Brown fat cell compositions and methods

230237/2

BROWN FAT CELL COMPOSITIONS AND METHODS

FIELD OF THE INVENTION

[0001] This invention relates to the field of cell culture and more specifically to the culture anduse of stem cells.

RELATED APPLICATIONS

[0002] This application claims priority to and benefit of U.S. Provisional application61/502,508 filed on June 29 2011, U.S. Provisional application 61/632,122 filed on January 18,2012, and U.S. Provisional application 61/632,516 filed on January 25, 2012.

BACKGROUND

[0003] Brown fat is one of two types of adipose tissue found in the human body. Duringembryogenesis, brown fat is derived from the differentiation of the mesoderm. Brown fat isinvolved in development and homeostasis by providing metabolic tissue capable of providingheat. Brown fat helps regulate metabolism and nonshivering thermogenesis. In addition, brownfat plays a larger role than white adipose tissue in regulating metabolism. Brown fat makes up 5percent of the body mass of a human neonate, and less than 1 percent of the body mass of anadult.

[0004] Metabolic activity of brown fat decreases with increased body mass index. Similarly,metabolic activity of brown fat decreases with increased body fat percent.

[0005] Stem cells have been identified and isolated in various tissues, and white adipose tissuestem cells have been isolated, expanded and shown to have functional therapeutic characteristics.To date, no stem cell population population has been identified in brown adipose tissue.

SUMMARY OF THE INVENTION

[0006] Described are methods of developing cell lines, such as stem cell lines, for therapeutic or cosmetic use. For example, the cell lines can be used to treat a wide range of degenerative and metabolic disorders including, but not limited to, obesity, diabetes, hypertension, and cardiac 230237/2 deficiency. Also described are methods of using such cell lines to screen for compounds that play a role in regulating a variety of processes, such as, but not limited to, methylation, homeostasis, and genes involved in the regulation of metabolism, thermogenesis, activation, and/or maintenance of brown and white fat levels in the body.

[0007] In one aspect, the invention features a method of generating a stem cell. In oneembodiment the steps include obtaining brown fat tissue; isolating a stem cell from the brown fattissue; and culturing the stem cell in a medium not comprising an animal product, therebygenerating a stem cell. In another embodiment the brown fat tissue is from a sample obtainedfrom a subject. In another embodiment the stem cell is positive for one or more of the followingcell surface markers: CD63, CD90, HLA ABC, CD105, CD73, CD166, CD 9, CD44. In anotherembodiment the stem cell is negative for one or more of the following cell surface markers:CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD 117. In still yet another embodiment thestem cell is autogeneic. In another embodiment the stem cell is allogeneic. In anotherembodiment the medium includes Low Glucose DMEM without phenol red, 0.5-20% HumanPlatelet Lysate, 1X NEAA, 1X Glutamax, 1X Gentamycin, and 1000 units of Heparin.

[0008] In another aspect, the invention features a method of generating a cellular extract ofstem cells, progenitor cells or differentiated cells such as activated brown fat adipocytes (e.g.,brown fat adipocytes expressing UCP-1 and/or PRDM16) and administrating this extract into fatdeposits within the subject, thereby treating obesity.

[0009] Another aspect of the invention is a method of treating obesity in a subject. In oneembodiment the method includes the steps of removing brown fat from a subject; andadministering the brown fat to a fat deposit within the subject, thereby treating obesity in thesubject. In one embodiment the fat deposit is a white fat deposit. In another embodiment the fatdeposit is a brown fat deposit.

[0010] In another aspect, the invention features a method of identifying a compound involvedin metabolism regulation, brown fat activation and/or maintenance, comprising contacting anautologous stem cell, progenitor cell or differentiated cell produced by a method described hereinwith a test compound; determining the level of metabolic activity, thermogenesis, and/oractivation of certain genes such as PRDM-16 myf-5 UCP-1, CYC1, NDUFA11, NDUFA13, - 2 - 230237/2 CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, CIDEA, Cox8b, Glut4, of the stem cell,progenitor cell, or differentiated cell in the presence of the test compound; and comparing thelevel of metabolic activity, thermogenesis and/or gene activity of the stem cell, progenitor cell ordifferentiated cell in the presence of the test compound to a level of metabolic activity,thermogenesis and/or gene activity of the stem cell, progenitor cell or differentiated cell in theabsence of the test compound, wherein a level of metabolic activity, thermogenesis and/or geneactivity in the presence of the test compound that is different from a level of metabolic activity,thermogenesis and/or gene activity in the absence of the test compound identifies the testcompound as a compound involved in metabolism regulation or brown fat activation,thermogenesis and/or maintenance.

[0011] In yet another aspect the invention relates to a method of treating a metabolic disorderin a subject. In one embodiment the method includes the steps of removing a stem cell from adonor; differentiating the stem cell into a brown adipocyte in a medium not comprising ananimal product; and administering the brown adipocyte to a fat deposit within the subject,thereby treating the metabolic disorder in the subject. In another embodiment the metabolicdisorder is selected from the group consisting of obesity, central obesity, diabetes, hypertension,cardiac deficiency, ischemic cardiac disease, high blood pressure, triglyceride dyslipidemia, HDL dyslipidemia, cholesterol dyslipidemia, elevated fasting plasma glucose, leptindysregulation, and adipon dysregulation. In still another embodiment the brown adipocyte isadministered by subcutaneous injection. In still yet another embodiment the brown adipocyte isadministered by systemic injection. In another embodiment the medium contains adifferentiation agent selected from the group consisting of insulin, dexamethasone,isobutylmethylxanthine, and indomethacin.

