obtained by crosslinking these biocompatible molecules intermolecularly and / or intramolecularly. Examples of hydrogel precursors include fibrinogen and thrombin, collagen, gelatin, hyaluronic acid, alginic acid, and pectin. It is preferable that the hydrogel precursor contains fibrinogen and thrombin.

[0024] The hydrogel is preferably a fibrin gel, which is a gel composed of fibrin. Fibrin is a component produced when thrombin acts on fibrinogen, releasing A and B chains from the N-terminuses of the Aα and Bβ chains. Fibrin is formed by bringing fibrinogen into contact with thrombin.

[0025] The hydrogel may be a gel containing stem cells and a cationic polymer. The stem cells and cationic polymer may be dispersed within the hydrogel.

[0026] The order in which the stem cells, cationic polymer, and hydrogel precursor are mixed is not particularly limited. Any two of the stem cells, cationic polymer, and hydrogel precursor may be mixed first, followed by the remaining two. Alternatively, the stem cells, cationic polymer, and hydrogel precursor may be mixed simultaneously or nearly simultaneously.

[0027] The gelation process may be carried out by mixing stem cells with a solution containing a cationic polymer and an aqueous medium, and a solution containing a hydrogel precursor and an aqueous medium. In this specification, "aqueous medium" means a liquid in which water is an essential component. The aqueous medium may be water, physiological saline such as phosphate-buffered saline (PBS), or liquid medium such as Dulbecco's Modified Eagle medium (DMEM). The liquid medium may be a mixed medium obtained by mixing two or more types of media. The pH of the aqueous medium may be 6.0 to 8.0, 6.8 to 7.8, or 7.2 to 7.6. The aqueous medium used in the gelation process may contain serum (e.g., fetal bovine serum (FBS)) and antibiotics as needed.

[0028] The stem cell seeding density is, for example, 1.0 × 10⁻⁶ based on the total volume of the hydrogel (or mixture). 4 cells / mL or more, or 1.0 × 10 5 cells / mL or more, and 1.0 × 10 8 cells / mL or less, or 1.0 × 10 7 The seeding density may be less than or equal to cells / mL. The seeding density indicates the number of cells per 1 mL of hydrogel (or mixture) at the time of mixing with the cationic polymer and hydrogel precursor.

[0029] The concentration of the cationic polymer may be 1 μg / mL or more, 2 μg / mL or more, 3 μg / mL or more, 5 μg / mL or more, 8 μg / mL or more, 10 μg / mL or more, 13 μg / mL or more, 15 μg / mL or more, or 18 μg / mL or more, based on the total amount of hydrogel (or mixture). The concentration of the cationic polymer may be 60 μg / mL or less, 55 μg / mL or less, 50 μg / mL or less, 45 μg / mL or less, 40 μg / mL or less, 35 μg / mL or less, 30 μg / mL or less, 25 μg / mL or less, or 22 μg / mL or less, based on the total amount of hydrogel.

[0030] The concentration of the hydrogel precursor may be 0.1 mg / mL or higher, 0.2 mg / mL or higher, 0.3 mg / mL or higher, 0.4 mg / mL or higher, 0.5 mg / mL or higher, 0.6 mg / mL or higher, 0.7 mg / mL or higher, 0.8 mg / mL or higher, 0.9 mg / mL or higher, or 1.0 mg / mL or higher, based on the total amount of hydrogel (or mixture). The concentration of the hydrogel precursor may be 10.0 mg / mL or lower, 8.0 mg / mL or lower, 6.0 mg / mL or lower, 5.0 mg / mL or lower, 3.0 mg / mL or lower, 1.0 mg / mL or lower, 0.8 mg / mL or lower, or 0.6 mg / mL or lower, based on the total amount of hydrogel (or mixture). When using a hydrogel precursor (a combination of fibrinogen and thrombin) that forms a hydrogel by mixing two compounds, the concentration of at least one of them (for example, fibrinogen) may be within the range of the above-mentioned hydrogel precursor concentrations. For example, if the hydrogel precursor contains fibrinogen and thrombin, the fibrinogen concentration may be 4.0-8.0 mg / mL or 5.0-7.0 mg / mL based on the total volume of the hydrogel (or mixture), and the thrombin concentration may be 1-5 U / mL or 2-4 U / mL based on the total volume of the hydrogel (or mixture).

