Fig. 3: Engineered dual WNT3A and RSPO1 carrying exosome.

a Schematic illustration of an exosome engineered to carry WNT and RSPO protein (Created in BioRender. Chan, Y. (2025) https://BioRender.com/yxnywpj). b WNT3A and RSPO1 levels on exoWT, exoWNT3A^WLS, exoRSPO1, and exoWNT3A/RSPO1). c Images with the STORM system showing WNT3A and RSPO1 on exoWNT3A/RSPO1. Exosomes were stained with antibodies specific for WNT3A, RSPO1, and CD63. d Immunogold-labeling of WNT3A and RSPO1 on exosomes with anti-WNT3A antibody (10-nm gold particle, blue arrow) and anti-RSPO1 antibody (6-nm gold particle, green arrow). e TOPFlash reporter assay in HEK293FT cells treated with exoWT, exoWNT3A^WLS+exoRSPO1, or exoWNT3A/RSPO1 at the same particle number (1 × 10⁹) or CHIR99021 (3 μM). The relative fold change in activity normalized to exoWT control is shown (n = 3). f Bright-field image and immunofluorescence staining of AFP, ALB, and HNF4α in HE cells after exoWNT3A/RSPO1 treatment for 6 days. g Expression level of LGR5, MYC, ALB, AFP, and HNF4α in HE cells after different treatments for 6 days. The relative fold-change normalized to exoWT control is shown (n = 3). h Bright-field images of hepatic organoids cultured with exoWT, exoWNT3A/RSPO1 or CHIR99021 (3 μM) for 10 days. i Expression level of LGR5 and AXIN2 in hepatic organoids after different treatments for 10 days. The relative fold-change normalized to exoWT control is shown (n = 3). j PCA of transcriptome profiles of hepatic organoids under different treatments for 10 days (n = 2). k Venn diagram showing the intersection of differentially expressed genes (DEGs) between exoWNT3A/RSPO1 vs. exoWT and CHIR99021 vs. exoWT. l GSEA of common and specific DEGs in k against the KEGG gene dataset. m Bright-field images of hepatic organoids treated with exoWT, or exoWNT3A/RSPO1 in the presence of DMSO, SB202580 (10 μM), or PAN-RAS-IN-1 (10 μM). All statistical data are presented as mean ± SD. Two-tailed unpaired Student’s t tests are used to compare the two groups. The p values are calculated and shown as: ns p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3 indicates 3 biological replicates using independent cell experiments.