Patent Description:
The prevalence of metabolic disorders, such as obesity, has increased dramatically over the past several decades and has become a pandemic. By <NUM>, more than <NUM>% of Americans will suffer from obesity, resulting in over a <NUM> billion dollar loss in economic productivity. Obesity is a major risk factor for type II diabetes mellitus, hypertension, cardiovascular disease, osteoarthritis, and certain forms of cancer. Current therapeutic approaches, such as caloric restriction and exercise, which rely mainly on patient's will power to reduce energy intake and/or increase energy expenditure, are of limited effectiveness in obese patients. Bariatric surgery is the only clinically proven therapy in terms of weight loss and decreased morbidity/mortality in patients with a body mass index (BMI) over <NUM>; however, it has associated risks, high costs, and requires proper management of patient's nutrition and physical activity. Despite efforts from researchers and medical professionals worldwide who have been trying to address obesity and other metabolic disorders, there is still a need for alternative ways to increase energy expenditure that could augment the current therapeutic options for treating obese patients and patients with other metabolic disorders.

<NPL>, can be seen as closest prior art document and discloses differentiating brown adipose stem cells in a differentiation medium including dexamethasone, isobutylmethylxanthine, rosiglitazone, and T3. The BADSC and a scaffold are required for three-dimensional formation. <NPL>, can also be seen as closest prior art document and discloses differentiating brown adipose stem cells in an adipogenic cocktail including dexamethasone and isobutylmethylxanthine. The BADSC and a scaffold are required for a three-dimensional formation (or hydrogel, or alginate microsphere). <CIT> also discloses differentiating brown adipose stem cells in a differentiation medium (differentiation factor cocktail) and the production of cell aggregates composed of adipose stem cells (ASC) and endothelial cells. <CIT> discloses no non-naturally occurring three-dimensional brown adipose derived stem cell aggregates, but differentiating brown adipose stem cells in an adipogenic induction medium including dexamethasone, isobutylmethylxanthine, rosiglitazone, and T3. <CIT> serves a different object, namely modulating heat loss in a neonatal human subject and discloses differentiating brown adipose stem cells in a differentiation medium including dexamethasone, isobutylmethylxanthine, and rosiglitazone. The natural BADS are cultured as a three dimensional sheets employing a 'self-assembly' culture methodology and biocompatible, preferably micro-sized, carriers. <CIT> discloses cell aggregates consisting of adipose stem cells and endothelial cells. During the aggregation process, the endothelial cells migrate to the middle of the aggregate while the adipose stem cells remain on the outside. This particular 3D conformation is important because it promotes collagen production, close cell-cell association, and rounded cell shape, which in turn promotes vascularization of the endothelial cells and differentiation of the adipose stem cells to brown fat. The aggregates comprise BADSC composed of adipose stem cells (ASC) and, necessarily, endothelial cells. The importance of using endothelial cells when producing the aggregates is emphasized.

This section provides a general summary of the disclosure and is not comprehensive of its full scope or all of its features.

Provided herein is a non-naturally occurring three-dimensional brown adipose derived stem cell aggregate, which is not part of the invention. The three-dimensional brown adipose derived stem cell aggregate comprises brown adipose-derived stem cells that express one or more brown adipocyte genes in the absence of differentiation medium.

Also provided herein is an encapsulation system, which is not part of the invention, comprising a non-naturally occurring three-dimensional brown adipose derived stem cell aggregate. The three-dimensional brown adipose derived stem cell aggregate comprises brown adipose-derived stem cells that express one or more brown adipocyte gene in the absence of differentiation medium.

Also provided herein is a method of making a non-naturally occurring three-dimensional brown adipose derived stem cell aggregate according to claim <NUM>. The method comprises: loading brown adipose derived stem cells grown in a two-dimensional (2D) culture into a non-adherent culture plate, and centrifuging the non-adherent culture plate to uniformly position the brown adipose- derived stem cells in the non-adherent culture plate, thereby forming three-dimensional brown adipose derived stem cell aggregates, wherein the aggregates consist of brown adipose derived stem cells.

Also provided herein is a method of making a three-dimensional brown adipose tissue in an encapsulation system. 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 the 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.

Also provided herein is a method of treating a patient with a disorder. 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.

In addition to the illustrative examples and features described herein, further aspects, examples, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

The patent or patent application contains at least one drawing executed in color.

