Non-naturally occurring three-dimensional (3D) brown adipose-derived stem cell aggregates, and methods of generating and using the same

Abstract

To provide non-naturally occurring three-dimensional (3D) brown adipose-derived stem cell aggregates, and methods of making and using the same.SOLUTION: The present application provides non-naturally occurring 3D brown adipose-derived stem cell (BADSC) aggregates, methods of making the 3D BADSC aggregates, and methods of using the 3D BADSC aggregates. Also provided herein is an encapsulation system comprising non-naturally occurring 3D brown adipose-derived stem cell aggregates. The 3D brown adipose-derived stem cell aggregate may comprise brown adipose-derived stem cells that express one or more brown adipocyte gene in the absence of differentiation medium.SELECTED DRAWING: None

Description

Technical Field

[0001] Field This application provides non-naturally occurring 3D brown adipose-derived stem cell (BADSC) aggregates, methods of making the 3D BADSC aggregates, and methods of using the 3D BADSC aggregates.

[0002] Related Application This application claims the benefit of U.S. Provisional Application No. 62 / 840,096, filed Apr. 29, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0003] Background The prevalence of metabolic disorders (e.g., obesity) has increased dramatically over the past several decades and is spreading globally. By 2030, it is estimated that over 50% of Americans will be affected by obesity, resulting in over $500 billion in lost economic productivity. Obesity is a major risk factor for type II diabetes, hypertension, cardiovascular disease, osteoarthritis, and certain forms of cancer. Current treatment approaches, such as calorie restriction and exercise, rely heavily on the self-discipline of patients attempting to reduce energy intake and / or increase energy expenditure, and have limited effectiveness in obese patients. Bariatric surgery is the only clinically proven treatment for patients with a body mass index (BMI) over 40 in terms of weight loss and reduced morbidity / mortality; however, it has associated risks, high costs, and requires appropriate management of the patient's nutritional intake and physical activity. Despite the efforts of researchers and medical professionals worldwide to address obesity and other metabolic disorders, there is still a need for alternative methods of increasing energy consumption that can expand the current treatment options for treating patients with obesity and other metabolic disorders.

Summary of the Invention

Means for Solving the Problems

[0004] Abstract This section provides an overview of the present disclosure and is not intended to cover its entire scope or all of its features.

[0005] Non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates are provided herein. The three-dimensional brown adipose tissue-derived stem cell aggregates comprise brown adipose tissue-derived stem cells that express one or more brown adipocyte genes in the absence of a differentiation medium.

[0006] Encapsulation systems comprising non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates are also provided herein. The three-dimensional brown adipose tissue-derived stem cell aggregates comprise brown adipose tissue-derived stem cells that express one or more brown adipocyte genes in the absence of a differentiation medium.

[0007] Methods for making non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates are provided herein. The method comprises loading brown adipose tissue-derived stem cells grown in two-dimensional (2D) culture into a non-adhesive culture plate; and centrifuging the non-adhesive culture plate to evenly distribute the brown adipose tissue-derived stem cells within the non-adhesive culture plate, thereby forming a three-dimensional brown adipose tissue-derived stem cell aggregate.

[0008] Methods for making three-dimensional brown adipose tissue in an encapsulation system are also provided herein. The method comprises forming a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate; loading the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into the encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; and differentiating the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium.

[0009] Methods for treating patients with disorders are also provided herein. The method includes forming a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate; loading the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the disorder.

[0010] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative examples and features described herein, further aspects, examples, objects, and features of the present disclosure will become fully apparent from the drawings and the detailed description and claims. In certain embodiments, for example, the following are provided: (Item 1) A non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate, wherein the three-dimensional brown adipose tissue-derived stem cell aggregate comprises brown adipose tissue-derived stem cells that express one or more brown adipose cell genes in the absence of a differentiation medium. (Item 2) The one or more brown adipose cell genes are selected from the group consisting of PPARα, PPARγ, PGC1β, PRDM16, CEBPD, CEBPB, CEBPΑ, TFAM, PGC1α, and PGC1β, the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to Item 1. (Item 3) The aggregate is formed in a non-adherent environment, the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to Item 1. (Item 4) The aggregate produces an extracellular biologic agent selected from the group consisting of exosomes, microRNAs, cytokines, proteins, and adipokines, the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to Item 1. (Item 5) An encapsulation system comprising an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate according to item 1. (Item 6) The encapsulation system according to item 5, which is selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel. (Item 7) The encapsulation system according to item 5, which is an encapsulated medical device. (Item 8) A method for producing an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate, the method comprising: loading brown adipose tissue-derived stem cells grown in two-dimensional (2D) culture into a non-adhesive culture plate; and centrifuging the non-adhesive culture plate to evenly distribute the brown adipose tissue-derived stem cells within the non-adhesive culture plate, thereby forming a three-dimensional brown adipose tissue-derived stem cell aggregate. A method comprising the above. (Item 9) Before the loading step, culturing the brown adipose tissue-derived stem cells in two-dimensional (2D) culture using a growth medium under normoxic or hypoxic conditions. The method according to item 8, further comprising the above. (Item 10) A method for producing three-dimensional brown adipose tissue in an encapsulation system, the method comprising: forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into the encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; and differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium. A method comprising the above. (Item 11) The encapsulation system is the method according to item 10, selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel. (Item 12) The encapsulation system is the method according to item 10, which is an encapsulated medical device. (Item 13) The first differentiation medium is the method according to item 10, which contains dexamethasone, IBMX, and T3. (Item 14) The second differentiation medium is the method according to item 10, which contains T3 and rosiglitazone. (Item 15) A method for treating a patient with a disorder, the method comprising: forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the disorder. A method comprising the above steps. (Item 16) The encapsulation system is the method according to item 15, selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel. (Item 17) The encapsulation system is the method according to item 15, which is an encapsulated medical device. (Item 18) The first differentiation medium is the method according to item 15, which contains dexamethasone, IBMX, and T3. (Item 19) The second differentiation medium contains T3 and rosiglitazone, and the method according to item 15. (Item 20) The disorder is a metabolic disorder, an endocrine disorder, a cardiovascular disorder, or a liver disease, and the method according to item 15. (Item 21) The metabolic disorder is obesity or diabetes, and the method according to item 20.

