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. 2025 Aug 21;28(9):113419. doi: 10.1016/j.isci.2025.113419

Exosomes derived from 3D-cultured hUCMSCs exhibit superior hepatoprotection against ALF

Huixin Tang 1,2,4, Wang Lu 1,2,4, Qi Yang 1,2, Yizhi Zhang 3, Xiaoxuan Liu 1,2, Shanshan Li 1,2, Manman Xu 1,2, Zhongping Duan 1,2, Li Bai 1,2,∗, Yu Chen 1,2,5,∗∗
PMCID: PMC12446390  PMID: 40978151

Summary

Most recently, exosomes (Exos) derived from 3D-cultured cells exhibit great advantages over 2D-Exos. Nevertheless, it remains to be explored whether 3D (tissue engineering liver) exosomes are superior to 2D-Exos in protecting mice from acute liver injury (ALF) and the underlying mechanism. Herein, we demonstrated that 3D-Exos manifested higher yields, protein concentration and RNA content compared to 2D-Exos. Remarkably, 3D-Exos exhibited superior hepatoprotection against ALF, as shown by reduced TBiL levels, improved liver architecture and alleviated inflammation. According to miRNA sequencing and KEGG enrichment analysis, the differentially expressed genes between 2D- and 3D-Exos could be enriched in “Hippo-YAP” and “autophagy” pathways. Moreover, the levels of YAP/TAZ and autophagy markers were more elevated in 3D-Exos-treated mice. Specifically, inhibiting YAP and autophagy signaling destroyed the hepatoprotection conferred by 3D-Exos. Collectively, 3D-Exos exert superior hepatoprotection against ALF by up-regulating YAP and autophagy signaling. Our finding provides powerful support for administering 3D-Exos to treat ALF efficiently.

Subject areas: Biochemistry, Tissue Engineering, Cell biology

Graphical abstract

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Highlights

  • •

    3D-Exos manifest higher yields, RNA content, and protein concentration

  • •

    3D-Exos exhibit superior hepatoprotection against ALF compared to 2D-Exos

  • •

    2D/3D-Exos DEGs associate with Hippo-YAP and autophagy pathways

  • •

    3D-Exos trigger more enhanced YAP/TAZ and autophagy signaling

Biochemistry; Tissue Engineering; Cell biology

Introduction

Acute liver failure (ALF) refers to a group of clinical syndromes in patients without underlying liver diseases, which is characterized by jaundice, ascites, coagulation dysfunction and hepatic encephalopathy. ALF is triggered by diverse etiologies such as viruses, alcohol and drugs, with rapid progression and high mortality.1,2 The treatment strategies for ALF consist of comprehensive medical treatment, artificial liver support therapy and liver transplantation. Nevertheless, these therapies face challenges such as limited efficacy, strict indication requirements or critical shortage of liver donors.1,3 In this context, it is imperative for researchers in this field to develop effective therapies for ALF.

Stem cell transplantation brings new hope for patients with ALF. Emerging reports have documented the hepatoprotective effects of stem cell-based therapies, especially MSCs, on multiple liver diseases, including ALF.4,5,6,7,8 However, cumulative evidence shows that stem cell transplantation faces some dilemmas, such as dedifferentiation risk, poor survival, inadequate homing, and tumor formation.9,10 In view of this, exosome-based cell-free therapy comes into being and quickly attracts wide attention from researchers.10,11 Exosomes are extracellular vesicles which can be released by almost all cells. The functional cargoes carried by exosomes, such as DNA, mRNA, miRNA, and proteins, can be effectively transferred into target cells, thereby regulating their biological function.12,13 Several studies have demonstrated the hepatoprotective effects of stem cell-derived exosomes on ALF.14,15,16 However, stem cells were cultured in a traditional single-layer (two dimension, 2D) mode in these studies. Although 2D culture can effectively expand stem cells, these cells grow in a circumstance that is markedly different from their in vivo microenvironment, especially lacking exposure to the extracellular matrix (ECM) and physiological circumstances; therefore, resulting in abnormal cell metabolism and protein expression. In view of this, three-dimensional (3D) culture technology of stem cells has gradually entered the field of view of researchers. Compared with 2D culture, 3D culture provides a preferable microenvironment for the proliferation of stem cells, such as the complex cell-cell and cell-ECM interactions, thereby improving their biological characteristics.17,18,19

The 3D tissue engineering liver is constructed based on decellularization and recellularization technology which has been creatively established by Yunfang Wang and our team.20 Briefly, liver cells are eluted from the livers through enzymatic digestion, and the resultant scaffold, which is composed of almost all known hepatic ECM components and matrix-bound soluble signals (e.g., growth factors/cytokines), was recellularized with hUCMSC suspension. In this circumstance, hUCMSCs will be implanted into the scaffold and cultured in a 3D system. Notably, this model reestablished cell-cell and cell-ECM communications, which are essential for maintaining cell activity and function.20 In this setting, cells grow and proliferate in a microenvironment that mimics the natural liver as much as possible.