Various aspects of 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 are disclosed and described in this specification and can be better understood by reference to the accompanying figures, in which:.

<FIG> show thirteen human brown adipose-derived mesenchymal stem cell (BADSC) populations (BF-<NUM> to BF-<NUM>) isolated from subcutaneous supraclavicular and mediastinal adipose tissue biopsies 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).

Certain exemplary aspects of the present disclosure will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use 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 of ordinary skill 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 that the scope of the various examples of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary aspect may be combined with the features of other aspects. Such modifications and variations are intended to be included within the scope of the present disclosure.

Non-naturally occurring three-dimensional BADSC aggregates (not part of the invention) or "BAGs" are disclosed herein. The BAGs are 3D structures formed from BADSC after the BADSC 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 a more efficient and consistent differentiation and are easier to load into encapsulation systems. Furthermore, uniform aggregation provides a more accurate cell number and a more accurate dosage.

BADSCs grown in 2D are the natural state of the BADSC whenever the cells expand in tissue adherent cell culture flasks. BADSCs cultured in growth media in 2D are multipotent and function as a stem cell. The BADSC grown in 2D cannot form aggregates because they attach to the cell culture flask and then differentiate into an unwanted non-adipose cell type and ultimately induce apoptotic cascade and cell death.

BADSC cannot form cell aggregates in 2D culture, but whenever the BADSC are removed from their 2D tissue adherent environment and placed in a non-adherent environment, the cells form 3D aggregates, as described above. The BADSC when aggregated form clusters of cells that are able to communicate with each other and their environment in 3D.

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 if differentiated in AD-<NUM>. AD-<NUM> is a serum based differentiation medium composed of DMEM low
glucose (Gibco, Thermo Fisher Scientific) supplemented with <NUM>% fetal bovine serum (FBS, HyClone, GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, UK), <NUM> dexamethasone (MP Biomedicals, Santa Ana, California, USA), <NUM> <NUM>-isobutyl-<NUM>-methylxanthine (IBMX, Sigma-Aldrich, St. Louis, Missouri, USA), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> indomethacin (Sigma-Aldrich), <NUM> triiodothyronine (T3, Sigma-Aldrich), <NUM> rosiglitazone (Sigma-Aldrich), <NUM> units/ml of penicillin, <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific), and <NUM> L-glutamine (Gibco, Thermo Fisher Scientific).

The BAGs can become brown adipose tissue if differentiated in AD-<NUM>. AD-<NUM> is a two-step xeno-free, serum free, chemically defined differentiation medium. In a first step, BADSC are grown in a first differentiation medium, AD-<NUM> DIFF-<NUM> culture medium, which comprises DMEM / Ham's F12 Media (<NUM>:<NUM>) (Lonza Group AG, Basel, Switzerland), <NUM> HEPES Buffer (Lonza Group AG), <NUM> L-glutamine (Gibco, Thermo Fisher Scientific), <NUM> dexamethasone (MP Biomedicals), <NUM> IBMX (Sigma-Aldrich), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> T3 (Sigma-Aldrich), <NUM>µg/ml apo-transferrin (Sigma-Aldrich), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific). In a second step, after three days the AD-<NUM> DIFF-<NUM> culture medium was replaced with a second differentiation medium, AD-<NUM> DIFF-<NUM>, a xeno-free, serum free, chemically defined differentiation medium, which comprises DMEM / Ham's F12 Media (<NUM>:<NUM>) (Lonza Group AG), <NUM> HEPES Buffer (Lonza Group AG), <NUM> L-glutamine (Gibco, Thermo Fisher Scientific), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> T3 (Sigma-Aldrich), <NUM>µg/ml apo-transferrin (Sigma-Aldrich), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific) and <NUM> rosiglitazone.

These BAGs can serve as cell factories that can produce white or brown extracellular biologics (e.g., exosomes, microRNAs, cytokines, proteins, adipokines).

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

The formation of BAGs resulted in an increased expression of transcription factors and co-factors from the CEBP and PPAR families, which are master regulators of adipogenesis and browning (<FIG>). "Browning" refers to the BAGs ability to express UCP-<NUM> post-differentiation in AD-<NUM> medium.

