Renal Protective Effects of Extracellular Vesicle-Encapsulated Tumor Necrosis Factor-α-Induced Protein 6 Derived from Mesenchymal Stem Cells

Immunology Bioengineering Basic Medical Sciences Nephrology Cell Biology Biochemistry Life Sciences Medicine
mesenchymal stem cells inflammation renal protection extracellular vesicles TNF-α-induced protein 6 M2 macrophage polarization regulatory T cells

I. Research Background and Academic Significance

In recent years, the incidence of acute kidney injury (AKI) has been continuously rising worldwide. AKI not only leads to acute loss of renal function but is increasingly recognized as being closely related to the occurrence and progression of chronic kidney disease (CKD). Numerous epidemiological and basic studies have shown that a considerable proportion of AKI patients eventually progress to CKD, constituting the clinical challenge of “AKI-to-CKD progression.” The main scientific questions in this field include inflammatory responses, microvascular rarefaction, hypoxic reaction, transforming growth factor β1 (TGF-β1) signaling, and epithelial–mesenchymal transition, all of which synergistically drive the development of renal interstitial fibrosis — the common endpoint of CKD.

At present, there are no effective therapies to block or reverse the progression from AKI to CKD, making the development of novel intervention strategies a pressing direction in nephrology. Mesenchymal stem cells (MSCs), as multipotent cells with extensive immunoregulatory and tissue repair capabilities, are regarded as highly promising tools for treating renal injury, reducing inflammation, and inhibiting fibrosis. Recent studies have found that the therapeutic effects of MSCs are mainly attributed to their secretion of various active factors (the “secretome”), including soluble proteins, nucleic acids, lipids, and molecules encapsulated in extracellular vesicles (EVs).

Among the known secreted factors from MSCs, tumor necrosis factor-α induced protein 6 (TSG-6) has been demonstrated to possess outstanding anti-inflammatory and anti-fibrotic activities, playing a central role in various tissue injury models. However, major gaps remain regarding how TSG-6 is secreted by MSCs, its mechanism of action, and how to optimize its secretion/encapsulation. This study addresses these cutting-edge scientific questions, focusing on the role of TSG-6 in the transition from AKI to CKD, aiming to elucidate its function and secretion mechanisms, and to explore new strategies to enhance TSG-6 secretion, thereby providing both theoretical and experimental support for clinical translation.

II. Paper Source and Authors

This research article, titled “Renal protective effects of extracellular vesicle-encapsulated tumor necrosis factor-α-induced protein 6 derived from mesenchymal stem cells,” was authored by Keisuke Morimoto, Ayumu Nakashima, Naoki Ishiuchi, Kisho Miyasako, and others, mainly affiliated with Hiroshima University and Twocells Company, among other institutions. The paper was published on April 18, 2025, as an Original Research article in the 43rd volume, issue 5 of the journal Stem Cells (DOI: 10.1093/stmcls/sxaf022).

III. Overall Study Design and Detailed Experimental Workflow

1. Overview of the Research Workflow

Starting from MSCs, this study enhanced the expression/encapsulation of TSG-6 in MSCs and their extracellular vesicles using gene engineering and chemical approaches. The anti-inflammatory and anti-fibrotic effects were systematically evaluated in a rat model of acute renal ischemia–reperfusion injury (IRI). The experiments were divided into three major modules: in vitro molecular and cellular experiments, animal model interventions, and mechanistic validation, incorporating a variety of technologies such as flow cytometry, molecular biology, and immunohistochemistry, all characterized by a clear multilevel and innovative design.

2. Detailed Experimental Steps

a) Preparation of TSG-6 Overexpressing MSCs

  • Subjects and Methods: Human bone marrow-derived MSCs (provided by RIKEN BRC) were cultured under standard conditions. The TSG-6 gene (TNFAIP6) was introduced using an adeno-associated virus (AAV) vector to generate TSG-6-overexpressing MSCs (TSG-6 MSCs). The control group (Null MSCs) received the empty AAV vector.
  • Novelty: The use of AAV for genetic modification avoids certain safety risks associated with other viral vectors. Optimization based on multiplicity of infection (MOI) ensured stable and high expression of TSG-6 mRNA.

