Aregs exhibit robust non-and anti-adipogenic properties that arise with age and involve RA signaling (2022)
Figure 1

Figure 1. CD142+ ASPCs constitute a robust ASPC subpopulation defined by a stable non-adipogenic phenotype and a highly specific transcriptomic signature

t-SNE cell map of integrated scRNA-seq datasets (see Methods) visualizing the main identified subpopulations of mouse subcutaneous ASPCs: adipose stem cells (ASCs) in green, pre-adipocytes (PreAs) in red, F3(CD142)+ ASPCs in blue, as well as Cilp+ ASPCs, endothelial and immune cells (see also Appendix Fig. 1);

Clustering tree of the Seurat-based clustering result of the integrated analysis described in A, visualizing the relationships between clustering at different resolutions of the three main ASPC subpopulations as well as Cilp+ ASPCs, demonstrating a high stability of the F3(CD142)+ ASPC cluster;

Gene expression heatmap of the top CD142+ markers (Appendix Table S1) across bulk RNA-seq samples of freshly isolated total, CD142− and CD142+ ASPCs; log normalised expression scaled by row;

Correlation of the logFC of top CD142+ markers (Appendix Table S1) across scRNA-seq, bulk RNA-seq and mass spectrometry data; logFC was defined as the log2FC of freshly isolated CD142+ over CD142− ASPCs for bulk RNA-seq and mass spectrometry, and as the average logFC of F3(CD142)+ over the remaining cells in scRNA-seq across integrated datasets described in A (Methods);

Representative fluorescence microscopy images of total, CD142- and CD142+ ASPCs isolated with the use of the respective anti-CD142 antibodies: SinoBiological (SB), SinoBiological-LL (SB-LL), BiOrbyt (BO) and R&D Systems (RnD) (Appendix Fig. S2B, Appendix Fig. S5A, Methods), after in vitro adipogenic differentiation;

Fraction of differentiated cells per ASPC type shown in E, as quantified by the "adiposcore"; marker shapes correspond to different anti-CD142 antibodies used for isolation as indicated, n=9-15, 3-4 biological replicates, 3-5 independent wells for each;

Boxplot showing the distribution of the "CD142+ score" based on the expression of the top CD142+ ASPC markers, n=4-7, 1-5 biological replicates, 1-4 independent wells for each (Appendix Table S1, Methods);

Boxplot showing the distribution of the "white fat cell differentiation score" based on the expression of the genes linked to the GO term "white fat cell differentiation" (GO:0050872), n=9-12, 2 biological replicates, 1-8 independent wells for each (Methods);

Boxplot showing the distribution of the "negative regulation of fat cell differentiation score" based on the expression of the genes linked to the GO term "negative regulation of fat cell differentiation" (GO:0045599) ), n=9-12, 2 biological replicates, 1-8 independent wells for each (Methods);

Representative fluorescence microscopy images of CD142-, 20% CD142+ and (5-7%) CD142+ ASPCs isolated following the sorting strategy shown in Appendix Fig. S7A, after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails (Methods);

Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J, as quantified by the "adipo- score"; marker shapes correspond to different biological replicates, n=8-17, 3-5 biological replicates, 2-5 independent wells for each;

Representative fluorescence microscopy images of male- and female-derived total, CD142- and CD142+ ASPCs, isolated following the sorting strategy shown in Appendix Fig. S8A after in vitro adipogenic differentiation;

Fraction of differentiated cells per ASPC type and per sex shown in L, as quantified by the "adiposcore"; bar colour shading corresponds to male- and female-derived cells as indicated; marker shapes correspond to different biological replicates, n=9-10, 4 biological replicates, 2-3 independent wells for each;

Fraction of differentiated cells per ASPC type and per mouse genetic background shown in Appendix Fig. S10A, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=9-24, 3-5 biological replicates, 3-6 independent wells for each.

Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 100 µm; bar colours: total ASPCs - brown, CD142- ASPCs - yellow, CD142+ ASPCs - blue, 20% CD142+ ASPCs - turquoise;

*P ≤ 0.05, ***P ≤ 0.001, pairwise two-sided t-test (F, K, M, N) or one-way ANOVA and Tukey HSD post hoc test (G-I), for statistical details see Methods.