[0012] Still yet another aspect of the invention is a method of identifying a compound involvedin metabolism regulation. In one embodiment the method includes the steps of isolating a stemcell from the brown fat tissue; culturing the stem cell in a medium not comprising an animalproduct; contacting the stem cell with a test compound; determining the level of metabolicactivity of the stem cell in the presence of the test compound; and comparing the level ofmetabolic activity of the stem cell in the presence of the test compound to a level of metabolicactivity of the stem cell in the absence of the test compound, wherein a level of metabolic - 3 - 230237/2 activity in the presence of the test compound that is different from a level of metabolic activity inthe absence of the test compound identifies the test compound as a compound involved inmetabolism regulation. In one embodiment the determining step includes measuringadipogenesis. In another embodiment the method further comprises detecting the levels of oneor more of PRDM16, BMP7, UCP1, UCP2, or MYF5. In still yet another embodiment the testcompound is selected from the group consisting of protein, antibody, peptide, mutein,polynucleotide, nucleic acid aptamer, and small molecule.

[0013] An aspect of the invention relates to differentiating a stem cell into a brown fat cell. Inone embodiment the method includes contacting a stem cell with a medium not comprising ananimal product, thereby generating a brown fat stem cell. In another embodiment the mediumincludes an agent selected from the group consisting of: insulin, dexamethasone, isobutylmethylxanthine, indomethacin, T3, rosiglitazone, FNDC5 and combinations thereof. Instill yet another embodiment the medium includes insulin, dexamethasone,isobutylmethylxanthine, and indomethacin. In yet another embodiment the brown fat stem cellexpresses PRDM-16, PGC-1, or a combination thereof. In still yet another embodiment thebrown fat tissue is from a sample obtained from a subject.

BRIEF DESCRIPTION OF DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies ofthis patent or patent application publication with color drawing(s) will be provided by the Officeupon request and payment of the necessary fee. The present teachings described herein will bemore fully understood from the following description of various illustrative embodiments, whenread together with the accompanying drawings. It should be understood that the drawingsdescribed below are for illustration purposes only and are not intended to limit the scope of thepresent teachings in any way.

[0015] FIG. 1 is a schematic diagram depicting exemplary methods of injecting brown fat cellsor brown fat precursor cells into a subject.

[0016] FIG. 2 is a micrograph of a cell culture ready for passaging. - 4 - 230237/2 [0017] FIG. 3 are images showing gene expression by RT-PCR in white adipose depots and brown adipose depots.

[0018] FIGS. 4 A and B are fluorescent micrographs of cells stained with stem cell markers.

[0019] FIGS. 5A-F is a series of graphs showing detection by flow cytometry of various celltype markers.

[0020] FIG. 6 is a micrograph showing presence of fat droplets in cell culture.

[0021] FIGS. 7A and B are micrographs of cells stained with oil red O. FIG. 7A shows largefat droplets (200 pm) and FIG. 7B shows small fat droplets (50 pm).

[0022] FIGS. 8A and B are micrographs of cells stained with markers of differentiation. FIG.8A shows staining with alizarin red, a marker of osteogenesis. FIG. 8B shows staining withosteocalcin antibody and alcian blue, to show chondrogenesis.

[0023] FIG. 9 is a scanning electron micrograph of exosomes isolated from brown fat cells.

[0024] FIG. 10 shows a timecourse of glucose levels and body weight in mice treated withbrown fat progenitor cells.

[0025] FIG. 11 shows a timecourse of triglyceride levels and cholesterol levels in mice treatedwith brown fat progenitor cells.

[0026] FIG. 12 shows a timecourse of leptin levels and adipon levels in mice treated withbrown fat progenitor cells.

DETAILED DESCRIPTION

[0027] The invention is based, at least in part, on the discovery that cells, such as autologous ornonautologous brown fat cells or stem cells, can be isolated and used to modify metabolismand/or thermogenesis in a subject, and/or to treat a wide range of disorders in a subject, such asmetabolic disorders. (Fig. 1) - 5 - 230237/2 [0028] As described herein, brown fat cells and brown fat stem cells can be used therapeutically to treat a number of metabolic disorders. In some embodiments, the metabolicdisorder is selected from central obesity (waist circumference of 40 inches or greater in males or36 inches or greater in females); triglyceride dyslipidemia (1.7 mmol/L (150 mg/dl) or greater);HDL dyslipidemia (less than 40 mg/dl in males, less than 50 mg/dl in females); blood pressure(130/85 mmHg or greater); and elevated fasting plasma glucose (6.1 mmol/L (110 mg/dl) orgreater).

[0029] The cells isolated can be from, for example, an autologous source or an allogeneicsource. However, other sources also can be used.

[0030] Cellular products can be made from these cultured stem cells derived from mediastinalfat depots, such as cellular extracts. These cellular extracts then can be used therapeutically.

[0031] Cells or stem cells described in this application isolated from brown fat depots also canbe used to screen compounds (including naturally occurring compounds) that increase the levelsof brown fat, metabolism, and or gene expression profiles that favor higher metabolic activity.Similarly, cells or stem cells described herein can be used to identify compounds (includingnaturally occurring compounds) that decrease the levels of brown fat, metabolism, and or geneexpression profiles that disfavor higher metabolic activity. In some embodiments, the screeningis small molecule screening.

[0032] As used herein, “treatment” means any manner in which one or more of the symptomsof a disease or disorder are ameliorated or otherwise beneficially altered. As used herein,amelioration of the symptoms of a particular disorder refers to any lessening, whether permanentor temporary, lasting or transient, of the symptoms, which can be attributed to or associated withtreatment by the compositions and methods of the present invention.

[0033] The terms “effective amount” and “effective to treat,” as used herein, refer to an amountor a concentration of one or more of the compositions described herein utilized for a period oftime (including acute or chronic administration and periodic or continuous administration) that iseffective within the context of its administration for causing an intended effect or physiologicaloutcome. - 6 - 230237/2 [0034] As used herein, the term “subject” means an animal, human or non-human, to whomtreatment according to the methods of the present disclosure is provided. Veterinary and non-veterinary applications are contemplated. The term includes, but is not limited to, mammals.Typical subjects include humans, farm animals, and domestic pets such as cats and dogs. Insome embodiments, the subject is a mammal. In some embodiments, the subject is a humansubject, e.g., an obese human subject. In some embodiments, the subject is a non-humanmammal, e.g., an experimental animal, a companion animal, or an animal that is raised for food.

