Preparation of browning and control media

The control media was prepared by adding 10% fetal bovine serum (FBS), 1% Penicillin Streptomycin, 20 mM HEPES, 50 μg mL−1 sodium ascorbate, 1 μM insulin into Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMAX Supplement, pyruvate; 10569010, ThermoFisher). The browning media was prepared by adding 1 μM Dexamethasone, 500 μM Isobutylmethylxanthine, 50 μM Indomethacin, 1 μM Rosiglitazone and 1 μM CL316243, and 250 nM triiodothyronine (T3), and 25 ng mL−1 VEGF into control media.

Mouse WAT harvesting, processing, and culture (native inguinal WAT) for autologous re-implant-ation experiments (Figs 2 and 3)

All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee (IACUC) and all experiments were performed in accordance with relevant guidelines/regulations. The mice used in this study were the C57BL/6 strain (Taconic) and were maintained on a high fat diet (Research Diets) starting at 6 weeks of age. Mice were 22 weeks of age at the time of the initial surgery to extract WAT. During surgical procedures, mice were anesthetized with isoflurane and maintained on a warm water circulating pad to maintain temperature. To extract inguinal WAT, a small 1 cm incision was made on the midline of posterior dorsum adjacent to the inguinal depot. The inguinal WAT depots extend from the dorsolumbar region to the gluteal region of the mouse and are anatomically distinct from surrounding tissue and muscle63. Skin was gently lifted and retracted and a portion of the inguinal WAT depot (approximately 0.3–0.5 grams) was lifted with forceps and excised using a scalpel. Tissues were transferred aseptically to tubes containing phosphate buffered saline (PBS) and weighed. The incisions were closed with wound clips and mice were allowed to recover with a pain-relieving drug (buprenorphine, 5 mg/kg) administered for two days.

After briefly rinsing the explanted tissues in PBS, the tissues were transferred into DMEM, gently cut into small pieces (~2–5 mm) using #11 scalpel blades and rinsed 3 times in 30 mL PBS at 37 °C. Tissue culture flasks (75 cm2) were used to incubate the tissues and 12 mL of each type of media was added into each flask. The adipose tissues were incubated in a 37 °C incubator with 5% CO 2 and the culture media were changed every two days for a duration of three weeks. After the culture period, a portion of the live tissue fragments were used for viability and mitochondrial staining and another portion of the tissues were fixed in 4% paraformaldehyde (PFA) for 24 hours and stored in PBS for immunostaining. The rest of cultured tissues (approximately 0.2–0.3 grams) were reimplanted into the same mice where they were initially explanted. 8 weeks after reimplantation, the animals were euthanized by carbon dioxide inhalation, and the tissues were taken out and fixed in 4% PFA for 24 hours and stored in PBS. Implanted tissues were able to be identified by the naked eye (Fig. 3d) as they do not fully integrate, and no translocation was observed.

Native interscapular BAT and inguinal WAT were also excised from euthanized mice to serve as control tissues in staining procedures. The control tissues were not cultured in any media and they were fixed in 4% PFA immediately after explantation for 24 hours and stored in PBS.

Viability and mitochondrial staining

To perform viability and mitochondrial staining, we transferred several tissue fragments per mouse into 96 well plates, and then incubated for 1 hour at 37 °C in a solution of DMEM with Calcein AM (Life Technologies) to label cytoplasm in live cells, NucBlue Live Cell Stain ReadyProbes reagent (Life technologies, Hoechst 33342 Special Formulation) to label nuclei, and MitoTracker Deep Red FM (Life Technologies) to label active mitochondria. Fragments were then rinsed in DPBS, transferred to a glass slide, sandwiched under a coverslip, and then imaged immediately using a Leica DMi6000b widefield fluorescent microscope. For dual live/dead staining, dead nuclei were stained using Ethidium homodimer and all nuclei were stained using Hoescht blue. Cells were imaged using a Leica DMi6000b widefield fluorescent microscope. Manual counting was performed on maximum intensity projection images of z-stacks (10X magnification), using the Leica LAS AF software.

H&E and IHC staining and quantitative analysis

Following dissection or after a specified culture period, tissues were rinsed 3X in PBS and subsequently fixed in 10% neutral buffered formalin overnight at room temperature. Tissues were paraffin embedded, sectioned, and stained by HistoWiz Inc (Brooklyn, NY). To quantify percent UCP1/tissue area and mean lipid droplet area, we used Aperio Imagescope (Leica Biosystems) to acquire representative 10x images from each IHC stain for WAT and BAT inguinal tissue, and processed those images through automated segmentation and measurement of DAB positive areas using an ImageJ macro (available upon request).

Whole mount staining of adipose tissue fragments

Following dissection or after a specified culture period, tissue fragments were fixed in fresh 4% paraformaldehyde solution in phosphate buffered saline (PBS) overnight, then transferred to PBS. The fixed whole mount adipose tissues were stained with anti-UCP1 antibody produced in rabbit (U6382, Sigma-Aldrich), based on the protocol described by Xue et al.64, which was optimized for the best result in our study (see Supp. Experimental Procedures for details).