[0031] A hydrogel can be obtained by incubating the mixture to allow the gelation reaction to proceed. The incubation temperature of the mixture may be 20°C or higher, 25°C or higher, 30°C or higher, or 35°C or higher, and may be 40°C or lower, or 38°C or lower, or 30°C to 37°C. The incubation time of the mixture may be 5 minutes or higher, 10 minutes or higher, 15 minutes or higher, or 20 minutes or higher, and may be 40 minutes or lower, or 30 minutes or lower.

[0032] The container used in the gelation process is not particularly limited, and any known cell culture container can be used. Examples of containers used in the gelation process include dishes, well inserts, low-adhesion plates, and plates with bottom shapes such as U-shaped or V-shaped.

[0033] <Preliminary culture process> The method according to this embodiment may further include a step of performing a preliminary culture of the hydrogel as needed before the culture step (differentiation induction culture) (preliminary culture step).

[0034] Preliminary culture can be performed by culturing the hydrogel in a liquid medium that does not contain T3 and insulin. Growth medium can be used as the medium for preliminary culture. The growth medium may be, for example, the medium exemplified as a liquid medium in this specification, and may be high-glucose DMEM. The pH of the medium may be 6.0 to 8.0, 7.0 to 8.0, or 7.2 to 7.6. The growth medium may further contain serum and antibiotics, etc.

[0035] The culture temperature during the preliminary culture may be 20°C or higher, 25°C or higher, 30°C or higher, or 35°C or higher, and may be 40°C or lower, or 38°C or lower. The duration of the preliminary culture may be, for example, 1 to 3 days or 2 days. After the preliminary culture, the culture process can be carried out by replacing the culture medium for the preliminary culture with a medium containing T3 and insulin.

[0036] <Culture process> In the culture process, stem cells and a hydrogel containing a cationic polymer are cultured (differentiation induction culture) in a medium containing T3 and insulin, which are brown adipose tissue differentiation inducers, to induce differentiation of the stem cells in the hydrogel into brown adipose cells.

[0037] Differentiation into brown adipocytes can be confirmed by brown adipocyte differentiation markers. Specifically, differentiation into brown adipocytes can be confirmed by measuring UCP1 content and lipid content, measuring the expression levels of the UCP1 gene, Cidea gene, and PRDM16 gene, and measuring mitochondrial content and oxygen consumption rate.

[0038] In the method according to this embodiment, the hydrogel used in the culture process contains a cationic polymer, thereby promoting the differentiation induction of stem cells into brown adipocytes. The following is considered to be the mechanism by which this effect is obtained: T3 and insulin, which are brown adipocyte differentiation inducers, have negative zeta potentials and can be adsorbed by cationic polymers. Electrostatic interactions between the cationic polymer to which T3 and insulin are adsorbed and negatively charged molecules present on the cell surface (such as heparan sulfate proteoglycan (HSPG)) attract the T3 and insulin adsorbed to the cationic polymer to stem cells, thereby promoting the uptake of these brown adipocyte differentiation inducers into stem cells. As a result of easier uptake of brown adipocyte differentiation inducers into cells, differentiation into brown adipocytes is thought to be promoted, but the mechanism is not limited to this.

[0039] The culture medium used for differentiation induction culture (differentiation induction medium) contains T3 and insulin.

[0040] The concentration of T3 in the culture medium may be 10 nM or higher, 50 nM or higher, or 100 nM or higher, and may be 200 nM or lower, 150 nM or lower, or 130 nM or lower, based on the total volume of the culture medium.

[0041] The insulin may be bovine insulin. The insulin concentration in the culture medium may be 10 nM or higher, 50 nM or higher, 100 nM or higher, 300 nM or higher, 500 nM or higher, 700 nM or higher, or 800 nM or higher, based on the total volume of the culture medium, and may be 2000 nM or lower, 1600 nM or lower, 1200 nM or lower, 1000 nM or lower, or 900 nM or lower.

[0042] The culture medium may contain components other than T3 and insulin. Other components may include serum, dexamethasone, indomethacin, 3-isobutyl-1-methylxanthine (IBMX), peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists (such as rosiglitazone), and antibiotics (such as penicillin-streptomycin).