[0035] As used herein, an “isolated” or “purified” cell is a cell substantially free ofcontaminating components from a cell culture or tissue source from which the cell is derived.“Substantially free” means that a preparation of a selected cell has less than about 50%, (e.g.,less than about 40%, 30%, 20%, or 10%) of non-selected components. Such a non-selectedcomponent is also referred to herein as “contaminating component.” When the isolated cells arerecombinantly produced, they can be substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, (e.g., less than about 10% or 5%) of the volume of the cellpreparation.

[0036] As used herein, “stem cells” are cells capable of both self-renewal and differentiationinto many different cell lineages (is pluripotent). “Progenitor cells” refers to a subset of stemcells with phenotypes similar to that of a stem cell. A progenitor cell is capable of self-renewaland is typically multipotent. As used herein, “differentiated cells” refers to a subset of cells withphenotypes of a mature cell type specific to a particular tissue or organ system.

Methods of Obtaining Cells [0037] In general, cells useful in the methods described herein can be obtained from a subject,such as by isolating the cells from a subject, or clinical grade embryonic stem cells can beobtained from a commercial source. In certain embodiments, the cells are part of a tissuesample, such as a brown fat sample, from a subject. The tissue sample can be administereddirectly into a subject or the cells can be isolated from the tissue and processed as describedherein. In other embodiments, the cells are stem cells, such as from a bone marrow biopsy, andcan be processed as described herein. - 1 - 230237/2 [0038] Certain cells that can be used in the methods described herein are unipotent, multipotent, or pluripotent cells and can be of mesodermal origin. In some embodiments, thecells are brown adipose progenitor cells, mature brown fat cells or stem cells. Brown adiposeprogenitor cells, mature brown fat cells or stem cells can be identified by determining thepresence or absence of one or more cell surface expression markers. Exemplary cell surfacemarkers that can be used to identify a brown adipose progenitor cell, mature brown fat cell orstem cell include, but are not limited to, MYF5, UCP-1, PRDM-16, SSEA-4, Sca-1, CD45, Mac-1, CD29 (integrin β1), CD105 (Endoglin), CD166 (ALCAM), desmin, vimentin, and c-kit. Inyet other methods, the cells are brown adipose cells.

[0039] In any of the methods described herein, the cells can be autologous, syngeneic,allogeneic, or xenogeneic.

[0040] Cells suitable for use in the methods described herein can be found in a variety oftissues and organs including, but not limited to, for example, skeletal muscle, cardiac muscle,smooth muscle, prostate, dermis, the cardiovascular system, mammary gland, liver, neonatalskin, calvaria, bone marrow, the intestine, adipose tissue (e.g., white adipose tissue, brownadipose tissue), peripheral blood, mobilized peripheral blood, and umbilical cord. Cells can beisolated from such tissues and organs in a number of known ways, including, e.g., biopsy,apheresis, or liposuction.

[0041] In some embodiments, cells are obtained from adipose tissue, e.g., white or brownadipose deposits. For example, brown adipose tissue can be harvested using known means (e.g.,by biopsy) from specific anatomical regions of a subject, such as cervical-supraclavicularregions, superior mediastinal regions, and regions superficial or lateral to sternocleidomastoidmuscles. Brown adipose tissue can be identified in a number of ways, such as by imaging (e.g.,by PET scan). The brown adipose tissue can be subjected to minimal processing (such as bywashing with buffer, e.g., DPBS, and mechanically separated into portions) and administeredinto a subject as described herein.

[0042] In other methods, cells, such as brown fat cells, mononuclear cells, progenitor cells, orstem cells, can be isolated from contaminating components of the tissue sample. For example,the tissue sample can be treated with an enzyme such as collagenase (e.g., Type I, II, or III), - 8 - 230237/2 dispase, hyaluronidase, or elastase and cells can be isolated by filtration or centrifugation.

Alternatively, the tissue sample can be treated chemically, such as with EDTA, and cells can be isolated using, e.g., mechanical disruption (e.g., vortexing). The isolated cells can be washed with a suitable buffer, such as DPBS.

[0043] In some methods, cells are isolated from a bone marrow sample of a subject. Forexample, a bone marrow sample can be obtained using needle aspiration or other knowntechnique. In certain instances, cells can be isolated from a bone marrow sample using a Ficoll -Hypaq density gradient.

[0044] In yet other methods, cells are isolated from skin of a subject. For example, a punchbiopsy can be used to obtain a skin sample. In one exemplary method, a punch biopsy is used toobtain a 0.5 cm piece of skin, which is washed three times with DPBS, and the dermis isremoved from the biopsy. The skin is then cut into small (about 3 mm) sections and plated ontothe wells of a 6-well tissue culture plate with a sterile cover slip over the tissue and the cellscultured.

Methods of Culturing Cells [0045] In certain methods, cells obtained as described herein are maintained in a suitableculture medium, e.g., a culture medium not comprising an animal-based product (such as bovineserum or calf serum) in order to obtain a larger population of cells. The culture methods caninclude allowing the cells to undergo sufficient rounds of doubling, e.g., to produce either aclonal cell strain or a heterogeneous cell strain of desired size, e.g., a sufficient number toprovide a therapeutic effect to a subject, or a sufficient number to establish a stable cell line. Forexample, cells can be cultured at 37°C for a period of 3-6 weeks after an initial biopsy.

[0046] The culture media used in the methods described herein do not include animal-basedproducts. An exemplary culture medium suitable for the methods described herein can includehuman platelet lysate and an anticoagulant, such as heparin or acid-citrate-dextrose. Anotherexemplary culture medium includes human pooled AB type serum and an anticoagulant. Otherexamples of culture medium can include Modified Eagle Medium (MEM) (such as Dulbecco’sMEM or alpha MEM) supplemented with glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, L-glutamine, and an antibiotic. One exemplary culture - 9 - 230237/2

medium includes: DMEM (low glucose, without phenol red); 0.5-20% human platelet lysate; 1X non-essential amino acids (NEAA); 1X Glutamax; 1X gentamycin, and 1000 units of heparin.

