Fig. 3: SWAT cells are multipotent mesenchymal progenitors with high canonical Wnt activity.
From: Wnt signaling preserves progenitor cell multipotency during adipose tissue development
a, Schematic of the cell separation assay with Percoll density gradient centrifugation. b, Image of cells after density centrifugation. c, Separated cell layers from b, re-seeded in multi-well plates. Images were taken 72 h after seeding. d, qRT–PCR assessment of ADIPOQ and MGP messenger RNA levels in A-LD and A-HD from 7-d adipogenic-induced cells (n = 2). e, Scatter-plot of each detected gene’s TPM values between the high- and low-density cells measured by bulk RNA-seq. Genes highlighted in blue were the 121 genes enriched in the cluster 5/ADIPOQ+ cells, as identified by differential expression analysis of single-cell RNA-seq presented in Fig. 2f and genes highlighted in red were the 114 genes enriched in the cluster 3/SWAT cells. f, Schematic of the experiment assessing multipotent and proliferative potential of A-HD cells obtained from 7-d adipogenic-induced cells. For low-seeding-density plating, 10,000 cells were plated per well in a 96-well plate; for high-seeding-density plating, 30,000 cells were plated per well. g, mRNA levels of adipogenic (ADIPOQ), chondrogenic (COMP) and osteogenic (FRZB) lineage markers of A-HD after 3-d lineage differentiation (n = 2). h, Oil Red O staining of A-HD after 10-d adipogenic induction. Scale bar, 50 μm. i, Alizarin red S staining of A-HD after 10-d osteogenic induction. Scale bar, 50 μm. j, Phase images of A-HD after growth for indicated days in progenitor growth medium. Scale bar, 100 μm. k, Cell counts of A-HD after indicated days in progenitor growth medium (n = 3). l, qRT–PCR assessment of MGP mRNA levels in A-HD cells collected after indicated days in progenitor growth medium. m, Schematic of cell separation assay with STF luciferase reporter. n, STF luciferase levels in A-LD and A-HD cells before and after cell separation (n = 3, P values are determined by one-way analysis of variance (ANOVA)). RLU, relative light units. All data plots are shown as mean ± s.d. Each dot represents a data point from a biological replicate.