[0043] Examples of serum include fetal bovine serum (FBS). The concentration of serum in the culture medium may be, for example, 1 v / v% or more, 3 v / v% or more, 5 v / v% or more, 7 v / v% or more, or 9 v / v% or more, based on the total volume of the culture medium, and may be 15 v / v% or less, 13 v / v% or less, or 11 v / v% or less.

[0044] The concentration of dexamethasone in the culture medium may be, for example, 10 nM (0.01 μM) or more, 50 nM (0.05 μM) or more, 0.1 μM or more, 0.2 μM or more, or 0.4 μM or more, based on the total volume of the culture medium, and may be 5 μM or less, 3 μM or less, 1 μM or less, 0.8 μM or less, or 0.6 μM or less.

[0045] The concentration of indomethacin in the culture medium may be, for example, 10 nM or more, 20 nM or more, 40 nM or more, 60 nM or more, 80 nM or more, 100 nM or more, or 120 nM or more, based on the total amount of the culture medium, and may be 1000 nM or less, 800 nM or less, 600 nM or less, 400 nM or less, 200 nM or less, 160 nM or less, or 130 nM or less.

[0046] The concentration of IBMX in the culture medium may be, for example, 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, 220 μM or more, or 240 μM or more, based on the total amount of the culture medium, and may be 2000 μM or less, 1500 μM or less, 1000 μM or less, 800 μM or less, 600 μM or less, 400 μM or less, 300 μM or less, or 260 μM or less.

[0047] The concentration of rosiglitazone in the culture medium may be, for example, 100 nM or higher, 300 nM or higher, 500 nM or higher, 700 nM or higher, or 900 nM or higher, based on the total volume of the culture medium, and may be 100 μM or less, 50 μM or less, 10 μM or less, 6.0 μM or less, 2.0 μM or less, 1.8 μM or less, 1.6 μM or less, or 1.2 μM or less.

[0048] For inducing differentiation into brown adipocytes, a liquid medium containing T3, insulin, and other components as needed can be used. Examples of liquid media include Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The liquid medium may also be a mixed medium prepared by mixing two types of media. The liquid medium may be high-glucose DMEM. The medium for inducing differentiation into brown adipocytes can be prepared using T3, insulin, other components as needed, and their salts.

[0049] The culture temperature may be, for example, 20°C or higher, 25°C or higher, 30°C or higher, or 35°C or higher, and may be 40°C or lower, or 38°C or lower, or 30°C to 37°C. The pH of the culture medium may be 6.0 to 8.0, 7.0 to 8.0, or 7.2 to 7.6. The culture time may be 1 day or more, 3 days or more, 5 days or more, 7 days or more, 9 days or more, 11 days or more, or 13 days or more, and may be 20 days or less, 18 days or less, 16 days or less, or 14 days or less.

[0050] The culture vessel (support) is not particularly limited and may be, for example, a dish, a well insert, a low-adhesion plate, or a plate with a bottom shape such as U-shaped or V-shaped. As a culture vessel, for example, a vessel equipped with a substrate (permeable membrane) that allows liquid to pass through but does not allow cells in liquid to pass through may be used. Examples of containers equipped with a permeable membrane include, but are not limited to, cell culture inserts such as Transwell® insert, Netwell® insert, Falcon® cell culture insert, and Millicell® cell culture insert.

[0051] The hydrogel may be cultured while attached to the support, cultured without being attached to the support, or cultured after being separated from the support during the culture process. When culturing the hydrogel without being attached to the support, or when culturing after being separated from the support during the culture process, it is preferable to use a plate with a bottom shape such as a U-shape or V-shape that inhibits adhesion to the support, or a low-adsorption plate.

[0052] [Hydrogel] The hydrogel according to this embodiment comprises brown adipose tissue and a cationic polymer. The hydrogel according to this embodiment can be obtained by the manufacturing method described above. The cationic polymer may be as described above.