[0047] In some embodiments, the cells can be maintained in the culture medium understandard conditions, such as described in, e.g., Freshney (1994) Culture of Animal Cells, aManual of Basic Technique, third edition, Wiley-Liss, New York.

[0048] In certain embodiments, the cells are cultured in human platelet lysate or human pooledAB type serum derived from subjects who have been exposed to cold temperatures, e.g., atemperature suitable to generate a sympathetic nervous system response in the subject.

[0049] In other embodiments, the cells can be maintained in a hypoxic environment (e.g., 0 toabout 5% O2 at 37°C).

[0050] In certain embodiments, cultured cells can express MYF-5, BMP7, and/or PRDM16,SSEA4. In other embodiments, cells cultured from muscle tissue can express SCA1 and notexpress c-Kit or CD45.

Methods of Differentiating Cells [0051] In certain embodiments, cells obtained as described herein are maintained in a suitabledifferentiation medium, e.g., a medium not comprising an animal-based product. For example,stem cells can be maintained in a differentiation medium for a time sufficient to result in thedifferentiation of the stem cells into brown adipocyte progenitor cells or brown adipocytes. Inparticular embodiments, cells are maintained in a differentiation medium for a time sufficient toresult in the differentiation of the stem cells into MYF5 expressing cells. In certain instances,cells are maintained in the differentiation medium for, e.g., 1-5 days, 5-10 days, 10-14 days, 14-21 days, 21-28 days, or longer.

[0052] In some embodiments, the differentiation medium includes one or more chemical orhormone differentiation inducers and/or thiazolidinediones (as described in, e.g., Klien et al., J.Biol. Chem. 274:34795-34802 (1999); Hauner et al, J. Clin. Invest. 84:1663-1670 (1989)). Insome instances, the differentiation medium includes insulin, dexamethasone, isobutylmethylxanthine, and indomethacin and rosiglitazone. One exemplary differentiation - 10 - 230237/2 medium includes DMEM (low glucose without phenol red); 0.5% - 20% Human Platelet Lysate;1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 10 ug/mL of insulin; 1 uMdexamethasone; 200 uM indomethacin; and 0.5 mM isobutylmethylxanthine. In anotherembodiment, the differentiation medium includes BMP7. In another embodiment, thedifferentiation medium includes DMEM (low glucose without phenol red); 0.5% - 20% HumanPlatelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin;0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nMT3, and 1 uM rosiglitazone. In another embodiment, the differentiation medium includes DMEM(low glucose without phenol red); 0.5% - 20% Human Platelet Lysate; 1X NEAA; 1XGlutamax; 1X gentamycin; 1000 units of heparin;0.5 mM isobutylmethylxanthine, 125 nMindomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, and 1 uM rosiglitazone and 20nM FNDC5.

[0053] In some instances, the cells are maintained in differentiation medium until one or moremarkers of brown adipocyte differentiation are detected. Non-limiting examples of markers ofdifferentiation include the expression of cell death-inducing DFF45-like effector A (CIDEA),Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein(e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A,ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4.

[0054] In some embodiments, the methods include exposing the cells to cold shock byculturing or differentiating the cells during daily cycles of 4 °C - 25C for one hour followed byculture at standard conditions (37 °C, 5% CO2 or hypoxic conditions).

[0055] In some embodiments, the methods include evaluating the level of adipogenesisfollowing maintenance in a differentiation medium. Adipogenesis can be evaluated bymeasuring, e.g., lipid accumulation (e.g., using oil red-o (ORO) staining), cell morphology (e.g.,using visual, e.g., microscopic, inspection of the cells), or cell thermodynamics (e.g., cytochromeoxidase activity, Na+-K+-ATPase enzyme units, or other enzymes involved in brown adipocytethermogenesis). In addition, in some embodiments, functional brown fat adipogenesis followingdifferentiation can be determined by fatty acid uptake assays or oxygen consumption rate.

Further Processing Methods - 11 - 230237/2 [0056] In some embodiments, cells obtained as described herein can be subjected to additionalprocessing before administration into a subject. In certain embodiments, a cell obtained asdescribed herein can be recombinantly modified to express one or more genes, e.g., a geneinvolved in brown adipogenesis. For example, a cell can be transfected with one or more nucleicacids encoding DFF45-like effector A (CIDEA), Type II deiodinaie, PPAR gamma coactivator(PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16,CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b,Glut4 or combinations of the genes, prior to administration into a subject.

[0057] In other embodiments, the cells can be pluripotent stem cells artificially derived (e.g.,differentiated or partially differentiated) from a non-pluripotent cell. For example, skin cells,such as dermal fibroblasts, can be reprogrammed to a pluripotent state by transfection withOCT4, Nanog, or SSEA4. Such cells are known in the art as induced pluripotent stem cells. Inother embodiments the cells can be further manipulated after becoming pluripotent bytransfection with with one or more nucleic acids encoding DFF45-like effector A (CIDEA), TypeII deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g.,UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3,DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4 or combinations of the genes, (ie. PPARG2,CEBPB, PRDM16) [0058] In further embodiments, a cell can be mitotically inactivated prior to administration intoa subject. Without wishing to be bound by theory, it is believed that upon administration to asubject, cells obtained and/or treated as described herein express certain factors that may have aparacrine effect on surrounding cells and tissues. Accordingly, mitotic inactivation preventsfurther cell division while allowing the inactivated cells to maintain the paracrine effect. Incertain embodiments, cells can be treated with chemical or gamma irradiation sufficient tomitotically inactivate the cells.

