A direct tissue-grafting approach to increasing endogenous brown fat

Concept of exBAT

The three steps of exBAT are: 1) harvesting of host WAT; 2) exposure of WAT fragments to browning factors via single-step culture; and 3) re-implantation of converted BAT within subcutaneous WAT (Fig. 1). While only small amounts of BAT can have a significant impact on metabolism48, large amounts of viable WAT can be obtained by plastic surgeons using well-established harvesting techniques (i.e. liposuction)49,50. Moreover, compared to traditional processes that involve sorting and purification of isolated progenitor cells followed by long periods of cell-culture expansion, the exBAT procedure is quick, because a single browning step acts on whole tissue fragments to convert WAT to BAT mass, which is ready for direct implantation.

Figure 1
figure1

Concept of tissue-engineering therapeutic approach to increase endogeneous brown fat via a single-step ex vivo browning method. Illustration of 3-step process for increasing brown adipose tissue (BAT) in humans through ex vivo browning: (1) subcutaneous white adipose tissue (WAT) is harvested by liposuction or excision and cultured as tissue fragments; (2) WAT fragments are exposed to chronic browning stimuli (i.e. browning factors in the media) to convert the WAT to BAT, in a process that takes approximately 1 to 3 weeks; (3) the converted BAT fragments are autologously reimplanted within WAT.

Development and characterization of ex vivo browning process in mice

We first developed a mouse model to test whether whole WAT fragments could be converted to BAT ex vivo (Fig. 2a). We first excised a small piece (~0.5 mL) of subcutaneous WAT from the left inguinal depot, located along the rear flank of the mouse above the hindlimb. Next, we gently minced the WAT tissue into fragments of approximately 2 to 5 mm in diameter to mimic the size of fragments obtained during fat harvesting procedures in humans and suspended the fragments in either browning media or control media (i.e. basal media without browning factors). Working with whole pieces of tissue is simpler than working with individual cell populations such as adipocyte progenitors, which are frequently isolated from adipose tissue depots and require expansion in 2D culture9,16. We used a cocktail of rosiglitazone (PPARγ agonist), isobutylmethylxanthene (IBMX, phosphodiesterase inhibitor), T3 (thyroid hormone), indomethacin (COX inhibitor), CL316,243 (β3 adrenoreceptor agonist), and vascular endothelial growth factor (VEGF) (see Methods for details). This single-step cocktail was found to induce browning over a duration of 1–3 weeks ex-vivo while maintaining cell viability, compared to multi-step induction which was previously performed for browning of adipocyte progenitors51,52.

Figure 2
figure2

Single-step browning of mouse WAT tissue ex vivo. (a) Illustration of experimental design for studies of ex vivo browning and autologous transplantation in mice: (1) subcutaneous WAT from the left inguinal depot is excised from anesthetized mouse; (2) WAT fragments are gently minced into 2–5-mm fragments; (3) WAT fragments are cultured in media for 1–3 weeks in control or browning media; 4) fragments are removed from media and washed with PBS; (5) fragments are re-implanted subcutaneously adjacent to the right inguinal WAT depot. (b) Live-cell and mitochondrial staining of inguinal WAT fragments both immediately after harvest (left) and one week of culture with browning factors (middle); native interscapular BAT fragments immediately after harvest (right). Epifluorescence images show staining for calcein AM (green, indicates cytoplasm in live cells), Mitotracker (red, indicates active mitochondria in live cells), and Hoescht (blue, indicates nuclei). Scale bars are 150 µm (top row) and 30 µm (middle and bottom rows). (c) Dual live-dead staining after three weeks in culture. Top panel shows representative image of tissues cultured in control media and middle panel shows a representative image of tissues cultured in browning media (scale bar 100 is µm for both). Ethidium homodimer (red) labels dead nuclei and Hoescht (blue) labels all nuclei. Bottom panel shows quantification of live/dead ratio for tissues cultured in browning media and control media for 3 weeks. Graphs display Mean +/− SEM; n = 7 for browning media, n = 11 for control media (each sample came from independent culture experiment); p = 0.9364 using Student’s t-test. (d) H&E staining and UCP1 immunohistochemistry of inguinal WAT fragments cultured in control or browning media for 10 days. Scale bars are 50 µm. Quantification of percent UCP1 per tissue area, quantified by automated analysis of DAB positive areas in images of 10-micron sections (p < 0.001, Student’s t-test), is shown on the right. Error bars show SEM (n = 10).

