Non-naturally occurring three-dimensional (3D) brown adipose-derived stem cell aggregates and methods of producing and using 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 The present application relates to naturally occurring 3D brown adipose-derived stem cell (BADSC) aggregates, Methods of making BADSC aggregates and methods of using said 3D BADSC aggregates are provided.

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

[0003] background The prevalence of metabolic disorders (e.g., obesity) has increased dramatically over the past few decades and is spreading worldwide. By 2030, it is estimated that more than 50% of Americans will suffer from obesity, resulting in a loss of more than $500 billion in economic productivity. Obesity is a major risk factor for type 2 diabetes, hypertension, cardiovascular disease, osteoarthritis, and certain forms of cancer. Current treatment approaches, such as calorie restriction and exercise, rely heavily on patient discipline to reduce energy intake and / or increase energy expenditure, limiting their effectiveness in obese patients. Weight loss surgery is the only clinically proven treatment for weight loss and reduced morbidity / mortality in patients with a body mass index (BMI) greater than 40; 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 remains a need for alternative methods of increasing energy expenditure that could augment current therapeutic options for treating patients with obesity and other metabolic disorders. Summary of the Invention [Means for solving the problem]

[0004] Abstract This section provides a summary of the disclosure and does not exhaust its entire scope or all of its features.

[0005] Provided herein are non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, which comprise brown adipose-derived stem cells that express one or more brown adipose-derived genes in the absence of a differentiation medium.

[0006] Also provided herein is an encapsulation system comprising non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, the three-dimensional brown adipose-derived stem cell aggregates comprising brown adipose-derived stem cells that express one or more brown adipose-derived genes in the absence of a differentiation medium.

[0007] Provided herein is a method for producing a non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate, comprising the steps of: loading brown adipose-derived stem cells grown in two-dimensional (2D) culture into a non-adherent culture plate; and centrifuging the non-adherent culture plate to distribute the brown adipose-derived stem cells uniformly within the non-adherent culture plate, thereby forming a three-dimensional brown adipose-derived stem cell aggregate.

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

[0009] Also provided herein is a method for treating patients with disorders.The method comprises: forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates; loading the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into an encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; and delivering the brown adipose tissue to the patient with 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 more fully apparent from the drawings and detailed description, and from the claims. In certain embodiments, for example, the following are provided: (Item 1) A non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate, wherein said three-dimensional brown adipose-derived stem cell aggregate comprises brown adipose-derived stem cells that express one or more brown adipose-derived genes in the absence of a differentiation medium. (Item 2) 2. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate of item 1, wherein the one or more brown adipocyte genes are selected from the group consisting of PPARα, PPARγ, PGC1β, PRDM16, CEBPD, CEBPB, CEBPA, TFAM, PGC1α, and PGC1β. (Item 3) 2. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate of item 1, wherein the aggregate forms in a non-adherent environment. (Item 4) 2. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate of item 1, wherein the aggregate produces an extracellular biologic selected from the group consisting of exosomes, microRNA, cytokines, proteins, and adipokines. (Item 5) 10. An encapsulation system comprising the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates of item 1. (Item 6) 6. The encapsulation system of item 5, 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. (Item 7) Item 6. The encapsulation system of item 5, wherein the encapsulation system is an encapsulated medical device. (Item 8) 1. A method of making non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, said method comprising: Loading brown adipose-derived stem cells grown in two-dimensional (2D) culture into non-adherent culture plates; and centrifuging the non-adherent culture plate to distribute the brown adipose-derived stem cells uniformly within the non-adherent culture plate, thereby forming three-dimensional brown adipose-derived stem cell aggregates; A method that encompasses (Item 9) culturing the brown adipose-derived stem cells in two-dimensional (2D) culture using a growth medium under normoxic or hypoxic conditions prior to the loading step; The method according to item 8, further comprising: (Item 10) 1. A method for producing three-dimensional brown adipose tissue in an encapsulated system, the method comprising: forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates; loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into said encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; and differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; A method that encompasses (Item 11) 11. The method of claim 10, 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. (Item 12) 11. The method of claim 10, wherein the encapsulation system is an encapsulated medical device. (Item 13) Item 11. The method of item 10, wherein the first differentiation medium comprises dexamethasone, IBMX, and T3. (Item 14) 11. The method of claim 10, wherein the second differentiation medium comprises T3 and rosiglitazone. (Item 15) 1. A method of treating a patient having a disorder, said method comprising: forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates; loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into an encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; and delivering said brown adipose tissue to said patient with said disorder; A method that encompasses (Item 16) 16. The method of claim 15, 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. (Item 17) Item 16. The method of item 15, wherein the encapsulation system is an encapsulated medical device. (Item 18) Item 16. The method of item 15, wherein the first differentiation medium comprises dexamethasone, IBMX, and T3. (Item 19) 16. The method of item 15, wherein the second differentiation medium comprises T3 and rosiglitazone. (Item 20) 16. The method of claim 15, wherein the disorder is a metabolic disorder, an endocrine disorder, a cardiovascular disorder, or a liver disease. (Item 21) 21. The method of claim 20, wherein the metabolic disorder is obesity or diabetes. [Brief explanation of the drawings]

[0011] This patent or patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] Various aspects of non-naturally occurring 3D brown adipose-derived stem cell (BADSC) aggregates, methods of making said 3D BADSC aggregates, and methods of using said 3D BADSC aggregates are disclosed and described herein and may be better understood by reference to the accompanying drawings.