In the present work, we aimed to characterize exosomes derived from 2D- and 3D-cultured hUCMSCs (2D- and 3D-Exos), and then we attempted to compare the efficacy of 2D- and 3D-Exos on Concanavalin A (ConA)-induced ALF and dissect the potential molecular mechanisms. For this purpose, hUCMSCs were cultured in a 2D and 3D system (tissue engineering liver), respectively, and then the supernatant was harvested for exosomes isolation. Exosomes from 2D- and 3D-cultured hUCMSCs were identified, and then administered into normal mice followed by ConA challenge. The hepatic damage was compared between ALF mice treated with 2D- and 3D-Exos. To dissect the molecular mechanism responsible for the discrepant efficacy of 2D- and 3D-Exos, we performed miRNA sequencing and GO/KEGG enrichment analysis. Furthermore, we validated the expression of relevant signaling molecules based on the results of KEGG enrichment analysis. This study showed that 3D-Exos exhibit better hepatoprotection against ConA-induced liver injury by promoting YAP/TAZ and autophagy signaling. Our finding provides insights and powerful support for treating ALF with 3D-Exos.

Results

The characterization of 2D- and 3D-cultured hUCMSC-Exos

First, we conducted the 2D- (conventional culture) and 3D cultures (cultured in a tissue engineering liver system) of hUCMSCs, respectively, then analyzed and compared the features of 2D- and 3D-Exos. Transmission electron microscopy (TEM) revealed that both 2D- and 3D-Exos displayed a classic “cup-shaped” structure with a bilayer membrane (Figure 1A). Western blot analysis showed that the exosome surface proteins, including CD63 and TSG101, were expressed in both 2D- and 3D-Exos. And obviously, enhanced the expression of CD63 and TSG101 was noticed in 3D-Exos compared with that in 2D-Exos (Figure 1B). Nanoparticle tracking analysis (NTA) demonstrated that the diameters of 2D- and 3D-Exos were about 135 nm, which are within the normal range of exosomes (Figure 1C). Together, these results support that 2D- and 3D-cultured hUCMSC-Exos have been successfully isolated.

Figure 1.

Figure 1

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Characterization of 2D-Exos and 3D-Exos

(A) The morphology of 2D-Exos and 3D-Exos by TEM analysis.

(B) Representative western blot for exosome markers (CD63 and TSG101). M represents a protein marker.

(C) Particle sizes and numbers of 2D- and 3D-Exos according to NTA.

(D) Difference in protein concentration between 2D- and 3D-Exos.

(E) Difference in RNA content between 2D- and 3D-Exos. Data are represented as mean ± SEM. Scale bars: 200 nm.

To characterize the exosomes from hUCMSCs cultured in a conventional 2D system and 3D system, respectively, we harvested the supernatants from hUCMSCs at the same cell counts. NTA and BCA assays were utilized to determine the yields of 2D- and 3D-Exos. According to NTA data, the concentration of 2D- and 3D-Exos was 1×1010 and 1.5×1010 particles/mL, respectively. BCA assay showed that the protein concentration of 3D-Exos was significantly higher than that of 2D-Exos (Figure 1D). Also, RNA content was remarkably higher in 3D-Exos than 2D-Exos (Figure 1E). Collectively, the exosomes from hUCMSCs cultured in a conventional 2D system and 3D system exhibit markedly different features, and 3D-Exos possess higher yield, protein concentration, and RNA content.

3D-Exos exhibit superior hepatoprotection against ConA-induced acute liver failure than 2D-Exos

Next, we analyzed the therapeutic efficacy of 2D- and 3D-Exos on ConA-induced acute liver failure. The levels of liver biochemical indexes, including ALT, AST, and TBIL, were significantly increased in response to ConA treatment. However, the levels of these indices were obviously decreased in mice treated with hUCMSC-Exos. Remarkably, the levels of ALT, AST, and TBIL were lower or exhibited a decreased trend in 3D-Exos compared with 2D-Exos (Figure 2A). Macroscopic changes (Figure 2B) and H&E staining (Figure 2C) manifested that hepatic damage was obviously alleviated in the livers of mice treated with hUCMSC-Exos plus ConA compared with those treated with ConA only. Notably, hepatic damage was evidently less severe in mice treated with 3D-Exos compared with 2D-Exos. We also compared the inflammation extent in mice treated with 2D- or 3D-Exos. The mRNA levels of inflammatory genes, including IL-1β, GSDMD, CASP-1, and NLRP3, were significantly lower in the liver tissues from mice treated with 3D-Exos compared to 2D-Exos (Figure 2D). Therefore, hUCMSC-Exos cultured in a 3D system exert enhanced hepatoprotective effect against ConA-induced acute liver failure.

