3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGF\u03b2RII-SMADS pathway

Ning Wang,Yingjie Fu,Xiajing Li,Keqiang Ma,Yueming Qiu ,Zihang Zhong ,Shuai Zhang,Yimeng Ou,Honglin Chen,Weizhong Gu ,Xianglian Tang,Tingting Guo,Jinsong Li,Jie Cao,Shoupei Liu,Chuxin Chen,Bailin Wang,Haibin Wu,Mark Α Zern ,Qicong Chen,Yuyou Duan

doi:10.1186/s12951-021-01138-2

Abstract

BackgroundExosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo.ResultsIn vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFβ-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFβRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFβRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFβRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis.ConclusionsOur results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFβRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.Graphical

Highlights

Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver
Our results showed that miR-6766-3p in the 3D-human embryonic stem cell (hESC)-Exosomes inactivates smads signaling by restraining TGFβRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESCExosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease
Characterization of 2D and 3D hESC‐derived Exosome It has been reported that 3D culture can maintain cell stemness [22,23,24,25], we passed hESCs from 2D culture into ultralow attachment culture plates to form hESC spheroids to determine whether the stemness of hESCs was improved by 3D culture

Summary

Introduction

Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. It has been shown that pluripotent stem cells (PSC), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC), exert great potential for liver regeneration because of their unparalleled ability to differentiate [12, 13]; the risk to form teratomas would limit the transplantation of PSC-derived cells [14]. Accumulating evidence demonstrate that a wide range of immunosuppressive and paracrine factors generated by stem cells display efficient effects in reversing fibrosis and enhancing liver cell function [17]. In an attempt to enhance the effects of stem cell therapeutics, our group has suggested that stem cell based-exosomes could provide therapeutic effects in cell-free treatment of liver diseases [18]

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