Conceived and designed the experiments: Y-BZ Z-LG. Performed the experiments: Y-BZ X-HZ Z-LH C-SL JL. Analyzed the data: Y-BZ Y-RG B-LL D-YX. Contributed reagents/materials/analysis tools: Y-BZ X-HZ S-BX LP. Wrote the paper: Y-BZ Z-LG.
The authors have declared that no competing interests exist.
Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid–derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1–receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra–expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine–induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.
Fulminant hepatic failure is a serious clinical condition that is associated with a high mortality rate. Orthotopic liver transplantation is the treatment of choice for FHF and end-stage liver disease
A recent study has reported that stem cells derived from second-trimester amniocentesis were pluripotent, with the capacity to differentiate into multiple lineages, including representatives of all three embryonic germ layers
Recent studies have shown that FHF is an inflammatory disease; this conclusion is supported clinically by elevated serum levels of immuno-inflammatory cytokines, including IL-1, TNF, IL-6
IL-1Ra is a naturally occurring cytokine and a member of the IL-1 family whose only known function is to prevent a biological response to IL-1 by competing for its receptor. The balance between endogenous IL-1 and IL-1Ra in vivo is an important determinant of the host response to infection
Based on this concept, we tested whether transfusion of IL-1Ra-expressing AF-MSCs could protect damaged livers in a rat FHF model by suppressing excessive immunoinflammatory responses and promote regeneration after cell transplantation.
AF-MSCs retained a fibroblastic morphology after repeated passages (
(a) Morphological characterization of AF-MSCs. (b) MSCs were transduced with lentivirus green fluorescence protein (LV-GFP) or LV- human Interleukin-1–receptor antagonist (LV-hIL-1Ra) and the transduction efficiency of AF-MSCs was detected by flow cytometry using a GFP marker. (c) Fluorescence-activated cell sorting analysis of rat AF-MSCs. Percentage in the panels represents mean fluorescence intensity of the cells expressing each marker. (d) hIL-1Ra concentrations in the supernatants of MSCs, GFP/MSCs, and hIL-1Ra/MSCs. (e) LV-mediated expression of hIL-1Ra by MSCs over time.
AF-MSCs transduced with lentiviral vectors(Lv)-IL-1Ra were designated as IL-1Ra/MSCs. 96.53% of IL-1Ra/MSCs were positive for GFP on day 3 after transduction(
There was no significant difference between the survival rates in any of the rodent groups until 96 hours after cell transplantation. However, the survival rates of the lentiviral vectors -IL-1Ra(LV/IL-1Ra) and MSC transduced with lentiviral vectors-IL-1Ra (IL-1Ra/MSC) groups were higher than those of the physiological saline (PS) and MSC transduced with lentiviral vectors-GFP (GFP/MSC) groups after 96 hours of cell perfusion. The 21-day survival rates of rats in the PS, GFP/MSC, Lv/IL-1Ra, and IL-1Ra/MSC groups were 22.2%, 30.0%, 63.6%, and 69.2%, respectively (
LV-IL-1Ra, Lentiviral-Interleukin-1 receptor antagonist; MSCs, mesenchymal stem cells; PS, physiological saline; GFP, green fluorescence protein.
The degree of hepatic injury in our rat FHF model was assessed by histological analyses (
Increased infiltration of mononuclear cells(white arrow), vacuolar degeneration, and necrosis(black arrow) were observed in the PS group(B, F, H), Increased infiltration of mononuclear cells were observed in the green fluorescent protein (GFP)/MSC group(F), Increased vacuolar degeneration, and necrosis were observed in the LV-IL-1Ra group (H), as compared to the interleukin-1 receptor antagonist/mesenchymal stem cell (IL-1Ra/MSC) group (D). Original magnification ×200 (A, C, E, G), ×400 (B, D, F, H). (b) There were significantly lower alanine aminotransferase (ALT) levels in IL-1Ra/MSC rats than those in the PS and green fluorescent protein (GFP)/MSC groups by day 5 after cell transplantation. *
Serum levels of ALT were measured on days 3, 5, 7, 11, and 14 after cell transplantation in order to evaluate the extent of hepatocellular lesions. On day 5, the IL-1Ra/MSC group had significantly lower levels of ALT (448.7±112.6 IU/L), than the PS group (657.3±147.4 IU/L) (
In order to explore the mechanism of proliferation promoted by IL-1Ra in hepatocytes, immunohistochemical analyses were conducted using anti-BrdU antibodies. Seventy-two hours after cell transplantation, a significant increase in BrdU-positive hepatocytes was noted in the IL-1Ra/MSC group (27.3±1.23%) compared to the PS, GFP/MSC and LV-IL-1Ra groups (18.32±1.21%, 19.9±1.33% and 23.5±1.34%, respectively;
(a) Immunohistochemical staining of bromodeoxyuridine (BrdU) incorporation and 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) assay on days 3 and 5 after cell perfusion. Representative immunohistochemistry at 72 hours (A, D). (b) Graph of the average percentage of BrdU-positive nuclei at the indicated times after cell transplantation. TUNEL-positive cells on days 3 and 5 (B, E). (c) Graph of the average percentage of TUNEL-positive cells. Original magnification ×200 (A, B, D, E), ×400 (C, F). *Versus physiological saline (PS) and green fluorescent protein/mesenchymal stem cell (GFP/MSC) groups,
On day 5 after cell transplantation, a clearly lower percentage of apoptotic liver cells was noted in the IL-1Ra/MSCrats compared to the PS and GFP/MSC rats (18.3±1.5% vs 27.6±1.2% and 24.5±1.8%;
IL-1Ra/MSCs improved liver function by producing hIL-1Ra and down-regulating the inflammatory responses activated by interleukin-1 in vivo. In order to ascertain that hIL-1Ra is expressed in IL-1Ra/MSCs in vivo, we measured serum hIL-1Ra concentrations by ELISA (
(a) Serum hIL-1Ra concentrations were determined by enzyme-linked immunosorbent assay (ELISA).