[0013] [Figure 1A-C] Figures 1A-1M show 13 human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-1-BF-13) isolated from biopsies of subclavian and mediastinal adipose tissue and evaluated for their ability to differentiate into brown adipocytes. These BADSC populations were evaluated via bright field (top panels) and Oil Red O staining (ORO) (middle panels). Figure 1A shows human BADSC population BF-1. Figure 1B shows human BADSC population BF-2. Figure 1C shows human BADSC population BF-3.

[0014] [Figure 1D-F]Figures 1A-1M show 13 human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-1 to BF-13) isolated from biopsies of subclavian and mediastinal adipose tissue and evaluated for their ability to differentiate into brown adipocytes. These BADSC populations were evaluated via bright field (top panels) and Oil Red O staining (ORO) (middle panels). Figure 1D shows human BADSC population BF-4. Figure 1E shows human BADSC population BF-5. Figure 1F shows human BADSC population BF-6.

[0015] [Figure 1G-I] Figures 1A-1M show 13 human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-1 to BF-13) isolated from biopsies of subclavian and mediastinal adipose tissue and evaluated for their ability to differentiate into brown adipocytes. These BADSC populations were evaluated via bright field (top panels) and Oil Red O staining (ORO) (middle panels). Figure 1G shows human BADSC population BF-7. Figure 1H shows human BADSC population BF-8. Figure 1I shows human BADSC population BF-9.

[0016] [Figure 1J-K] Figures 1A-1M show 13 human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-1 to BF-13) isolated from biopsies of subclavian and mediastinal adipose tissue and evaluated for their ability to differentiate into brown adipocytes. These BADSC populations were evaluated via bright field (top panels) and Oil Red O staining (ORO) (middle panels). Figure 1J shows human BADSC population BF-10. Figure 1K shows human BADSC population BF-11.

[0017] [Figure 1L-M]Figures 1A-1M show 13 human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-1 to BF-13) isolated from biopsies of subclavian and mediastinal adipose tissue and evaluated for their ability to differentiate into brown adipocytes. These BADSC populations were evaluated via bright field (top panels) and Oil Red O staining (ORO) (middle panels). Figure 1L shows human BADSC population BF-12. Figure 1M shows human BADSC population BF-13.

[0018] [Figure 2A] Figure 2A shows UCP-1 expression in BADSC population BF-1 before differentiation in AD-1 culture medium (PreDiff AD-1); BADSC population BF-1 after differentiation in AD-1 culture medium (PostDiff AD-1); BADSC population BF-1 before differentiation in AD-2 culture medium (PreDiff AD-2); and BADSC population BF-1 after differentiation in AD-2 culture medium (PostDiff AD-2). Human brown adipose tissue was used as a positive control (human BAT). Human white adipose tissue was used as a negative control (SubQ WAT and Visc. WAT).

[0019] [Figure 2B] FIG. 2B shows the expression of UCP1 mRNA via qPCR in the BADSC population BF-1 after 15 days of differentiation in either StemPro™, AD-1, or AD-2 culture medium.

[0020] [Figure 2C] FIG. 2C shows FABP4 mRNA expression via qPCR in the BADSC population BF-1 after 15 days of differentiation in either StemPro™, AD-1, or AD-2 culture medium.

[0021] [Figure 2D]FIG. 2D shows adipsin mRNA expression via qPCR in the BADSC population BF-1 after 15 days of differentiation in either StemPro™, AD-1, or AD-2 culture medium.

[0022] [Figure 2E] FIG. 2E shows leptin mRNA expression via qPCR in the BADSC population BF-1 after 15 days of differentiation in either StemPro™, AD-1, or AD-2 culture medium.

[0023] [Figure 2F-K] Figure 2F shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for perilipin (green) using an antibody that binds to perilipin. Figure 2G shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for perilipin (green) using an IgG control antibody. Figure 2H shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for UCP1 (red) using an antibody that binds to UCP1. Figure 2I shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for UCP1 (red) using an IgG control antibody. Figure 2J shows BADSC population BF-1 differentiated in AD-2 culture medium. Fifteen days after induction of differentiation, cells were fixed, immunostained for perilipin and UCP1, and counterstained with DAPI (blue). Figure 2K shows the BADSC population BF-1 differentiated in AD-2 culture medium. Fifteen days after induction of differentiation, cells were fixed, immunostained for perilipin and UCP1, and counterstained with DAPI (blue).