[0059] In some embodiments, a cell extract or lysate can be prepared from a cell describedherein and administered to a subject. For example, cells can be obtained and cultured, and about106 to about 1010 cells can be collected and used to prepare a cell-free extract using knownmethods. In one exemplary method, an extract is prepared by subjecting the collected cells to - 12 - 230237/2 freeze-thaw cycles (e.g., 1, 2, 3, 4, 5, or more freeze-thaw cycles) using an ethanol/dry ice bath.

The extract is then centrifuged (e.g., at about 14,000 rpm) to remove insoluble material. The extract can then be administered to a subject.

Expression Methods [0060] In certain embodiments, a cell described herein can be recombinantly modified toexpress one or more genes. Such nucleic acids can be incorporated into an expression vector.Expression vectors comprising a nucleic acid sequence described herein can be administered inany effective carrier, e.g., any formulation or composition capable of effectively delivering thecomponent gene to cells in vivo. Approaches include insertion of the gene in viral vectors,including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, poxvirus,alphavirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viralvectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, forexample, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated),polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers,as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo.

[0061] In some embodiments, the expression vector is a viral vector containing one or morenucleic acid sequences (e.g., cDNA). Retrovirus vectors can be used as a recombinant genedelivery system for the transfer of exogenous genes in vivo, particularly into humans as is knownto one skilled in the art. ( Protocols for producing recombinant retroviruses and for infectingcells in vitro or in vivo with such viruses can be found in Ausubel, et al., eds., Current Protocolsin Molecular Biology, Greene Publishing Associates (1989), Sections 9.10-9.14, and otherstandard laboratory manuals. Another viral gene delivery system useful in the present methodsutilizes adenovirus-derived. Yet another viral vector system useful for delivery of nucleic acidsis the adeno-associated virus (AAV) as is known to one skilled in the art..

[0062] In addition to viral transfer methods, such as those illustrated above, non-viral methodscan also be employed to express a nucleic acid into a cell described herein. Typically non-viralmethods of gene transfer rely on the normal mechanisms used by mammalian cells for the uptakeand intracellular transport of macromolecules. In some embodiments, non-viral gene deliverysystems can rely on endocytic pathways for the uptake of the subject gene by the targeted cell. - 13 - 230237/2

Exemplary gene delivery systems of this type include liposomal derived systems, poly-cationicconjugates such as polyamine and polylysine, and artificial viral envelopes. Other embodimentsinclude plasmid injection systems as known to one skilled in the art. Other non-viral vectorsinclude a scaffold/matrix attached region (S/MAR)-based vector. In particular embodiments, acell described herein is transfected with an S/MAR-PRDM16 construct, an S/MAR-BMP-7/PRDM16 construct, or an S/MAR-BMP-7 construct. In another embodiment the cell istransfected with an S/MAR construct containing one or more nucleic acids encoding DFF45-likeeffector A (CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta,uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11,NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4 or combinationsof the genes or a combination of them (i.e. PPARG2, CEBPB, PRDM16).

[0063] In some embodiments, a nucleic acid can be expressed using naked DNA constructsand/or DNA vector based constructs as is known to one skilled in the art. In some embodiments,DNA vectors can be introduced into target cells via conventional transformation or transfectiontechniques. As used herein, the terms “transformation” and “transfection” are intended to referto a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into atarget cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene gun, sonoporation, or magnetofection.

[0064] All the molecular biological techniques required to generate an expression constructdescribed herein are standard techniques that will be appreciated by one of skill in the art.

Methods of Administration [0065] Methods described herein can include implanting tissue or cells, e.g., cells obtained orisolated as described herein, into a subject to be treated. The cells can be differentiated brownadipocytes (e.g., isolated brown adipocytes or differentiated adipocytes produced as describedherein), or can be stem cells or undifferentiated cells, which cells, or their progeny (i.e., daughtercells), will differentiate into brown adipocytes after implantation. These methods are useful,e.g., for modifying metabolism in a subject as described herein.

[0066] Prior to administration into a subject, the cells can be washed (e.g., in isotonic PBS) toremove any contaminants, including contaminating components of tissue sample, culture media - 14 - 230237/2 or differentiation media, before implantation. The number of required cells is variable and depends on a variety of factors, including but not limited to, the cell type used, the site of implantation of the cells (for example, the number of cells that can be used can be limited by the anatomical site of implantation), and the age, surface area, and clinical condition of the subject.

In some embodiments, at least about 105, 106, 107, 108, 109, or about 1010 cells are implanted intothe subject.

[0067] Methods for implanting cells within a subject are known in the art, e.g., using a deliverysystem configured to allow the introduction of cells into a subject. In general, the deliverysystem can include a reservoir containing cells and a needle in fluid communication with thereservoir. Such delivery systems are also within the scope of the invention. Generally, suchdelivery systems are maintained in a sterile manner. Various routes of administration andvarious sites (e.g., renal sub capsular, subcutaneous, central nervous system (includingintrathecal), intravascular, intrahepatic, intrasplanchnic, intraperitoneal (including intraomental),intramuscularly implantation) can be used.

[0068] The cells can be in a pharmaceutically acceptable carrier, with or without a scaffold,matrix, or other implantable device to which the cells can attach (examples include carriers madeof, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin,gelatin, and combinations thereof). Initially 1,000,000 cells where seeded onto porousextracellular scaffolds and cultured for 5 days. Differentiation into brown adipose was initiatedby adding DMEM (low glucose without phenol red) containing 10% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, - 15 - 230237/2 1 nM T3, 1 uM rosiglitazone. and the cells were grown in this medium for 2 days followed by further differentiation for anadditional 18 days in media composed of: DMEM (low glucose without phenol red); 10% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin;0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone 20 nM FNDC5.

[0069] Cells implanted using scaffolding were shown under scanning electron microscopy toattach to the scaffold. Further measurements of fatty acid uptake by the cells showed that thecells were metabolically active.

[0070] In particular instances, the cells are implanted into white fat or brown fat in the subject,and/or into or near an anatomical region containing white fat or brown fat.