Brief Description of the Drawings

[0011] This patent or patent application includes drawings made in at least one color. Copies of the patent or patent application publication with color drawings are provided by the Office upon payment of the claims and the necessary fees.

[0012]

[0013]

Figure 1A-C

[0014]

Figure 1D-F

[0015]

Figure 1G-I

[0016]

Figure 1J-K

[0017]

Figure 1L-M

[0018]

Figure 2A

[0019]

Figure 2B

[0020]

Figure 2C

[0021]

Figure 2D

[0022]

Figure 2E

[0023]

Figure 2F-K

[0024]

Figure 2L-Q

[0025]

Figure 2R

[0026]

Figure 3A-C

[0027]

Figure 3D-F

[0028]

Figure 3G-I

[0029]

Figure 4A

[0030]

Figure 4B

[0031]

Figure 4C

[0032]

Figure 4D

[0033]

Figure 4E

[0034]

Figure 4F

[0035]

Figure 4G

[0036]

Figure 4H

[0037]

Figure 4I-L

[0038]

Figure 4M-N

[0039]

Figure 4O-P

[0040]

Figure 4Q-R

[0041]

Figure 4S-T

[0042]

Figure 4U-V

[0043]

Figure 5A

[0044]

Figure 5B

[0045]

Figure 5C

DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description Certain exemplary aspects of the present disclosure are described herein to provide an overall understanding of the structure, function, manufacture, and principles of use of the non - naturally occurring three - dimensional brown adipose tissue - derived stem cell aggregates and methods disclosed herein. One or more examples of these aspects are illustrated in the accompanying drawings. Those skilled in the art will understand that the non - naturally occurring three - dimensional brown adipose tissue - derived stem cell aggregates and methods specifically described herein and illustrated in the accompanying drawings are non - limiting exemplary aspects, and that the scope of the various examples of the present disclosure is defined only by the claims. Features illustrated or described in connection with one exemplary aspect may be combined with features of other aspects. Such modifications and variations are intended to be included within the scope of the present disclosure.

[0047] Non - naturally occurring three - dimensional brown adipose tissue - derived stem cell aggregates and methods of making the non - naturally occurring 3D BAG

[0048] Non - naturally occurring three - dimensional BADSC aggregates or “BAGs” are disclosed herein. The BAGs are 3D structures formed from BADSCs after the BADSCs are removed from their two - dimensional (2D) cultures in cell - adhesive tissue culture flasks, added to non - adhesive culture plates, and centrifuged. After centrifugation, the aggregates are homogeneous. Homogeneous cell aggregates provide more efficient and consistent differentiation and easier loading into encapsulation systems. Further, uniform aggregation provides more accurate cell numbers and more accurate dosages.

[0049] BADSCs grown in 2D are in their natural state whenever the cells expand in a tissue-adhesive cell culture flask. BADSCs cultured in a growth medium in 2D are multipotent and function as stem cells. The BADSCs grown in 2D cannot form aggregates. This is because they adhere to the cell culture flask and then differentiate into undesired non-fat cell types and ultimately induce the apoptotic cascade and cell death.

[0050] BADSCs cannot form cell aggregates in 2D culture, but whenever the BADSCs are removed from their 2D tissue-adhesive environment and placed in a non-adhesive environment, as described above, the cells form 3D aggregates. When aggregated, the BADSCs form clusters of cells that can interact with each other and communicate via their cross-linking in 3D.

[0051] The BAGs can expand and further aggregate to become artificial brown adipose tissue or artificial white adipose tissue. The BAGs can become white adipose tissue when differentiated in AD-1. AD-1 consists of 10% fetal bovine serum (FBS, HyClone, GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, UK), 5 μM dexamethasone (MP Biomedicals, Santa Ana, California, USA), 500 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, It is a serum-based differentiation medium composed of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with 860 nM insulin (Gibco, Thermo Fisher Scientific), 125 nM indomethacin (Sigma-Aldrich), 1 nM triiodothyronine (T3, Sigma-Aldrich), 1 μM rosiglitazone (Sigma-Aldrich), 100 units / ml penicillin, 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific), and 2 mM L-glutamine (Gibco, Thermo Fisher Scientific).