The levels of IL-1Ra expression observed in IL-1Ra/MSC rats are consistent with the time-line for peak expression after lentiviral transduction in vitro (
In order to determine whether exogenous IL-1Ra is derived from in situ expression of the transgene in the liver, we investigated hIL-1Ra expression 24 hours before treatment, 72 hours after treatment, and 14 days after treatment with Lv/IL-1Ra or IL-1Ra/MSCs. As shown in
IL-1Ra mRNA expression in IL-1Ra/MSC and Lv-IL-Ra rats was upregulated within 48 hours after cell transplantation and remained for up to two weeks but was undetectable in the PS and GFP/MSC rats during the same period of time. hIL-1Ra in mRNA levels was also not detected 24 hours before treatment (
In order to determine whether IL-1Ra can attenuate inflammatory responses in FHF rats, we measured concentrations of the serum inflammatory cytokine IL-6, IL-1β and TNF-α using ELISA after cell perfusion (
In order to determine whether MSCs over-expressing IL-1Ra can be incorporated into liver tissues and differentiate into hepatocytes, rats with FHF used in the survival study were killed 4 weeks after cell transplantation, and the GFP-positive cells were quantified. Cryostat sections were examined for GFP expression and localization with a fluorescence microscope. Minimal expression of GFP was seen in GFP/MSC and IL-1Ra/MSC rats on day 5 after cell transplantation. However, 4 weeks after cell transplantation, the number of GFP-positive cells had increased to approximately 8–14 cells/field in IL-1Ra/MSC rats (
(a, b) Expression of albumin in GFP-labeled MSCs engrafted in liver tissues, detected by immunofluorescence histochemistry of tissues from IL-1Ra/MSC and GFP/MSC rats. A circle refers to no albumin expression in GFP-labeled MSCs in IL-1Ra/MSC and GFP/MSC rats. (c) Histological appearance of the liver 4 weeks after cell perfusion. Apparent hepatocyte degeneration and increased infiltration of mononuclear cells were detected in the green fluorescent protein/mesenchymal stem cell (GFP/MSC) rats (A, B), but not in the IL-1Ra/MSC rats (C, D). Original magnification ×200 (A, C) ×400 (B, D). Original magnification ×400. Alb, albumin; DAPI, 4′,6-diamidino-2-phenylindole.
We then determined the degree of hepatic differentiation by graft MSCs using double-fluorescent immunohistochemistry to detect the expression of albumin (red) in GFP-positive (green) cells. Among the GFP-labeled cells, the percentages of double-positive cells were approximately 38.5±3.7% and 34.5±3.1% in IL-Ra/MSC and GFP/MSCs rats, respectively (
Notably, conspicuous infiltration of mononuclear cells was detected in the GFP/MSC rats four weeks after cell transplantation but not in the IL-1Ra/MSC rats (
We have shown that delivering IL-1Ra-expressing MSCs into FHF rats leads to the attenuation of hepatic inflammatory responses, the regeneration of liver cells, the suppression of apoptosis in hepatocytes, and the migration of MSCs into injured livers; these responses result in significant improvements in liver function and survival.
