Effect of Adipose-Derived Stem Cells Embedded in Fibrin Gel on Post-Hepatectomy Liver Regeneration: A Therapeutic Model Inducing a Paracrine Effect

doi:10.21203/rs.3.rs-4783251/v1

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Effect of Adipose-Derived Stem Cells Embedded in Fibrin Gel on Post-Hepatectomy Liver Regeneration: A Therapeutic Model Inducing a Paracrine Effect

https://doi.org/10.21203/rs.3.rs-4783251/v1

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We investigated the potential of adipose-derived stem cells (ADSCs) in preventing post-hepatectomy liver failure, emphasizing the necessity of direct administration using a scaffold to harness their paracrine effects. A fibrin gel scaffold was employed for ADSCs (gelADSCs) to assess their therapeutic impact on liver regeneration in both in vitro and in vivo settings. Experiments were conducted on C57BL/6 mice with normal livers and those with chronic hepatitis. We also explored the role of extracellular vesicles (EVs) secreted by ADSCs in conjunction with fibrin gel. GelADSCs showed sustained release of hepatocyte growth factor, vascular endothelial growth factor, and stromal cell–derived factor 1 for at least 7 days in vitro. In vivo, gel ADSCs significantly enhanced postoperative liver regeneration by upregulating the cell cycle and fatty acid oxidation in both normal and chronically hepatitis-affected mice. The therapeutic effects of gel ADSCs were notably superior to those of intravenously administered ADSCs, especially in mice with chronic hepatitis. Increased EV secretion associated with fibrin gel use was significantly linked to enhanced liver regeneration post-surgery through the promotion of fatty acid oxidation. The findings underscore the enhanced therapeutic potential of gelADSCs, particularly in the context of chronic hepatitis, compared to intravenous administration.

Biological sciences/Stem cells/Mesenchymal stem cells

Health sciences/Anatomy/Gastrointestinal system

Health sciences/Gastroenterology/Hepatology

Health sciences/Medical research/Translational research

hepatectomy

liver regeneration

adipose-derived stem cell

paracrine effect

fibrin gel

Post-hepatectomy liver failure (PHLF) is a relatively rare but potentially life-threatening complication of hepatectomy. The incidence of PHLF is reported to range from 8–12%, with 2–10% mortality, depending on the volume of the resected liver ¹. Although liver transplantation is the only treatment for PHLF that is definitive and curative, it is potentially limited by donor availability and the invasiveness of surgery.

Adipose-derived stem cells (ADSCs) have been reported to have tissue reparative effects in various organ dysfunctions ². The use of ADSCs post-hepatectomy is reported to be a potential preventive measure against PHLF ³. The actions of ADSCs include endocrine, paracrine, and autocrine effects, with paracrine effects being particularly important for tissue repair ^4,5. Paracrine effects refer to the mechanism by which ADSCs administered in a scaffold at the site of damage secrete various kinds of cytokines, promoting tissue repair by acting directly on the damaged tissue ⁴. For example, in animal models of bone or skin defects, tissue repair has been achieved by administering ADSCs to the defect site within a scaffold ^6,7. However, the paracrine effects of ADSCs on liver regeneration in hepatectomy models have not been fully investigated, because in animal experiments ADSCs are commonly administered by intravenous injection. It has been reported that ADSCs injected into the circulation are mostly trapped in the lungs and do not reach the target organ (i.e., the liver) ^8,9. Sid‑Otmane et al. reported that intravenous administration of ADSCs induced an “endocrine-like” effect, since ADSCs which were intravenously administered exhibited therapeutic effects at sites remote from the lungs ⁴. In other words, intravenous administration may not be an optimal delivery mode in which ADSCs can exert their paracrine effects.