[0052] When the above BAG differentiates in AD-2, it can become brown adipose tissue. AD-2 is a two-step xeno-free, serum-free, chemically defined differentiation medium. In the first step, BADSC is grown in the AD-2 DIFF-1 culture medium, which is the first differentiation medium and contains DMEM / Ham's F12 medium (1:1) (Lonza Group AG, Basel, Switzerland), 25 mM HEPES buffer (Lonza Group AG), 2 mM L-glutamine (Gibco, Thermo Fisher Scientific), 1 μM dexamethasone (MP Biomedicals), 100 μM IBMX (Sigma-Aldrich), 860 nM insulin (Gibco, Thermo Fisher Scientific), 0.2 nM T3 (Sigma-Aldrich), 10 μg / ml apo-transferrin (Sigma-Aldrich), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Gibco, Thermo Fisher Scientific). In the second step, after 3 days, the above AD-2 DIFF-1 culture medium is replaced with AD-2 DIFF-2, which is the second differentiation medium, a xeno-free, serum-free, chemically defined differentiation medium that contains DMEM / Ham's F12 medium (1:1) (Lonza Group AG), 25 mM HEPES buffer (Lonza Group AG), 2 mM L-glutamine (Gibco, Thermo Fisher Scientific), 860 nM insulin (Gibco, Thermo Fisher Scientific), 0.2 nM T3 (Sigma-Aldrich), 10 μg / ml apo-transferrin (Sigma-Aldrich), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Gibco, Thermo Fisher Scientific) and 100 nM rosiglitazone.

[0053] These BAGs can act as cell factories that can generate white or brown extracellular biologics (e.g., exosomes, microRNAs, cytokines, proteins, adipokines).

[0054] Gene expression of 3D BAG

[0055] BAGs upregulate adipocyte markers (PPARα, PPARγ, PGC1β, PRDM16, CEBPd, CEBPb, CEBPa, and TFAM) and the brown adipocyte marker (PGC1α) in the absence of a differentiation medium.

[0056] The formation of BAGs results in increased expression of transcription factors and cofactors derived from the CEBP and PPAR families, which are the main regulators of adipogenesis and browning (Figure 3 A to 3I ). “Browning” refers to the ability of BAGs to express UCP-1 after differentiation in AD-2 medium.

[0057] The early adipocyte differentiation transcription factors CEBPD and CEBPB were increased after 24 hours in 3D culture, whereas CEBPA was significantly increased after 48 hours in 3D culture. Both PPARα (a main regulator of fatty acid oxidation) and PGC1α (a regulator of mitochondrial respiration and thermogenesis in brown adipocytes) were increased after 24 hours in 3D culture, whereas no significant increase was observed in the expression of PPARγ, PRDM16, TFAM, or PGC1β.

[0058] These data suggest that the above BAGs have started to follow the path of brown adipocyte differentiation in the absence of an adipocyte differentiation medium, as the formation of 3D BAGs tilts BADSC aggregates towards adipogenesis and the brown adipose phenotype.

[0059] Encapsulation system as a delivery system for BAT

[0060] Transplanting brown adipose tissue (BAT) into humans to increase BAT mass and / or activity has emerged as a potential way to increase energy consumption through energy waste. This approach of transplanting BAT into humans can be used to treat metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases. Accordingly, there is a need for a method of delivering BAT for transplantation using 3D BAGs loaded into an encapsulation system. This method is disclosed herein.

[0061] Several different encapsulation systems can be loaded with BAGs and used to deliver BAT for transplantation (e.g., alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, Afibromer TM polymers (Sigilon Therapeutics, Cambridge, Massachusetts, USA), PEG-based hydrogels, non-hydrogel beads, and Matrigel).

[0062] The encapsulation systems described herein enable the BAGs to generate extracellular factors (e.g., proteins, cytokines, microRNAs, cytokines, exosomes, and cell-specific secretomes) that can interact with the host environment.

[0063] The encapsulation systems described herein are manufactured from transplantable-grade materials or biologics and are selected for long-term biocompatibility.

[0064] The encapsulation systems described herein provide bidirectional exchange of nutrients and molecules (e.g., glucose, fatty acids, cytokines, adipokines, and hormones).

[0065] In certain examples, the encapsulation system can be an encapsulated medical device. In other examples, the encapsulated medical device can be an FDA-approved immunoprotective and readily recoverable encapsulated medical device (e.g., Encaptra® Drug Delivery System) (Viacyte, San Diego, California, USA). This device is manufactured from a transplant-grade material specifically selected for long-term biocompatibility and allows for bidirectional exchange of nutrients and molecules (e.g., glucose, fatty acids, and hormones). The encapsulated medical device provides a barrier between the host and the transplanted cells and thus should prevent immune rejection of BAT while increasing safety and preventing the transplanted cells from migrating out of the encapsulated medical device.

[0066] Method for fabricating 3D BAT in an encapsulation system

[0067] A method for fabricating 3D BAT in an encapsulation system is disclosed herein. The method includes (1) forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate, (2) loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into the encapsulation system, (3) differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium, and (4) differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium. The "first differentiation medium" may also be referred to herein as the AD-2 DIFF-1 culture medium. The "second differentiation medium" may also be referred to herein as the AD-2 DIFF-2 culture medium.

