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Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration

Year: 2026

Type: article

Abstract: R-spondin1 plays a pivotal role in potentiating canonical WNT/β-catenin signaling, a pathway critical for maintaining stem cell homeostasis and driving tissue regeneration. Here, we present a scalable, serum-free protocol for producing R-spondin1-loaded small extracellular vesicles (evRSPO1) from engineered HEK293 cells, with multimodal characterization of vesicle quality and bioactivity. We also describe steps for in vitro functional validation of the vesicles using mouse intestinal organoids and for in vivo therapeutic applications in mouse intestinal regeneration via oral delivery. For complete details on the use and execution of this protocol, please refer to Yang et al. 1 1. Yang, L. ∙ Wang, X. ∙ Wei, X. ... Oral Delivery of R-spondin1-Loaded Small Extracellular Vesicles Activates WNT Signalling Pathway to Accelerate Intestinal Injury Repair and Reverse Ageing J. Extracell. Vesicles. 2026; 15 , e70226 Crossref Scopus (3) Google Scholar , 2 2. Yang, L. ∙ Wang, S. ∙ Qiao, Z. ... Dual-ligand engineered exosome regulates WNT signaling activation to promote liver repair and regeneration Nat. Commun. 2025; 16 :9019 Crossref Scopus (19) PubMed Google Scholar

Source: STAR Protocols

DOI: 10.1016/j.xpro.2026.104891

Authors: Lingyan Yang, Ting Jiang, Shixiang Wang, Yue Yang, Zhiping Qiao, Yun-Shen Chan