This study was conducted to evaluate the paracrine effect of ADSCs in the setting of hepatectomy. We utilized a fibrin gel (FG) as a scaffold for administering ADSCs for the three reasons. First, FG has already been reported to be an excellent scaffold that provides a favorable environment for ADSCs ^10,11. Second, FG is widely used in surgical practice to prevent bleeding and bile leakage after liver resection ¹². Therefore, the safety and usefulness of FG in the setting of hepatectomy have been sufficiently verified. Third, since FG can expand itself, it is suitable for covering the liver, a three-dimensional structure. In this study, we created a mouse liver resection model and aimed to verify the paracrine effects of ADSCs on liver regeneration by administering ADSCs to the liver surface, using FG as a scaffold. We also evaluated the therapeutic potential of our method by comparing it with that of intravenously administered ADSCs.

1. GelADSC displayed favorable survival and cytokine secretion in vitro.

In this study, gelADSC refers to 1.0 × 10⁶ ADSCs embedded in 2-fold diluted FG. First, we evaluated the in vitro dynamics of gelADSC. Histological examination of gelADSC cultured in vitro for 7 days revealed a considerable number of ADSCs within FG (Fig. 1a). TUNEL staining demonstrated a substantial population of viable ADSCs (Fig. 1b). Subsequently, the assessment of FG's influence on the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and stromal cell-derived factor-1 (SDF-1) revealed consistently higher levels from gelADSC compared to those cultured without FG, and the levels of secreted cytokines continued to increase until day 7 (Fig. 1c). Furthermore, western blot (WB) analysis revealed that the expression of hypoxia inducible factor-1 α (HIF-1α) was heightened in the gelADSC group on days 3, 5, and 7 (Fig. 1d; the uncropped membrane was shown in Supplementary Fig. S1). These results suggest that gelADSC provided a favorable scaffold on which ADSCs were able to survive for at least 7 days, with the hypoxic condition potentially upregulating cytokine expression.

2. ADSCs survived in FG in hepatectomized mice.

Next, the dynamics of gelADSC in vivo were evaluated. GelADSC were extracted from hepatectomized mice on POD 7. Histological analysis using hematoxylin & eosin (H&E) staining (Fig. 2a) and TUNEL staining (Fig. 2b) revealed that, similar to the in vitro results, the majority of ADSCs within FG remained viable in vivo on POD 7. Furthermore, the in vivo distribution of ADSCs was assessed by labeling them with iron and tracking them by magnetic resonance imaging (MRI). Appropriate labeling was confirmed by Berlin staining (Fig. 2c). In the gelADSC group, FG on the liver surface was detected as a low-intensity signal on T2-weighted imaging (T2WI), reflecting the presence of iron (Fig. 2d). Notably, on PODs 1, 3, and 7, the liver exhibited a low-intensity signal on T2WI in the ivADSC group compared with the livers in the control group and the gelADSC group. Taken together, ADSCs remained within FG in the hepatectomized mice for a duration of 7 days, demonstrating continuous viability.

3. GelADSC promoted liver regeneration after hepatectomy in normal liver through upregulation of the cell cycle and fatty acid oxidation.

In the gelADSC group, gelADSC were placed on the surface of the remnant liver after 70% partial hepatectomy (Fig. 3a). The remnant liver to body weight ratio (LTBR) significantly increased in the gelADSC group (mean: 0.034) compared with the control group (mean: 0.029) on POD 2 (Fig. 3b). Immunostaining of proliferating cell nuclear antigen (PCNA) on thin sections of the liver on POD 2 showed a significant increase in PCNA-positive cells in the gelADSC group compared with the control group (Fig. 3c). Pathway analysis using RNA-seq revealed a significant upregulation of the cell cycle pathway in the gelADSC group (Supplementary Fig. S2a). H&E staining on POD 2 revealed less accumulation of lipid droplets in the gelADSC group (Fig. 3c). A cholesterol assay and triglyceride assay using liver samples demonstrated a significantly lower triglyceride content in the gelADSC group (Fig. 3d). RNA-seq analysis revealed a significant upregulation of fatty acid oxidation in the gelADSC group (Supplementary Fig. S2b). Polymerase chain reaction (PCR) analysis of mRNA extracted from liver samples showed significant upregulation of carnitine O-octanoyltransferase (CROT), carnitine palmitoyltransferase 1a (CPT-1a), and peroxisome proliferator-activated receptors α (PPARα), which are involved in fatty acid oxidation, in the gelADSC group. In contrast, no significant differences were observed in the mRNA expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1c (SREBP-1c), which are involved in fatty acid synthesis (Fig. 3e). In addition, in the 90% hepatectomy model, the gelADSC group exhibited significantly improved survival compared with that of the control group (p = 0.0013; Supplementary Fig. S3).