[0068] Treatment method

[0069] Methods for treating patients having a disorder are disclosed herein. Methods for treating patients having a metabolic disorder, an endocrine disorder, a cardiovascular disorder, and a liver disease are disclosed herein. Examples of metabolic disorders can include, but are not limited to, diabetes and obesity. Examples of endocrine disorders can include, but are not limited to: acromegaly, Addison's disease, adrenal cancer, adrenal disorders, anaplastic thyroid cancer, Cushing's syndrome, De Quervain's thyroiditis, diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes, maturity onset diabetes of the young), follicular thyroid cancer, goiter, Graves' disease, growth disorders, growth hormone deficiency, Hashimoto's thyroiditis, heart disease, Hurthle Cell Thyroid Cancer, hyperglycemia, hyperparathyroidism, hyperthyroidism, hypoglycemia, hypoparathyroidism, hypothyroidism, low testosterone, medullary thyroid cancer, MEN 1, MEN 2A, MEN 2B, menopause, metabolic syndrome, obesity, osteoporosis, papillary thyroid cancer, parathyroid disorders, pheochromocytoma, pituitary disorders, pituitary tumors, polycystic ovary syndrome, borderline diabetes, reproduction, painless thyroiditis, thyroid cancer, thyroid disorders, thyroid nodules, thyroiditis, Turner syndrome, insulin resistance, hypertension, central obesity, hypertriglyceridemia (e.g., high serum triglycerides), dyslipidemia, low serum HDL, lipodystrophy. Examples of cardiovascular disorders can include, but are not limited to: coronary artery disease, peripheral artery disease, carotid artery disease, peripheral arterial (arterial) disease, aneurysm, atherosclerosis, renal artery disease, Raynaud's disease (Raynaud's phenomenon), Buerger's disease, peripheral venous disease, cerebrovascular disease (e.g., stroke), venous thrombosis, and blood clotting disorders, cardiomyopathy, hypertensive heart disease (e.g., heart disease secondary to hypertension or high blood pressure). Examples of liver diseases can include, but are not limited to, simple fatty liver disease, non-alcoholic steatohepatitis (NASH), and alcohol-related fatty liver disease (ALD).

[0070] Methods for treating patients with metabolic disorders are disclosed herein. The methods include the steps of forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the metabolic disorder.

[0071] Methods for treating patients with obesity are disclosed herein. The methods include the steps of forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the obesity.

[0072] Methods for treating patients with endocrine disorders are disclosed herein. The methods include the steps of forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the endocrine disorder.

[0073] A method for treating a patient having a cardiovascular disorder is disclosed herein. The method includes the steps of forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the cardiovascular disorder.

[0074] A method for treating a patient having a liver disease is disclosed herein. The method includes the steps of forming an artificially created three-dimensional brown adipose tissue-derived stem cell aggregate; loading the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into an encapsulation system; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; differentiating the artificially created three-dimensional brown adipose tissue-derived stem cell aggregate into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient having the liver disease.

[0075] Materials and Methods of the Invention

[0076] Various aspects of the invention according to the present disclosure include, but are not limited to, the aspects enumerated in the items listed below: Item 1. An artificially created three-dimensional brown adipose tissue-derived stem cell aggregate, wherein the three-dimensional brown adipose tissue-derived stem cell aggregate comprises brown adipose tissue-derived stem cells that express one or more brown adipocyte genes in the absence of a differentiation medium. Item 2. The artificially created three-dimensional brown adipose tissue-derived stem cell aggregate according to Item 1, wherein the one or more brown adipocyte genes are selected from the group consisting of PPARα, PPARγ, PGC1β, PRDM16, CEBPD, CEBPB, CEBPΑ, TFAM, PGC1α, and PGC1β. Item 3. The aggregate is a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to any one of Items 1 to 2, which is formed in a non-adhesive environment. Item 4. The aggregate is a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to any one of Items 1 to 3, which generates an extracellular biologic agent selected from the group consisting of exosomes, microRNAs, cytokines, proteins, and adipokines. Item 5. An encapsulation system comprising the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate according to any one of Items 1 to 4. Item 6. The encapsulation system according to Item 5, which is selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel. Item 7. The encapsulation system according to Item 5, which is an encapsulated medical device. Item 8. A method for producing a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate, the method comprising: loading brown adipose tissue-derived stem cells grown in two-dimensional (2D) culture into a non-adhesive culture plate; and centrifuging the non-adhesive culture plate to uniformly arrange the brown adipose tissue-derived stem cells in the non-adhesive culture plate, thereby forming a three-dimensional brown adipose tissue-derived stem cell aggregate. A method comprising. Item 9. The method according to Item 8, further comprising culturing the brown adipose tissue-derived stem cells in two-dimensional (2D) culture using a growth medium under normoxic or hypoxic conditions before the loading step. Item 10. A method for producing a three-dimensional brown adipose tissue in an encapsulation system, the method comprising: forming a non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate; loading the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregate into the encapsulation system; The step of differentiating the above-mentioned non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; and The step of differentiating the above-mentioned non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into brown adipose tissue in a second differentiation medium, A method comprising the above. Item 11. The encapsulation system is selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel, according to the method described in Item 10. Item 12. The encapsulation system is an encapsulated medical device, according to the method described in Item 10. Item 13. The first differentiation medium contains dexamethasone, IBMX, and T3, according to the method described in any one of Items 10 to 12. Item 14. The second differentiation medium contains T3 and rosiglitazone, according to the method described in any one of Items 10 to 13. Item 15. A method for treating a patient with a disorder, the method comprising: The step of forming non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates; The step of loading the above-mentioned non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into an encapsulation system; The step of differentiating the above-mentioned non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; The step of differentiating the above-mentioned non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; and The step of delivering the above-mentioned brown adipose tissue to the above-mentioned patient with the above-mentioned disorder, A method comprising the above. Item 16. The encapsulation system is selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel, according to the method described in Item 15. Item 17. The encapsulation system is the method according to item 15, which is an encapsulated medical device. Item 18. The first differentiation medium contains dexamethasone, IBMX, and T3, and is the method according to any one of items 15 to 17. Item 19. The second differentiation medium contains T3 and rosiglitazone, and is the method according to any one of items 15 to 18. Item 20. The disorder is a metabolic disorder, an endocrine disorder, a cardiovascular disorder, or a liver disease, and is the method according to any one of items 15 to 19. Item 21. The metabolic disorder is obesity or diabetes, and is the method according to item 20.