The early adipocyte differentiation transcription factors CEBPD and CEBPB were increased after <NUM> hours in 3D culture whereas CEBPA was significantly increased after <NUM> hours in 3D culture. PPARα, a master regulator of fatty acid oxidation, and PGC1α, a regulator of mitochondrial respiration and heat production in brown adipocyte, were both increased after <NUM> hours in 3D cultures whereas, no significant changes in the expression of PPARy, PRDM16, TFAM or PGC1β were observed.

These data suggest that the formation of 3D BAGs commits BADSC aggregates to adipogenesis and a brown adipose phenotype and so the BAGs have started down the path of brown adipose differentiation in the absence of adipocyte differentiation medium.

Transplanting brown adipose tissue (BAT) into humans, in order to increase BAT mass and/or activity, has emerged as a potential way to increase energy expenditure by energy wasting. This approach of transplanting BAT into humans can be used to treat metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases. Therefore, a method according to claims <NUM> to <NUM> of delivering BAT for transplantation using 3D BAGs loaded into encapsulation systems was sought and is disclosed herein.

Several different encapsulation systems (not part of the invention) can be loaded with BAGs and used to deliver the BAT for transplantation such as alginate microcapsules, cellulose hydrogels, red blood cells, porous polymer membranes, 3D biological scaffolds, Afibromer™ polymers (Sigilon Therapeutics, Cambridge, Massachussetts, USA), PEG-based hydrogels, non-hydrogel beads, and matrigel.

The encapsulation systems described herein allow the BAGs to produce extracellular factors that can interact with the host environment, such as proteins, cytokines, microRNAs, cytokines, exosomes, and cell specific secretome.

The encapsulation systems described herein are manufactured from implantable- grade materials or biologies and are selected for long-term biocompatibility.

The encapsulation systems described herein provide a bidirectional exchange of nutrients and molecules such as glucose, fatty acids, cytokines, adipokines, and hormones.

In certain examples, the encapsulation system can be an encapsulation medical device. In other examples, the encapsulation medical device can be an FDA-approved, immune-protecting, easily retrievable encapsulation medical device, such as the Encaptra® Drug Delivery System (Viacyte, San Diego, California, USA). This device is manufactured from implant-grade materials specifically selected for long-term biocompatibility and allows for bidirectional
exchange of nutrients and molecules such as glucose, fatty acids, and hormones. The encapsulation medical device provides a barrier between the host and the transplanted cells and therefore should prevent immune rejection of BAT while increasing safety and preventing transplanted cells to migrate out of the encapsulation medical device.

Disclosed herein is a method of making 3D BAT in an encapsulation system according to claims <NUM> to <NUM>. An embodiment of the method comprises (<NUM>) forming non-naturally occurring three-dimensional brown adipose derived stem cell aggregates, (<NUM>) loading the non-naturally occurring three-dimensional brown adipose derived stem cell aggregates into the encapsulation system, (<NUM>) differentiating the non-naturally occurring three-dimensional brown adipose derived stem cell aggregates into brown adipose tissue in a first differentiation medium, and (<NUM>) 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" can also be referred to herein as AD- <NUM> DIFF-<NUM> culture medium. The "second differentiation medium" can also be referred to herein as AD-<NUM> DIFF-<NUM> culture medium.

Methods of treating patients with disorders are disclosed herein, being not part of the invention. Methods of treating patients with metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases 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 <NUM> diabetes, type <NUM> diabetes, gestational diabetes, maturity onset diabetes of the young), follicular thyroid cancer, goiters, 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 <NUM>, MEN 2A, MEN 2B, menopause, metabolic syndrome, obesity, osteoporosis, papillary thyroid cancer, parathyroid diseases, pheochromocytoma, pituitary disorders, pituitary tumors, polycystic ovary syndrome, prediabetes, reproduction, silent thyroiditis, thyroid cancer, thyroid diseases, 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 artery (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 blood clots, and blood clotting disorders, cardiomyopathy, hypertensive heart disease (e.g., diseases of the heart secondary to high blood pressure or hypertension). Examples of liver disease can include, but are not limited to, simple fatty liver disease, nonalcoholic steatohepatitis (NASH), and alcohol-related fatty liver disease (ALD).

Disclosed herein is a method of treating a patient with a metabolic disorder. 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 a metabolic disorder.

Disclosed herein is a method of treating a patient with obesity. 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 obesity.

Disclosed herein is a method of treating a patient with an endocrine disorder. 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 an endocrine disorder.

Disclosed herein is a method of treating a patient with a cardiovascular disorder. 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 a cardiovascular disorder.