[0024] [Figure 2L-Q]Figure 2L shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for mitochondria (green) using an antibody that binds to mitochondria. Figure 2M shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for mitochondria (green) using an IgG control antibody. Figure 2N shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for UCP1 (red) using an antibody that binds to UCP1. Figure 2O shows BADSC population BF-1 differentiated in AD-2 culture medium. 15 days after induction of differentiation, cells were fixed and immunostained for UCP1 (red) using an IgG control antibody. Figure 2P shows BADSC population BF-1 differentiated in AD-2 culture medium. Fifteen days after induction of differentiation, cells were fixed, immunostained for mitochondria and UCP1, and counterstained with DAPI (blue). Figure 2Q shows the BADSC population BF-1 differentiated in AD-2 culture medium. Fifteen days after induction of differentiation, cells were fixed, immunostained for mitochondria and UCP1, and counterstained with DAPI (blue).

[0025] [Figure 2R] Figure 2R shows quantification of the adipocyte differentiation efficiency (% differentiation) of the BADSC population BF-1, defined as the percentage of perilipin-positive cells by quantifying the total number of cells per field using DAPI. Figure 2R also shows quantification of the brown adipocyte differentiation efficiency (% brown) of the BADSC population BF-1, defined as the percentage of perilipin-positive cells that are positive for UCP1.

[0026] [Figure 3A-C]Figure 3A shows the gene expression levels of the adipocyte marker (PPARα) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3B shows the gene expression levels of the adipocyte marker (PPARγ) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3C shows the gene expression levels of the brown adipocyte marker (PGC1a) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours.

[0027] [Figure 3D-F] Figure 3D shows the gene expression levels of the adipocyte marker (PGC1b) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3E shows the gene expression levels of the adipocyte marker (PRDM16) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3F shows the gene expression levels of the adipocyte marker (CEBPd) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours.

[0028] [Figure 3G-I]Figure 3G shows the gene expression levels of adipocyte marker (CEBPb) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3H shows the gene expression levels of adipocyte marker (CEBPa) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours. Figure 3I shows the gene expression levels of adipocyte marker (TFAM) determined by qPCR for (1) BADSC population BF-1 in 2D, (2) BADSC population BF-1 in 3D at 24 hours, and (3) BADSC population BF-1 in 3D at 48 hours.

[0029] [Figure 4A] Figure 4A shows a schematic diagram of the three-step 3D brown adipocyte differentiation protocol in an encapsulated system.

[0030] [Figure 4B] FIG. 4B shows a photomicrograph (5× magnification) of a BAG 24 hours after formation.

[0031] [Figure 4C] FIG. 4C shows a photomicrograph (5× magnification) of the BAG after harvesting.

[0032] [Figure 4D] FIG. 4D shows a photograph of an encapsulated medical device, Encaptra® EN20.

[0033] [Figure 4E] FIG. 4E shows a photograph of an encapsulated medical device, Encaptra® EN20, loaded with a BAG.

[0034] [Figure 4F]FIG. 4F shows a photomicrograph (10× magnification) of live BAG differentiating inside an encapsulated medical device, Encaptra® EN20.

[0035] [Figure 4G] Figure 4G shows a section of a BAG differentiating inside an encapsulated medical device, Encaptra® EN20, which was visualized using hematoxylin and eosin staining.

[0036] [Figure 4H] Figure 4H shows a section of a BAG differentiating inside an encapsulated medical device, Encaptra® EN20, shown using bright field.

[0037] [Figure 4I-L] Figure 4I shows a section of BAG differentiating within an encapsulated medical device, Encaptra® EN 20. The section was immunostained for perilipin (green), UCP1 (red), and counterstained with DAPI (blue). Figure 4J shows a section of BAG differentiating within an encapsulated medical device, Encaptra® EN20. The section was immunostained with DAPI (blue). Figure 4K shows a section of BAG differentiating within an encapsulated medical device, Encaptra® EN20. The section was immunostained for perilipin (green). Figure 4L shows a section of BAG differentiating within an encapsulated medical device, Encaptra® EN20. The section was immunostained for UCP1 (red).

[0038] [Figure 4M-N]Figure 4M shows the gene expression level of an adipocyte marker (FABP4) determined by qPCR. The RNA used for qPCR was collected from the BAG at D0 (undifferentiated) and from the BAG located in the encapsulated medical device, Encaptra® EN20, at D25 (see Figure 4A). Figure 4N shows the gene expression level of an adipocyte marker (adipsin) determined by qPCR. The RNA used for qPCR was collected from the BAG at D0 (undifferentiated) and from the BAG located in the encapsulated medical device, Encaptra® EN20, at D25 (see Figure 4A).