The therapeutic effect of gelADSC was further evaluated by comparing the gelADSC group and the ivADSC group. The LTBR on POD 2 was comparable between the two groups (mean: 0.033) (Fig. 3f). The histological analyses revealed that the number of PCNA-positive cells and the extent of lipid accumulation were comparable between the two groups (Fig. 3c). In the ivADSC group, the expression of Cpt-1a, Crot, and PPARα mRNAs was significantly downregulated in comparison with that in the gelADSC group (Fig. 3e). In summary, gelADSC contributed to post-hepatectomy liver regeneration by upregulating the fatty acid oxidation and cell cycle pathways. Moreover, the therapeutic impact of gelADSC was comparable to that of ivADSC, although the expression of mRNAs associated with fatty acid oxidation was upregulated in the gelADSC group.

4. GelADSC promoted liver regeneration after 70% hepatectomy in liver with chronic hepatitis by upregulating the cell cycle and fatty acid oxidation.

We investigated the therapeutic effects of gelADSC for hepatectomy in mice with chronic hepatitis. Five weeks of preoperative thioacetamide (TAA) administration successfully induced liver fibrosis, which was confirmed by Masson’s trichrome staining (Fig. 4a). The LTBR did not differ significantly between the control group and the gelADSC group on POD 2, but it did on POD 3 (mean: 0.034 vs. 0.037) (Fig. 4b). H&E staining demonstrated reduced intracellular lipid droplets in the gelADSC group (Fig. 4c). Immunostaining for PCNA revealed an increase in the positive cells in the gelADSC group (Fig. 4c). Furthermore, PCR analysis demonstrated significant upregulation of enzymes involved in fatty acid oxidation in the gelADSC group compared to the control group (Fig. 4d).

The therapeutic effect was further evaluated by comparing the gelADSC group with the ivADSC group. The LTBR on POD 3 was significantly increased in the gelADSC group (mean: 0.033 vs. 0.037) (Fig. 4e). The histological analyses revealed that the number of PCNA-positive cells was increased in the gelADSC group, although lipid accumulation was comparable between the two groups (Fig. 4c). The mRNA expression of Cpt-1a, Crot, and PPARα was significantly increased in the gelADSC group (Fig. 4d).

In summary, gelADSC promoted post-hepatectomy liver regeneration by upregulating the cell cycle and fatty acid oxidation in the presence of chronic hepatitis. In this model, the therapeutic impact of gelADSC was superior not only to the control group but also to the ivADSC group.

5. The hepatic distribution of ADSCs administered through a tail vein decreased in the presence of chronic hepatitis.

The post-hepatectomy distribution of ADSCs in a chronic hepatitis model mouse was evaluated by MRI. In the gelADSC group, FG containing iron-labeled ADSCs was consistently enhanced by a low-intensity signal for 7 postoperative days (Fig. 5a), just as it was after hepatectomy for normal liver (Fig. 2d). In contrast, in the ivADSC group, the liver did not display a low-intensity signal as it did after hepatectomy for normal liver. This finding was confirmed by the comparison of the liver signal intensity relative to that of muscle between the two ivADSC groups (i.e., the ivADSC group mice with normal liver and those with chronic hepatitis). The signal intensity was significantly higher in the livers of the ivADSC group with chronic hepatitis than in the livers of the ivADSC group with normal livers (Fig. 5b). This result may suggest that the number of intravenously administered ADSCs that reached the liver was smaller in the chronic hepatitis model than in the normal liver model.