Disclosed herein is a method of treating a patient with liver disease. 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 liver disease.

In addition to the definitions previously set forth herein, the following definitions are relevant to the present disclosure:
The singular forms "a," "an," and "the" include plural references, unless the context clearly dictates otherwise.

A "<NUM>-dimensional (2D) culture" refers to cells spreading throughout the surface of a cell culture plate and adhering to the surface of the cell culture plate.

A "<NUM>-dimensional (3D) culture" refers to cells that do not adhere to the surface of a cell culture plate and instead associate with each other, thereby forming cellular aggregates.

Any numerical range recited in this specification describes all sub-ranges of the same numerical precision (i.e., having the same number of specified digits) subsumed within the recited range. For example, a recited range of "<NUM> to <NUM>" describes all sub-ranges between (and including) the recited minimum value of <NUM> and the recited maximum value of <NUM>, such as, for example, "<NUM> to <NUM>," even if the range of "<NUM> to <NUM>" is not expressly recited in the text of the specification. Accordingly, the Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range of the same numerical precision subsumed within the ranges expressly recited in this specification. All such ranges are inherently described in this specification such that amending to expressly recite any such sub-ranges will comply with written description, sufficiency of description, and added matter requirements, including the requirements under <NUM> U. § <NUM>(a) and Article <NUM>(<NUM>) EPC. Also, unless expressly specified or otherwise required by context, all numerical parameters described in this specification (such as those expressing values, ranges, amounts, percentages, and the like) may be read as if prefaced by the word "about," even if the word "about" does not expressly appear before a number. Additionally, numerical parameters described in this specification 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 numerical parameters described in this specification will necessarily possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of the parameter.

Any patent, publication, or other disclosure material identified herein is incorporated by reference into this specification in its entirety unless otherwise indicated, but only to the extent that the incorporated material does not conflict with existing descriptions, definitions, statements, or other disclosure material expressly set forth in this specification. As such, and to the extent necessary, the express disclosure as set forth in this specification supersedes any conflicting material incorporated by reference. Any material, or portion thereof, that is said to be incorporated by reference into this specification, but which conflicts with existing definitions, statements, or other disclosure material set forth herein, is only incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. Applicants reserve the right to amend this specification to expressly recite any subject matter, or portion thereof, incorporated by reference herein.

The 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 the materials and methods contemplated are now described. Other features, objects and advantages of the present disclosure will be apparent from the description. In the description examples, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, 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 present disclosure belongs. In the case of conflict, the present description will control.

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

BADSCs were isolated from fresh brown adipose tissue and were cultured for up to three passages. Cells were expanded in growth medium (GM) composed of Dulbecco's modified Eagle's medium (DMEM) low glucose (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), supplemented with <NUM>% human platelet lysate (Xcyte™ Plus Xeno-Free Supplement, iBiologics, Phoenix, Arizona, USA), <NUM>% GlutaMAX™ Supplement (Gibco, Thermo Fisher Scientific), <NUM>% Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA, Gibco, Thermo Fisher Scientific), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific). Cells were seeded at a density of <NUM> cells/cm<NUM> and medium was replaced every other day.

Adipocyte differentiation was induced two days after cells reached full confluency by addition of brown adipocyte differentiation medium <NUM> (AD-<NUM>). AD-<NUM> is a serum based differentiation medium composed of DMEM low glucose (Gibco, Thermo Fisher Scientific) supplemented with <NUM>% fetal bovine serum (FBS, HyClone, GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, UK), <NUM> dexamethasone (MP Biomedicals, Santa Ana, California, USA), <NUM> <NUM>-isobutyl-<NUM>-methylxanthine (IBMX, Sigma-Aldrich, St. Louis, Missouri, USA), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> indomethacin (Sigma-Aldrich), <NUM> triiodothyronine (T3, Sigma-Aldrich), <NUM> rosiglitazone (Sigma-Aldrich), <NUM> units/ml of penicillin, <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific), and <NUM> L-glutamine (Gibco, Thermo Fisher Scientific).

In order to develop a transplantable brown adipose tissue (BAT) for human applications, differentiation protocols applicable to cellular therapy in humans were sought.