Figure 2.

Figure 2

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3D-Exos exert enhanced hepatoprotection against ConA-induced acute liver failure

(A) Comparison in the levels of ALT, AST, and TBIL among ConA-treated mice with or without exosome transfer (n = 13–15).

(B) Representative macroscopic changes of the livers from ConA-treated mice with or without exosome transfer (n = 5).

(C) Representative H&E staining of the livers from ConA-treated mice with or without exosome transfer (n = 5).

(D) The mRNA expression of inflammatory genes, including IL-1β, GSDMD, CASP-1, and NLRP3, in the livers from ConA-treated mice with or without exosome transfer (n = 3–5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, data are represented as mean ± SEM. n: number of animals. Scale bars: 50 μm.

miRNA sequencing of 2D- and 3D-cultured hUCMSC-Exos

miRNAs are vital cargoes carried by exosomes. Numerous studies have documented the pivotal role of miRNAs in protective effects conferred by exosomes. Herein, we hypothesized that miRNAs are responsible for the better efficacy of 3D-Exos than 2D-Exos. For this reason, miRNA sequencing was performed using 2D- and 3D-Exos. Figure 3A displayed the differentially expressed miRNAs between 2D- and 3D-Exos. We also performed Gene Ontology (GO) and KEGG enrichment analysis for the target genes of these miRNAs, and the top 20 of enrichment results were displayed in Figures S3 and 3B.

Figure 3.

Figure 3

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miRNA sequencing of 2D- and 3D-exosomes and KEGG enrichment analysis of differentially expressed genes (DEGs)

(A) Volcano plots of differentially expressed miRNAs between 2D- and 3D-Exos.

(B) The top 20 of the KEGG enrichment analysis results of differentially expressed genes.

(C) Column diagram of differentially expressed miRNAs between 2D- and 3D-Exos.

3D-Exos trigger the up-regulated Yes-associated protein/ signaling in ConA-treated mice compared to 2D-Exos

Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), core members of the Hippo signaling pathway, hold a pivotal place in the regeneration and repair of damaged liver.21 Specially, recent studies have confirmed the critical role of YAP/TAZ over-expression in improving ALF.22,23,24 According to our miRNA sequencing data, the Hippo signaling pathway was in the list of top 20 enrichment pathways of DEGs between 2D- and 3D-Exos. Therefore, we conjectured that YAP/TAZ signaling may promote the tissue repair and functional improvement in ALF mice upon 3D-Exos treatment. Real-time PCR analysis manifested that the expression of YAP, TAZ, and TEAD2 was higher in 3D-Exos-treated livers as compared to 2D-Exos, although the difference was not statistically significant (Figure 4A). At the protein level, enhanced the expression of YAP and TAZ was noticed in 3D-Exos-treated livers as compared to 2D-Exos (Figure 4B). Together, 3D-Exos treatment leads to enhanced YAP/TAZ signaling and improved liver architecture in ALF compared to 2D-Exos.

Figure 4.

Figure 4

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3D-Exos treatment leads to up-regulated YAP/TAZ signaling compared to 2D-Exos

(A) The gene expression of Hippo signaling molecules, including YAP, TAZ, and TEAD2 in the livers of ConA-treated mice with or without exosomes transfer (n = 3–5).

(B) The protein levels of Hippo signaling molecules, including YAP and TAZ in the livers of ConA-treated mice with or without exosome transfer. Data are represented as mean ± SEM. n: number of animals.

3D-Exos lead to enhanced autophagy signaling in ConA-treated mice compared to 2D-Exos

Autophagy is a lysosome-mediated degradation of excess or aberrant long-lived cytoplasmic proteins and organelles.25,26 Cumulative evidence has demonstrated that autophagy is critically involved in the pathophysiological process of ALF.27,28,29 Our results of miRNA sequencing displayed that the autophagy signaling pathway was among the top 20 enrichment pathways of DEGs between 2D- and 3D-Exos. Thus, we speculated that autophagy may be responsible for superior hepatoprotection in 3D-Exos-treated mice. To test this speculation, we detected and compared the expression of autophagy-related markers in ALF mice treated with 2D- and 3D-Exos. According to our data, the mRNA levels of ULK1, ATG5, ATG16L1, ATG12, and LC3B were significantly higher or exhibited an elevated tendency in the livers of mice treated with 3D-Exos (Figure 5A). At the protein level, enhanced the expression of ATG16L1 and LC3B was observed in 3D-Exos-treated livers as compared to 2D-Exos (Figure 5B). Therefore, 3D-Exos treatment brings about enhanced autophagy signaling and preferable liver protection in ALF compared to 2D-Exos.