6. Extracellular vesicles play an important role in liver regeneration by gelADSC.

Finally, we examined the impact of extracellular vesicles (EVs) released from ADSCs on the liver regeneration after hepatectomy. In in vitro experiments, ADSCs secreted more EVs when encapsulated in FG than when cultured without it, and EV secretion increased as time progressed (Fig. 6a). Blood samples from hepatectomized mice treated with gelADSC showed the presence of EV in serum from 8–16 hours post-hepatectomy, persisting for at least 48 hours (Fig. 6b). In this experiment, anti-CD63 antibody was used as an EV marker since this antibody does not have reactivity with mouse tissue. Subsequently, we inhibited ADSCs’ EV secretion by siRNA-mediated knockdown of ALIX (referred to as ADSC^siALIX) ⁹. The qPCR analysis confirmed the reduction of ALIX gene expression (Fig. 6c), and the attenuation of EV secretion was observed through reduced expression of EV markers such as TSG101 and syntenin in WB analysis (Fig. 6c; the uncropped membrane was shown in Supplementary Fig. S1). The secretion of cytokines, specifically HGF and SDF-1, by ADSCs was significantly reduced upon silencing ALIX mRNA, although the VEGF secretion remained unchanged (Fig. 6d). The impact of EVs on hepatectomy-induced liver regeneration was assessed using gelADSC^iALIX. The LTBR on POD 2 was significantly decreased in the gelADSC^iALIX group relative to that in the gelADSC^iControl group (mean: 0.035 vs. 0.038) (Fig. 6e). Moreover, the expression of CROT and PPARα mRNAs was significantly reduced in the gelADSC^iALIX group (Fig. 6f). The findings suggest a significant contribution of EVs to post-hepatectomy liver regeneration, by upregulating the cell cycle and fatty acid oxidation along with the release of important cytokines.

The importance of the paracrine effect of ADSCs has been reported in various organ dysfunction models ². To obtain a paracrine effect, ADSCs must be administered in the proximity of the damaged organ. However, in a liver damage model, the administration of ADSCs directly to the liver surface is technically demanding, due to the need for general anesthesia and laparotomy, and thus intravenous administration of ADSC is often employed alternatively. The major disadvantage of intravenous administration of ADSCs is that the majority of ADSCs are trapped in the lungs ⁸ ⁹: a case of pulmonary embolism probably attributable to intravenously administered ADSC has been reported ¹³. Therefore, the paracrine effect of ADSCs in liver dysfunction models has been insufficiently validated. In this context, Hu et al. emphasized the need for evaluating appropriate ADSC administration routes to treat liver damage ¹⁴. In this study, we investigated the effectiveness of a therapeutic model expected to exert a paracrine effect by administering ADSCs to the surface of the liver using FG as a scaffold in a liver resection model.

The therapeutic mechanism of gelADSC is summarized in Fig. 7. Our in vitro experiments showed continuous secretion of VEGF, HGF, and SDF-1, crucial for liver regeneration ^15-17, for over 7 days when ADSCs were enclosed in FG. The crucial period for liver regeneration after hepatectomy is reported to be 7 days post-surgery ¹⁸, suggesting that gelADSC is an effective therapy for liver regeneration following liver resection. Additionally, increased HIF-1α expression was noted in gelADSC, which may have led to the enhanced secretion of VEGF, HGF, and SDF-1 ^19-21. Taking these results together, gelADSC present a promising model for ADSCs’ paracrine effects on post-hepatectomy liver regeneration.