[0039] [Figure 4O-P] Figure 4O shows the gene expression level of an adipocyte marker (PPAR g) determined by qPCR. The RNA used for qPCR was collected from the BAG at D0 (undifferentiated) and from the BAG located within the encapsulated medical device, Encaptra EN20, at D25 (see Figure 4A). Figure 4P shows the gene expression level of an adipocyte marker (CEBPa) determined by qPCR. The RNA used for qPCR was collected from the BAG at D0 (undifferentiated) and from the BAG located within the encapsulated medical device, Encaptra EN20, at D25 (see Figure 4A).

[0040] [Figure 4Q-R] Figure 4Q shows the gene expression levels of an adipocyte marker (leptin) as determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A). Figure 4R shows the gene expression levels of a brown adipocyte marker (UCP1) as determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A).

[0041] [Figure 4S-T] Figure 4S shows the gene expression level of a brown adipocyte marker (PG C1a) determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A). Figure 4T shows the gene expression level of a brown adipocyte marker (ELOVL3) determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A).

[0042] [Figure 4U-V] Figure 4U shows gene expression levels of a brown adipocyte marker (CI DEA) as determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A). Figure 4V shows gene expression levels of a brown adipocyte marker (COX10) as determined by qPCR. RNA used for qPCR was collected from the BAG on D0 (undifferentiated) and from the BAG placed in the encapsulated medical device, Encaptra® EN20, on D25 (see Figure 4A).

[0043] [Figure 5A] FIG. 5A is a graph showing glucose tolerance test (GTT) results for mice implanted with BAT encapsulated in Matrigel compared to mice implanted with Matrigel alone.

[0044] [Figure 5B] 5B-C are tables representing data generated in the GTT experiment described in FIG. 5A.

[0045] [Figure 5C] 5B-C are tables presenting data generated in the GTT experiment described in FIG. 5A. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description Certain exemplary aspects of the present disclosure are now described to provide a general understanding of the structure, function, production and use principles of the non-naturally occurring three-dimensional brown adipose-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-derived stem cell aggregates and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary aspects, and the scope of various examples of the present disclosure is defined only by the claims.Features illustrated or described in connection with one exemplary aspect can 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-derived stem cell aggregates and methods for producing said non-naturally occurring 3D bags

[0048] Disclosed herein are non-naturally occurring three-dimensional BADSC aggregates or "BAGs." The BAGs are 3D structures formed from BADSCs after they are removed from their two-dimensional (2D) culture in cell-adherent tissue culture flasks, added to non-adherent culture plates, and centrifuged. After centrifugation, the aggregates are uniform. Uniform cell aggregates provide more efficient and consistent differentiation and allow easier loading into encapsulation systems. Furthermore, uniform aggregates provide more accurate cell counts and more precise dosages.

[0049] BADSCs grown in 2D are the natural state of BADSCs whenever cells are expanded in tissue-adherent cell culture flasks. BADSCs cultured in 2D in growth medium are multipotent and function as stem cells. BADSCs grown in 2D cannot form aggregates because they adhere to the cell culture flask and then differentiate into undesirable non-adipocyte cell types, ultimately inducing apoptotic cascades and cell death.

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

[0051] The BAGs can be expanded and further aggregated to become artificial brown adipose tissue or artificial white adipose tissue. The BAGs can become white adipose tissue when differentiated in AD-1 medium. AD-1 medium contains 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, The serum-based differentiation medium consisted of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific), and 2 mM L-glutamine (Gibco, Thermo Fisher Scientific), and consisted of 100 nM erythrocyte maturation medium (100 nM erythrocyte maturation medium, ...

[0052] The BAG can become brown adipose tissue when differentiated in AD-2, a two-step, xeno-free, serum-free, chemically defined differentiation medium. In the first step, BADSCs are grown in the primary differentiation medium, AD-2 DIFF-1 culture medium, which 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 penicillin, and 100 μg / ml 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, which is composed of 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), and 100 nM HCl). Scientific), 0.2 nM T3 (Sigma-Aldrich), 10 μg / ml apo-transferrin (Sigma-Aldrich), 100 units / ml penicillin and 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific) and 100 nM rosiglitazone).

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

[0054] Gene expression of 3D BAG

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

[0056] The formation of BAG results in increased expression of transcription factors and cofactors from the CEBP and PPAR families, which are master regulators of adipogenesis and browning ( Figure 3 A~3I "Browning" refers to the ability of BAG 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 master 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 increases in the expression of PPARγ, PRDM16, TFAM, or PGC1β were observed.

[0058] These data suggest that the formation of 3D BAGs commits BADSC aggregates toward adipogenesis and the brown fat phenotype, and thus the BAGs are initiated down the pathway of brown fat differentiation in the absence of adipocyte differentiation medium.