[0071] Where non-immunologically compatible cells are used (e.g., where non-autologouscells are administered to a subject), an immunosuppressive compound, e.g., a drug or antibody,can be administered to the subject at a dosage sufficient to achieve inhibition of rejection of thecells. Dosage ranges for immunosuppressive drugs are known in the art. Dosage values mayvary according to factors such as the disease state, age, sex, and weight of the individual.

Subjects - 16 - 230237/2 [0072] The methods and compositions described herein are useful for the treatment of metabolic disorders. Generally, the methods include administering an effective amount of cells described herein to a subject in need thereof, including a subject that has been diagnosed to be in need of such treatment. Subjects can includemammals [0073] In some embodiments, the methods include identifying a subject in need of treatment(e.g., a subject having or at risk of developing a metabolic disorder), and administering to thesubject an effective amount of tissue or cells described herein. In certain instances, the subject isdiagnosed as being an overweight or obese subject, e.g., with a body mass index (BMI) of 25-29or 30 or above or a subject with a weight related disorder. A subject in need of treatment withthe methods described herein can be selected based on the subject's body weight or body massindex. In some embodiments, the methods include evaluating the subject for one or more of:weight, adipose tissue stores, adipose tissue morphology, insulin levels, insulin metabolism,glucose levels, thermogenic capacity, and cold sensitivity. In some embodiments, subjectselection can include assessing the amount or activity of brown adipose tissue in the subject andrecording these observations.

[0074] The evaluation can be performed before, during, and/or after the administration of thecells described herein. For example, the evaluation can be performed at least 1 day, 2 days, 4, 7,14, 21, 30 or more days before and/or after the administration of cells described herein.

Metabolism and Metabolic Disorders [0075] Metabolism is a cascade of chemical reactions that regulate the mechanisms by whichliving organisms regulate, maintain and respond to intrinsic and extrinsic factors that affect theirability to maintain, grow, and reproduce. An imbalance of this metabolic activity can lead todownstream cellular events that can give rise to a number of degenerative disorders. Suchdisorders can bring about not only symptoms of a wide range of diseases, but can interfere withproper methylation of the imprinting pattern of male and female germ cells, thus establishing agenetic predisposition in the new generation to degenerative disorders.

[0076] In certain embodiments, the tissue or cells described herein are administered to asubject to modify, e.g., increase, metabolic activity in the subject. In certain embodiments, the - 17 - 230237/2 subject has a disorder such as, but not limited to, obesity, osteoarthritis, hypertension, diabetes, an auto-immune disorder, stroke, kidney failure, neoplasia, or a cardiac deficiency such as ischemic heart disease. In particular instances, the administration of cells described herein treat the disorder.

[0077] In other embodiments, subjects can be screened for levels of adipogenesis markers,such as DFF45-like effector A (CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4,and/or BMP7, and the information used as a diagnostic tool for obesity, hypertension, diabetes orischemic cardiac disease. For example, an adipose tissue sample or a blood sample can beobtained from a subject and the level of DFF45-like effector A (CIDEA), Type II deiodinaie,PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1),PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2,LHX8, COX8A, CYFIP2, Cox8b, Glut4, and/or BMP7 can be measured in the sample. A levelof DFF45-like effector A (CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4,and/or BMP7 below a predetermined level indicates the subject has, or is at risk of developing, ametabolic disorder, such as obesity, hypertension, diabetes or ischemic cardiac disease. Thepredetermined level can be the level of a marker in a corresponding sample from a subject nothaving the metabolic disorder. In yet other embodiments, the levels of brown fat and white fat ina subject can be measured, e.g., using PET scan, and a ratio of brown fat to white fat can bedetermined. A ratio of brown fat to white fat less than a predetermined level can indicate thesubject has, or is at risk of developing, a metabolic disorder, such as obesity, hypertension,diabetes or ischemic cardiac disease.

Methods of Screening for Adipogenesis Compounds [0078] Cells isolated or cultured as described herein can be used to screen for compounds thatmodify, e.g., increase or reduce, metabolic activity. In some embodiments, a cell, e.g., a brownadipocyte progenitor cell, differentiated cell or stem cell, can be contacted with a test compoundand the level of a marker for adipogenesis, e.g., a marker described herein (e.g., PRDM16, - 18 - 230237/2 BMP7, UCP1 or MYF5) can be measured. A test compound that increases the level of a markerfor adipogenesis relative to a cell not contacted with the test compound is identified as acompound that increases metabolic activity, whereas a test compound that reduces a marker foradipogenesis is identified as a compound that decreases metabolic activity. In anotherembodiment the cell is a white adipocyte and it is transfected with a construct expressing areporter gene (i.e. GFP, luciferase) under the control of DFF45-like effector A (CIDEA), Type IIdeiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g.,UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3,DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4 or a combination of them. A test compound thatincreases the level of a marker for adipogenesis relative to a cell not contacted with the testcompound is identified as a compound that increases metabolic activity, whereas a testcompound that reduces a marker for adipogenesis is identified as a compound that decreasesmetabolic activity [0079] In one exemplary method, a cell described herein is transfected with an expressionvector that includes a nucleic acid encoding DFF45-like effector A (CIDEA), Type II deiodinaie,PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1),PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2,LHX8, COX8A, CYFIP2, Cox8b, Glut4, BMP7, MYF5 or a combination of them, under thecontrol of an inducible promoter. Nonlimiting examples of such promoters include chemically-regulated promoters (i.e tetracycline, steroids and metals), or temperature specific promoters thatwill activate and promote expression at a permissive temperature, such as below normal bodytemperature of a subject.

[0080] In one example, a temperature specific promoter is placed in a viral or non viral vectorupstream of a nucleic acid encoding DFF45-like effector A (CIDEA), Type II deiodinaie, PPARgamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, orCIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A,CYFIP2, Cox8b, Glut4 or a combination of them, MYF5 and/or BMP7. A cell isolated fromadipose tissue (white or brown), bone marrow, skin, muscle, or umbilical cord is transfected withthe vector. The cell is then cultured under the permissive temperature (which allows gene - 19 - 230237/2 expression from the promoter) in the presence or absence of a test compound, and the effect of the test compound on adipogenesis is determined.