Figure 5.

Figure 5

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The 3D-Exos treatment brings about the enhanced autophagy signaling compared to 2D-Exos

(A) The gene expression of autophagy signaling molecules, including ATG5, ULK1, ATG12, ATG16L1, and LC3B in the livers of ConA-treated mice with or without exosome transfer (n = 4–5).

(B) The protein levels of autophagy signaling molecules, including ATG16L1 and LC3B, in the livers of ConA-treated mice with or without exosome transfer. Data are represented as mean ± SEM. n: number of animals.

The inhibition of Yes-associated protein and autophagy signaling destroys the hepatoprotection conferred by 3D-Exos

To confirm the critical role of YAP and autophagy in the hepatoprotection conferred by 3D-Exos, we administered YAP and autophagy inhibitors, respectively, into 3D-Exos-treated ALF mice, and then hepatic damage was assessed. According to H&E staining, the administration of YAP or an autophagy inhibitor aggravated the hepatic damage in 3D-Exos-treated mice, as shown by more massive necrosis and inflammatory responses (Figure 6). In other words, YAP or autophagy inhibition destroyed the hepatoprotection conferred by 3D-Exos. This finding provides further support for the pivotal place of YAP or autophagy signaling in the hepatoprotection conferred by 3D-Exos.

Figure 6.

Figure 6

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The inhibition of YAP or autophagy signaling destroys the hepatoprotection conferred by 3D-Exos

Representative H&E staining of the livers from Exo-treated ALF mice with or without YAP or autophagy inhibitor (n = 5). Exo, 3D-Exos; YAPINT, YAP inhibitor; Spautin, autophagy inhibitor. n: number of animals. Scale bars: 100 μm, 50 μm.

Discussion

In the present work, we utilized a tissue engineering liver model to conduct 3D culture of hUCMSCs, and compared the signature and therapeutic effects of exosomes derived from 2D- and 3D-cultured hUCMSCs. Moreover, we dissected the potential mechanism responsible for the divergent efficacy of 2D- and 3D-Exos on ConA-induced ALF. And we found: (1) Exosomes derived from hUCMSCs cultured in a tissue engineering liver system (3D-Exos) manifest higher yield, protein concentration and RNA content than 2D-Exos; (2) 3D-Exos exhibit superior hepatoprotection against ConA-induced ALF than 2D-Exos; (3) miRNA sequencing and KEGG enrichment analysis showed that DEGs between 2D- and 3D-Exos can be enriched in “Hippo-YAP” and “autophagy” pathways; (4) the levels of key signaling molecules closely related to YAP/TAZ and autophagy pathways are more elevated in the livers of mice treated with 3D-Exos compared with 2D-Exos; and (5) YAP or autophagy inhibition destroys the hepatoprotection conferred by 3D-Exos. Collectively, exosomes derived from 3D-cultured hUCMSCs exert superior hepatoprotection against ConA-induced acute liver failure by up-regulating YAP/TAZ and autophagy signaling. To the best of our knowledge, this study was conducted successfully 3D culture of hUCMSCs using a tissue engineering liver. In this setting, we characterized exosomes derived from 2D- and 3D-cultured hUCMSCs, and compared the efficacy of these two Exos on acute liver failure. Specifically, we demonstrated that 3D-Exos exhibit superior hepatoprotection against ALF than 2D-Exos, and dissected the potential molecular mechanisms from the perspective of YAP/TAZ and autophagy signaling.

First, we successfully constructed a tissue-engineering liver to conduct 3D culture of hUCMSCs. This ex vivo model was innovatively established by Professor Yunfang Wang and our team.20 We have previously established models of nonalcoholic fatty liver disease (NAFLD) and drug-induced liver injury based on this technology, which are utilized to investigate the pathogenesis of these diseases and to screen therapeutic drugs.30,31,32 Compared with other 3D culture methods such as spheroids and organoids, this model exhibits some advantages: strong proliferative capacity, no need for Matrigel, and dynamic perfusion.20,33 We then characterized the exosomes from 2D- and 3D-cultured hUCMSCs. According to our data, enhanced the expression of exosome markers, including CD63 and TSG101, was detected by western blot. In addition, the yield of exosomes is higher in 3D-cultured hUCMSCs compared with that in 2D-cultured hUCMSCs. Moreover, the protein concentration and RNA content are also elevated in 3D-cultured hUCMSCs relative to those in 2D-cultured hUCMSCs. These findings support the advantages of the 3D-culture system over the 2D-culture system. Specifically, we demonstrated the advantages of 3D-Exos over 2D-Exos.