[0059] Encapsulation systems as delivery systems for BAT

[0060] Transplantation of brown adipose tissue (BAT) into humans to increase BAT mass and / or activity has emerged as a potential method to increase energy expenditure by energy waste. This approach of transplanting BAT into humans can be used to treat metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases. Therefore, a method for delivering BAT for transplantation using 3D BAG loaded into an encapsulation system was sought. This method is disclosed herein.

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

[0062] The encapsulation system described herein allows BAGs to produce 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 fabricated from implantable grade materials or biologics, selected for long-term biocompatibility.

[0064] The encapsulation systems described herein provide for the bidirectional exchange of nutrients and molecules (eg, 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, easily reversible encapsulated medical device (e.g., the Encaptra® Drug Delivery System) (Viacyte, San Diego, California, USA). This device is fabricated from implant-grade materials 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 implanted cells, thus increasing safety and preventing immune rejection of BAT while preventing migration of implanted cells from the encapsulated medical device.

[0066] Methods for producing 3D BAT in an encapsulated system

[0067] Disclosed herein is a method for producing 3D BAT in an encapsulated system. The method includes (1) forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, (2) loading the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into the encapsulated system, (3) differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium, and (4) differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium. The "first differentiation medium" may also be referred to herein as AD-2 DIFF-1 culture medium. The "second differentiation medium" may also be referred to herein as AD-2 DIFF-2 culture medium.

[0068] Treatment method

[0069] Disclosed herein are methods of treating patients with disorders. Disclosed herein are methods of treating patients with metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases. Examples of metabolic disorders may include, but are not limited to, diabetes and obesity. Examples of endocrine disorders may 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, Hürthle 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 disease, pheochromocytoma, pituitary disorders, pituitary tumors, polycystic ovary syndrome, borderline diabetes mellitus, reproduction, painless thyroiditis, thyroid cancer, thyroid disease, 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 may 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., high blood pressure or heart disease secondary to hypertension). Examples of liver diseases may include, but are not limited to, simple fatty liver disease, non-alcoholic steatohepatitis (NASH), and alcohol-related fatty liver disease (ALD).

[0070] Disclosed herein is a method for treating the patient with metabolic disorder.Method comprises: forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregate;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is loaded into an encapsulation system;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a first differentiation medium;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a second differentiation medium;And the brown adipose tissue is delivered to the patient with metabolic disorder.

[0071] Disclosed herein is a method for treating the patient with obesity.Method described above comprises: forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregate;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is loaded into an encapsulation system;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a first differentiation medium;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a second differentiation medium;And the brown adipose tissue is delivered to the patient with obesity.

[0072] Disclosed herein is a method for treating the patient with endocrine disorder.Method comprises: forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregate;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is loaded into an encapsulation system;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a first differentiation medium;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a second differentiation medium;And delivers the brown adipose tissue to the patient with endocrine disorder.

[0073] Disclosed herein is a method for treating the patient with cardiovascular disease.Method described comprises: forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregate;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is loaded into an encapsulation system;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a first differentiation medium;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a second differentiation medium;And the brown adipose tissue is delivered to the patient with cardiovascular disease.

[0074] Disclosed herein is a method for treating the patient with liver disease.Method comprises: forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregate;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is loaded into an encapsulation system;The non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a first differentiation medium;AndThe non-naturally occurring three-dimensional brown adipose derived stem cell aggregate is differentiated into brown adipose tissue in a second differentiation medium;AndThe brown adipose tissue is delivered to the patient with 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, those listed in the following enumerated items: Item 1. A non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate, wherein the three-dimensional brown adipose-derived stem cell aggregate comprises brown adipose-derived stem cells that express one or more brown adipose-derived genes in the absence of a differentiation medium. Item 2. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate of Item 1, wherein the one or more brown adipocyte genes are selected from the group consisting of PPARα, PPARγ, PGC1β, PRDM16, CEBPD, CEBPB, CEBPA, TFAM, PGC1α, and PGC1β. Item 3. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate according to any one of Items 1 to 2, wherein the aggregate forms in a non-adhesive environment. Item 4. The non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate of any one of Items 1 to 3, wherein the aggregate produces an extracellular biologic selected from the group consisting of exosomes, microRNA, cytokines, proteins, and adipokines. Item 5. An encapsulation system comprising the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregate according to any one of Items 1 to 4. Item 6. The encapsulation system according to Item 5, 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. Item 7. The encapsulation system according to Item 5, wherein the encapsulation system is an encapsulated medical device. Item 8. A method for producing non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, the method comprising: Loading brown adipose-derived stem cells grown in two-dimensional (2D) culture into non-adherent culture plates; and centrifuging the non-adherent culture plate to distribute the brown adipose-derived stem cells uniformly within the non-adherent culture plate, thereby forming three-dimensional brown adipose-derived stem cell aggregates; A method that encompasses Item 9. The method of Item 8, further comprising culturing the brown adipose-derived stem cells in two-dimensional (2D) culture using a growth medium under normoxic or hypoxic conditions prior to the loading step. Item 10. A method for producing three-dimensional brown adipose tissue in an encapsulated system, the method comprising: forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates; loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into said encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; and differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; A method that encompasses Item 11. The method of item 10, 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. Item 12. The method of Item 10, wherein the encapsulation system is an encapsulated medical device. Item 13. The method according to any one of Items 10 to 12, wherein the first differentiation medium contains dexamethasone, IBMX, and T3. Item 14. The method according to any one of Items 10 to 13, wherein the second differentiation medium contains T3 and rosiglitazone. Item 15. A method of treating a patient having a disorder, the method comprising: forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates; loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into an encapsulation system; differentiating said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; and delivering said brown adipose tissue to said patient having said disorder; A method that encompasses Item 16. The method of item 15, 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. Item 17. The method of Item 15, wherein the encapsulation system is an encapsulated medical device. Item 18. The method according to any one of Items 15 to 17, wherein the first differentiation medium contains dexamethasone, IBMX, and T3. Item 19. The method according to any one of Items 15 to 18, wherein the second differentiation medium contains T3 and rosiglitazone. Item 20. The method according to any one of Items 15 to 19, wherein the disorder is a metabolic disorder, an endocrine disorder, a cardiovascular disorder, or a liver disease. Item 21. The method according to Item 20, wherein the metabolic disorder is obesity or diabetes.