Fluorescent imaging and quantitative analysis

Fluorescent images were acquired using Leica TCS SP5 confocal microscopes using LAS AF software (Leica Microsystems). We maintained consistent exposure settings across different samples and acquired wide-area z-stacks across tissue fragments using an automated stage (Supplementary Videos 1–4). To quantify UCP1 intensity for individual samples, we calculated the mean UCP1 intensity across all z-slices. Multiple tissue fragments were quantified for each condition, and controls without primary antibody were used to normalize for background fluorescence. To quantify UCP1 and lipid volume fraction for individual samples, we binarized each image in the z-stack with Otsu’s method while taking into account the slightly dimmer signal in the deeper slices using a custom-written MATLAB script (available upon request). We visually checked this binarization for each image in all the z-stacks (Supplementary Fig. 1). Volume fractions were then calculated from the binarized z-stacks, the top-half and bottom-half volumes being comparable within each z-stack.

Allogeneic injection of adipose tissue fragments

All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee (IACUC) and all experiments were performed in accordance with relevant guidelines/regulations. The mice used in this study were the C57BL/6 strain (Taconic) and were maintained on a high fat diet (Research Diets) starting at 6 weeks of age. Donor mice (12 weeks old) were sacrificed using CO 2 asphyxiation, and visceral epididymal fat pads were dissected. Both fat pads were harvested from each donor mouse; one for culture in control media and one for culture in browning media. Fat pads were placed in DMEM and rinsed 3x with PBS. Each fat pad was gently broken up into 2–5 mm pieces using forceps to mimic the size of fragments obtained by traditional fat harvesting techniques, which cannot be easily performed on small reservoirs of fat in mice58. The pieces were then passed through a sterile 19-gauge needle to further break up the pieces. Fat was cultured for three weeks using the same media as described previously with media changes once every 2 days. After 3 weeks, fat was rinsed 3x in PBS and ~0.2 mL minced fat was loaded into a sterile 19-gauge needle and subcutaneously injected above the right rear hindlimb. Mice were allowed to recover for 1 week, and were monitored for any adverse reactions.

Metabolic phenotyping (indirect calorimetry and MRI)

Following the injection, mice were transferred to the mouse metabolic phenotyping core facility at Columbia University and allowed to acclimate for 1 week. Mice were kept un a high-fat diet (Research Diets) beginning at 6 weeks of age and maintained on this diet throughout the duration of the experiment. Food and cages were changed weekly and body weights were measured weekly.

Comprehensive Lab Animal Monitoring System (Oxymax/CLAMS, Columbus Instruments) was used for metabolic phenotyping at 1, 11, and 15 weeks post-implantation (see Supp. Experimental Procedures for details).

We used EchoMRI-100H (EchoMRI™; Echo Medical Systems) to measure percent fat mass of the mice (see Supp. Experimental Procedures for details). Measurements were taken before placement in the metabolic chambers, after one week in the chambers at room temperature, and after one week in the chambers at 8 °C. This sequence of MRI measurements was repeated each of the three runs in the metabolic chambers (i.e. 1, 11, and 15 weeks post-implantation).

Human specimens

Human subcutaneous adipose tissue samples were obtained under a protocol approved by the Columbia University Medical Center IRB for research use of de-identified discarded tissue from autologous fat transfer procedures (Columbia University Medical Center Protocol #AAAN7112) and all experiments were performed in accordance with relevant guidelines/regulations. Patients reviewed and signed informed-consent documents describing the use of their discarded adipose tissue and de-identified data for research into browning of adipose tissue. Samples were obtained from the abdominal region using standard fat harvesting procedures, which involved local injection of tumescent anesthesia for 15 min followed by manual harvesting using a 3 mm cannula and 20 cc syringe. Approximately 6 cc of leftover tissues were retrieved within 4 hours of removal, transferred to a biosafety cabinet, rinsed 3 times with 100 mL DPBS, then transferred to culture. The adipose fragments were approximately 1–3 mm in size and did not require further mincing or processing for culture.

Human citrate synthase assay, Western blot, qPCR

Citrate synthase activity assays to assess tissue mitochondrial function were performed as described previously33. Protein lysate (8 μg) was used for the citrate synthase activity assay following the manufacturer’s protocol (Sigma CS070). Tissue samples were homogenized in tissue lysis buffer provided with the kits to obtain protein lysates. After centrifugation and removal of the lipid layer, protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific Inc.).

For gene expression assays, total RNA was extracted using Trizol (Invitrogen), treated with DNase (ThermoScientific) and reverse transcribed to cDNA (AppliedBiosystems) according to manufacturer’s instructions. Gene expression, normalized to 36B4, was analyzed by quantitative real-time RT-PCR (Sybr Green, 384-well plates) using the QuantStudio 6 PCR System (ThermoFisher). Primer sequences are available upon request.

To perform immunoblot analysis, homogenized tissue was lysed in protein lysis buffer (Sigma) containing protease and phosphatase inhibitors. Standard western blotting was performed using rabbit polyclonal antibodies to Ucp1 (1:500; Abcam; ab23841) and GAPDH (1:3000; CellSignaling; #2118). HRP linked Goat anti-Rabbit (1:3000; Biorad; #170–6515) was used as secondary antibody. Proteins were detected by chemiluminescence (Roche) and images were acquired using FusionFx (Peqlab).

Statistical analysis

One and two-way ANOVA tests and Bonferroni/Tukey post hoc tests were performed using Graphpad Prism 7 software. Student’s t-test was performed using Graphpad Prism 7 software.