b) Preparation of Extracellular Vesicles and Conditioned Medium

  • Work Flow: MSC culture supernatant was centrifuged (300×g and 2000×g sequentially to remove debris), followed by ultracentrifugation at 189,600×g to isolate extracellular vesicles (EVs). TSG-6 MSCs and Null MSCs yielded TSG-6 MSC-EVs and Null MSC-EVs, respectively. Conditioned media (CM) from both cell types were also collected for subsequent experiments.
  • Quality Control: Flow cytometry and Western blotting were employed to detect EV-specific markers (CD9, CD63, CD81), and nanoparticle size analysis and transmission electron microscopy characterized their size and morphology.

c) Animal Model and Intervention

  • Experimental Subjects: Eight-week-old male Sprague–Dawley (SD) rats were subjected to left renal ischemia for 1 hour followed by reperfusion to establish the IRI model, and were grouped as Sham, PBS control, Null MSC, TSG-6 MSC, and I3C MSC (treatment group, see below).
  • Intervention: After reperfusion, 5×10^5 MSCs (or PBS) were injected via the abdominal aorta. Animals were sacrificed at days 7 and 21, and left kidney tissues were collected for evaluation of inflammation and fibrosis.

d) Chemically Induced High Expression of TSG-6

  • Method and Innovation: Indole-3-carbinol (I3C, a plant-derived AHR agonist found in broccoli) was added to the MSC culture medium to activate and upregulate TSG-6 expression through the aryl hydrocarbon receptor (AHR) pathway. After dose optimization, 200μM was selected as a safe and effective concentration for cell treatment.

e) In Vitro Functional Experiments

  • Macrophage Polarization Assay: THP-1 monocytes were induced to differentiate into macrophages and then exposed to different CMs or EVs; CD163 (M2 marker) and CD11c (M1 marker) were detected via Western blotting or flow cytometry.
  • Regulatory T Cell Induction Assay: CD4+ naïve T cells were isolated from human peripheral blood and induced with MSC conditioned medium. Foxp3 expression was detected by Western blotting to determine the proportion of Treg cells.
  • TSG-6 Knockdown Experiment: MSCs were transfected with siRNA and treated with I3C to further test the essential role of TSG-6 expression in MSC function.

f) Tissue and Molecular Assessment

  • Immunohistochemistry and Fibrosis Staining: Kidney tissues were sectioned and stained for α-SMA, Collagen I, CD3, CD68, CD163, and Foxp3; Masson’s trichrome staining assessed the area of interstitial fibrosis.
  • Molecular Detection: Real-time qPCR and immunoblotting were used to detect molecular markers such as TSG-6, TGF-β, α-SMA, and Rap1.

3. Features and Innovative Approaches

  • AAV-mediated targeted TSG-6 overexpression in MSCs is robust and safe
  • I3C chemical modulation of MSC secretion of TSG-6 shows great potential for clinical translation
  • Systematic evaluation of the role of MSC EVs as TSG-6 carriers clarifies the “EVs-TSG-6-immunoregulation” pathway
  • Includes multi-scale studies (cellular, molecular, animal), rigorous mechanistic validation, and logical experimental arrangement

IV. Detailed Main Research Results

1. TSG-6 Is Secreted Mainly via Extracellular Vesicles

The study found that AAV-mediated overexpression of TSG-6 resulted in a stable increase in TSG-6 mRNA in MSCs, but intracellular TSG-6 protein was not significantly elevated, and free TSG-6 protein in the culture medium was below the ELISA detection limit. This clearly indicates that TSG-6 is mainly and rapidly secreted in EVs by MSCs, rather than stored in the cytoplasm. The TSG-6 protein level in EVs increased over time, while intracellular TSG-6 in MSCs remained relatively unchanged.

2. TSG-6 Overexpression Does Not Alter Fundamental MSC Properties

Assessment of surface markers and differentiation capacity showed that TSG-6 overexpression did not alter the MSC phenotype (CD29/CD44/CD73/CD90, etc.) or their adipogenic and osteogenic differentiation abilities, ensuring retention of essential properties for cell therapy.

3. TSG-6 MSCs Significantly Inhibit Renal Fibrosis in the Animal Model

In the rat IRI model, injection of TSG-6 MSCs more effectively inhibited TGF-β and α-SMA protein expression in renal tissue than Null MSCs. Masson’s trichrome staining and immunohistochemistry showed significant statistical reductions in fibrosis area and the proportions of collagen I and α-SMA positive regions, indicating that TSG-6 enhances the anti-fibrotic efficacy of MSCs.