BADSCs were isolated from fresh brown adipose tissue and were cultured for up to three passages. Cells were expanded in GM composed of DMEM low glucose (Gibco, Thermo Fisher Scientific), supplemented with <NUM>% human platelet lysate (Xcyte™ Plus Xeno-Free Supplement, iBiologics), <NUM>% GlutaMAX™ Supplement (Gibco, Thermo Fisher Scientific), <NUM>% Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA, Gibco, Thermo Fisher Scientific), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific). Cells were seeded at a density of <NUM> cells/cm<NUM> and medium was replaced every other day.

Adipocyte differentiation was induced two days after cells reached full confluency by addition of brown adipocyte differentiation medium <NUM> (AD-<NUM>). AD-<NUM> is a two-step xeno-free, serum free, chemically defined differentiation medium. In a first step, BADSC are grown in a first differentiation medium, AD-<NUM> DIFF-<NUM> culture medium, which comprises DMEM / Ham's F12 Media (<NUM>:<NUM>) (Lonza Group AG, Basel, Switzerland), <NUM> HEPES Buffer (Lonza Group AG), <NUM> L-glutamine (Gibco, Thermo Fisher Scientific), <NUM> dexamethasone (MP Biomedicals), <NUM> IBMX (Sigma-Aldrich), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> T3 (Sigma-Aldrich), <NUM>µg/ml apo-transferrin (Sigma-Aldrich), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific). In a second step, after three days the AD-<NUM> DIFF-<NUM> culture medium was replaced with a second differentiation medium, AD-<NUM> DIFF-<NUM>, a xeno-free, serum free, chemically defined differentiation medium, which comprises DMEM / Ham's F12 Media (<NUM>:<NUM>) (Lonza Group AG), <NUM> HEPES Buffer (Lonza Group AG), <NUM> L-glutamine (Gibco, Thermo Fisher Scientific), <NUM> insulin (Gibco, Thermo Fisher Scientific), <NUM> T3 (Sigma-Aldrich), <NUM>µg/ml apo-transferrin (Sigma-Aldrich), <NUM> units/ml of penicillin and <NUM>µg/ml of streptomycin (Gibco, Thermo Fisher Scientific) and <NUM> rosiglitazone.

In some examples, AD-<NUM> can comprise human platelet lysate. In other examples, AD-<NUM> does not comprise human platelet lysate.

The BADSC populations were differentiated in a xeno-free, serum free, chemically defined brown differentiation medium (AD-<NUM> DIFF-<NUM> and AD-<NUM> DIFF-<NUM>) using a two-step method described above and its potency in generating brown adipocytes was compared to an FBS-based differentiation medium (AD-<NUM>) and to a commercially available adipogenic medium (StemPro™ Adipogenesis, Gibco, Thermo Fisher Scientific).

As demonstrated by the expression of the adipocyte markers FABP4 and adipsin (<FIG> and <FIG>), AD-<NUM> and AD-<NUM> adipogenic media were comparably efficient at converting BADSC into adipocytes and more efficient than the commercial adipogenic medium, StemPro™ (Gibco, Thermo Fisher Scientific). Although AD-<NUM> and AD-<NUM> were equivalent in promoting adipocyte differentiation, adipocytes obtained in the xeno-free, serum free, chemically defined medium AD-<NUM> were morphologically larger and contained larger lipid droplets (<FIG> show cells cultured in AD-<NUM>; data not shown for cells cultured in AD-<NUM>). Differentiation using the AD-<NUM> medium allowed for a much higher brown adipocyte differentiation than the AD-<NUM> or the commercial adipogenic media.

Results also show that UCP1 gene expression was over <NUM> fold higher in AD-<NUM> and over <NUM> fold higher in AD-<NUM> as compared to the commercial adipogenic medium, StemPro™ (<FIG>). Additionally, the expression of the white specific marker leptin was <NUM> fold lower in AD-<NUM> than AD-<NUM> confirming the superior efficiency of AD-<NUM> to direct BADSC to a brown adipose phenotype (<FIG>).

Immunocytochemistry analysis of BADSC population BF-<NUM> differentiated in AD-<NUM> for <NUM> days showed that the adipocyte conversion rate i.e. the percentage of cell positive for the adipocyte marker perilipin, is very high with over <NUM>% of the cells differentiating into adipocytes (<FIG> and <FIG>). <NUM>% of the differentiated cells (perilipin+ cells) co-expressed the brown specific marker UCP1 (<FIG> and <FIG>). This data confirmed the expression of UCP1 at the protein level (<FIG>; <FIG> and <NUM>) and showed high yield of brown adipocyte conversion in the xeno-free, chemically defined differentiation medium. As expected, UCP1 protein localized in mitochondria as shown by the overlapping signals obtained when co-immunostaining differentiated BADSC for UCP1 and mitochondria (<FIG>).