Our previous studies have indicated that human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a tool for tissue engineering and cell therapy for hepatic tissues in vitro and in vivo
In a recent publication, researchers reported that IL-1Ra is a naturally occurring anti-inflammatory protein; it competitively blocks the binding of IL-1α and IL-1β to type IL-1 receptors, but exerts no agonist activity
Our recent studies have shown that hepatocyte regeneration and hepatic differentiation were insufficient in mesenchymal stem cells under hepatic injury conditions
Siegel also reported that AF-MSCs were being used as a new tool to study human genetic diseases and are suitable for transfection with exogenous genes
In the present study, we first expanded AF-MSCs over-expressing IL-1Ra ex vivo and then transfused these stem cells into the portal vein of rats with FHF. IL-1Ra was shown to promote proliferation and suppress hepatocyte apoptosis on day 5 after cell transplantation. Immunohistochemical analyses showed that IL-1Ra/MSCs have potent anti-apoptotic and mitogenic effects on hepatocytes. Studies have also demonstrated that IL-1Ra functions as a negative regulator of cell senescence through inhibition of IL-1 signaling and prevents apoptosis in ex vivo expansion of human limbal epithelial cells
Our findings also indicated that transfusion of IL-1Ra/MSCs into rats with FHF significantly improved liver function and reduced hepatocellular damage, ultimately improving the 21-day survival rate. Other studies have shown that transfused MSCs were in the sinusoid for the first week, migrated into the liver parenchyma 1 week after engraftment, and further differentiated into hepatocyte-like phenotypes in two weeks
Although our previous study showed that BM-MSCs represent a promising source of autologous cells for cell therapies related to liver failure
The major risks to be considered in research with HIV-1 based lentivirus vectors are the potential for generation of replication-competent lentivirus (RCL) and the potential for oncogenesis. IL-1Ra/MSCs possess the same proliferation and self-renewal capacity as AF-MSCs. These results demonstrate that modifications to the lentiviral vectors optimized the vector as a tool and conferred superior biosafety for the treatment of diverse terminal liver diseases. In this context, it should be noted that our single-injection gene delivery system using a lentiviral vector was proven to be a safe approach, and no tumors or other lesions were observed in the relevant organs.
In summary, our studies showed that IL-1Ra–expressing AF-MSCs participate in the suppression of local inflammatory responses in the liver and systemic immuno-inflammatory responses. IL-1Ra expressed by MSCs not only protects cell grafts from the serum of patients with FHF injury, but also accelerates hepatocyte and MSC graft regeneration and suppresses apoptosis in hepatocytes after cell-perfusion in FHF models. In addition, IL-1Ra promotes MSC migration and incorporation into liver tissue. These MSC grafts subsequently differentiate into hepatocyte-like cells and are instrumental in increasing the survival rate of rats with FHF. This approach to managing the uncontrolled hepatic immunoactivation associated with FHF using MSCs that are genetically-modified to over-express IL-1Ra may offer potentially better outcomes for the treatment of FHF. However, preclinical trials are still needed to determine the safety and efficacy of these cells before they are widely available to patients. Evidence for the inhibition of IL-1 activity by AF-MSCs with IL-1Ra included non-selective immunosuppression and suppression of inflammation. The actions of AF-MSCs expressing IL-1Ra should be further studied in preclinical trials to determine whether such treatment interferes with antiviral immunity in hosts with liver injury from HBV or HCV infections. These cells should also be further tested to determine their biosafety. Such studies should address the issues of insertional mutagenesis and/or oncogenicity in an animal model, as well as inappropriate suppression of inflammation and biodistribution of these cells in non-target tissues. In summary, well-designed preclinical trials should be conducted to explore the clinical applicability of AF-MSCs expressing IL-1Ra.
Syngenic male S-D rats (8–12 weeks of age, weighing approximately 200 g; the Animal Center of Sun Yat-Sen University, Guangzhou, China) were used in all of the experiments in order to exclude any effects of immunologic interference. The animals were kept in the animal facilities at Sun Yat-Sen University, and the experiments were conducted in accordance with the guidelines approved by the China Association of Laboratory Animal Care.