Next, we compared the efficacy of exosomes coming from 2D- and 3D-cultured hUCMSCs on acute liver failure. As expected, both 2D- and 3D-Exos protect mice against ConA-induced ALF, as shown by reduced levels of liver function indexes and improved liver architecture. Notably, 3D-Exos exhibit a superior hepatoprotection against ConA-induced ALF compared with 2D-Exos, as manifested by lower levels of hepatic function indexes and better-preserved liver architecture. In view of the critical role of inflammasome-mediated inflammatory responses in acute liver injury,34 we also assessed and compared the inflammation extent in mice treated with 2D- or 3D-Exos. The mRNA levels of IL-1β, GSDMD, CASP-1, and NLRP3 were obviously reduced in 3D-Exos-treated mice compared to those in 2D-Exos-treated mice. Therefore, 3D-Exos provide a preferable protective effect against ConA-induced ALF as compared to 2D-Exos. The efficacy advantages of 3D-Exos over 2D-Exos have been reported in periodontitis and experimental colitis,35 osteochondral repair,36 and cerebral ischemia.37 As far as acute liver failure is concerned, this is the first work to compare the therapeutic effects of 2D- and 3D-Exos on ALF.

So, why are the hepatoprotective effects of 3D-Exos superior to 2D-Exos? In view of the pivotal role of miRNAs in the protective effects conferred by exosomes as mentioned in the introduction, we speculated that miRNAs carried by 3D-Exos hold a crucial place in the better hepatoprotection conferred by 3D-Exos. To testify to this speculation, we performed miRNA sequencing for 2D- and 3D-Exos. KEGG enrichment analysis showed that “MAPK signaling pathway” occupies the first place of the top 20 enrichment results of DEGs between 2D- and 3D-Exos. Hence, we detected and compared the expression of key molecules in the MAPK signaling pathway in the livers of mice treated with 2D- and 3D-Exos. Unfortunately, there is no significant difference in the expression of MAPK signaling molecules between 2D- and 3D-Exos treatment groups (data not shown). Similarly, there is also no obvious difference in the expression of “PI3K-AKT” signaling molecules between 2D- and 3D-Exos-treated mice (data not shown). So, we ruled out “MAPK” and “PI3K-AKT” signaling pathways, and there should be other signaling pathways responsible for the superior protection conferred by 3D-Exos.

The Hippo pathway is an evolutionarily conserved signaling pathway. It is critically involved in tissue regeneration, organ development, epithelial homeostasis, wound healing, and immune modulation. As the major transcriptional coactivator and core component of the Hippo signaling pathway, YAP/TAZ directs the expression of multiple downstream genes by binding to TEAD transcription factors. YAP/TAZ activity is mainly regulated by phosphorylation. When the Hippo pathway is activated, YAP/TAZ activity is inhibited by LATS1/2-mediated phosphorylation. Conversely, when the Hippo pathway is inactivated, dephosphorylated YAP/TAZ translocates into the nucleus and binds to TEAD to induce gene expression.21,38,39 Multiple studies have confirmed that YAP signaling plays crucial roles in diverse liver diseases, including fibrosis,40 and hepatocellular carcinoma.41 In terms of ALF, YAP has been demonstrated to play a key role in the hepatoprotection conferred by liver progenitor cells transplantation against CCl4-induced acute liver injury.38 Conversely, Yang Y and colleagues reported that activated Notch1 and YAP signaling in liver macrophages is closely related to D-GalN/LPS-induced acute liver injury, whereas the dual inhibition of Notch1 and YAP in macrophages promotes M2 polarization and alleviates liver damage.42 Our data showed that mice treated with 3D-Exos have less severe liver damage and higher levels of YAP/TAZ gene and protein expression compared with 2D-Exos, supporting that 3D-Exos exert the superior hepatoprotection against ALF by enhanced YAP/TAZ signaling as compared to 2D-Exos.

Autophagy is a lysosome-mediated intracellular degradation process. It holds a fundamental place in cell differentiation, development, survival, and homeostasis maintenance by eliminating cellular components such as defective organelles and aggregates of misfolded protein.25,43 Mounting evidence supports that the dysfunction of autophagy in liver parenchymal and non-parenchymal cells can result in diverse liver diseases, such as non-alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis, and hepatocellular carcinoma.25,44,45,46,47 Moreover, multiple studies have reported the pivotal but divergent roles of autophagy in ALF. For example, IRGM/Irgm1 was documented to alleviate inflammatory injury-induced ALF by enhancing autophagy.48 On the other hand, magnesium isoglycyrrhizinate was reported to ameliorate ConA-induced ALF by inhibiting autophagy.49 In the present work, enhanced autophagy was noticed in the livers of mice treated with Exos. Especially, 3D-Exos-treated livers manifested stronger activation of autophagy signaling, as manifested by elevated gene or protein levels of autophagy key markers including ATG5, ULK1, ATG12, ATG16L1, or LC3B. Therefore, 3D-Exos exert superior hepatoprotection against ALF by enhanced autophagy signaling as compared to 2D-Exos.