The results in <FIG> demonstrate that that the <NUM>-step AD-<NUM> differentiation medium (AD-<NUM> DIFF-<NUM> and AD-<NUM> DIFF-<NUM>) promotes a stronger brown adipocyte differentiation compared to AD-<NUM> differentiation medium and the commercial adipogenic media.

Non-naturally occurring <NUM>-dimensional BADSC aggregates or BAGs were formed in non-adherent culture plates, such as AggreWellTM 400Ex <NUM>-well plates (StemCell Technologies, Vancouver, British, Columbia, Canada).

BADSCs were first cultured in 2D using growth media under normoxia or hypoxia until <NUM>% confluency. The non-adherent plates were coated with a rinsing solution, such as AggreWellTM rinsing solution (StemCell Technologies) following manufacturer's instructions. After washing the non-adherent plates with GM, <NUM> of cell suspension containing <NUM> million cells/ml in GM was loaded to each well of the non-adherent plates. The non-adherent plates were then centrifuged at <NUM> for <NUM> minutes using a swinging bucket centrifuge to allow the cells to settle uniformly into the microwells, resulting in a density of <NUM> cells per microwell, thus creating uniform cellular aggregates. Without centrifugation, the non-naturally occurring <NUM>-dimensional brown adipose-derived stem cell aggregates or BAGs will not be uniform.

The BAGs were then cultured in a non-adherent culture plate, such as AggreWellTM 400Ex <NUM>-well plates, at <NUM> in normoxia or hypoxia and <NUM>% humidity for <NUM> hours in GM prior to harvest. Approximately <NUM> BAGs were collected per non-adherent plate by gentle pipetting and resuspended in <NUM>µl of GM.

A differentiation protocol to efficiently differentiate BADSC into functional brown adipocytes in 3D culture within an encapsulation system, such as an encapsulation medical device, has been developed. This method, summarized in <FIG>, consists of <NUM> steps: (<NUM>) forming non-naturally occurring three-dimensional BADSC aggregates (BAGs) in growth medium (≈<NUM>/aggregate) (<FIG>) and loading of BAGs into an encapsulation system, such as an encapsulation medical device (<FIG>); (<NUM>) further differentiating the BAGS into brown adipose tissue (BAT) using the xeno-free, serum free, chemically defined AD-<NUM>-DIFF-<NUM> medium; and (<NUM>) differentiating the BAGs into brown adipose tissue using the xeno-free, serum-free, chemically defined AD-<NUM>-DIFF-<NUM> medium (<FIG>).

In step <NUM>: BAGs were formed in AggreWellTM 400Ex <NUM>-well plates (StemCell Technologies) using BADSC population BF-<NUM>. The optimal cell plating density, in order to generate uniform BAGs, was determined to be <NUM> cells per microwell. BAGs were subsequently loaded into the encapsulation system, such as an encapsulation medical device. The BAGs suspension was loaded into an encapsulation device such as one Encaptra® EN20 (ViaCyte) encapsulation device, using a Sureflo® <NUM> 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 for <NUM> hours in <NUM> of GM in a <NUM> tissue culture dish. At that point, the BAGs merged and filled the entire volume of the encapsulation device. The resulting BAGs were highly uniform in size and shape, and uniform within and between experiments. Size can be easily modified by adjusting the cell seeding concentration formed in AggreWell™ 400Ex <NUM>-well plates (StemCell Technologies). The optimal cell plating density in order to generate uniform BAGs was determined to be <NUM> cells per microwell.

In step <NUM>: the BAGs within the encapsulation medical device were differentiated for <NUM> days in vitro in a first differentiation medium called AD-<NUM> DIFF-<NUM> medium.

In step <NUM>: the BAGs within the encapsulation medical device were further differentiated for <NUM> days in vitro in a second differentiation medium called AD-<NUM> DIFF-<NUM> medium.

