Small extracellular vesicles from human adipose-derived mesenchymal stromal cells: a potential promoter of fat graft survival

Fig. 1. Characterizations of human adipose-derived mesenchymal stromal cells (hADMSCs). a Morphological features of the fourth-passage hADMSCs under light microscopy (× 100 magnification, scale bar =75 μm). b Representative images of Oil Red O staining (left: non-induced control group; right: induced group; × 200 magnification, scale bar = 75 μm). c Representative images of the alkaline phosphatase calcium cobalt staining (left: non-induced control group; right: induced group; × 200 magnification, scale bar =75 μm). d Flow cytometry analysis of cell surface marker CD29, CD90, CD31, and CD45 was showed in hADMSCs

Fig. 2. Characterizations of hADMSC-sEVs. a Gross observation: the number of T75 flasks for culturing cells at one time. b The gross visible sEVs precipitate was obtained from a. c NTA measured the size distribution of sEVs. d The ratio of the number of sEVs particles(× 108) to 1 × 106 cells. e The ratio of protein amount (μg) to 1 × 106 cells. f The potential of hADMSC-sEVs was measured by Zeta potential. g Representative transmission electron microscopy image of ADMSC-sEVs with double circular vesicle structures was shown by directional arrow (× 100,000 magnification, scale bar = 200 nm). h Morphological characterization of sEVs with three-dimensional shapes under scanning electron microscopy (× 10,000magnification; scale bar = 10 μm). i Nanoflow analysis of the hADMSC-sEVs’ surface protein marker (CD9 and CD81), and IgG as positive controls

Fig. 3. hADMSC-sEVs internalization to HUVECs. hADMSC-sEVs (labeled with PKH67 dye, green) and HUVECs (nuclei stained with DAPI) were co-incubated for 6 h and 12 h, respectively. In the control group, PKH67 dye was co-incubated with HUVECs for 6 h and 12 h, respectively. Representative fluorescence images were shown above(× 1000 magnification; scale bar = 25 μm)

Fig. 4. hADMSC-sEVs improved the fat graft survival rate in the nude mouse model. a Gross observation of the grafted fat demonstrated that the hADMSC-sEVs group (the left side of each picture) had larger graft sizes and heavier weight compared to that in the control group (the right side of each picture). b, c 1, 2, and 3 months after fat grafting, the weight (measured by the balance) and volume (measured by the liqiud outflow method) of grafted fat were significantly better in the hADMSC-sEVs group compared to the control group (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)

Fig. 5. Histological changes in adipose tissue over time. a HE staining revealed that the grafted fat in the hADMSC-sEVs groups exhibited better survival and morphologic integrity (evidenced by the presence of intact and nucleated adipocytes and the presence of cysts and vacuoles) compared to the control group. b, c Semiquantitative scale of integrity and cysts/vacuoles of HE staining (***p < 0.001,****p < 0.0001)

Fig. 6. hADMSC-sEVs promoted neovascularization in the nude mice fat grafting model (rows means different staining and lines represents different groups and months). CD34 was selectively expressed in the small vessel endothelium membrane. The VEGF-kinase ligand/receptor signaling system plays a key role in vascular development and regulation of vascular permeability. The VEGFR2 was mainly localized in membrane. Signs of proliferation (nuclei stained by Ki-67) in the vessel endothelium (blue line at the bottom left of each figure represents scale bar; scale bar = 50 μm)