[0077] definition

[0078] In addition to the definitions set forth herein above, the following definitions are relevant to the present disclosure.

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

[0080] "Two-dimensional (2D) culture" refers to cells that are spread across the 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 cellular aggregates.

[0082] Any numerical range described herein describes all subranges of the same numerical precision (i.e., having the same number of specified digits) encompassed within the described range. For example, a described range of "1.0 to 10.0" describes all subranges (such as "2.4 to 7.6") between the described minimum value of 1.0 and the described maximum value of 10.0 (inclusive), even if the "2.4 to 7.6" range is not explicitly recited in the body of the specification. Applicants therefore reserve the right to amend this specification, including the claims, to explicitly recite any subrange of the same numerical precision encompassed within the range explicitly recited herein. All such ranges are essentially described herein, such that amending to explicitly recite any such subranges complies with the requirements of description, sufficiency of description, and additional matter, including those under 35 U.S.C. §112(a) and EPC Article 123(2). Additionally, unless expressly specified otherwise or required otherwise by context, all numerical parameters described herein (e.g., those expressing values, ranges, amounts, percentages, etc.) can be read and understood as if preceded by the word "about," even if the word "about" does not explicitly appear before the number. Further, the numerical parameters described herein should be construed in light of the number of reported significant digits, numerical precision, and by applying ordinary rounding techniques. It is also understood that the numerical parameters described herein necessarily possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of such parameters.

[0083] Any patent, publication, or other disclosure material identified herein is incorporated by reference in its entirety unless otherwise indicated, but only to the extent that the incorporated material does not contradict existing descriptions, definitions, statements, or other disclosure material explicitly set forth herein. Thus, and to the extent necessary, the express disclosure as set forth herein supersedes any conflicting material incorporated by reference. Any material, or portion thereof, that is said to be incorporated by reference herein but that contradicts existing definitions, statements, or other disclosure material set forth herein is only incorporated to the extent that no contradiction arises between the incorporated material and the existing disclosure material. Applicant reserves the right to amend this specification to explicitly describe any subject matter, or portion thereof, incorporated herein by reference.

[0084] Details of one or more aspects of the present disclosure are set forth in the accompanying Examples below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, specific examples of contemplated 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 also includes the plural unless the context clearly dictates otherwise. 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, the present description will control. [Example]

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

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

[0087] BADSCs were isolated from fresh brown adipose tissue and cultured for up to three passages. Human platelet lysate (XcyteTM Plus Xeno-Free Supplement, iBiologics, Phoenix, Arizona, USA), 1% GlutaMAX TM Cells were expanded in growth medium (GM) consisting of Dulbecco's Modified Eagle's Medium (DMEM) Low Glucose (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 1% Minimal 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 grown at a density of 3500 cells / cm. 2 The cells were seeded at a density of 1000 and the medium was changed every other day.

[0088] Adipocyte differentiation was induced 2 days after the cells reached full confluency by adding brown adipocyte differentiation medium 1 (AD-1). AD-1 was cultured in 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). This serum-based differentiation medium is composed of 100% soluble erythrocytes (Cat. No. 1001 ...

[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 for up to three passages. Human platelet lysate (Xcyte TM Plus Xeno-Free Supplement, iBiologics), 1% GlutaMAX TMCells were expanded in GM consisting of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with 1% Minimal 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 grown at 3500 cells / cm. 2 The cells were seeded at a density of 1000 and the medium was changed every other day.