[0077] Definitions

[0078] In addition to the definitions presented earlier in this specification, the following definitions are relevant to this disclosure.

[0079] The singular forms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0080] "Two-dimensional (2D) culture" refers to cells that spread across the entire surface of a cell culture plate and adhere to the surface of the cell culture.

[0081] "Three-dimensional (3D) culture" refers to cells that do not adhere to the surface of a cell culture plate but instead associate with each other, thereby forming cell aggregates.

[0082] Any numerical range recited in this specification recites all sub-ranges of the same numerical precision that are included within the recited range (i.e., having the same digits in a particular digit place). For example, a recited range of "1.0 to 10.0" recites all sub-ranges (e.g., such as "2.4 to 7.6") between the recited minimum value 1.0 and the recited maximum value 10.0 (including both ends), even if the range of "2.4 to 7.6" is not explicitly recited in the body of this specification. Thus, the applicant retains the right to amend this specification, including the claims, to explicitly recite any sub-range of the same numerical precision that is included within the range explicitly recited in this specification. All such ranges are essentially recited in this specification so that amending to explicitly recite any such sub-range complies with the requirements of description, sufficiency of description, and addition of matter, including the requirements under 35 U.S.C. § 112(a) and Article 123(2) of the EPC. Also, unless explicitly specified or otherwise required by the context, all numerical parameters recited in this specification (e.g., those representing values, ranges, amounts, percentages, etc.) are to be read and construed as if the word "about" preceded them, even if the word "about" does not explicitly appear before the number. Further, the numerical parameters recited in this specification are to be interpreted in view of the reported significant digits, numerical precision, and by applying ordinary rounding techniques. It is also understood that the numerical parameters recited in this specification necessarily have inherent variability characteristic of the underlying measurement techniques used to determine the numerical values of those parameters.

[0083] Any patents, publications, or other disclosure materials identified herein are incorporated by reference herein in their entirety, but only to the extent that the incorporated materials are not inconsistent with existing descriptions, definitions, statements, or other disclosure materials expressly set forth herein. Thus, and to the extent necessary, the explicit disclosure as set forth herein supersedes any conflicting materials incorporated by reference. Any materials or portions thereof that are said to be incorporated by reference herein but that conflict with existing definitions, statements, or other disclosure materials set forth herein are incorporated only to the extent that no conflict arises between the incorporated materials and the existing disclosure materials. Applicants reserve the right to amend this specification to expressly recite any subject matter or portion thereof incorporated by reference herein.

[0084] Details of one or more aspects of the present disclosure are set forth in the following appended examples. Any materials and methods similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, but specific examples of the intended materials and methods are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the detailed description. In the illustrative examples, the singular form also includes the plural unless the context otherwise expressly dictates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this description will control.

Examples

[0085] The present disclosure will be more fully understood by reference to the following examples. These examples provide illustrative and non-limiting aspects of the present invention.

[0086] Example 1 - Differentiation of BADSCs into brown adipocytes in a differentiation medium containing fetal bovine serum

[0087] BADSCs were isolated from fresh brown adipose tissue and cultured up to passage 3. The cells were 10% human platelet lysate (XcyteTM Plus Xeno-Free Supplement, iBiologics, Phoenix, Arizona, USA), 1% GlutaMAX TM Dulbecco's Modified Eagle Medium (DMEM) low glucose (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with Supplement (Gibco, Thermo Fisher Scientific), 1% Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA, Gibco, Thermo Fisher Scientific), 100 units / ml penicillin and 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific). Cells were expanded in growth medium (GM) composed of this. The cells were seeded at a density of 3500 cells / cm 2 and the medium was changed every other day.

[0088] Adipocyte differentiation was induced 2 days after the cells reached full confluence by adding brown adipocyte differentiation medium 1 (AD-1). AD-1 is a serum-based differentiation medium composed of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, HyClone, GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, UK), 5 μM dexamethasone (MP Biomedicals, Santa Ana, California, USA), 500 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, St. Louis, Missouri, USA), 860 nM insulin (Gibco, Thermo Fisher Scientific), 125 nM indomethacin (Sigma-Aldrich), 1 nM triiodothyronine (T3, Sigma-Aldrich), 1 μM rosiglitazone (Sigma-Aldrich), 100 units / ml penicillin, 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific), and 2 mM L-glutamine (Gibco, Thermo Fisher Scientific).

[0089] Example 2 - Differentiation of BADSCs into brown adipocytes in a two-step serum-free chemically defined differentiation medium

[0090] To develop transplantable brown adipose tissue (BAT) for human applications, a differentiation protocol applicable to cell therapy in humans was sought.

[0091] BADSCs were isolated from fresh brown adipose tissue and cultured up to passage 3. The cells were cultured in 10% human platelet lysate (Xcyte TM Plus Xeno-Free Supplement, iBiologics), 1% GlutaMAX TMExpanded in GM consisting of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with Supplement (Gibco, Thermo Fisher Scientific), 1% minimum essential medium non-essential amino acids (MEM-NEAA, Gibco, Thermo Fisher Scientific), 100 units / ml penicillin and 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific). Cells were seeded at a density of 3500 cells / cm 2 and the medium was changed every other day.