We performed live-cell staining on whole tissue fragments cultured in the presence of browning media (Fig. 2b,c). Initially, the WAT fragments displayed cytoplasmic and mitochondrial staining around large lipid droplets, as well as in branching vascular structures (Fig. 2b, left panel). After one week in browning media, tissues displayed higher cell density with smaller and increased number of lipid droplets, consistent with formation of BAT-like tissue (Fig. 2b, middle and right panels). Assessment of cell viability, which could affect the efficacy of the implant in vivo, showed that viability after 3 weeks of ex vivo browning was comparable to that of control media under the same duration (Fig. 2c).

To further test the extent to which we could achieve ex vivo browning from subcutaneous WAT fragments, we performed immunohistochemistry on samples that had been cultured in both browning and control media for 10 days. Samples cultured in BAT media exhibited smaller lipid droplets and significantly higher UCP1 staining (Fig. 2d, p < 0.001), consistent with what would be expected of native BAT.

Autologous re-implantation into mice and persistence of BAT-like phenotype in vivo

Next, we examined whether the BAT-like properties of tissues cultured in browning media would persist weeks after re-implantation. After 3 weeks of ex vivo culture, we subcutaneously implanted approximately 0.2 mL of autologous converted BAT on the right inguinal WAT depot and extracted the tissue again from the implant site after 8 weeks.

After 8 weeks of implantation, tissue fragments cultured in control media during the ex vivo process retained a WAT-like appearance (Fig. 3a). However, we observed that tissues cultured in browning media during the ex vivo period, after 8 weeks of implantation, continued to exhibit a brown color consistent with BAT which contains a high density of iron-rich mitochondria (Fig. 3a). We performed immunostaining for UCP1 (with counterstaining to label lipid droplets and cell nuclei) on both whole-tissue fragments immediately before re-implantation and on extracted tissue after 8 weeks of implantation. As negative and positive controls, native uncultured inguinal WAT and interscapular BAT tissue fragments were also stained (Fig. 3b, left panels). Tissues cultured in control media prior to re-implantation retained a WAT-like appearance (Fig. 3b, middle panels). By comparison, tissues cultured in browning media exhibited high levels of UCP1 signal, numerous small lipid droplets, and a high density of cell nuclei (Fig. 3b, right panels), both in pre-implant whole-tissue fragments and extracted tissues after 8 weeks of implantation.

Figure 3
figure3

Autologous re-implantation of converted BAT, and analysis after 8 weeks in mice. Experimental design is shown in Fig. 2a. (a) Macroscopic images of native inguinal WAT, native interscapular BAT, and WAT fragments that were cultured for three weeks in browning or control media, imaged before (pre) and after (post) 8 weeks re-implantation. Scale bar is 3 mm. (b) Confocal microscopy of native inguinal WAT, native interscapular BAT, and WAT fragments that were cultured for three weeks in browning or control media, imaged before (pre) and after (post) re-implantation. Scale bars are 50 µm. Images stained for UCP1 expression (red), and counterstained with LipidTox (green) and Sytox nuclear stain (blue). (c) Mean UCP1 intensity (left), UCP1 volume fraction (middle), and lipid volume fraction (right) measurements from 3D confocal images of whole-mount stained tissues after 3 weeks of culture (pre-implant) and 8 weeks after reimplantation (post-implant). Error bars indicate SEM. Compared to control media, UCP1 intensity for fragments cultured in browning media was significantly higher both before (p < 0.0001) and after (p < 0.0001) reimplantation, as determined by one-way ANOVA and Bonferroni post hoc tests. UCP1 volume fraction (middle) and lipid volume fraction (right) were also statistically significant both before (p < 0.0001 and p < 0.0001) and after (p = 0.0012 and p < 0.0001) reimplantation. (d) Image of reimplanted tissue that was cultured for 3 weeks in browning media, 8 weeks following reimplantation. The implanted tissue formed a fat pad that became vascularized (arrowheads) within the surrounding subcutaneous WAT. (e) High magnification confocal images of tissues cultured in both control media (left) and browning media (right), showing channel networks of putative capillaries within explanted tissues. Scale bars are 30 µm. Images stained for UCP1 expression (red) and counterstained with Sytox nuclear stain (blue); greyscale displays transmitted light.