The therapeutic potential of gelADSC was subsequently assessed in vivo using a mouse hepatectomy model, revealing enhanced liver regeneration via upregulation of fatty acid oxidation and the cell cycle. Our results show that gelADSC contributed to lipolysis (i.e., fatty acid oxidation) rather than lipogenesis, consistent with a previous report ²². The intravenous administration of ADSCs is a potent therapy for liver regeneration after hepatectomy of normal liver, despite the reports of entrapment by the lung ^8,9. Sid‑Otmane et al. reported that intravenously administered ADSCs exert their therapeutic effect by an “endocrine-like” mechanism, where ADSCs trapped in the lung remotely function to repair damaged organs ⁴. In the current study, the therapeutic impact of ivADSCs was comparable to that of gelADSC. However, our MRI results showed that gelADSC and ivADSCs showed different distribution of ADSCs. In the gelADSC group, ADSCs remained in FG on the surface of the liver for at least 7 days after hepatectomy, potentially enhancing their paracrine effect. In contrast, in the ivADSC group, the MRI signal in the liver was enhanced at a comparatively low level for 7 days postoperatively. Previous studies suggested that intravenously administered ADSCs remain in liver for only 2 to 3 days ⁸, which seems contrary to our MRI findings. However, we need to be cautious in the interpretation of MRI because the presence of ADSCs was indirectly evaluated as the presence of iron. Overall, gelADSCs exhibited a paracrine effect, promoting liver regeneration via fatty acid oxidation and cell cycle, and ivADSCs also displayed comparably potent effects in normal liver hepatectomy models.

The therapeutic effect of gelADSC was also evaluated in mice with underlying chronic hepatitis because, in current surgical practice, approximately 54% of the patients undergoing hepatectomy have chronic hepatitis ²³. In a mouse model of underlying chronic hepatitis, gelADSC demonstrated significant upregulation of fatty acid oxidation and cell cycle compared to both the control and ivADSC groups. Interestingly, our MRI findings indicate that the distribution of intravenously administered ADSCs differed according to the presence or absence of underlying chronic hepatitis. Specifically, fewer ADSCs reached the liver in the chronic hepatitis model compared to the normal liver model. This discrepancy may explain why ivADSCs showed inferior therapeutic potential to gelADSCs in the chronic hepatitis model, unlike in the normal liver model. Therefore, especially in patients with chronic hepatitis, gelADSCs could be a preferable therapeutic option for liver regeneration post-hepatectomy over ivADSC.

EVs have been considered crucial to liver regeneration by gelADSC. Hypoxia has been reported to promote the secretion of EVs ²⁴. Our study found that ADSCs secrete significantly more EVs when embedded in FG (Fig. 6a), suggesting a hypoxic environment in FG favored EV release. Interestingly, EVs appeared in the serum of hepatectomized mice treated with gelADSC from 8–16 hours until at least 48 hours after surgery (Fig. 6b). A previous report described that, when ADSCs was intravenously administered, the amount of serum EV markedly increased at 4 hours after administration, rapidly decreasing thereafter ⁹. The continuous release of EVs from gelADSC may have contributed to enhanced liver regeneration after hepatectomy. Moreover, inhibiting the release of EVs from ADSC in vivo led to a significant decrease in HGF and SDF-1 secretion in vitro, reduced LTBR levels, and downregulated mRNA levels related to fatty acid oxidation. This underscores the therapeutic role of EVs in gelADSC. Indeed, the concept of using EVs instead of ADSCs has already been reported, due to EVs’ lack of immunogenicity ²⁵. Nevertheless, the application of gelADSC led to a time-dependent and persistent increase in EV secretion (Fig. 6a), offering a clinical advantage in liver regeneration post-hepatectomy.

In clinical scenarios like living-donor liver transplantation, gelADSC holds promise beyond post-hepatectomy use. Addressing concerns like small-for-size syndrome ²⁶, administering gelADSC to the grafted liver could facilitate liver regeneration. Although a large area must be covered by gelADSC, considering the size of the human liver, Mori et al.'s cell spray method ²⁷, offers a potential solution. This technique, previously used to spray FG onto the heart, ensures consistent distribution on the liver surface.

The current study has some limitations. First, due to the use of ADSCs of human origin, the therapeutic potential of gelADSC as an allograft or autograft was not evaluated. Second, the validity of using TAA to induce chronic hepatitis is controversial. The pathogenesis of chronic hepatitis in the human being is quite different. Third, we need to be careful in interpreting MRI images. It remains unclear how long ADSCs can retain iron after being administered in vivo.

In conclusion, the therapeutic impact of ADSCs’ paracrine effect in the post-hepatectomy setting was validated using FG as a scaffold, highlighting their superiority over intravenously administered ADSCs, particularly in the context of underlying chronic hepatitis.