4. TSG-6 MSCs Suppress Renal Inflammatory Cell Infiltration and Promote Immune Regulation

Inflammatory assessments showed that the number of infiltrating T-lymphocytes (CD3) and macrophages (CD68) in renal tissue was significantly reduced in the TSG-6 MSC group, while immunosuppressive M2 macrophages (CD163) and regulatory T cells (Foxp3) increased. This demonstrates that TSG-6 not only inhibits inflammation but also positively regulates immune tolerance.

5. Validation of TSG-6 EVs in Immune Cell Modulation

In vitro experiments showed that EVs containing TSG-6 upregulated CD163 expression and downregulated the M1 marker CD11c in macrophages, promoting polarization toward immunosuppressive M2. Conditioned medium depleted of EVs lost this effect. Furthermore, TSG-6-enriched MSC conditioned medium promoted CD4+ T cell conversion into Foxp3+ Tregs, highlighting the key role of TSG-6 in remodeling the immune microenvironment.

6. I3C Enhances MSC Expression of TSG-6 and Anti-fibrotic Function

AHR agonist I3C increased MSC TSG-6 mRNA expression in a dose-dependent manner. At safe concentrations, I3C-treated MSCs also demonstrated superior anti-fibrotic and anti-inflammatory effects in IRI rats compared to untreated MSCs, including significant reductions in α-SMA and collagen I expression and tissue fibrosis area. Specific knockdown of TSG-6 sharply weakened the protective effects of I3C-treated MSCs, directly confirming its dominant functional role.

7. Assessment of Paracrine Activity and Safety

Rap1 protein is essential for maintenance of MSC paracrine function. MSCs treated with AAV or I3C showed no significant changes in Rap1 expression, indicating that genetic or pharmacological intervention does not impair the inherent therapeutic activity of MSCs.

V. Conclusions and Profound Implications

This study systematically demonstrates:

  1. TSG-6 is the core effector of MSC function, secreted primarily in EVs that directly modulate immune cells to suppress inflammation and fibrosis.
  2. Enhancing TSG-6 expression — whether through gene engineering or I3C-based small molecule induction — can robustly improve the therapeutic efficacy of MSCs, bringing us closer to standardized and high-efficiency MSC therapy.
  3. The “EVs-TSG-6” axis represents a novel molecular mechanism for disease regulation and shows promise as a new therapeutic target for renal fibrosis and immune-related diseases.
  4. I3C, as a safe small molecule drug, offers a low-cost, highly accessible approach for clinical translation and personalized MSC intervention.

VI. Highlights and Distinctive Features

  • Clearly demonstrates that TSG-6 encapsulation in EVs is the key mode of action
  • Dual strategies using AAV and I3C to enhance MSC efficacy, with methodological innovation and translational potential
  • Elucidates the direct regulatory roles of EVs-TSG-6 in macrophage polarization and Treg induction, enriching new immunoregulatory mechanisms
  • Multi-scale, interdisciplinary experimental system ensures both scientific validity and application value
  • Lays the theoretical and technical foundation for the standardized, large-scale production of effective MSCs and their EVs, filling existing quality control gaps concerning heterogeneity and batch-to-batch variation

VII. Other Important Information

  • Part of the results have been previously presented at the American Society of Nephrology Annual Meeting.
  • The research team collaborated with Twocells Company, and the study was supported by Japanese research funds.
  • Supplementary materials and detailed data can be provided by the corresponding authors upon reasonable request.

VIII. Overall Evaluation and Outlook

This study not only elucidates, at a mechanistic level, a novel model by which MSC-secreted TSG-6, encapsulated in EVs, regulates immunity and inhibits fibrosis, but also provides practical and feasible strategies for promoting MSC/EV-based cell therapy and targeted regulation in clinical applications. The optimization of MSC quality and TSG-6 content via both AAV and I3C approaches points to a pathway toward greater standardization and large-scale production. This makes an important contribution to addressing key challenges in cell therapy, such as the control of heterogeneity, clarity of mechanisms, and application standardization. It represents a milestone in developing new solutions for the prevention and treatment of renal diseases—especially in AKI-CKD progression—and demonstrates broad potential for future clinical translation and widespread application.