Using 3D confocal imaging and segmentation of image stacks through whole-mount stained tissues (Supplementary Fig. 1 and Videos 14), we quantified UCP1 immunostaining intensity, UCP1 volume fraction, and lipid volume fraction before and after re-implantation (Fig. 3c). Compared to tissues in the control media, tissues cultured in the browning media exhibited significantly higher UCP1 intensity, significantly higher UCP1 volume fraction, and significantly lower lipid volume fraction, both before and after implantation (Fig. 3c). These trends were consistent with phenotypes of the converted BAT in both pre- and post-implant states, and consistent with what we observed for native BAT vs. WAT phenotypes.

Finally, within the whole-tissue fragment, we also observed functional blood vessels as indicated by red blood cell-filled vessels that were visible by eye in the grafted tissue (Fig. 3d). The intact vascular structures within the engrafted converted exBAT were also visible under epifluorescence and brightfield microscopy after 8 weeks of re-implantation (Fig. 3e).

Successful conversion of human subcutaneous WAT into exBAT

We next sought to investigate whether human subcutaneous WAT could also be converted to BAT with our single-step ex vivo browning method. For these studies, we collected excess subcutaneous WAT samples via fat harvesting from the abdominal region of patients who underwent autologous fat grafting procedures (see Materials and Methods for details). Tissues were cultured in the same media and with the same browning factors as previously described for mouse tissues. After 3 weeks of ex vivo culture, we observed that human WAT tissue fragments cultured in browning media developed significant UCP1 expression and smaller lipid droplets (Fig. 4a, top panel, Supplementary Fig. 2d–f). Tissues cultured in control media retained a WAT-like phenotype, similar to native human WAT that was not cultured (Fig. 4a, middle and bottom panels). Immunohistochemistry demonstrated significantly increased UCP1 intensity, UCP1 volume fraction, and significantly decreased lipid volume fraction in exBAT cultured in browning compared to control conditions, comparable with the expression in native tissues (Supplementary Fig. 3). We assessed mRNA expression via qPCR for a variety of genes known to be differentially expressed in native BAT relative to WAT. In the converted exBAT, UCP1 mRNA levels were preferentially upregulated, while DIO2, leptin, and PRDM16 mRNA levels were downregulated (Fig. 4b), concordant with previous reports of human tissues in vitro53. Accordingly, UCP1 protein content (Fig. 4c), and citrate synthase activity (Fig. 4d), a measure of mitochondrial metabolic activity, were significantly increased under browning conditions, indicating that human exBAT possess not only cellular and molecular but also functional characteristics of thermogenically active fat (p < 0.0001).

Figure 4
figure4

Single-step browning of human WAT tissue ex vivo. Experimental design was shown in Fig. 1. (a) Human WAT fragments cultured in browning media (top panel) and control media (middle panel) for 3 weeks and native human WAT (not cultured, bottom panel). Tissue was stained for UCP1 expression (red) and counterstained with LipidTox (green) and Sytox nuclear stain (blue). Scalebars are 50 µm. (b) RNA expression levels in human fragments cultured in control versus browning media. Left to right: UCP1, PRDM16, DIO and LEPTIN. (c) Western blot analysis of UCP1 protein expression in human WAT fragments cultured in control (left) or browning (right) media for 2 weeks. Protein expression is normalized to GAPDH. Compared to control tissues, UCP1 protein expression in tissues cultured in browning media was significantly greater (p < 0.0001). (d) Citrate synthase analysis of UCP1 activity in control media and browning media. Student’s t-test performed (p < 0.0001).