1. Human ADSCs and FG preparation

Human ADSCs of fourth passage (Lonza Japan, Tokyo) were cultured at 37 °C in a 5% CO₂ atmosphere for a few days. FG was purchased from CSL Behring (King of Prussia, PA, USA).

2. Assessment of cytokine secretion and survival of ADSCs in FG

First, to assess the ADSCs’ survival within FG, gelADSC was cultured for 7 days in DMEM (high glucose) with 2% FBS. After formalin fixation, gelADSC was paraffin-embedded for H&E staining to evaluate morphology. TUNEL staining was utilized to assess the proportion of viable cells using an apoptosis kit (Medical Biological Lab, Nagoya, Japan). Optical microscopy (Keyence, Osaka, Japan) was used for examination of the images.

Furthermore, we evaluated the dynamics of cytokine secretion by gelADSC by culturing it in high-glucose DMEM containing 2% FBS until day 7. No medium change was performed during the incubation period. Supernatants were collected at five time points (days 1, 2, 3, 5, 7). The control group consisted of ADSCs dispersed in high-glucose DMEM containing 2% FBS without the use of FG and underwent the same collection. The concentrations of VEGF, HGF, and SDF-1 per 1.0 × 10⁵ ADSCs in the supernatants were evaluated by ELISA (Abcam). Then, to assess the unique environment within the FG, WB analysis of HIF-1α (Abcam) was performed using the samples obtained on days 3, 5, and 7.

3. Mouse hepatectomy models and ADSC administration

This study was approved by the Animal Experiments Committee, Osaka University (approval number 03-001-003). Animal care was in accordance with institutional guidelines. The animal studies were conducted in accordance with the ARRIVE guidelines. Male 7-week-old C57BL/6 mice, which were purchased from Clea Japan (Tokyo, Japan), were acclimatized for 1 week before experiments and housed with a 12-hour dark/light cycle.

A 70% partial hepatectomy was performed as previously described, with the mice under general anesthesia using medetomidine, midazolam, and butorphanol ²⁸. In the gelADSC group (n=5), FG containing 1.0 × 10⁶ ADSCs was attached to the remnant liver surface immediately after liver resection. In the ivADSC group (n=5), the same number of ADSCs were injected into a tail vein and FG without ADSCs was placed on the liver. In the control group (n=5), FG without ADSCs was placed. 90% partial hepatectomy was performed as a fatal hepatic failure model ²⁹. In this model, the survival of the mice was examined every 6 hours and compared between the gelADSC group (n=16) and the control group (n=16).

We also evaluated the effect of gelADSC on liver with chronic hepatitis. The mice were injected intraperitoneally with 150 mg/kg TAA twice a week for 5 weeks in accord with our previous report ³⁰. The liver fibrosis induced by TAA was confirmed by staining with Masson trichrome. A 70% partial hepatectomy was performed 2 to 3 days after the final TAA administration.

In each hepatectomy model, the liver and serum samples were collected and assessed at several time points.

4. Cholesterol and triglyceride assays

Lipid was extracted from a mouse liver sample by the method of Folch et al. ³¹. Triglyceride and cholesterol were measured using a LabAssay Triglyceride kit (Fujifilm Wako Shibayagi Corp., Gunma, Japan) and a LabAssay Cholesterol kit (Fujifilm Wako Shibayagi Corp.), respectively, according to the manufacturer’s protocol.

5. Iron labeling and MRI evaluation

ADSCs were labeled with iron by overnight incubation in high-glucose DMEM containing ferucarbotran (25 μg Fe/mL) and poly-L-lysine (0.75 mg/mL) ³². The iron labeling was confirmed by Berlin blue staining. The iron-labeled ADSCs administered to mice were detected using 7T- MRI. All MRI experiments were performed on a horizontal 7T-MRI (PharmaScan 70/16 US, Bruker BioSpin, Ettlingen, Germany) equipped with a volume coil with a 30-mm inner diameter. T2WI was obtained with rapid acquisition with a relaxation enhancement (RARE) sequence. The sequence parameters were as follows: repetition time (TR) = 2300 ms, echo time (TE) = 33 ms, the field of view (FOV) = 25.6 × 25.6 mm², slice thickness = 1 mm, matrix size = 128 × 128, the number of averages = 1, and in-plane resolution = 200 × 200 µm². The relative liver intensity on MRI was evaluated by dividing liver intensity by muscle intensity (Supplementary Fig. S4).

