The “Design” page of the iGEM team at Lanzhou University describes in detail the purpose, working principle and effects of engineered human umbilical cord mesenchymal stem cell-derived exosomes by our team.On this page, we focuse on the overall system design of these exosomes, specifically including: the strategy for the construction and preparation of the engineered exosomes, and the methodology for evaluating the synergistic anti-fibrotic effects produced by co-loading two miRNAs (miR-455-3p and miR-148a-5p) at different ratios within a hepatic stellate cell fibrosis model.

The choice to use stem cell exosomes rather than stem cells themselves for treating end-stage liver disease represents a shift in therapeutic strategy. The underlying logic traces an evolution from the initial, simplistic idea of directly "replacing" damaged cells with stem cells to the more advanced paradigm of utilizing exosomes as "precision-regulated" intelligent therapeutics.

The original rationale was straightforward: stem cell transplantation could potentially slow or even reverse the degenerative liver process, as the cells might replace the patient's damaged hepatocytes and stimulate regeneration and repair mechanisms in the surrounding diseased tissue. However, this cell replacement approach encountered significant obstacles in practice. Studies revealed that transplanted stem cells faced a complex and hostile pathological microenvironment, resulting in low engraftment rates and short-term stability of the engrafted cells [1]. More critically, introducing uncontrollable risks inherent to using live cells. These risks include potential tumorigenicity, immune rejection, and unpredictable cellular behavior [2].

It was the profound reflection on these challenges that prompted a pivotal shift in the scientific perspective. The research focus diversified: some efforts turned from naive stem cells to engineered stem cells, positing that genetic modifications could potentially reduce rejection risks and the need for lifelong immunosuppression [3]. Concurrently, another significant shift moved the focus from the cells themselves to their mode of action. Research indicated that extracellular vesicles secreted by mesenchymal stem cells (MSCs) exhibit therapeutic effects similar to the parent MSCs. This is because a portion of the paracrine effects of MSCs originates from exosomes; indeed, a significant part of the therapeutic action of MSCs in specific disease contexts is mediated by the paracrine effects of functional molecules—such as miRNAs, mRNAs, and proteins—carried by these exosomes.[4]

The technological complexity and risks associated with engineered cell therapies led us to bypass the hazards and uncertainties of transplanting live cells. Instead, we directly extracted their key therapeutic messengers—exosomes—to develop an "acellular therapy." This choice offers multiple advantages. As nanoscale vesicles, exosomes fundamentally avoid the risks of live cells, significantly enhancing safety. More importantly, they are not passive carriers but intelligent delivery systems with innate "homing" capabilities, actively accumulating at injury sites. The miRNAs, proteins, and other molecules encapsulated within their lumen form a sophisticated set of regulatory instructions that can simultaneously inhibit hepatic stellate cell activation, reduce inflammation, and promote hepatocyte regeneration, thereby reversing fibrosis through synergistic multi-pathway actions.

Ultimately, this approach points to a higher dimension: engineering. Since natural exosomes are already highly efficient, we explored whether they could be upgraded through engineering to enhance their capacity to carry specific therapeutic miRNAs (such as miR-455-3p and miR-148a-5p). This transforms exosomes from natural messengers into designable, quantifiable, and mechanistically clear targeted therapeutics, truly achieving a paradigm shift from "cell therapy" to "cell-derived precision pharmacotherapy."

The system design of this project aims to construct an engineered human umbilical cord mesenchymal stem cell (huMSC) exosome co-loaded with two miRNAs and to systematically evaluate its synergistic therapeutic effect on liver fibrosis. The entire design revolves around three key issues: the strategy for constructing the engineered exosomes, the validation of the synergistic effect of the dual miRNAs, and the determination of the optimal synergistic ratio.

First, during the construction phase, the project utilizes commercially sourced huMSC exosomes as the base carrier to circumvent potential issues related to quality and batch-to-batch variability associated with self-produced exosomes. To efficiently load miR-455-3p and miR-148a-5p—miRNAs with demonstrated anti-fibrotic potential—into the exosomes, we employed and compared two in vitro loading strategies: 1) Loaded using the ExoLoad® Exosome Nucleic Acid Loading Kit method.（kit method）, and 2) co-incubation, a physical method for active miRNA import. This comparison was conducted to identify a reliable and highly efficient exosome loading protocol. Through these strategies, the high-efficiency co-loading of therapeutic miRNAs is achieved, enabling synergistic anti-fibrotic action and thereby overcoming the limitation of low therapeutic molecule content in natural exosomes.

In the validation phase, the project will establish an in vitro fibrosis model using TGF-β1-induced human hepatic stellate cells (LX-2) to simulate the disease state. A comprehensive set of experimental groups will be systematically arranged, including negative and positive blank controls, a natural exosome control, single miRNA-exosome groups (delivering miR-455-3p or miR-148a-5p individually), and experimental groups involving the co-delivery of the two engineered exosomes at different ratios (e.g., 1:1, 1:2, 2:1). This design allows for a multi-faceted evaluation of the intervention effects.

Finally, in the evaluation and optimization phase, the expression levels of hepatic stellate cell activation markers, such as α-SMA, will be quantitatively measured using techniques like Western Blot to precisely assess the anti-fibrotic effect of each group. Through systematic comparison of data from the single miRNA groups and the dual miRNA co-delivery groups at different ratios, this project will focus on analyzing whether a synergistic effect exists between the two miRNAs. Based on this analysis, the optimal ratio for maximizing the therapeutic effect will be identified, thereby providing a theoretical and experimental foundation for treating end-stage liver disease.

[1] Heydari Z, Najimi M, Mirzaei H, et al. Tissue Engineering in Liver Regenerative Medicine: Insights into Novel Translational Technologies. Cells. 2020;9(2):304. Published 2020 Jan 27. doi:10.3390/cells9020304.

[2] Nadi A, Moradi L, Ai J, Asadpour S. Stem Cells and Hydrogels for Liver Tissue Engineering: Synergistic Cure for Liver Regeneration. Stem Cell Rev Rep. 2020;16(6):1092-1104. doi:10.1007/s12015-020-10060-3.

[3] Tolosa L, Pareja E, Gómez-Lechón MJ. Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases?. Transplantation. 2016;100(12):2548-2557. doi:10.1097/TP.0000000000001426.

[4] Matsuzaka Y, Yashiro R. Therapeutic Strategy of Mesenchymal-Stem-Cell-Derived Extracellular Vesicles as Regenerative Medicine. Int J Mol Sci. 2022;23(12):6480. Published 2022 Jun 9. doi:10.3390/ijms23126480.