[0092] Adipocyte differentiation was induced 2 days after the cells reached full confluence by adding brown adipocyte differentiation medium 2 (AD-2). AD-2 is a two-step xeno-free, serum-free, chemically defined differentiation medium. In the first step, BADSCs were grown in the first differentiation medium, AD-2 DIFF-1 culture medium, which consists of DMEM / Ham's F12 medium (1:1) (Lonza Group AG, Basel, Switzerland), 25 mM HEPES buffer (Lonza Group AG), 2 mM L-glutamine (Gibco, Thermo Fisher Scientific), 1 μM dexamethasone (MP Biomedicals), 100 μM IBMX (Sigma-Aldrich), 860 nM insulin (Gibco, It contains Thermo Fisher Scientific), 0.2 nM T3 (Sigma - Aldrich), 10 μg / ml apo - transferrin (Sigma - Aldrich), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Gibco, Thermo Fisher Scientific). In the second step, after 3 days, the AD - 2 DIFF - 1 culture medium was replaced with a second differentiation medium, AD - 2 DIFF - 2 (xeno - free, serum - free chemically defined differentiation medium). This medium contains DMEM / Ham’s F12 medium (1:1) (Lonza Group AG), 25 mM HEPES buffer (Lonza Group AG), 2 mM L - glutamine (Gibco, Thermo Fisher Scientific), 860 nM insulin (Gibco, Thermo Fisher Scientific), 0.2 nM T3 (Sigma - Aldrich), 10 μg / ml apo - transferrin (Sigma - Aldrich), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Gibco, Thermo Fisher Scientific) and 100 nM rosiglitazone.

[0093] In some examples, AD - 2 may contain human platelet lysate. In other examples, AD - 2 does not contain human platelet lysate.

[0094] Differentiate the above - mentioned BADSC population using the two - step method described above in a xeno - free, serum - free chemically defined brown differentiation medium (AD - 2 DIFF - 1 and AD - 2 DIFF - 2), and its efficacy in generating brown adipocytes was compared with an FBS - based differentiation medium (AD - 1) and a commercially available adipogenesis medium (StemPro TM Adipogenesis, Gibco, Thermo Fisher Scientific).

[0095] As shown by the expression of the adipocyte markers FABP4 and adiponectin (Figure 2 C and Figure 2D)、AD-1 and AD-2 adipogenic media are equally efficient in converting BADSCs into adipocytes and are more efficient than the commercially available adipogenic medium, StemPro TM (Gibco, Thermo Fisher Scientific). etc. AD-1 and AD-2 were equivalent in promoting adipocyte differentiation, but adipocytes obtained in the xeno-free, serum-free, chemically defined medium AD-2 were morphologically larger and contained larger lipid droplets (Figure 1 A to 1M shows cells cultured in AD-2; data are not shown for cells cultured in AD-1). Differentiation using the AD-2 medium enabled much higher brown adipocyte differentiation than AD-1 or the commercially available adipogenic medium.

[0096] The results also show that UCP1 gene expression was more than 200-fold higher in AD-1 and more than 3500-fold higher in AD-2 when compared to the commercially available adipogenic medium, StemPro TM (Figure 2B). Furthermore, the expression of leptin, a white-specific marker, was 1.5-fold lower in AD-2 than in AD-1. This confirms the superior efficiency of AD-2 in directing BADSCs towards the brown adipocyte phenotype (Figure 2 E ).

[0097] Immunocytochemical analysis of the BADSC population BF-1 differentiated in AD-2 for 15 days showed that the adipocyte conversion rate, i.e., the percentage of cells positive for the adipocyte marker perilipin, was very high, exceeding 80% of the cells differentiating into adipocytes (Figure 2 F to 2K and 2 R ). 98% of the differentiated cells (perilipin+ cells) co-expressed UCP1, a brown-specific marker (Figure 2 F to 2K and 2 R ). This data confirms the expression of UCP1 at the protein level (Figure 2 H and 2 I; Figures 2N and 2O) showed a high yield of brown adipocyte conversion in a xeno-free chemically defined differentiation medium. As predicted and as shown by the superimposed signals, mitochondrial-localized UCP1 protein was obtained when differentiating BADSCs were co-immunostained for UCP1 and mitochondria (Figure 2 N to 2Q ).

[0098] Figure 2A~ 2R The results in showed that the two-step AD-2 differentiation medium (AD-2 DIFF-1 and AD-2 DIFF-2) promoted stronger brown adipocyte differentiation compared to the AD-1 differentiation medium and a commercially available adipogenic medium.

[0099] Example 3 - Method for producing 3D BAG

[0100] A non-natural three-dimensional BADSC aggregate or BAG was formed in a non-adherent culture plate (e.g., AggreWell TM 400Ex 6-well plate (StemCell Technologies, Vancouver, British, Columbia, Canada).

[0101] BADSCs were first cultured in 2D using a growth medium under normoxic or hypoxic conditions until 80% confluence. The non-adherent plate was coated with a rinse solution (e.g., AggreWell TM rinse solution (StemCell Technologies)) according to the manufacturer's instructions. After washing the non-adherent plate with GM, a 12 ml cell suspension containing 2.4 million cells / ml in GM was loaded into each well of the non-adherent plate. The non-adherent plate was then centrifuged at 500 g for 5 minutes using a swinging-bucket centrifuge to allow the cells to precipitate uniformly into its microwells, resulting in a density of 1000 cells / microwell and thus enabling the creation of uniform cell aggregates. Without centrifugation, the non-natural three-dimensional brown adipose-derived stem cell aggregates or BAGs are not uniform.