6. EV isolation from cell culture supernatants and mouse blood samples

EVs were isolated from cell culture supernatants and mouse blood samples as described previously ³³. The existence of EVs was confirmed by the use of WB to demonstrate the upregulated expression of several markers associated with EVs, including syntenin, TSG101, and ALIX (Abcam). First, we investigated the impact of FG on the secretion of EVs from ADSCs. 1.0 × 10⁵ ADSCs were incubated with or without FG for 7 days using high-glucose DMEM containing 2% FBS, and the culture supernatant was collected on days 1, 2, 3, 5, and 7. Next, we evaluated the impact of EVs on liver regeneration after hepatectomy by inhibiting EV release from ADSCs. A previous study reported that introducing ALIX-siRNA to ADSCs can inhibit their EV secretion ⁹. The successful introduction of ALIX-siRNA (ADSCs^siALIX) was confirmed by qPCR analysis and WB analysis. ADSCs^siALIX were cultured for 48 hours in Mesenchymal Stem Cell Growth Medium 2 (PromoCell, Heidelberg, Germany). After a 48-hour incubation, the supernatant was subjected to ELISA analysis to evaluate cytokine secretion, and the cells (ADSCs^siALIX) were embedded in FG (gelADSC^siALIX) and administered to 70%-hepatectomy model mice.

7. RT-PCR

Total RNA extraction and real-time RT-PCR was performed in accordance with our previous report ³⁰. Relative expression levels of mRNAs were determined as the ratio of specific mRNA to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The primers used in the experiments are listed in Supplementary Table S1.

8. Western blot analysis

Total protein extracts were obtained in accordance with our previous report ³⁰. The primary antibodies included anti-HIF-1α, ALIX, TSG101, syntenin (Abcam), and CD63 (BD Biosciences), as well as an anti-β actin antibody (Sigma-Aldrich, Tokyo, Japan). The detection of the antigen–antibody complex was achieved using an ECL Prime Western Blotting Detection kit (GE Healthcare).

9. Histological analysis

The paraffin-embedded sections were stained with H&E or Masson’s trichrome. Additional sections were subjected to immunohistochemical labeling using a monoclonal anti-PCNA antibody and an LSABTM kit, a system for automated labeled streptavidin–biotinylated antibody immunostaining (both from DAKO, Glostrup, Denmark).

10. Statistical analyses

The data are presented as mean ± SDs. The means of continuous variables were compared using Student’s t test or analysis of variance followed by Tukey’s test. P-values <0.05 were considered significant. All statistical analyses were calculated with JMP software (JMP, version 13.2.1).

ACKNOWLEDGEMENTS

This study was supported by diligent staff at the Center for Medical Research and Education and the Center of Medical Innovation and Translational Research, Graduate School of Medicine, Osaka University. This study was funded by Rohto Pharmaceutical Co., Ltd.

AUTHOR CONTRIBUTIONS

HI, YT, SK (Shogo Kobayashi), and HE have made substantial contribution to the conception of the work; HI, AH, SS to the acquisition and analysis of the data; HI, YT, SK (Shogo Kobayashi), SK (Shunbun Kita), KS, YI, DY, TN, HT, DH, TK, KT to the interpretation of the data; HI, YT, TK, SS, IS, SM, YD, and HE to the drafting and revising the manuscript.

DATA AVAILABILITY STATEMENTS

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

ADDITIONAL INFORMATION

Conflict of interest disclosure statement: HI received a research fund from Rohto Pharmaceutical Co., Ltd., and SK (Shogo Kobayashi) received honoraria from AstraZeneca and Taiho. The other authors (YT, AH, SK (Shunbun Kita), KS, YI, DY, TN, HT, DH, TK, KT, TK, SS, IS, SM, YD, HE) declare no competing financial interests.

