Fig 2: Characterization of serum exosomes. (A) TEM images confirmed the presence of exosomes. Scale bar=200 nm. (B) Exosomes-enriched protein markers including CD9 and TSG101 were analyzed in exosomes (E) and exosome-depleted supernatant (EDS) by Western blot analysis. (C) The sizes of serum exosomes were determined via the NTA characterization system.

Fig 3: Western blot analysis. Western blot was performed on cExo and ncExo samples, both from exosome-fractions (fractions 7–9) and from a non-exosome-fraction (fraction 14). Normal human keratinocyte lysate was used as control. (a) CD9 was positive in all exosome-samples and in the lysate, but negative in the non-exosome-fraction. (b) Flotillin was positive in all exosome-fractions and lysate, but with varying signal strength, and no signal was detected in the non-exosome-fraction. (c) Annexin V was negative in all fractions, but positive in the lysate. (d) EpCAM was negative in all samples (expected at 40 kDa, dotted box). (e) HSP70, heat shock protein 70, was negative in all samples except for cell lysate control. (f) GRP94, an endoplasmic reticulum protein that commonly contaminates vesicle isolates, was also negative in all samples except for cell lysate control. d3, d4 and d5 refer to donors 3, 4 and 5.

Fig 4: PEP displays exosomal characteristics and drives in vitro activities of skin progenitors. (A): Representative transmission electron microscope image for PEP exosomes. (Scale; 1 bar represents 200 nm). (B): Western blot analysis for the expression of CD63, CD9 and Alix on PEP exosomes from three separate current good manufacturing practices lots. Total protein staining was used as a loading control validated by GAPDH probing. (C): Size distribution of PEP exosomes obtained using NTA (NanoSight). The exosomes were averagely 129.4 nm wide. (D): In vitro angiogenesis assay using co-culture of human dermal fibroblast (hFB) with GFP-tagged human umbilical vascular endothelial cells (HUVEC) in presence of VEGF, PEP or Suramin. (E): Quantification of average network length in 6 hours increments over a 6 day period. (F): Organoid differentiation assay of human keratinocytes treated PEP or serum free media at day 24. H&E staining was performed to all the groups. Red arrow: Organized differentiated keratinocyte with multiple epidermis-like layers. Blue arrow: unorganized keratinocytes differentiation. (G): Scratch assay evaluating the migration of primary rabbit dermal fibroblast treated with FBS, PEP or serum free media. Representative pictures of wound closure for FBS vs. PEP vs. serum free media at 0 h and 32 h. (H): Scratch assay testing the migration of human keratinocytes treated with FBS, PEP or PBS. Representative pictures of wound closure for FBS vs. PEP vs. PBS at 0hr and 72 h. (I): Graph showing quantification of fibroblast wound closure, performed every 2 h over a 32 h period. (J): Graph showing quantification of keratinocytes wound closure, performed every 3 h over a 72 h period. Scale bar in A&D: 200 µm, Scale bar in B&C: 50 µm. N= 8 in D-J, 2-tailed paired Student's t test for each group compared with control group. ***p < 0.001, ****p < 0.0001.

Fig 5: Characterization of iMSC-sEV. a Representative image of iMSC-sEV observed by TEM. Scale bar = 100 nm. b The particle size distribution of iMSC-sEV measured by nano-flow cytometer. c Western blotting showing the expression of exosomal markers including CD9, TSG101, and Alix in iMSC-sEV, but not the negative marker GM130