[0092] Adipocyte differentiation was induced 2 days after the cells reached full confluency 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 consisted 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, The AD-2 DIFF-1 medium was incubated in a 5% COOH medium containing 0.2 nM T3 (Sigma-Aldrich), 10 μg / ml apo-transferrin (Sigma-Aldrich), 100 units / ml penicillin, and 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific). In a second step, after 3 days, the AD-2 DIFF-1 culture medium was replaced with a second differentiation medium, AD-2 DIFF-2 (a 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 penicillin and 100 μg / ml streptomycin (Gibco, Thermo Fisher Scientific), and 100 nM rosiglitazone.

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

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

[0095] As indicated by the expression of adipocyte markers FABP4 and adipsin ( Figure 2 C and Figure 2D), AD-1 and AD-2 adipogenic media were equally efficient in converting BADSCs into adipocytes, and a commercially available adipogenic medium, StemPro TM (Gibco, Thermo Fisher Scientific) Ta. Although AD-1 and AD-2 were equivalent in promoting adipocyte differentiation, adipocytes obtained in the xeno-free, serum-free, chemically defined medium AD-2 were morphologically larger and contained larger lipid droplets (Figure 1 A~1M (The arrow indicates cells cultured in AD-2; data not shown for cells cultured in AD-1.) Differentiation using AD-2 medium allowed for much higher brown adipocyte differentiation than AD-1 or commercial adipogenic media.

[0096] The results also showed that UCP1 gene expression was significantly increased in a commercially available adipogenic medium, StemPro TM The expression of leptin, a white-specific marker, was 1.5-fold lower in AD-2 than in AD-1, confirming the superior efficiency of AD-2 in directing BADSCs toward the brown adipose phenotype (Figure 2B). 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, with over 80% of the cells differentiating into adipocytes ( Figure 2 F~2K and 2 R ) 98% of the differentiated cells (perilipin+ cells) co-expressed the brown-specific marker UCP1 (Figure 2 F~2K and 2 R ) This data confirmed the expression of UCP1 at the protein level (Figure 2 H and 2 I; Figures 2N and 2O), demonstrated high yields of brown adipocyte conversion in a xeno-free, chemically defined differentiation medium. As expected, mitochondria-localized UCP1 protein was obtained when differentiated BADSCs were co-immunostained for UCP1 and mitochondria, as indicated by the overlaid signals (Figure 2). N~2Q ).

[0098] Figure 2A~ 2R The results in this study show that the two-step AD-2 differentiation media (AD-2 DIFF-1 and AD-2 DIFF-2) promote stronger brown adipocyte differentiation compared to AD-1 differentiation media and commercial adipogenic media.

[0099] Example 3 - Method for making a 3D BAG

[0100] Non-naturally occurring three-dimensional BADSC aggregates, or BAGs, are grown on non-adherent culture plates (e.g., AggreWell TM The cells were formed in 400Ex 6-well plates (StemCell Technologies, Vancouver, British Columbia, Canada).

[0101] BADSCs were first cultured in 2D using growth medium under normoxic or hypoxic conditions until 80% confluency. Non-adherent plates were rinsed with a rinsing solution (e.g., AggreWell TM The non-adherent plate was coated with a rinsing solution (StemCell Technologies) according to the manufacturer's instructions. After washing the non-adherent plate with GM, 12 ml of a 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 basket centrifuge to allow the cells to uniformly settle into the microwells, resulting in a density of 1,000 cells / microwell and thus creating uniform cell aggregates. Without centrifugation, the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates (BAGs) are not uniform.

[0102] The BAGs are then plated onto non-adherent culture plates (e.g., AggreWell) in GM. TM The cells were cultured in 400Ex 6-well plates at 37°C in normoxia or hypoxia and 95% humidity for 24 hours before harvesting. Approximately 28,200 BAG per non-adherent 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 encapsulated system

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

[0105] In step 1: BAGs were formed in AggreWell™ 400Ex 6-well plates (StemCell Technologies) using BADSC population BF-1. The optimal cell seeding density was determined to be 1,000 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., an 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 time, the BAGs combined to fill the entire volume of the encapsulation device. The resulting bags are highly uniform in size and shape, both within and between experiments. TM This can be easily modified by adjusting the cell seeding density formed in 400Ex 6-well plates (StemCell Technologies). The optimal cell seeding density for generating uniform BAGs was determined to be 1000 cells per microwell.

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

[0107] In step 3: The BAGs within 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 non-naturally occurring brown adipose-derived stem cell aggregates, including the BADSC population BF-1, efficiently differentiated into brown adipocytes in 3D within Encaptra®-encapsulated medical devices. BADSC BF-1 cells differentiating within the encapsulated medical devices formed tissue-like structures visualized by hematoxylin and eosin staining that were highly enriched for brown adipocytes (UCP1- and perilipin-positive) containing a high content of mitochondria (Figure 4G). ~4L These cells express high levels of adipocyte markers (e.g., FABP4, adipsin, PPARg, CEBPa, and leptin) when compared to undifferentiated BAG (Figure 4). M~4Q )) and brown-specific markers (e.g., UCP1, PGC1a, CIDEA, ELOVL3, and COX10 (Figure 4 R~4V )) is expressed.