[0053] The content of the cationic polymer may be 1 μg / mL or more, 2 μg / mL or more, 3 μg / mL or more, 5 μg / mL or more, 8 μg / mL or more, 10 μg / mL or more, 13 μg / mL or more, 15 μg / mL or more, or 18 μg / mL or more, based on the total amount of hydrogel. The concentration of the cationic polymer may be 60 μg / mL or less, 55 μg / mL or less, 50 μg / mL or less, 45 μg / mL or less, 40 μg / mL or less, 35 μg / mL or less, 30 μg / mL or less, 25 μg / mL or less, or 22 μg / mL or less, based on the total amount of hydrogel.

[0054] [Differentiation Induction Promoter] The differentiation-inducing agent according to this embodiment is a differentiation-inducing agent from stem cells to brown adipocytes, and contains a cationic polymer as an active ingredient. The stem cells, brown adipocytes, and cationic polymer may be as described above. As specific embodiments of the differentiation-inducing agent, the embodiments described as a method for producing brown adipocytes from stem cells and embodiments of the hydrogel can be applied without limitation.

[0055] The differentiation-inducing agent may be, for example, a liquid or a solid. A liquid differentiation-inducing agent may be a solution containing a cationic polymer and an aqueous medium. The aqueous medium may be the aqueous medium described above. [Examples]

[0056] The present invention will be described more specifically below based on examples. However, the present invention is not limited to these examples.

[0057] <Chemicals and Reagents> Fibrinogen (derived from bovine plasma, F8630), thrombin (derived from bovine plasma, T4648), fibronectin (derived from human plasma, F2006), collagen type IV (derived from human placenta, C7521), poly-L-lysine (PLL, P4707), poly-L-lysine FITC conjugate (P3543), phosphate-buffered saline powder (PBS, D5652), Clostridium histolyticum collagenase ( Type I (C0130), Triton X-100 (T8787), bovine serum albumin (BSA, 3294), dexamethasone (D4902), indomethacin (I7378), 3-isobutyl-1-methylxanthine (IBMX, I5879), insulin (I6634), rosiglitazone (R2408), 3,3,5-triiodo-L-thyronine sodium salt (T3, T6397), and Hoechst 33324 (H3570) were obtained from Sigma-Aldrich, Inc. (St. Louis, Missouri, USA).

[0058] Tripan Blue (T10282), MitoTracker TM Deep Red FM (M7212), UCP1 polyclonal antibody (PA1-24894), Qubit HS DNA Assay Kit (Q3285), anti-rabbit secondary antibody Alexa Fluor® 647, Nile Red (N1142), PicoPure TM The RNA isolation kit (KITO204), penicillin, and streptomycin were obtained from Thermo Fisher Scientific, Inc. (Waltham, Massachusetts, USA).

[0059] Gelatin (077-03155), 4% paraformaldehyde (16310-245), Na2SO3 (198-03412), and trypsin (207-192-83) were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan).

[0060] Collagen type I FAM conjugate (AS-85111) was obtained from Funakoshi (Tokyo, Japan). Anti-heparan sulfate (370255-S) was obtained from AMS Biotechnology (Abingdon, UK). Dulbecco's modified Eagle medium (DMEM) high glucose (08458-16) was obtained from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Anti-mouse heparan sulfate antibody (370255-S) was obtained from AMS Biotechnology (Europe) Limited (Abingdon, UK). Human UCP1 ELISA kit (MBS451508) was obtained from MyBioSource (San Diego, USA). Oxoplate 96-well round-bottom OxoPlate (OP96U) was obtained from PreSens Precision Sensing (Regensburg, Germany). The Live / Dead® viability assay kit (PK-CA707-30002) was obtained from PromoKine (Heidelberg, Germany). Phosphate-buffered saline (PBS, 14249-24) was obtained from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Fetal bovine serum (FBS, 10270-106) was obtained from Gibco. Adipocyte differentiation medium (811D-250) was obtained from Cell Applications Inc. (San Diego, USA). Collagen type I (derived from bovine dermis, Atelocell, IPC-50) was obtained from Koken Co., Ltd. (Tokyo, Japan). Laminin (354259) was obtained from Corning Corporation (Arizona, USA). Gellan gum (8H1121A) was obtained from Sansho Co., Ltd. (Osaka, Japan). The phalloidin-iFluor 594 reagent (ab176757) was obtained from Abcam (Cambridge, UK). The RNase-free DNase set (79254) was obtained from Qiagen (Hilden, Germany). iScript TMThe cDNA synthesis kit was obtained from Bio-Rad (California, USA). The TaqMan gene expression assay (Applied Biosystems) was obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

[0061] <Production of brown adipocytes> (Isolation of dedifferentiated fat cells (DFAT)) DFAT was obtained using the method described in a previous report (A.S. Karanfil, F. Louis, Y. Sowa, M. Matsusaki, ECM proteins and cationic polymers coating promote dedifferentiation of patient-derived mature adipocytes to stem cells, Biomater. Sci. 11 (2023) 7623-7638).