We also validated the pivotal role of YAP and autophagy signaling in the hepatoprotection conferred by 3D-Exos by administering YAP or an autophagy inhibitor. After the administration of YAP or an autophagy inhibitor, the hepatoprotection conferred by 3D-Exos is obviously weakened, as manifested by aggravated hepatic damage assessed by H&E staining in inhibitor-treated mice. This finding provides powerful support for the crucial role of YAP and autophagy signaling in the hepatoprotection conferred by 3D-Exos.

Remarkably, YAP and autophagy signaling are cross-linked. In hepatic ischemia reperfusion injury, the protective effect of YAP is strongly dependent on the activation of autophagy.50 Nevertheless, Lee YA et al. uncovered that YAP is a driver of tissue remodeling and carcinogenesis when autophagy is impaired.51 In our work, the gene and protein levels of both YAP/TAZ- and autophagy-related signaling molecules are up-regulated in the livers of mice treated with 3D-Exos, supporting the positive relationship between YAP and autophagy signaling. Nevertheless, the precise regulatory mechanism between YAP and autophagy signaling needs to be further explored.

In sum, we demonstrated that 3D-Exos exert superior hepatoprotective effects on ConA-induced ALF than 2D-Exos by up-regulating YAP/TAZ and autophagy signaling. Our findings will provide powerful support for administering 3D-Exos to treat ALF efficiently.

Limitations of the study

This work is a preliminary study with some limitations. First, we did not culture hUCMSCs in different 3D systems, and compared the features of exosomes from these systems to validate the advantages of our tissue engineering liver system. Second, it needs to be further determined which miRNA carried by 3D-Exos promotes better hepatoprotection than 2D-Exos. Third, the precise regulatory mechanism between YAP and autophagy should be validated by overexpression and knockdown of YAP and autophagy signaling molecules. Finally, it is important to note that this study was conducted exclusively in male animals, which limits the generalizability of our findings to female populations. While this approach controlled for potential confounding effects of estrogen cycles, it precludes conclusions about whether the observed mechanism is sex-dependent. Thus, our results may represent a male-specific phenomenon, and future work should include both sexes to assess the broader applicability of these findings.

Resource availability

Lead contact

Further information and requests for resources and reagents, as well as datasets and protocols, should be directed to and will be fulfilled by Yu Chen (chybeyond1071@ccmu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Acknowledgments

This work was supported by the National Key Research and Development Program (2022YFC2304402); the Construction Project of High-level Technology Talents in Public-Health (Discipline leader-01-12); the Beijing Hospitals authority’s Ascent Plan (DFL20221501); the Beijing Municipal Natural Science Foundation (7232081); the Scientific Research Project of Beijing Youan Hospital, CCMU, 2024 (BJYAYY-YN2024-10); the Beijing You’an Hospital Construction of Talent Pool Program (YARCKC2024001); and the Open project of Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research (BJYAHKF2023004); the Chinese Foundation for Hepatitis Prevention and Control—Tian Qing Liver Disease Research Fund Subject (TQGB20210013).

Author contributions

Y.C. and L.B. conceived the study. H.T. and W.L. participated in the study design and carried out the experiments. Y.Z., S.L., Q.Y., and X.L. assisted in cell cultures and animal experiments. H.T., W.L., and M.X. analyzed the data. L.B. and Y.C. were responsible for the interpretation of data. H.T. wrote the article, Z.D., L.B., and Y.C. polished the article, and Z.D. and Y.C. provided financial support.

Declaration of interests

The authors have no competing interests to declare.

