1. Volume 27, Issue 1, Pages 213-225.e5 (April 2019)
Mature Human White Adipocytes Cultured under Membranes Maintain Identity, Function, and Can Transdifferentiate into Brown-like Adipocytes 
Matthew J. Harms, Qian Li, Sunjae Lee, Cheng Zhang, Bengt Kull, Stefan Hallen, Anders Thorell, Ida Alexandersson, Carolina E. Hagberg, Xiao-Rong Peng, Adil Mardinoglu, Kirsty L. Spalding, Jeremie Boucher 
Cell Reports 
Volume 27, Issue 1, Pages e5 (April 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 213-225.e5DOI: (10.1016/j.celrep.2019.03.026)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
Membrane Culture of Adipocytes Maintains Viability and Preserves Mature Adipocyte Gene Expression
(A) Model depicting the use of membranes to culture mature adipocytes.
(B) mRNA levels of adipogenic genes from mouse subcutaneous adipocytes cultured in different methods for 7 days. D0, freshly isolated adipocytes snap frozen without culture; explants; CC, adipocyte ceiling cultures in T25 flasks; floating, floating adipocytes; MAAC, membrane mature adipocytes aggregate cultures (mean ± SD; n = 4; CC, n = 3).
(C) mRNA levels of adipogenic genes from human subcutaneous D0 cells, adipose tissue explants, CC, and MAAC cultured for 2 weeks (mean ± SD; D0, n = 6; explant, n = 5; CC, n = 4; MAAC, n = 6).
(D) Viability (calcein-AM and propidium iodide) staining of human adipocytes before culture (D0) and after culture for 7 and 14 days under membranes.
(E) Average percentage of viable cells after 7 and 14 days relative to freshly isolated subcutaneous adipocytes from lean and obese patients measured by staining with Hoechst 33342/PI (mean ± SD; lean, n = 2; obese, n = 3).
(F) Western blot analysis of PPARγ, HSL, HIF1α, and β-Actin in fresh human adipose tissue, isolated adipocytes (D0), explants, and MAAC cultured for 1 and 2 weeks. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
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4. Figure 2
Unbiased RNA-Seq Indicates That the Gene Expression Profile of MAAC Is the Most Similar to Uncultured Adipocytes
(A) mRNA levels of adipogenic genes of adipose tissue snap frozen at the hospital, Day 0 isolated adipocytes (D0), explants, floating adipocytes, differentiated preadipocytes (PA), and MAAC cultured for 1 or 2 weeks (mean ± SD; adipose tissue, n = 5; D0 adipocytes, n = 5; explant 1 week, n = 4; explant 2 weeks, n = 4; floating 1 week, n = 4; floating 2 weeks, n = 4; undifferentiated preadipocytes, n = 6; differentiated preadipocytes 1 week, n = 4; differentiated preadipocyted 2 weeks, n = 8; MAAC 1 week, n = 4; MAAC 2 weeks, n = 4). *p < 0.05, **p < 0.01, ***p <
(B) Imaging comparing preadipocytes and MAAC at D0 and after 14 days in culture. Green, Bodipy (lipid); red, Hoechst (nuclei).
(C) Principal component analysis of gene expression profiles from different adipocyte culture methods. Dashed gray lines demarcate the different experimental groups.
(D) Hierarchical clustering based on average gene expression compared by Pearson’s correlation coefficients.
(E) Ranked similarity relative to D0 control adipocytes based on Pearson’s correlation coefficients.
(F) Number of identified differentially expressed genes (DEGs) in differentiated preadipocytes, floating adipocytes, and MAAC (false discovery rate [FDR], <1 × 10−8).
(G) Fold change in DEG in differentiated preadipocytes, floating adipocytes, and MAAC.
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5. Figure 3
Human MAAC Maintain Their Depot-Specific Gene Expression Patterns
(A) mRNA levels of adipogenic genes in human subcutaneous (subc.) and omental visceral (visc.) adipocytes freshly isolated (D0) and cultured under a membrane for 2 weeks (D14) (mean ± SD, n = 3).
(B) mRNA levels of subcutaneous and visceral-enriched genes in human subcutaneous and visceral adipocytes freshly isolated (D0) and cultured under a membrane for 2 weeks (D14) (mean ± SD, n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001
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6. Figure 4 Human Membrane-Cultured Adipocytes Remain Functional
(A) Western blot analysis of pAKT, AKT, and β-actin after a 10-minute insulin stimulation, comparing adipocytes that recovered under membranes overnight (control) or cultured for 7 days.
(B) Basal and insulin-stimulated glucose uptake in MAAC that recovered under membranes overnight (control) or cultured for 7 days (mean ± SD, n = 4).
(C) Basal and insulin-stimulated de novo lipogenesis of MAAC that recovered under membranes overnight (Ctl) or cultured for 7 days (D7) (mean ± SD, n = 4).
(D) Basal and forskolin- and isoproterenol-stimulated lipolysis in adipocytes cultured under membranes overnight (control) or for 7 days; (mean ± SD, n = 4).
(E) mRNA analysis of PPARγ, glucocorticoid receptor (GR), and liver X receptor (LXR) target genes in MAAC and explants after 7 days of culture in the presence of 10 μM of the PPARγ agonist rosiglitazone (Rosi), GR agonist dexamethasone (Dex), or LXR agonist GW3965 (mean ± SD, n = 4). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001
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7. Figure 5 MAAC Can Crosstalk with Macrophages
(A and B) Human IL-6 and IL-8 protein levels (A) and mouse TNFα protein levels (B) in the culture medium of human adipocytes, mouse macrophages, or adipocyte + macrophage co-cultures treated with vehicle (Ctl) or 10 ng/ml of LPS for 18 hours (mean ± SD, n = 4).
(C) Human IL-6 and IL-8 protein levels in the culture medium of human adipocytes treated with conditioned medium from mouse macrophages that had been cultured with 10 ng/ml or LPS for 18 hours and/or neutralizing TNFα antibody (mean ± SD, n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001
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8. Figure 6 MAAC Can Transdifferentiate into Brown-like Adipocytes
(A) Images comparing control MAAC and MAAC that were transduced with adenovirus encoding GFP after 72 hours. Blue, Hoechst (nuclei); red, cell mask deep red (plasma membrane); green, GFP.
(B) mRNA levels of GFP and PGC1α in MAAC, 7 days after transduction with adenovirus encoding GFP or PGC1α (mean ± SD, n = 4).
(C) mRNA levels of brown fat-enriched genes in MAAC 7 days after transduction with adenovirus encoding GFP or PGC1α (mean ± SD, n = 4).
(D) Western blot analysis of GFP, PGC1α, UCP1, and β-actin in MAAC 7 days after transduction with adenovirus encoding GFP or PGC1α.
(E) mRNA levels of UCP1 in MAAC and explants treated with FGF21, BMP7, BMP4, Irisin, Rosi, or T3 for 7 days (mean ± SD, n = 3).
(F) mRNA levels of UCP1 in MAAC 7 days after treatment with or without insulin and Rosi (mean ± SD, n = 3).
(G) mRNA levels of UCP1 in MAAC 7 days after transduction with adenovirus encoding GFP or PGC1α and treatment with or without insulin (mean ± SD, n = 4). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001
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