Funding statement: This study was funded by Rohto Pharmaceutical Co., Lt

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Competing interest reported. HI received a research fund from Rohto Pharmaceutical Co., Ltd., and SK (Shogo Kobayashi) received honoraria from AstraZeneca and Taiho. The other authors (YT, AH, SK (Shunbun Kita), KS, YI, DY, TN, HT, DH, TK, KT, TK, SS, IS, SM, YD, HE) declare no competing financial interests.

SupplementaryFigureS1.docx
Supplementary Fig. S1 The uncropped membranes of western blot analyses The uncropped membranes of the western blot analyses in Fig. 1d and Fig. 6c were shown (red squares).
SupplementaryFigureS2.tif
Supplementary Fig. S2 Pathway analysis based on RNA-seq RNA sequence analysis (RNA-seq) was performed at the NGS core facility of the Genome Information Research Center at the Research Institute for Microbial Diseases of Osaka University, Osaka, Japan. Library preparation was carried out using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing was conducted on a DNBSEQ-G400 sequencer from MGI, utilizing the 100-base single-read mode. After removing adapter sequences with Trimmomatic version 0.38, the reads were aligned to the human reference genome sequences (hg19) using TopHat2 version 2.1.1. To determine expression levels, the fragments per kilobase of exon per million mapped fragments (FPKMs) were calculated by using Cufflinks version 2.2.1. An analysis was conducted to compare RNA expression between the control group and the gelADSC group. Pathway analysis was performed using the KEGG database with the web application iDEP ( href="http://bioinformatics.sdstate.edu/idep96/">http://bioinformatics.sdstate.edu/idep96/). Data used for analysis are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE246897 with the accession number of GSE246897. The genes highlighted in red are upregulated in favor of the gelADSC group. The cell cycle pathway (a) and fatty acid degradation pathway (b) were upregulated in the gelADSC group compared with the control group
SupplementaryFigureS3.tif
Supplementary Fig. S3 The survival curve after 90% hepatectomy Kaplan-Meier survival curve demonstrating that the survival rate after 90% hepatectomy was significantly higher in the gelADSC group than in the control group (p = 0.0013)
SupplementaryFigureS4.tif
Supplementary Fig. S4 Evaluation of the signal intensity in liver relative to that in muscle on MRI images. The signal intensity in liver relative to that in muscle was calculated using ImageJ software. Signal intensity was measured by encircling the psoas muscle (left panel) and the liver (right panel). The signal intensity in liver relative to muscle was calculated by dividing the liver intensity by the psoas muscle intensity
SupplementaryTableS1Thebasesequenceofprimers.docx

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Effect of Adipose-Derived Stem Cells Embedded in Fibrin Gel on Post-Hepatectomy Liver Regeneration: A Therapeutic Model Inducing a Paracrine Effect

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Abstract

Figures

Introduction

Results

1. GelADSC displayed favorable survival and cytokine secretion in vitro.

2. ADSCs survived in FG in hepatectomized mice.

3. GelADSC promoted liver regeneration after hepatectomy in normal liver through upregulation of the cell cycle and fatty acid oxidation.

4. GelADSC promoted liver regeneration after 70% hepatectomy in liver with chronic hepatitis by upregulating the cell cycle and fatty acid oxidation.

5. The hepatic distribution of ADSCs administered through a tail vein decreased in the presence of chronic hepatitis.

6. Extracellular vesicles play an important role in liver regeneration by gelADSC.

Discussion

Methods

1. Human ADSCs and FG preparation

2. Assessment of cytokine secretion and survival of ADSCs in FG

3. Mouse hepatectomy models and ADSC administration

4. Cholesterol and triglyceride assays

5. Iron labeling and MRI evaluation

6. EV isolation from cell culture supernatants and mouse blood samples

7. RT-PCR

8. Western blot analysis

9. Histological analysis

10. Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

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