[0062] Mature adipocytes were isolated from human adipose tissue obtained from patients at Kyoto University Hospital. The human adipose tissue was washed with phosphate-buffered saline (PBS) containing 5% penicillin-streptomycin. The adipose tissue, 2-3 g per well of a 6-well plate, was minced, and a collagenase solution (concentration: 2 mg / mL, medium: Dulbecco's modified Eagle's medium (DMEM) containing 5% bovine serum albumin (BSA) and 1% penicillin-streptomycin) was added, followed by centrifugation at 37 °C and 250 rpm for 1 hour. After filtration and centrifugation, mature adipocytes were collected from the upper layer, and the stromal vascular fraction was collected from the bottom. After removing the intermediate liquid, washing was performed with PBS containing 5% BSA and 1% penicillin-streptomycin, and finally washing was performed with DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Then, the isolated mature adipocytes were seeded at 5.0×10 4 / cm 2At the seeding density, the cells were cultured in polystyrene flasks completely filled with DMEM containing 20% ​​FBS and 1% penicillin-streptomycin. The flask caps were tightly closed to prevent leakage of the medium, and the cells were incubated at 37°C for 1 week. After incubation, the medium was aspirated, and the DFAT obtained by trypsin treatment (0.25% Trypsin-EDTA) was detached. The obtained DFAT (passages 4-7) were used in subsequent experiments.

[0063] (Ethics statement) Adipose tissue was obtained from abdominal adipose tissue or liposuction from three human donors at Kyoto University Hospital (Kyoto, Japan). The three human donors were aged 41, 45, and 53 years, respectively, with BMIs of 22.40, 25.78, and 20.46. All uses were approved by the Osaka University Research Ethics Review Committee (Approval Number: L026).

[0064] (Preparation of fibrinogen solution) A fibrinogen stock solution (50 mg / mL) was dissolved in DMEM (containing 0% FBS and 1% penicillin-streptomycin), and the mixture was filtered through a 0.2 μm filter to prepare a fibrinogen solution.

[0065] (Preparation of thrombin solution) A stock thrombin solution (10 U / mL) was dissolved in DMEM (containing 10% FBS and 1% penicillin-streptomycin), and the solution was filtered through a 0.2 μm filter to prepare a thrombin solution.

[0066] (Preparation of polymer solution) Collagen type I (Col I), fibronectin (Fib), laminin (Lam), collagen type IV (Col IV), gelatin (Gel), gellan gum (GG), and poly-L-lysine (PLL) were prepared as polymers. Each polymer was dissolved in PBS (pH 7.4) and filtered through a 0.2 μm filter to prepare polymer solutions.

[0067] (Gelation process) DFAT, fibrinogen solution, and thrombin solution were mixed, and then a polymer solution was added. The seeding density of DFAT was 4.0 × 10⁻⁶ based on the total volume of the mixture. 6 The concentration was set to cells / mL. Fibrinogen was mixed to a final concentration of 6 mg / mL based on the total volume of the mixture. Thrombin was mixed to a final concentration of 3 U / mL based on the total volume of the mixture. Polymers other than PLL were mixed to a concentration of 50 μg / mL based on the total volume of the mixture. PLL was mixed to a concentration of 5, 10, or 20 μg / mL based on the total volume of the mixture.

[0068] A mixture of DFAT, fibrinogen, thrombin, and polymer was directly seeded into 96-well ultra-low adhesion round-bottom plates in 5 μL portions using a wide-mouth pipette tip. The mixture was incubated at 37°C for 20 minutes to gel and prepare a hydrogel. Subsequently, 80 μL of growth medium (high-glucose DMEM containing GM, 10% FBS, and 1% penicillin-streptomycin) was added to detach the droplets, and the hydrogel was transferred to a 24-well ultra-low adhesion plate.