[0081] Test compounds include, e.g., proteins (including antibodies), muteins, polynucleotides,nucleic acid aptamers, hormones (i.e Irisin) and peptide and nonpeptide small organic molecules.Test compounds can be isolated from natural sources, prepared synthetically or recombinantly,or any combination of the same. Particular, nonlimiting examples of test compounds include 2,4-dinitrophenol, Ephedrine, Sibutramine, FGF21, Bile acids, and Beta-3 adrenergic receptoragonists.

[0082] The materials, methods, and examples are illustrative only and not intended to belimiting. Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this inventionbelongs. Although methods and materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, suitable methods and materials aredescribed herein.

[0083] The disclosure is further illustrated by the following examples. The examples areprovided for illustrative purposes only. They are not to be construed as limiting the scope orcontent of the disclosure in any way.

EXAMPLES

Example 1 - Brown fat isolation processing [0084] In one example, a patient first underwent a 18F-fluorodeoxyglucose (18F-FDG) PET-CTscan to identify brown fat deposits. Prior to undergoing the PET-CT scan, the patient wasexposed to temperatures ranging from 1°C -25°C (to increase the uptake of F-FDG) for 1-2hours per day for a period of one month. Deposits identified within the cervical-supraclavicularand mediastinal area were biopsied using a needle. Brown fat biopsies were washed three timesin DPBS (-) and either treated with 0.075%-0.2% Collagenase Type IA with vigorous shaking at37°C for 30-60 minutes or treated with EDTA for 30 minutes and subjected mechanical tissuedisruption for 5-30 minutes. The tissue/cells were washed three times with DPBS (-), and 1 x106 - 1 x 1010 cells were injected into white fat deposits in the patient. Optionally, following - 20 - 230237/2 enzymatic or mechanical tissue/cell isolation, the tissue/cells were exposed to cold temperature (0°C - 4°C) for 1-48 hours prior to injection.

Example 2 - Brown fat culture [0085] In this example, cells were cultured prior to injection into a patient. The cells obtainedfrom Example 1 were plated into hyper flasks at a concentration of 1000 cells/cm2 in a culturemedium comprising in this embodiment: DMEM Low Glucose without phenol red0.5-20% Human Platelet Lysate

1X NEAA 1X Glutamax 1X Gentamycin1000 units of Heparin [0086] The cells were cultured for 3-6 weeks. The cells were then characterized by flow-cytometry and analyzed for the expression of one or more of the following markers: SCA-1,CD34, SSEA1, SSEA4, OCT4, CD31, Wnt5a, Telomerase activity, alpha-SMA, STRO-1,MYF5, PRDM16, or UCP1. The cells were then expanded to concentrations of 1 x 106 - 1 x1010 for implantation into white fat deposits of the patient.

Example 3 - Differentiation of cells into brown fat cells [0087] In this example, cells (such as stem cells or precursor brown adipocytes) obtained froma brown fat deposit of a patient were differentiated and subsequently injected into a patient. Thecells obtained from Example 1 were plated out at 1000 cells/cm2 in hyper flasks containing adifferentiation medium comprising, in this embodiment: DMEM Low Glucose with out phenol red0.5 - 20% Human Platelet Lysate

1X NEAA 1X Glutamax - 21 - 230237/2 1X Gentamycin 1000 units of Heparin10 ug/mL of insulin 1 uM Dexamethasone 200 uM Indomethacin 0.5mM Isobutylmethylxanthine

In an alternative embodiment the differentiation medium included:DMEM (low glucose without phenol red) 0.5% - 20% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone. and the cells were grown in this medium for 2-6 days followed by furtherdifferentiation for an additional 6-21 days in media composed of: DMEM (low glucose without phenol red); 0.5% - 20% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; - 22 - 230237/2 1000 units of heparin;0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone 20 nM FNDC5.

[0088] After being maintained in the differentiation medium for 10-30 days, cells werecharacterized by expression of DFF45-like effector A (CIDEA), Type II deiodinaie, PPARgamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, orCIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A,CYFIP2, Cox8b, Glut4. 1 x 106 - 1 x 1010 of the cells are then prepared for injection into thepatient.

Example 4 - Culturing and differentiating embryonic stem cells [0089] In this example clinical grade embryonic stem cells were first grown and expanded.

The cells were then differentiated into brown fat pre-adipocytes or fully differentiated brown fatadipocytes by exposing the cells, in one embodiment, to the following media: DMEM Low Glucose with out phenol red0.5 - 20% Human Platelet Lysate

1X NEAA 1X Glutamax 1X Gentamycin1000 units of Heparin10 ug/mL of insulin 1 uM Dexamethasone 200 uM Indomethacin - 23 - 230237/2 0.5 mM Isobutylmethylxanthine [0090] In another embodiment, the cells were grown for 2-6 days in a mediumcomprising: DMEM (low glucose without phenol red); 0.5% - 20% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone.

[0091] This is followed by further differentiation for an additional 6-21 days inmedia composed of: DMEM (low glucose without phenol red); 0.5% - 20% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mM isobutylmethylxanthine, - 24 - 230237/2 125 nM indomethacin, 5 uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone, 20 nM FNDC5.

[0092] Optionally, the cells were transfected, prior to differentiation or after differentiation,with a non-viral vector (scaffold/matrix attached region (S/MAR)) with a PRDM-16 gene byitself or in tandem with BMP-7 or MYF-5 or DFF45-like effector A (CIDEA), Type IIdeiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g.,UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3,DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4 or a combination of them driven by a strongpromoter such as CAGGS or PGK. The cells were then inactivated with a chemical basedmethod such as mitomycin C or gamma irradiation and implanted into a patient at concentrationsof 1 x 106 - 1 x 1010 cells.

