Fig 2: Therapeutic potential of exosome inhalation treatment in rats with pulmonary fibrosis.a Size analysis of fresh, frozen and lyophilized exosome particles by NanoSight. b Representative transmission electron micrograph (TEM) of LSC-Exo. Left scale bar = 0.2 µm. Right scale bar = 50 nm. c Immunoblot analysis of CD63, CD81, TSG101, and CD9 protein in LSC-Exo and MSC-Exo. d The experimental study schematic of the exosome study in SD rats; n = 12 biological independent animals per group. e Representative H&E staining Top: Scale bar = 100 µm Bottom: Scale bar = 50 µm. f Representative Gomori’s trichrome staining Scale bar = 100 µm (bottom); muscle fibers (red), collagen (blue), nuclei (black-purple) and erythrocytes (red). g Representative picrosirius red staining; Collagen types I and III (red); Scale bar = 50 µm. h Quantification of fibrosis by Ashcroft score; Each dot represents data from one animal; n = 12 biological independent animals. Ashcroft score was performed by averaging the score from one blinded and one non-blinded scorer. i Quantification of pulmonary hydroxyproline levels; *P = 0.05; each dot represents data from one animal; n = 4 biological independent animals. j Immunoblot analysis of matrix metalloproteinase 2 (MMP-2), alpha smooth muscle actin (aSMA), SMAD3 and GAPDH loading control. k Representative immunostaining of lung sections for aquaporin 5 (AQP5), alpha-smooth muscle actin (aSMA), and von Willebrand Factor (vWF). Scale bar = 75 µm. l–n Quantification of pixel intensity of AQP5 (l), a-SMA (m), and vWF (n). Each dot represents data from one animal; n = 4 biological independent animals. Throughout, data are mean ± s.d. P-value as indicated by non-parametric one-way ANOVA.

Fig 3: EV characterization from RL cell lines and sera. Nanotrack analysis (Nanosight, a) and TEM (c) for EVs isolated from RL cell line (Lipo246); Nanosight (b), TEM (d) for EVs isolated from serum. WB showing presence of Alix, CD9 and low level/absence of Calnexin in the EVs isolated form cell line (e), WB showing presence of CD9, CD81 and low level of ApoE and Albumin in the EVs isolated from sera (f)

Fig 4: rEV are separated from conditioned medium using density gradient centrifugation. a Schematic representation of rEV showing representative molecular components shared with sample EV, PA phosphatidic acid, PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol, PS phosphatidylserine, SM sphingomyelin. b Schematic overview of the production of rEV at the cellular level: (1) The gag-EGFP fusion protein inserts in regions of the plasma membrane enriched for tetraspanins CD9, CD63 and CD81 via its N-terminal MA domain containing a myristoyl group. (2) The gag-EGFP fusion protein oligomerizes and recruits ESCRT-1 proteins (TSG101) via the PTAP motive on its p6 domain. (3) Recruitment of ESCRT-2/3 proteins initiates the outward budding of the gag-EGFP containing plasma membrane. (4) ESCRT-3 mediated scission of the membranes finally causes release of rEV into the conditioned medium (CM)13. c Schematic overview of the workflow to separate rEV from CM of gag-EGFP transfected HEK293T cells. Seventy-two hour post transfection CM is collected from ~3 x 109 cells and concentrated to 1 mL. Concentrated CM is loaded on top of an OptiPrep density gradient (ODG) and centrifuged for 18 h at 100,000 × g. Density fractions of 1.086–1.119 g/mL are collected and pelleted for 3 h at 100,000 × g resulting in ~5 x 1011 rEV per harvest

Fig 5: Characterization of the purified exosomes from hiPSC-EC and HCMEC conditioned media. Exosomes isolated from cell culture media of hiPSC-ECs and HCMECs. (A) Western blot showed the detection of exosome markers CD9, CD63, and CD81 in isolated exosomes. (B) Glucose-regulated protein 94 (GRP94) and Golgi marker GM130 were not found in exosomes. (C) Flow cytometry analysis using exosome marker CD9. (D) The size distribution of isolated exosomes was measured by nanoparticle tracking analysis (NTA) in hiPSC-ECs and HCMECs.