[0069] (Preliminary culture process) 500 μL of GM was added as culture medium to each well containing the hydrogel, and the hydrogel was incubated in GM for 2 days.

[0070] (Culture process) After a two-day incubation period, the culture medium was completely replaced with Brown's Adipocyte Differentiation Medium (BAM). BAM is a medium containing 0.5 μM dexamethasone, 125 nM indomethacin, 250 μM IBMX, 850 nM bovine insulin, 1 μM rosiglitazone, 120 nM triiodothyronine (T3), 1% penicillin-streptomycin, and 10% FBS in high-glucose DMEM. The hydrogels were cultured in BAM for two weeks, with half the volume of medium replaced every two days during the culture period. To evaluate the induction of differentiation into brown adipocytes, hydrogels from day 14 of the differentiation-inducing culture were used.

[0071] <Evaluation Method> (Immunofluorescence imaging) Hydrogels obtained by incubation in BAM for 2 weeks were washed three times with PBS and fixed overnight in 4% paraformaldehyde PBS solution at 4°C to prepare the samples. To enhance permeability, the samples were treated with 0.05% Triton X-100 PBS solution for 15 minutes, and then incubated in 1% BSA PBS solution at room temperature for 1 hour to minimize nonspecific staining. Anti-UCP1 antibody diluted with 1% BSA (1:500 dilution) was applied to the samples overnight at 4°C. Subsequently, the samples were exposed to Alexa Fluor® 647 secondary antibody (1:200 dilution) at room temperature for 2 hours. Nile Red (final concentration: 50 ng / mL) was used to visualize intracellular lipid accumulation, and the nuclei were counterstained with Hoechst (final concentration: 10 ng / mL).

[0072] For mitochondrial staining, samples were washed three times with PBS, incubated for 30 minutes at 37°C in a 5% CO2 incubator in MitoTracker dye diluted with culture medium (high glucose DMEM containing 10% FBS, 1% penicillin-streptomycin, and no phenol red), and then washed with PBS.

[0073] All samples were washed with PBS and observed using an FV3000 confocal laser scanning microscope (CLSM) (Olympus, Tokyo, Japan). Z-stack images were acquired using the same procedure to compare UCP1 content, lipid accumulation, and mitochondrial volume, with maximum intensity projection performed while maintaining consistent exposure time and excitation power for all samples. Data were acquired using ImageJ software (Fiji for Mac OS X), with the total fluorescence intensity of UCP1, lipid droplets, and MitoTracker for each sample normalized to the total fluorescence intensity of Hoechst.

[0074] (RT-qPCR analysis) Gene expression was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR). This evaluation used a combination of six drop-shaped hydrogels (drops) per repeat. Total RNA from the drops was collected using PicoPure according to the manufacturer's instructions. TM RNA was isolated using an RNA isolation kit. The extracted RNA was processed using Nanodrop. TM Quantification was performed using the N1000 device (Thermo Fisher Scientific, MA, USA). The iScript cDNA synthesis kit was used to convert isolated RNA to DNA, following the manufacturer's instructions. For DNA amplification, cDNA was amplified using the Taqman Fast Advanced Mix with Taqman gene expression assays for UCP1, Cidea, PRDM16, and RPII (used as a housekeeping gene), following the manufacturer's protocol. cDNA synthesis and RT-qPCR reactions were performed using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, MA, USA). RT-qPCR analysis was performed on cells from three different donors, with a total of 3 to 9 replicates.

[0075] (UCP1 ELISA assay) After washing the samples three times with PBS, they were treated with trypsin-EDTA at 37°C until the fibrin gel dissolved. Cells were collected by centrifugation, washed three times with cold PBS, and then subjected to three freeze-thaw cycles. After removing cell debris by centrifugation at 1500g, 4°C for 10 minutes, the human UCP1 ELISA kit was used according to the manufacturer's instructions. Data normalization was performed by quantifying DNA in the ELISA lysates using the Qubit HS DNA assay.