Amniotic fluid MSCs were harvested from pregnant Sprague-Dawley rats at gestation day 13±1d as previously described by Hung-Chuan Pan et al
AF-hMSCs that stably express human IL-1Ra(hIL-1Ra) were established as previously described
Morphological characterization of AF-MSCs and IL-1Ra/MSCs compared over 3 weeks are described in
Following the third passage, AF-MSCs and IL-1Ra/MSCs were seeded at 1000 cells/cm2 in six-well plates (Corning, USA). Cells were detached by treatment with 0.25% Trypsin-0.02% EDTA and counted with a hemocytometer at days 3, 6, 9, 12, 15, 18, and 21. Dead cells were excluded by Trypan blue staining (Sigma-Aldrich, China). Both of these experiments were performed in triplicate for each point described in
RNA was extracted from 5×105 cells, including AF-MSCs and IL-1Ra/MSCs from passages 3, 6, and 9 using Trizol solution (TIANGEN Biotech Co., LTD., BeiJing, China). Primers used for amplification are described in
Hepatic differentiation was induced as in our previous study
Measurement of liver-specific marker(ALB and AFP) mRNA expression in AF-MSCs and IL-1Ra/MSCs by Semi-Quantitative RT-PCR(
The presence of liver-specific markers including ALB and AFP in both differentiated AF-MSCs and IL-1Ra/MSCs(
Fulminant hepatic failure was induced by an intraperitoneal injection of 1.5 g/kg galactosamine and 200 ug/kg lipopolysaccharides. On day 1 after the induction, rats with FHF were anesthetized with intraperitoneal injections of 6 ml/kg 5% chloral hydrate (Sigma-Aldrich) and 0.4 mg/kg xylazine (Sangon Biotech Co., Ltd, Shanghai ). The rats undergoing FHF induction were divided into four groups according to the material they would receive by transfusion: (i) physiological saline (PS) (ii) GFP/MSCs, (iii) IL-1Ra/MSCs, and (iv) Lentiviral vectors- IL-1Ra(LV/IL-1Ra) as a positive control. One day after FHF induction, either PS, 1×106 MSCs, or 5×107 transducing units(TU)LV-IL-1Ra in a volume of 0.8 ml were transfused into the portal vein of each recipient rat with 28-gauge needle over a period of 5 minutes. Rats were evaluated every 12 hours and killed if they appeared moribund.
Fifteen rats in each group were used for the survival study. Rats that had lived for more than 21 days after transplantation were considered to be survivors.
Rats were killed on days 1, 3, 5, 7, 9, 11 and 14 post-transfusion. Twelve hours before killing, a single dose (50 mg/kg) of BrdU (Sigma-Aldrich) was injected intraperitoneally. To detect serum hIL-1Ra levels on days 0, 2, 4, 6, 8, 10, 12 and 14 after transfusion, blood samples were collected from the tail vein 24 h before sacrificing. Liver tissues were excised and processed for further RNA analysis and western blot analysis. The remaining tissue was fixed and processed for histology and immunohistochemistry.
The plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after cell transfusion were measured in a biochemistry laboratory (AEROSET, Abbott, USA). The levels of plasma IL-1Ra, IL-6, IL-1β and TNF-α were detected using a commercially available ELISA kit according to the manufacturer’s instructions (IL-1Ra and IL-6, BioSource Corporation, Chantilly, Virginia, USA; IL-1β and TNF, Invitrogen, CA, USA).
Total RNA was isolated using Trizol solution (TIANGEN Biotech Co., LTD., BeiJing, China). Primers used for amplification are described in
Gene | Sequence | Product (bp) |
hIL-1Ra | F: |
356 |
R: |
||
GAPDH | F: |
170 |
R: |
Abbreviations: hIL-1Ra, human interleukin-1 receptor antagonist; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Hepatic tissues were homogenized, and whole cellular proteins were extracted. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cellular extracts (50 µg) was performed using 10% acrylamide gels, followed by electrophoretic transfer to polyvinylidene difluoride membranes (Sigma-Aldrich, PeiJing, China). The membranes were then probed with a rabbit anti-hIL-1Ra polyclonal primary antibody (GeneTex, Inc, USA), followed by a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, PeiJing, China). Positive signals were detected using the enhanced chemiluminescence method. Equal loading was assessed by β-actin.
Paraffin sections of hepatic tissues were prepared. Bromodeoxyuridine(BrdU) incorporation was determined using a mouse anti-BrdU antibody conjugated to peroxidase (1∶200, R&D Systems, ShangHai, China). Apoptosis was detected by in situ determination of DNA fragmentation using terminal deoxynucleotidyl transferase- mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) assay. The proliferative index and apoptotic index were quantitatively assessed by calculating the percentage of BrdU-labeled hepatocytes and TUNEL-positive per 1000 hepatocellular nuclei (200-fold magnification), respectively.
Cryostat sections of livers were prepared and directly estimated for GFP expression and localization using 4′, 6-diamidino-2-phenylindole (1∶10 000, DAPI, Sigma-Aldrich, ShangHai, China), counterstaining, and a fluorescence microscope (BX51, Olympus, TOKYO, Japan). The number of GFP-labeled hepatocytes per 1000 hepatocellular nuclei (200-fold magnification) was also determined. The presence of albumin cryostat sections were detected by immunofluorescent staining as previously described by
All data were expressed as(Means ± SD). Comparisons involving one independent factor (e.g., treatment) or two independent factors (e.g., treatment and time) were analyzed using one-way and two-way ANOVA, respectively, followed by Bonferroni post-hoc testing. Results from survival experiments were analyzed using the log-rank test and expressed as Kaplan-Meier survival curves. P-values less than 0.05 were considered to be statistically significant. Statistical analysis was performed using SPSS v16.0 software((SPSS, Chicago,IL, USA).
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We thank professor Peng Xiang for his help.