Figure 3.

Conventional adipocyte progenitors and HSCDA progenitors are more abundant in the gonadal fat of Ablated mice. (A) Gonadal and subcutaneous (SubQ) fat was recovered from transplant-naïve (black/white crosshatch bar), Competent (black/green crosshatch bar) and Ablated (solid black bar) mice at the end of the experiment at 18 weeks. The adipose tissue underwent collagenase digestion and stromal vascular cells were separated and stained with conventional adipocyte and HSCDA lineage marker antibodies and subjected to flow cytometric analysis (gating strategy shown in Supplementary Figure 4 ). The proportion of conventional adipocyte progenitors (Lin^NEG/CD29^POS/PDGFRα^POS cells) are shown here. (n=3 per cohort, * indicates p≤0.05) (B) Putative conventional adipocyte progenitors from the gonadal fat of Competent and Ablated mice were purified by flow cytometry sorting and plated on tissue culture plates. The cells from both cohorts spontaneously underwent adipogenic conversion over a period of 7-14 days. Representative 10x brightfield microscope images are shown. (C) The abundance of HSCDA progenitors was measured in the gonadal and SubQ fat of transplant-naïve, (black/white crosshatch bar), Competent (black/green crosshatch bar) and Ablated (solid black bar) mice as described in 3A, except that fluorescent antibodies to CD45, CD11b, CD29, PDGFRα and Sca1 were used instead of those described above. (n=3 per cohort, * indicates p≤0.05) (D) Putative HSCDA progenitors were isolated by flow cytometry sorting, plated in 3-dimensional fibrin clots and recovered after 5 days. The cells were then re-plated on tissue culture plates and treated with conventional pro-adipogenic culture media to induce adipogenesis for a period of 14 days. Representative 10x brightfield microscope images show robust adipogenic conversion of cells from Competent, but not Ablated mice.