STAR★Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
ATG16L1 Abcam Ab_187671
LC3II Abcam Ab_192890
YAP CST 8418t
TSG101 Abcam Ab_125011
CD63 Abclonal A19023
Chemicals, peptides, and recombinant proteins
ConA Sigma-Aldrich C2010-1G
YAP/TAZ inhibitor-2 MCE HY-147322
Spautin-1 MCE HY-12990
Deposited data
Accession code Gene Expression Omnibus GSE305065
Experimental models: Cell lines
hUCMSCs Tianjin Angsai Cell Gene Engineering Co., Ltd Cell Line:hUCMSCs-UC200030 P3
Software and algorithms
DEseq2 software TRANSCRIPTOMIC N/A
Prism 7.0 software Graphpad N/A
NTA software Particle Metrix N/A
cellSens imaging software Olympus America N/A
Other
Nanoparticle Tracking Analysis Particle Metrix Pmx110
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Experimental model and study participant details

Animals

Male C57BL/6J mice with body weight of 22∼25g, aged 6∼8 weeks or Sprague-Dawley rats (body weight 180∼220g, aged 6 weeks) were purchased from the Laboratory Animal Breeding Centre of SiBeiFu (Beijing, China). Mice were housed in a specific pathogen-free (SPF) environment under a standard 12h light/dark cycle. Animals were fed standard laboratory chow with free access to water. Animals were treated humanely in accordance with the requirement of the Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the Institutional Animal Care and Use Committee of Beijing YouAn Hospital, Capital Medical University (Approval NO. AEEI-2023-128).

Animal protocol

The experimental animals were treated as follows: (1) Control: C57BL/6J mice were given phosphate-buffered saline (PBS) as control. (2) The induction of acute liver failure: Mice were injected with ConA (Sigma-Aldrich, 15 mg/kg) via tail vein. (3) Exosome treatment: 2D- or 3D-cultured hUCMSC-derived exosomes (2×109 particles) were administered into normal mice via tail vein, followed by ConA induction. (4) YAP or autophagy inhibition: YAP inhibitor YAP/TAZ inhibitor-2 (10 mg/kg) or autophagy inhibitor Spautin (2mg/kg) was administered into 3D-Exo-treated ALF mice. Sera and liver tissues were harvested for analysis.

2D and 3D culture of hUCMSCs

For 2D culture, hUCMSCs (Tianjin Angsai Cell Gene Engineering Co., Ltd) were cultured in an exosome-free DMEM medium (Gibco). For the cell line, to confirm the successful isolation of hUCMSCs, verification was performed using methods such as flow cytometry, and the cell line was also tested for mycoplasma contamination.

For 3D culture, we first constructed the 3D tissue engineering liver according to previously reported method.20 SD rats were anesthetized, and the skin preparation was performed, followed by abdominal disinfection with 75% alcohol. Then the abdominal cavity was opened to expose the liver and portal vein. A disposable venous indwelling needle was inserted into the portal vein, which will serve as the perfusion entrance to the hepatic vascular system. Subsequently, the liver was decellularized by perfusing 1% sodium deoxycholate (1% SDC) buffer containing 20U/L phospholipase A2. After that, the liver was rinsed with saline. At this point, a cell-free liver scaffold with all extracellular matrix components preserved can be obtained. Then, this scaffold was dissociated, trimmed and ligated with some liver lobes retained. After that, the scaffold was suspended in a sterile complicated-shape bottle with DMEM medium preloaded. Afterwards, the bottle was connected to the peristaltic pump (MasterFlex, USA). The unit was placed into an incubator with 5% CO2 at 37 °C, and recirculated overnight (Figure S1). Twenty-four hours later, the medium was replaced with complete medium (DMEM+ 10% FBS). And hUCMSC suspension (about 1×107 cells each time, three times) was injected into the unit, and perfusion rate was set at 8 mL/min. In this circumstance, hUCMSCs will enter the scaffold. The supernatant was collected every 24 hours for exosome isolation.

The macroscopic changes of decellularized 3D tissue engineering liver scaffold were shown in Figure S2A, and the microscopic changes of this scaffold were manifested in Figure S2B. Recellularized tissue engineering liver under scanning electron microscope and transmission electron microscope were exhibited in Figures 2C and 2D.

Method details

Exosomes isolation and characterization

Exosomes were isolated using differential centrifugation according to previous methods with minor modification.52 Briefly, the supernatant of hUCMSCs was collected and centrifuged consecutively at 300×g for 10 min followed by 2000×g for 10 min to remove cell debris. The resultant supernatant was collected and centrifuged at 10,000×g for 30 min followed by 100,000×g for 90 min at 4°C. The pellet at the bottom of the ultracentrifuge tube was re-suspended in sterile PBS, and centrifuged at 100,000×g for 90 min at 4°C. The obtained exosomes were re-suspended in PBS and stored at -80°C.

The morphology of the exosomes was observed with transmission electron microscopy (TEM, Hitachi-HT7700, Tokyo, Japan). The particle concentration, size, and distribution were measured by nanoparticle tracking analysis (NTA, PARTICLE METRIX-ZetaVIEW, Germany). Exosomes markers were detected by western blot using primary antibodies against CD63 and TSG101.

