Fig. 2

CD34+ cells characterization in the fibrous septa and stroma. a Microdissection of AT septa and stroma. A piece of the whole AT was rinsed with PBS (image 1), and lobules were isolated one by one. Isolated lobules were precisely dissected using Dumont forceps and Vannas spring scissors under a Zeiss StemiV6 stereomicroscope at ×8 magnification. The septa surrounding the lobule were progressively lifted off (images 2–5) until its complete separation from the stroma (image 6). The aspect of dissected septa (fibrous membrane without mature adipocytes) and stroma (mature adipocytes without fibrous membrane) are shown in images 7 and 8, respectively, under a bright-field microscope with a ×40 magnification. b Representative microphotographs of dissected septa and stroma stained with picrosirius red (upper panel) and Bodipy/DAPI (lower panel). c Gene expression in non-adipose cells isolated from matched fibrous septa (blue) and stroma (black) from subcutaneous AT lobules determined by RT-qPCR analyses. The results are means ± s.e.m. of experiments performed on n = 5 independent donors, paired t test, *P < 0.05. d Representative dot plots obtained by flow cytometry analyses with SVF cells isolated from septa and stroma using anti-CD45, -CD31, and -CD34 antibodies and quantification of the CD45+, CD45−/CD34−/CD31−, CD45−/CD34+/CD31+,  and CD45−/CD34+/CD31− cell populations in the septa (blue) and stroma (black). The results are means ± s.e.m. of experiments performed on n = 7 independent donors, two-way ANOVA. e Representative histograms of fluorescence intensity of CD36, CD9, MSCA1, and CD271 on CD45−/CD34+/CD31− cells and f CD36 and CD9 mean fluorescence intensity (MFI) determined by flow cytometry analyses performed on matched fibrous septa (blue) and stroma (black) cells (n = 7, paired t test, *P < 0.05). g Repartition of CD45−/CD34+/CD31− progenitor cell subsets (i.e., −/−, −/CD271+, and MSCA1+) in matched septa and stroma determined by flow cytometry analyses. The results are expressed as log2 ratio of septa to stroma percentages and are means ± s.e.m. of experiments performed on n = 7 independent donors. One-way ANOVA followed by Dunnett’s multiple comparison test, **P < 0.01. h CD271 MFI determined by flow cytometry analyses on the −/CD271+ cells from septa (blue) and stroma (black) from n = 7 independent donors, paired t test, *P < 0.05