Example 5 - Brown fat human depot biopsy 18 [0093] In this example a patient underwent F-PET-CT scan to identify tissue that hasmetabolic activity. An area with high metabolic activity was identified within the humanmediastinum region of the patient. In addition, this patient was to undergo routine cardiothoracicsurgery. At the time of surgery the region was exposed, and within this region 5 x 5 cm brownfat depot was dissected out and placed into sterile saline.

[0094] A brown fat depot was discovered in the mediastinum that contains a population ofstem cells that express unique markers different than those found in other stem cells, includingstem cells isolated from white adipose depots. These newly identified stem cells were separatedfrom the mediastinum brown fat depot and cultured for greater than 20 passages and still retaineda normal karyotype. These cells also are capable of undergoing adipogenesis (white and brown),osteogenesis, and chondrogenesis.

Example 6 - Isolation of cells from human brown fat depot biopsy - 25 - 230237/2 [0095] The brown fat human depot isolated in Example 5 was washed 5 -10 times in sterile saline. The biopsy was cut using scissors until pieces are approximately 0.1 - 0.5 cm in size.

The material was washed 3 times by centrifuging at 1200 RPM for 5 minutes. The material wasthen digested using either collagenase or dispase for no longer than an hour in a shaking waterbath set at 37C. After digestion was complete the enzymatic reaction was terminated by addingequal volume of complete media. The material was then filtered using a 100 uM filter and thesingle cell suspension was washed 3 times with complete medium.

Example 7 - Culture of cells isolated from brown fat human depot biopsy [0096] The single cell suspension described in Example 6 was plated onto cell culture flasks ata concentration of 5,000 - 10,000 cells/cm2. After approximately 3-6 days the cells were readyfor passaging (Fig. 2). The cells after undergoing more than 10 passages showed a normalkaryotype. RT-PCR analysis of the cultured brown fat cells demonstrated that they were positivefor CEBPB, UCP-1, UCP-2, PPARG, PGC-1, PRDM-16. It is important to note that cellsisolated from white adipose depots were negative for PRDM-16, and PGC-1. (Fig. 3).Accordingly, PRDM-16 and/or PGC-1 expression can be used to differentiate between whiteadipose cells and brown adipose cells. Moreover, CEBPB, UCP-1, UCP-2, PPARG, PGC-1,PRDM-16 can be used as markers to identify brown adipose cells.

[0097] Staining of the cells showed that they are positive for the stem cell markers HCAM andCD90 (Figs. 4 A and B). The expanded cells also expressed exosomes (Fig. 9). Flow cytometryof the cell population demonstrated that the cells were negative for the following cell surfacemarkers: CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD 117. The cell population wasalso low for SSEA4, and Stro-1. The cells were positive for CD63, CD90, HLA ABC, CD105,CD73, CD166, CD 9, CD44 (Figs. 5A-F) which are markers for undifferentiated brown adiposestem cells.

Example 8 - Differentiation of cells into Adipose (white and brown) [0098] Expanded cells were plated onto cell culture plates and supplemented with adipogenicdifferentiation medium (Life Technologies). The media was changed every 3 days anddifferentiation was allowed to continue for 14 days. After 7 days in induction media, fat droplets - 26 - 230237/2 were visible within the culture (Fig. 6), indicating that the cells had begun differentiating into adipose cells.

[0099] Staining with oil red O demonstrated positive differentiation into adipose cells (Fig. 7).

[0100] Identification of positive brown fat droplets in the differentiation was observed by theaccumulation of smaller fat droplets (Fig. 7B) compared to those found in cells differentiatedfrom cells derived from white fat depots, which produce larger fat droplets (Fig. 7A).

In another embodiment stem cells were plated in 6-well dishes at a density of 50,000cells/well. White or brown adipogenesis differentiation medium was added. For brownadipogenesis, FNDC5 was added 6 days post induction as previously described. 0.3% Oil Red O(Sigma Aldrich) was used for staining to detect intracellular lipid accumulation.

Example 9 - Chondrogenic and Osteogenic Induction of cells [0101] Expanded cells were plated onto cell culture plates and supplemented with osteogenicand chondrogenic medium (Life Technologies). The media was changed every 3 days anddifferentiation was allowed to continue for 21 days. Confirmation of differentiation intochondroblasts and osteoblasts was observed by staining of the cells with alizarian red orosteocalcin antibody and alcian blue (Figs. 8 A and B). These data indicate that progenitor cellsisolated from brown fat depots maintain the ability to differentiate in to a variety of cell typesother than white adipose or brown adipose.

Example 10 - Therapeutic use of Brown Fat Progenitor Cells [0102] NOD-SCID mice are severely immune deficient and are used as a model system to testhuman cell-based therapies. Mice were raised on high fat/high carbohydrate chow throughoutthe test period. Mice were split into two groups: 10 untreated mice served as controls and 10mice received a single subcutaneous injection underneath the dorsal skin of about 1 millionhuman brown fat progenitor cells cultured as described in examples 5-9. The following analyteswere measured for four months following injection: plasma glucose, triglycerides, cholesterol,leptin, and adipon. Body weight also was monitored. Baseline is 23 +/-5 g in the control group - 27 - 230237/2 and 25+/-7 g in the treated group. The control group received a sham injection of carrier solution (DPBS) with no cells.

[0103] Compared to the control group that received a sham injection of carrier solution only(DPBS) with no cells, the experimental cohort which received a dose of 1,000,000 cellsdemonstrated a decrease in plasma glucose, triglycerides, cholesterol, and weight gain over afour month monitoring period. Adiponectin and leptin levels also correlated to improved levelsdue to the having received the cell dose. This is in sharp contrast compared to the untreatedcontrol mice that received the sham carrier solution (Figs. 10-12).

EQUIVALENTS

[0104] It is to be understood that while the disclosure has been described in conjunction withthe detailed description thereof, the foregoing description is intended to illustrate and not limitthe scope of the invention, which is defined by the scope of the appended claims. Other aspects,advantages, and modifications are within the scope of the following claims. - 28 -