Reverse transcription and SYBR green quantitative polymerase chain reaction (qPCR)

Total RNA from liver tissues was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Then, reverse transcription was performed using the AMV retrotranscriptase system (TaKaRa, Dalian, Liaoning, China). qPCR amplifications were run in triplicate on an ABI StepOne Plus System (Thermo Fisher Scientific) using a TB Green reaction mix (TaKaRa). In a 20 μl reaction volume, the following reagents were added: 1× TB Green PCR master mix, template cDNA, 0.5 mM of each primer, and ROX. The thermal cycling protocol was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The primers used in this work were designed by Primer 5.0 and are listed in Table S1. The relative expression of the target genes was calculated according to 2-ΔΔCT method and normalized to Gapdh expression.

Exosome small RNA extraction, library construction, miRNA sequencing and bioinformatics analysis

Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer’s instructions. Library construction was performed using QIAseq® miRNA Library Kit (Qiagen) according to the manufacturer’s protocol. miRNA sequencing was performed on an Illumina HiSeq2500 sequencer. Quality control was conducted on raw reads to obtain clean reads for differential expression analysis of miRNA between 2D- and 3D-exosomes, using DEseq2 software (p value < 0.05, |log2foldchange|> 1. Target genes for differentially-expressed miRNAs were predicted, and Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using Database for Annotation, Visualization and Integrated Discovery (http://david.niaid.nih.gov).53

Western blot analysis

Total proteins were extracted from frozen liver tissues using RIPA lysis buffer supplemented with Halt™ protease inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined using the bicinchoninic acid (BCA) method. Samples were then subjected to SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% BSA for 1 h at room temperature, followed by overnight incubation at 4 °C with the following primary antibodies: TSG101 (1:1000, Abcam), CD63 (1:1000, Abclonal), YAP (1:1000, CST), LC3II (1:1000, Abcam), ATG16L1 (1:1000, Abcam) and β-actin. The protein bands were visualized by Luminol ECL reagent (Thermo Fisher Scientific).

Liver histopathology

Liver tissues were fixed in 10% buffered formalin and embedded in paraffin for sectioning. And the sections were stained with hematoxylin & eosin according to the standard operating procedure. Histological images were captured using an Olympus Bx51 microscope (Olympus America, Melville, NY, USA) and processed with cellSens imaging software (version 1.4.1).

Transmission electron microscopy (TEM)

The isolated exosomes were suspended in 50-100 μl of 2% paraformaldehyde (PFA) solution (stable at 4°C for one week). For TEM sample preparation, 5-10 μl of the exosome suspension was loaded onto Formvar-carbon coated copper grids. The grids were then washed by placing them (Formvar film side down) on a 100 μl PBS droplet deposited on Parafilm, using fine-tip forceps for handling. Then the samples were prepared using negative staining combined with aldehyde fixation. TEM imaging was performed at 80 kV acceleration voltage (TEM, Hitachi-HT7700, Tokyo, Japan).

Nanoparticle tracking analysis (NTA) of exosomes

Samples were diluted with distilled water to achieve particle concentrations between 1×107/ml and 1×109/ml. Particle quantification and size distribution were determined using the PARTICLE METRIX-ZetaVIEW instrument (Particle Metrix, Germany) with a 405 nm laser. Image acquisition was performed at 30 frames per second for a duration of 1 minute. Particle movement was analyzed using NTA software (ZetaView 8.02.28). Detailed results and raw data are presented in the corresponding report.

Quantification and statistical analysis

Statistical analysis

The results are expressed as the mean ± standard error of the mean or median (Min, Max). Group comparisons were performed using Student’s t-test, the Mann–Whitney U test, and one-way ANOVA followed by Tukey’s multiple comparison tests, as appropriate. Statistics and graphs were generated using Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). p <0.05 was considered statistically significant.

Published: August 21, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.113419.

Contributor Information

Li Bai, Email: tender78@ccmu.edu.cn.

Yu Chen, Email: chybeyond1071@ccmu.edu.cn.

Supplemental information

Document S1. Figures S1–S3, Table S1 and Data S1
mmc1.pdf (1.6MB, pdf)
Table S2. Differently expressed miRNA between 2D- and 3D-Exos
mmc2.csv (9.1KB, csv)
Table S3. Target genes for differently expressed miRNA between 2D- and 3D-Exos
mmc3.csv (9.2MB, csv)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S3, Table S1 and Data S1
mmc1.pdf (1.6MB, pdf)
Table S2. Differently expressed miRNA between 2D- and 3D-Exos
mmc2.csv (9.1KB, csv)
Table S3. Target genes for differently expressed miRNA between 2D- and 3D-Exos
mmc3.csv (9.2MB, csv)

Data Availability Statement

Articles from iScience are provided here courtesy of Elsevier

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