[0102] Next, the above BAGs were cultured in a non-adhesive culture plate (e.g., AggreWell TM 400Ex 6-well plate) in GM at 37°C in normoxia or hypoxia and 95% humidity for 24 hours and then harvested. Approximately 28,200 BAGs per non-adhesive plate were collected by gentle pipetting and resuspended in 800 μl of GM.

[0103] Example 4 - Method for Producing Three-Dimensional Brown Adipose Tissue in an Encapsulation System

[0104] A differentiation protocol was developed to efficiently differentiate BADSCs into functional brown adipocytes in 3D culture within an encapsulation system (e.g., an encapsulated medical device). This method (summarized in Figure 4A) consists of three steps: (1) forming non-naturally occurring three-dimensional BADSC aggregates (BAGs) (approx. 160 μm / aggregate) in growth medium (Figures 4B, 4C) and loading the BAGs into an encapsulation system (e.g., an encapsulated medical device) (Figures 4D, 4E); (2) further differentiating the above BAGs into brown adipose tissue (BAT) using xeno-free, serum-free, chemically defined AD-2-DIFF-1 medium; and (3) differentiating the above BAGs into brown adipose tissue using xeno-free, serum-free, chemically defined AD-2-DIFF-2 medium (Figure 4F).

[0105] In Project 1: BAGs were formed in an AggreWellTM 400Ex 6-well plate (StemCell Technologies) using the BADSC group BF-1. The optimal cell seeding density was determined to be 1000 cells per microwell to generate uniform BAGs. The BAGs were then loaded into an encapsulation system (e.g., an encapsulated medical device). The BAG suspension was loaded into an encapsulation device (e.g., one Encaptra® EN20 (ViaCyte) encapsulation device) using a Sureflo® 20G catheter (Terumo Corporation, Tokyo, Japan). The device port was sealed with RTV Silicone Adhesive (NuSil Technology, Carpinteria, California, USA), and the encapsulated BAGs were cultured in 15 ml of GM in a 100 mm tissue culture dish for 24 hours. At that point, the BAGs coalesced to fill the entire volume of the encapsulation device. The resulting BAGs were very uniform in size and shape and were uniform within and between experiments. The size can be easily modified by adjusting the cell seeding density formed in the AggreWell TM 400Ex 6-well plate (StemCell Technologies). The optimal cell seeding density to generate uniform BAGs was determined to be 1000 cells per microwell.

[0106] In Project 2: The BAGs in the encapsulated medical device were differentiated in vitro for 3 days in a first differentiation medium called AD-2 DIFF-1 medium.

[0107] In Project 3: The BAGs in the encapsulated medical device were further differentiated in vitro for 20 days in a second differentiation medium called AD-2 DIFF-2 medium.

[0108] Immunocytochemical analysis showed that the artificially created brown adipose-derived stem cell aggregates containing the above BADSC population BF-1 efficiently differentiated into brown adipocytes in 3D inside the Encaptra® encapsulated medical device. The BADSC BF-1 cells differentiating within the encapsulated medical device formed tissue-like structures that were visualized by hematoxylin and eosin staining highly enriched in brown adipocytes (UCP1 positive and perilipin positive) containing a high content of mitochondria (Figure 4G ~4L ). These cells expressed high levels of adipocyte markers (e.g., FABP4, adiponectin, PPARg, CEBPa, and leptin (Figure 4 M to 4Q )) and brown-specific markers (e.g., UCP1, PGC1a, CIDEA, ELOVL3, and COX10 (Figure 4 R to 4V )) when compared to undifferentiated BAGs.

[0109] In conclusion, it was shown that artificially created BADSC aggregates represent a very promising source of transplantable brown adipose tissue to increase energy consumption and potentially treat metabolic, endocrine, cardiovascular, and liver disorders. Furthermore, the strategy of using encapsulation to rapidly deliver artificially created BADSC aggregates represents a safe delivery system and helps to accelerate the development of BAT therapies for human applications.

[0110] Example 5 - Evaluation of the Efficacy and Safety of BAG Delivered in Matrigel

[0111] Eight-week-old male SCID-beige mice (C.B-Igh-1b / GbmsTac-Prkdcscid-LystbgN7) (Taconic Biosciences) were individually housed at 25°C and fed a high-fat diet (HFT) containing 60% fat (D12492, 60 kcal% fat [mostly lard], 20 kcal% carbohydrate). These mice had metabolic syndrome and were unable to process glucose.

[0112] The encapsulation system was prepared by adding brown adipose tissue-derived stem cells (BADSCs) in 1 mL of 4 mg / mL Matrigel (Corning® Matrigel® Matrix High Concentration (HC), phenol-free *LDEV-free) at a concentration of 1.6×10 6 . The BADSCs were removed from their two-dimensional (2D) cultures in cell-adhesive tissue culture flasks, added to non-adhesive culture plates, and centrifuged. After centrifugation, the aggregates were uniform and added to Matrigel.

[0113] The encapsulation system (1 mL) was added to 20 wells of a 96-well plate (50 μL / well). After 1 hour of gelation, the encapsulation system became a solid disk in the culture well. Growth medium was added to the wells for 24 hours. The growth medium was removed from the wells, and then AD-2 DIFF-1 was added to the wells for 24 hours. The AD-2 DIFF-1 was removed from the wells, and then AD-2 DIFF-2 was added to the wells for 14 - 21 days. After several days of culture / differentiation, the encapsulation system containing BAT formed a spherical shape (i.e., beads). After differentiation in vitro, the size of the beads decreased to 20 - 30 μl.