[0076] (Measurement of oxygen consumption rate) After washing the polymer-mixed sample three times with PBS, it was transferred to a 96-well round-bottom OxoPlate (OP96U, PreSens Precision Sensing). Four drops per well were added to DMEM (10% FBS, 1% penicillin-streptomycin) without phenol red. For plate calibration, eight wells were designated as a 0% O2 standard (H2O with 10 mg / mL sodium sulfite) and a 100% O2 standard (breathing medium) according to the manufacturer's protocol. Oxygen concentration was measured on day 14 and again 24 hours later using the same plate. A plate reader equipped with two calibration standards and filter pairs for indicator (excitation 540 nm, emission 650 nm) and reference (excitation 540 nm, emission 590 nm) was used for the measurements. Calibration and oxygen levels were calculated according to the manufacturer's manual to determine the 24-hour oxygen consumption rate. Data normalization was performed by DNA quantification using the Qubit HS DNA assay.

[0077] (DNA quantification) Qubit TM DNA HS assay kit from Qubit TM DNA quantification was performed using a 2.0 fluorometer (Life Technologies, Thermo Fisher Scientific Inc.). Samples were washed with PBS and incubated with trypsin-EDTA until the fibrin gel lysed. Three freeze-thaw cycles were then performed in Eppendorf tubes. The assay was performed according to the manufacturer's instructions. DNA normalization of ELISA samples was performed by directly assaying the ELISA lysates.

[0078] (statistical analysis) ANOVA was used to determine statistical significance between datasets using ezANOVA software. A p-value of less than 0.05 was considered statistically significant.

[0079] <Evaluation Results> Figure 1 shows immunofluorescence imaging images of the nucleus, lipids, and UCP1 of hydrogels cultured under PLL conditions, as well as their merged images and high-magnification images of the merged images. The scale bars for the nucleus, lipids, UCP1, and their merged images in Figure 1 are 50 μm, and the scale bar for the high-magnification image of the merged images is 200 μm.

[0080] Figure 2 shows a comparison of the ratio of UCP1 content to DNA fluorescence intensity (UCP1 content(AF647) / DNA(Hoechst) fluorescence(AU)) and the ratio of lipid content to DNA fluorescence intensity (Lipid content(Nile Red) / DNA(Hoechst) fluorescence(AU)) in hydrogels cultured under differentiation induction. Results are shown as mean ± standard deviation (n=3, 3 independent samples from one donor). Statistical significance by Anova statistical test is indicated as *=p<0.05 for UCP1 and #=p<0.05 for lipids.

[0081] Figure 3 shows the results of Fold Change of UCP1 Concentration by ELIZA in hydrogels cultured using differentiation induction. Results are shown as mean ± standard error (n=3, 3 independent experiments from one donor). Statistical significance by the Anova test is indicated as *=p<0.05. White dots represent data points for each parallel experiment.

[0082] The results shown in Figures 2-3 indicate that the amount of UCP1 tended to increase when PLL, a cationic polymer, was used.

[0083] Figures 4-6 are graphs showing the results of analyzing the relative gene expression of UCP1, Cidea, and PRDM16, which are brown adipose tissue differentiation markers, using hydrogels cultured under differentiation induction.

[0084] Figure 4 shows the ratio of UCP1 gene expression level to RPII gene (housekeeping gene) expression level (UCP / RPII) in differentiated hydrogels. Results are shown as mean ± standard error. The left graph shows the mean ± standard deviation of N=4-9 experiments using cells from three different patients, and the right graph shows the results of n=3 experiments using cells from one patient. *=p<0.05, **=p<0.01, ***=p<0.001.

[0085] As shown in Figure 4, the relative expression level of the UCP1 gene was significantly higher when PLL was used compared to the other groups. The relative expression level of the UCP1 gene showed a 6 (±3)-fold increase when PLL was used compared to fibrin alone. It was confirmed that UCP1 gene expression was similarly induced when the PLL concentration was changed. The relative expression level of the UCP1 gene increased in a concentration-dependent manner.

[0086] Figure 5 shows the ratio of Cidea gene expression to RPII gene expression (Cidea / RPII). The left graph shows the mean ± standard deviation of N=4-6 experiments using cells from two different patients, and the right graph shows the results of n=3 experiments using cells from one patient. *=p<0.05, **=p<0.01.