Immunocytochemistry analysis showed that the non-naturally occurring brown adipose-derived stem cell aggregates comprising the BADSC population BF-<NUM> efficiently differentiated into brown adipocytes in 3D inside the Encaptra® encapsulation medical device. BADSC BF-<NUM> cells differenting in the encapsulation medical device formed a tissue-like structure visualized by hematoxylin and eosin staining highly enriched in brown adipocytes (UCP1 positive and Perilipin positive) containing high contents of mitochondria (<FIG>). These cells express high levels of adipocyte markers such as FABP4, adipsin, PPARg, CEBPa and leptin (<FIG>) and brown specific markers such as UCP1, PGC1a, CIDEA, ELOVL3, and COX10 (<FIG>) as compared to undifferentiated BAGs.

In conclusion, it was shown that non-naturally occurring BADSC aggregates represent a very promising source of transplantable brown adipose tissue to increase energy expenditure and to potentially treat metabolic disorders, endocrine disorders, cardiovascular disorders, and liver diseases. Moreover, the strategy to use encapsulation to deliver the non-naturally occurring BADSC aggregates represents a safe delivery system and will help accelerate the development of BAT therapies for human applications.

Male SCID-beige mice (C. B-Igh-1b/GbmsTac-Prkdcscid-LystbgN7), <NUM> weeks old (Taconic Biosciences) were singly housed at <NUM> and fed a high fat diet (HFT) comprising <NUM>% fat (D12492, <NUM> kcal% fat [primarily lard], <NUM> kcal% carbohydrate). These mice have metabolic syndrome and cannot process glucose.

An encapsulation system was prepared by adding <NUM> x <NUM><NUM> brown adipose derived stem cells (BADSCs) in <NUM> of <NUM>/mL matrigel (Corning® Matrigel® Matrix High Concentration (HC), Phenol-Red Free *LDEV-Free). The BADSCs were 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 were uniform and added to the matrigel.

The encapsulation system (<NUM>) was added to <NUM> wells of a <NUM>-well plate (<NUM>µL per well). After <NUM> hour of gelation, the encapsulation system became solid disks in the culture well. Growth media was added to the wells for <NUM> hours. The growth media was removed from the wells and AD-<NUM> DIFF-<NUM> was then added to the wells for <NUM> hours. The AD-<NUM> DIFF-<NUM> was removed from the wells and AD-<NUM> DIFF-<NUM> was then added to the wells for <NUM>-<NUM> days. After several days of culturing/differentiation, the encapsulation system comprising BAT forms a spherical shape (i.e. a bead). The beads contracted in size and reduced to <NUM>-<NUM>µl after the in vitro differentiation.

Forty beads were collected using a cell strainer. Forty beads comprise ~<NUM> x <NUM><NUM> total cells that make up the encapsulated BAT. The beads were transferred into a <NUM> conical vial, and placed on ice. <NUM>µl of cold <NUM>/mL matrigel was added to the beads, mixed well, and kept on ice.

A small skin incision (~<NUM>) was made near in the Interscapular brown fat pads of twenty-two (<NUM>) SCID-beige mice. If additional space was needed, then dorsal subQ sites were used. A spatula was used to lift the skin off the underneath white fat layer. The <NUM> beads in matrigel were delivered to the incision site in <NUM> of the <NUM> mice (<FIG>, Treatment group) using a modified <NUM> micropipette tip and the incision was sutured. Matrigel alone was delivered to the incision site in the other <NUM> mice (<FIG>, Control group) using a modified <NUM> micropipette tip and the incision was sutured.

Mice from the treatment group and mice from the control group were analyzed weekly to determine their ability to absorb glucose via a Glucose Tolerance Test (GTT). Prior to the analysis, these mice were fasted for <NUM> hours. After <NUM> hours, the mice were given an
intraperitoneal (IP) injection of glucose (<NUM>/g of body weight) and the amount of glucose that was absorbed was measured using a blood sample at <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> minutes post glucose injection.

<FIG> show that mice transplanted with BAT (treatment group) were better able to absorb glucose over the course of <NUM> minutes at <NUM>-weeks post-treatment (<NUM>-weeks post time induction of obesity) as compared to mice that were not transplanted with BAT (control group).

Claim 1:
A method of making a non-naturally occurring three-dimensional brown adipose derived stem cell aggregate consisting of brown adipose derived stem cells that express one or more brown adipocyte genes in the absence of differentiation medium, the method comprising:
loading brown adipose derived stem cells grown in a two-dimensional (2D) culture into a non-adherent culture plate; and
centrifuging the non-adherent culture plate to uniformly position the brown adipose derived stem cells in 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.