Allogeneic transplantation of exBAT in mice with diet-induced obesity

Finally, we performed preliminary metabolic phenotyping of exBAT transplantation in a diet-induced obesity (DIO) mouse model to mimic the potential target population in humans. Here, we used allogeneic transplantation, performed in previous DIO mouse models54,55,56,57,58, to minimize variations in stress and weight caused by the multiple surgeries of autologous re-implantation (Fig. 5a). To mimic the envisioned clinical procedure in humans, we developed a minimally invasive delivery method, injecting the converted browned tissue via a 19-gauge (1.1 mm × 25 mm) needle into the subcutaneous, lower dorsal region of recipient mice. For donor tissue, we used visceral WAT from epididymal regions of age- and sex-matched DIO mice which is elevated in obese human subjects25 and exhibits soft mechanical properties (compared to subcutaneous WAT) which made it suitable for needle injection; although epididymal WAT had previously not been as well demonstrated to convert to BAT as inguinal WAT, we found it to brown effectively using our single-step ex vivo method (Fig. 5b, Supplementary Fig. 4). As a control, we also injected visceral WAT cultured in control media, denoted “control WAT”. We injected ~0.2 mL of cultured tissue fragments, which was the yield from one donor fat pad; in this study, we tried just a single dosage to perform a preliminary assessment of the in vivo effect before a full-scale trial of therapeutic dosages and conditions in a future trial.

Figure 5
figure5

Metabolic testing of reimplanted exBAT in mice. (a) Illustration of experimental design for studies of ex vivo browning of epididymal WAT and allogeneic injection (the allogeneic implantation was designed to minimize stress and background weight loss of mice): (1) donor mouse is sacrificed via CO2 asphyxiation; (2) epididymal WAT fragments are harvested from sacrificed donor mouse via necropsy; (3) WAT fragments are minced by passaging through a 19 gauge needle; (4) WAT fragments are cultured for 3 weeks in media with and without browning factors; (5) fragments are removed from media and washed with PBS; (6) fragments are injected subcutaneously into age-matched, sex-matched recipient mouse. (b) H&E (top) and UCP1 IHC (bottom) staining of cultured tissues (10 days) prior to implantation (note: visceral adipose tissue). Scale bar: 50 µm. (c) Change in body mass 15–17 weeks post-implantation (see Supplementary Fig. 4 for entire weight-loss data). Drop in body weight is due to entry into metabolic chambers from week 15–16, and subsequently exposure to cold temperature from week 16–17. Error bars indicate SEM, n = 8 for each group. (d) Percent fat mass of total body mass for control and experimental groups. Percent fat mass is measured prior to entering the metabolic chambers (15 weeks post-implant, p = 0.056), after 1 week in the chambers at 25 °C (16 weeks post-implant), and after 1 more week in the chambers at 8 °C (17 weeks post-implant). Error bars indicate SEM, n = 8 for each group.

Throughout 17 weeks post-implantation, mice injected with control WAT and the converted BAT both exhibited weight losses after being moved to the metabolic chamber and after cold exposure, as expected. Mice receiving converted BAT exhibited lower body weight than those injected with control WAT throughout the entire 17 weeks in terms of mean weights, but no statistical significance was observed (Fig. 5c, Supplementary Fig. 5a). EchoMRI measurements of percent fat mass of the animals (Fig. 5d) were taken prior to entry into metabolic chambers, after 1 week in the chambers at room temperature, and after 1 more week in the chambers at 8 °C, and approached significant difference at 15 weeks post-implant (Fig. 5d, p = 0.056). Differences in VO2 and heat expenditure in mice 1 week following implantation were non-significant (Supplementary Fig. 5b,c). Changes in dosage or cell source could be tested in future experiments.



https://www.nature.com/articles/s41598-018-25866-y