[0114] Forty beads were collected using a cell strainer. The forty beads contained approximately 3.2×10 6 total cells that constituted the encapsulated BAT. The beads were transferred to a 1.5 mL conical vial and placed on ice. 100 μl of cooled 10 mg / mL Matrigel was added to the beads, mixed well, and maintained on ice.

[0115] A small skin incision (about 5 mm) was made near the brown adipose tissue pad between the scapulae of 22 SCID-beige mice. If additional space was needed, the dorsal subcutaneous site was used. Using a spatula, the skin was lifted from the underlying white adipose layer. The 40 beads in Matrigel were delivered to the incision site of 11 out of the 22 mice using a modified 1 mL micropipette tip (Figures 5A - C, treatment group), and the incision was sutured. Matrigel alone was delivered to the incision site of the other 11 mice using a modified 1 mL micropipette tip (Figures 5A - C, control group), and the incision was sutured.

[0116] The mice in the treatment group and the mice in the control group were analyzed once a week to determine their ability to absorb glucose via a glucose tolerance test (GTT). Before the above analysis, these mice were fasted for 24 hours. After 24 hours, the mice were given an intraperitoneal (IP) injection of glucose (1 mg / g body weight), and the amount of glucose absorbed was measured using blood samples at 0, 15, 30, 60, and 120 minutes after the glucose injection.

[0117] Figures 5A - C show that mice transplanted with BAT (treatment group) were able to absorb glucose better over a 60 - minute period 4 weeks after treatment (at 8 weeks after the induction of obesity) compared to mice not transplanted with BAT (control group).

[0118] Example 6 - Evaluation of the Efficacy and Safety of BAG Delivered in Matrigel

[0119] The mice included in the GTT experiment described in Example 5 were also monitored for their body weight. To measure body weight, the mice were weighed once a week for 3 months. Each week, the mice were placed on a scale set to zero and their body weight was recorded. Mice transplanted with BAT (treatment group) showed a lower overall body weight or a lower overall body weight gain compared to mice not transplanted with BAT (control group).

[0120] Notes on Illustrative Embodiments

[0121] This disclosure provides descriptions of various specific aspects for the purpose of illustrating various examples of the disclosure and / or its potential applications, and it is understood that variations and modifications will occur to those skilled in the art. Thus, it should be understood that the inventions described herein are not defined as broadly as they are claimed and are not more narrowly defined by the specific illustrative examples provided herein.

Claims

A method for producing an artificially non-existent three-dimensional brown adipose-derived stem cell aggregate consisting of one or more brown adipose cell genes-expressing brown adipose-derived stem cells in the absence of a differentiation medium, said method comprising: loading brown adipose-derived stem cells grown in two-dimensional (2D) culture into a non-adhesive culture plate; and centrifuging the non-adhesive culture plate to uniformly arrange the brown adipose-derived stem cells in the non-adhesive culture plate, thereby forming an artificially non-existent three-dimensional brown adipose-derived stem cell aggregate, said aggregate consisting of brown adipose-derived stem cells. A method comprising the above steps. The method according to claim 1, further comprising culturing the brown adipose-derived stem cells in two-dimensional (2D) culture in a normoxic or hypoxic state using a growth medium before the loading step. The method according to claim 1, further comprising the above step. After the centrifuging step and before recovering the artificially non-existent three-dimensional brown adipose-derived stem cell aggregate, culturing the artificially non-existent three-dimensional brown adipose-derived stem cell aggregate in a non-adhesive culture plate, such as an AggreWell™ 400Ex 6-well plate, in a normoxic or hypoxic state at 37 °C and 95% humidity for 24 hours in a growth medium. The method according to any one of the preceding claims, further comprising the above step. A method according to any one of the preceding claims, comprising: loading the artificially non-existent three-dimensional brown adipose-derived stem cell aggregate into an encapsulation system; differentiating the artificially non-existent three-dimensional brown adipose-derived stem cell aggregate into brown adipose tissue in a first differentiation medium; and differentiating the artificially non-existent three-dimensional brown adipose-derived stem cell aggregate into brown adipose tissue in a second differentiation medium. A method further comprising the above steps. The method according to claim 4, wherein the encapsulation system is selected from the group consisting of alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, polymers, PEG-based hydrogels, non-hydrogel beads, and Matrigel. Step of forming the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates in a growth medium at about 160 pm / aggregate prior to loading the non-naturally occurring three-dimensional brown adipose tissue-derived stem cell aggregates into the encapsulation system; In the first differentiation step, using a xeno-free, serum-free, chemically defined AD-2-DIFF-1 medium as the first differentiation medium; and In the second differentiation step, using a xeno-free, serum-free, chemically defined AD-2-DIFF-2 medium as the second differentiation medium The method according to any one of claims 4 to 5, comprising: In the first differentiation step, differentiating the aggregates in the encapsulation system in vitro in the first differentiation medium for 3 days; and In the second differentiation step, further differentiating the aggregates in the encapsulation system in vitro in the second differentiation medium for 20 days The method according to claim 6, comprising: Claim 8, wherein the encapsulation system is an encapsulation medical device; or The first differentiation medium contains dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and triiodothyronine (T3); or The second differentiation medium contains T3 and rosiglitazone, The method according to claim 4.

Images