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We have further validated that vesicles from iMSCs are actually derived from EVs to confirm the function of EVs. The effect of sEVs was first studied by being directly applied to the cultured cells. The number of cells was detected after 48 h. Staining of CD63 and GM130 was done and fluorescence microscopy was used to observe the localization of the two proteins. The results showed that the number of cells was significantly increased while the expression of IL-1 was at a level similar to that from which the pure sEVs were harvested (Figure 6B and D).

As we all know, due to the physical barrier and immunologic privilege of the skin, MSCs have great difficulty in delivering cargoes into the subcutaneous tissues. Therefore, we have to exert a considerable amount of force to make iMSCs deliver cargoes into the target area. To improve the penetration ability of the systemic delivery vectors, we chose **skin-on-top** to construct an epidermis-on-top three-dimensional (3D) cell culture model. The epidermis-on-top model retains the barrier function of skin while providing the pharmacological or physical barrier function of the skin. The lower body of the animals is immersed in PBS solution. Here we added purified sEVs or vesicles to the culture medium to validate their penetration ability. In vitro confocal microscope was used to observe the penetration of the fluorescent-labeled sEVs into the 3D cell model. The results showed that, whether vesicles contained proteins or not, when applied to the normal epidermis, no fluorescence was observed in the cells under the microscope (Figure 5A and the figure in original jurisdiction). We repeated the experiment five times and obtained the same results. However, when the sEVs were exposed to an acidified bath and the oocyte’s newly formed epidermis, sEVs were found to target the 3D model (Figure 5B and C). Fluorescent sEVs were observed to enter the layer of the basal epidermis, the outermost skin cells in the 3D model, and even the apical lumen. In some cases, the sEVs actually entered the oocyte layer.

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