BMP7-Loaded Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorate Liver Fibrosis by Targeting Activated Hepatic Stellate Cells

Abstract

Purpose

Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations.

Patients and Methods

To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 (BMP7) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results.

Results

Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects.

Conclusion

These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis.

Graphical Abstract

Keywords:

• hucMSCs
• sEVs
• aHSCs
• nanocarrier
• BMP7

Abbreviations

HucMSC, human umbilical cord mesenchymal stem cell; sEVs, small extracellular vesicles; hucMSC-sEVs, human umbilical cord mesenchymal stem cell-derived small extracellular vesicles; BMP7, gene bone morphogenic protein 7; CCL4, carbon tetrachloride; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay; aHSCs, activated hepatic stellate cells; ECM, excessive extracellular matrix; qHSCs, quiescent hepatic stellate cells; α-SMA, α-smooth muscle actin; COLI, collagen type I; LDs, lipid droplets; MSCs, mesenchymal stem cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin; CM: conditioned media; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis; PBS, phosphate-buffered saline; HE, Hematoxylin and eosin; ALT, alanine transaminase; AST, aspartate aminotransferase; HA, hyaluronic acid; IV-C, collagen type IV; PC-III, procollagen type III; PCNA, proliferating cell nuclear antigen.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Approval

This study was conducted in strict accordance with the ethical guidelines outlined in the Declaration of Helsinki. All study participants provided informed consent, and the study design and the research procedures were approved by Ethics Committee of LZU No. l Hospital (no. LDYYLL-2023-459).

Acknowledgments

We would like to thank the staff of the laboratory for their help with this project.

Author Contributions

All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82060119, 32171610) and Natural Science Foundation of Gansu province (Grant No.21JR1RA072, 20JR10FA661).