Fig 2: Adipogenic differentiation and proliferation in DLK1-/CD34+/CD24+ and DLK1-/CD34+/CD24- ASCs. Cells were seeded directly after sorting in 48-well plates and adipogenesis was induced 16 h later. Representative results for ASCs from 3 different donors are shown 9 days post induction of adipogenesis. (A) Oil Red-O (ORO) staining. (B) Western Blot analysis of perilipin protein level. (C) RT-qPCR gene expression analysis of PPAR?2, FABP4 and Perilipin. Gene expression was normalized to RPS18 as control. (D) BrdU incorporation assay. 1600 cells/96-well plate were seeded directly after sorting and BrdU incorporation was measured 6 days after adding BrdU. (E) Cell counting. 5000 cells/48-well plate were seeded and the relative cell numbers counted by FACS at the indicated time points. Representative results from 3 different donors are shown. Mean+SEM is shown; unpaired student´s t-test was performed to calculate significances; * p < 0.05 and *** p < 0.001.

Fig 3: CD24 Expression in ASCs. (A) CD24 gene expression in sorted CD24+ and CD24- ASCs as well as in cultivated ASCs, routinely isolated by the plastic-adherence protocol. Merged data from 4 different donors are shown, gene expression was normalized to RPS18, n = 3 for technical replicates. Mean+SEM is shown, unpaired student´s t-test was performed to calculate significances with *** p < 0.001. (B,C) Intracellular (left panel, permeabilized) and cell-surface (right panel, unpermeabilized) CD24 protein level measured by FACS for (B) sorted CD24- cells and (C) unsorted cultivated ASCs routinely isolated by the plastic-adherence protocol at passage 2. Unstained control (black, dashed lines) and CD24 stained cells (red, solid lines) are shown. (D) CD24 cell-surface expression measured by FACS on ASCs in the course of routine isolation by the plastic-adherence protocol and cultivation. (Left panel) Unsorted SVF cells (red) and plastic-adherent cells after cultivation for 6 days in serum-free ASC1 medium (P-1, light blue) are shown. (Right panel) SVF cells are shown in red. P-1 cells further cultivated for 1 week in serum-containing PM4 medium were referred to as P0 and are shown in dark blue. After freezing, these cells were re-thawed, taken into culture and referred to as ASC passage 1 (P1, orange) and further cultivated to passage 2 (P2, light green) and passage 3 (P3, dark green).

Fig 4: Isolation of DLK1-/CD34+/CD24+ and DLK1-/CD34+/CD24- cell populations from the SVF of human sWAT. (A) Fluorescence-activated cell sorting (FACS) scheme: Un-permeabilized and un-fixed cells in the SVF were sorted for cell surface tethered DLK1 and CD34 protein by staining with anti-DLK1 and anti-CD34 antibodies to obtain DLK1+/CD34- (3.91% ± 3.38%), DLK1+/CD34dim (20.53% ± 2.74%) and DLK1-/CD34+ (50.93% ± 8.04%) cells. These cells were subsequently sorted for cell surface tethered CD24 generating the DLK1-/CD34+/CD24+ (6.64% ± 5.76%) and DLK1-/CD34+/CD24- (92.65% ± 5.87%) fractions. n = 4. (B) Representative Dot Blots showing the sorting scheme to obtain DLK1-/CD34+/CD24+ and DLK1-/CD34+/CD24- cell populations.

Fig 5: Analysis of CD44+/CD22- stem cell population in theviable percentage of DU145 and PC3 cells.A, B. Mean fluorescence intensity of CD44+ and CD24+ cell populations in DU145 and PC3 comparing total and viable populations using Annexin V/PI assay. The values repres ent the mean ± SD (n = 6). *** p < 0.001, **** p = 0.0001, significantly different from total vs viable. ## p = 0.001, # p = 0.0001, significantly different from the total population control. %% p = 0.001, % p = 0.0001, significantly different from the viable supopulation control.