Figure 1.

BMDAs are generated from HSCs via the myeloid lineage. (a) Competitive transplants were performed with either haematopoietic or mesenchymal progenitor cells constitutively expressing GFP, mixed with GFP-naïve carrier cells. After 8 weeks, free-floating adipocytes were prepared from adipose tissue from the transplanted mice and analysed by flow cytometry. GFP^POS BMDAs were detected in the adipocyte fraction of mice transplanted with GFP-labelled HSC (gated green events) but not labelled BM mesenchymal stem cells. (b) Backgating of the GFP^POS events in Figure 1a confirmed their large size (high forward scatter) and low internal complexity (low forward scatter) common to unilocular adipocytes. (c) Images of GFP^POS adipocytes from mice transplanted with GFP^POS HSC confirmed their identity as unilocular adipocytes with a single nucleus and cytosolic GFP fluorescence. (d) Haematopoietic sub-population cells were isolated from the BM of GFP-expressing mice with magnetic bead bearing antibodies to specific lineages (B220 = B lymphocyte, CD5 = T lymphocyte, CD11b = myeloid and Ly-6 G = granulocytes). The separated GFP^POS BM cells were combined with GFP^NEG BM cells from wild type mice to ensure survival of recipients. After 8 weeks flow cytometry showed production of BMDAs only in mice transplanted with labelled (Cd11b^POS) myeloid cells. (e) Flow cytometry of free-floating adipocytes isolated from dual transgenic mice in which GFP expression (from an mTmG transgene) was controlled by the myeloid-specific lysozyme gene promoter (LysMcre). A population of GFP^POS adipocytes confirmed that some adipocytes were produced from the myeloid lineage. (f) Free-floating adipocytes were isolated from the LysMcre mTmG mice and subjected to imaging flow cytometry. Brightfield and fluorescence images confirmed the presence of GFP^POS unilocular (single LipidTOX stained lipid droplet) adipocytes in additional to GFP^NEG adipocytes