[0109] In conclusion, we have shown that non-naturally occurring BADSC aggregates represent a highly promising source of transplantable brown adipose tissue to increase energy expenditure and potentially treat metabolic, endocrine, cardiovascular, and liver disorders. Furthermore, the strategy of fast-delivering non-naturally occurring BADSC aggregates using encapsulation represents a safe delivery system and will help 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 (CB-Igh-1b / GbmsTac-Prkdcscid-LystbgN7) (Taconic Biosciences) were individually housed at 25°C and fed a 60% fat-containing high-fat diet (HFT) (D12492, 60 kcal% fat [primarily lard], 20 kcal% carbohydrates). These mice have metabolic syndrome and are unable to process glucose.

[0112] The encapsulation system was prepared by adding 1 mL of 4 mg / mL Matrigel (Corning® Matrigel® Matrix High Concentration (HC), phenol-free *LDEV-free) 6 Brown adipose-derived stem cells (BADSCs) were prepared by adding them to non-adherent culture plates and centrifuging them after removal from their two-dimensional (2D) culture in cell-adherent tissue culture flasks. After centrifugation, the aggregates were homogenous 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 encapsulated system containing BAT formed spherical shapes (i.e., beads). After in vitro differentiation, the beads reduced in size to 20-30 μl.

[0114] Forty beads were collected using a cell strainer, representing approximately 3.2 × 10 beads that comprise the encapsulated BAT. 6 The beads were transferred to a 1.5 mL conical vial and placed on ice. 100 μl of chilled 10 mg / mL Matrigel was added to the beads, mixed thoroughly and kept on ice.

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

[0116] The treated and control mice were analyzed weekly to determine their ability to absorb glucose via a glucose tolerance test (GTT). Prior to the analysis, the 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 glucose injection.

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

[0118] Example 6 – Evaluation of the efficacy and safety of BAG delivered in Matrigel

[0119] Mice included in the GTT experiment described in Example 5 are also monitored for their body weight. To measure body weight, mice are weighed once a week for three months. Each week, mice are placed on a zeroed scale and their weight is recorded. Mice transplanted with BAT (treated group) show lower total body weight or show lower total body weight gain compared to mice not transplanted with BAT (control group).

[0120] Notes regarding illustrative examples

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

Claims

1. 1. A method of producing non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates in the absence of a differentiation medium, comprising brown adipose-derived stem cells expressing one or more brown adipose-derived genes, wherein the one or more brown adipose-derived genes are selected from the group consisting of PPARα, PPARγ, PGC1β, PRDM16, CEBPD, CEBPB, CEBPA, TFAM, PGC1α, and PGC1β, said method comprising: Loading brown adipose-derived stem cells grown in two-dimensional (2D) culture into non-adherent culture plates; and centrifuging the non-adherent culture plate to distribute the brown adipose-derived stem cells uniformly within the non-adherent culture plate, thereby forming non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, wherein the aggregates consist of brown adipose-derived stem cells; A method that encompasses

2. culturing the brown adipose-derived stem cells in two-dimensional (2D) culture under normoxic or hypoxic conditions using a growth medium prior to the loading step; The method of claim 1 further comprising:

3. After the centrifuging step and prior to harvesting the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates, culturing the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates in a non-adherent culture plate in a growth medium at 37° C. under normoxia or hypoxia and 95% humidity for 24 hours. The method of claim 1 or claim 2, further comprising:

4. The method according to any one of claims 1 to 3, loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into an encapsulation system; differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a first differentiation medium; and further differentiating the non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into brown adipose tissue in a second differentiation medium; The method further comprises:

5. 5. The method of 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.

6. forming said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates in a growth medium at about 160 pm / aggregate prior to loading said non-naturally occurring three-dimensional brown adipose-derived stem cell aggregates into said encapsulation system; In the first differentiation step, a step of using a xeno-free, serum-free, chemically defined AD-2-DIFF-1 medium as the first differentiation medium; and a step of using a xeno-free, serum-free, chemically defined AD-2-DIFF-2 medium as the second differentiation medium in the second differentiation step; The method according to any one of claims 4 to 5, comprising:

7. In the first differentiation step, the aggregates in the encapsulation system are differentiated in vitro in the first differentiation medium for 3 days; and In the second differentiation step, the aggregates in the encapsulation system are further differentiated in vitro in the second differentiation medium for 20 days. The method of claim 6, comprising:

8. the encapsulation system is an encapsulated medical device; or the first differentiation medium comprises dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and triiodothyronine (T3); or the second differentiation medium comprises T3 and rosiglitazone; The method of claim 4.

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