[0087] As shown in Figure 5, the relative expression level of the Cidea gene increased when PLL was used compared to the other groups. It was confirmed that the relative expression level of the Cidea gene showed a similar trend to that of the UCP1 gene when PLL was used. It was also confirmed that Cidea gene expression was induced even when the PLL concentration was changed.

[0088] Figure 6 shows the ratio of PRDM16 gene expression level to RPII gene expression level (PRDM16 / RPII). The left graph shows the mean ± standard deviation of an N=4-6 experiment using cells from two different patients, and the right graph shows the results of an n=3 experiment using cells from one patient. =p<0.05. In the left graph, PLL was used at 10 μg / mL. Statistical significance is indicated by Student's t-test, where *=p<0.05. The white dots represent data points for each parallel experiment.

[0089] As shown in Figure 6, the relative expression level of the PRDM16 gene increased when PLL was used compared to the other groups. It was confirmed that the relative expression level of the PRDM16 gene showed a similar trend to that of the UCP1 and Cidea genes with the use of PLL. It was also confirmed that PRDM16 gene expression was induced even when the PLL concentration was changed. Similarly, it was confirmed that Cidea gene expression was induced even when the PLL concentration was changed.

[0090] Figure 7 shows immunofluorescence imaging images of the nucleus and mitochondria of hydrogel cultured under PLL conditions, as well as a merged image of these images.

[0091] Figure 8 shows the results of a comparison of the ratio of mitochondrial content (Mito Trakcer) / DNA (Hoechst) fluorescence (AU) to DNA fluorescence intensity in differentiated hydrogels. Results are shown as mean ± standard error (n=3, 3 independent experiments from one donor). Statistical significance by Anova test is indicated as *=p<0.05. When compared with the fibrin-only group on day 14 as a control, ●=p<0.05 and ●●=p<0.01.

[0092] As shown in Figure 8, mitochondrial content increased when PLL was used compared to the other groups.

[0093] Figure 9 shows the comparative results of 24-hour oxygen consumption in differentiated hydrogels. Results are shown as mean ± standard error (n=3, 3 independent experiments from one donor). Statistical significance by Anova test is indicated as *=p<0.05, **=p<0.01, and ***=p<0.001. White dots represent individual data points.

[0094] As shown in Figure 9, the oxygen consumption rate was higher when PLL was used compared to the other groups. The oxygen consumption rate when PLL was used was significantly higher (138 (±13)) compared to fibrin alone (100±13).

[0095] The results in Figures 8-9 further confirm the brown adipose tissue differentiation-inducing effect and increased metabolic activity of PLL.

Claims

1. A method for producing brown adipose tissue from stem cells, A method comprising a culture step of culturing stem cells and a hydrogel containing a cationic polymer in a medium containing 3,3',5-triiodo-L-thyronine and insulin, thereby inducing differentiation of the stem cells into brown adipose cells.

2. The method according to claim 1, wherein the cationic polymer is polylysine.

3. The method according to claim 1 or 2, wherein the hydrogel is a fibrin gel.

4. The method according to claim 1 or 2, wherein the stem cells are dedifferentiated adipocytes.

5. The method according to claim 1 or 2, wherein the concentration of the cationic polymer in the hydrogel is 5 μg / mL or more and 20 μg / mL or less.

6. The method according to claim 1 or 2, further comprising a gelation step, prior to the culture step, of gelling a mixture obtained by mixing stem cells, a cationic polymer, and a hydrogel precursor to form a hydrogel containing stem cells and a cationic polymer.

7. The method according to claim 6, wherein the hydrogel precursor comprises fibrinogen and thrombin.

8. A hydrogel containing brown adipose tissue and a cationic polymer.

9. The hydrogel according to claim 8, wherein the cationic polymer is polylysine.

10. The hydrogel according to claim 8 or 9, which is a fibrin gel.

11. The hydrogel according to claim 8 or 9, wherein the concentration of the cationic polymer in the hydrogel is 5 μg / mL or more and 20 μg / mL or less.

12. A differentiation inducer for stem cells into brown adipose tissue, A differentiation induction promoter containing a cationic polymer as an active ingredient.

13. The differentiation induction promoter according to claim 12, wherein the cationic polymer is polylysine.

Images