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Potential conflicts of interest: Drs. Fonsato and Herrera Sanchez received grants from Sis-Ter S.p.a. Drs. Camussi and Tetta is an employee of and received grants from Fresenius Medical Care.
Funded by Regione Piemonte, Piattaforme Biotecnologiche, Pi-Stem project, Converging Technologies NanoIGT.
Liver transplantation is currently the only effective therapy for fulminant liver failure, but its use is limited by the scarcity of organs for transplantation, high costs, and lifelong immunosuppression. Here we investigated whether human liver stem cells (HLSCs) protect from death in a lethal model of fulminant liver failure induced by intraperitoneal injection of D-galactosamine and lipopolysaccharide in SCID mice. We show that injection of HLSCs and of HLSC-conditioned medium (CM) significantly attenuates mouse mortality in this model. Histopathological analysis of liver tissue showed reduction of liver apoptosis and enhancement of liver regeneration. By optical imaging we observed a preferential localization of labeled HLSCs within the liver. HLSCs were detected by immunohistochemistry in large liver vessels (at 24 hours) and in the liver parenchyma (after day 3). Fluorescence in situ hybridization analysis with the human pan-centromeric probe showed that positive cells were cytokeratin-negative at 24 hours. Coexpression of cytokeratin and human chromosome was observed at 7 and, to a lesser extent, at 21 days. HLSC-derived CM mimicked the effect of HLSCs in vivo. Composition analysis of the HLSC-CM revealed the presence of growth factors and cytokines with liver regenerative properties. In vitro experiments showed that HLSC-CM protected human hepatocytes from apoptosis and enhanced their proliferation. Conclusion: These data suggest that fulminant liver failure may potentially benefit from treatment with HLSCs or HLSC-CM. (HEPATOLOGY 2013)
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Fulminant liver failure (FLF) is a life-threatening disease for which liver transplantation is the only definitive treatment,1 but the scarcity of donor livers and the timing of available organs often precludes transplantation. Liver regeneration could also be facilitated by using a bioartificial liver, but this approach is limited by the lack of availability of viable hepatocytes, required by the bioreactor.2 Stem cells, which can be expanded in vitro and cryopreserved, could form the basis of an ideal therapy.1 Liver stem cells, or even stem cells derived from other tissues, could potentially provide a source of human hepatocytes for regeneration of the injured liver.3, 4
In particular, mesenchymal stem cells (MSCs), shown to be capable of in vitro differentiation into hepatocytes,5 were investigated as a possible source of hepatocytes for liver regeneration. In addition, it has been shown that secretion of trophic molecules by MSCs may favor regeneration following acute liver injury.6
In a previous study, we isolated a population of human adult liver stem cells (HLSCs) expressing MSC markers and certain embryonic and hepatic cell markers, and having multipotent differentiation capabilities and regenerative properties.7 However, the therapeutic potential of HLSCs and HLSC-conditioned medium (CM) in FLF has not yet been evaluated. In this study we investigated the effect of HLSCs and HLSC-derived CM in a lethal model of liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in SCID mice.
Materials and Methods
Culture of HLSCs, MSCs, and Human Hepatocytes.
HLSCs were isolated from human cryopreserved normal hepatocytes and MSCs were obtained from Lonza (Basel, Switzerland) and were cultured as described in the online Supporting Information.7, 8
Preparation of CM.
Detailed protocols for the preparation of CM from HLSCs or MSCs9 are provided in the online Supporting Information.
CM, conditional medium; FLF, fulminant liver failure; GalN, D-galactosamine; HLSCs, human liver stem cells; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells.
The CM was analyzed for specific proteins, using multiplex biometric immunoassay, Bioclarma (Bio-Plex Human Cytokine Assay; Bio-Rad Laboratories, Hercules, CA) and data were confirmed by enzyme-linked immunosorbent assay (ELISA).
SCID Mice Model of FLF.
Studies were approved by the University of Torino Ethics Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Intramuscular injection of Zolazepam (0.2 mL/kg) and Xilazin (16 mg/kg) were used as anesthesia (40 µL/mouse). FLF was induced in male SCID mice (7-8 weeks old) (Charles River Laboratories, Milan, Italy), by intraperitoneal injection of GalN (600 mg/kg body weight) and LPS (125 ng per animal).10 Injection of GalN and LPS induced liver injury causing apoptosis and necrosis of hepatocytes with 100% lethality at 8 hours.
Thirty minutes after GalN/LPS administration, mice received different treatments. The following groups were studied: group 1, FLF mice intravenously injected with vehicle alone (n = 18); group 2, healthy mice intraperitoneally injected with vehicle instead of GalN/LPS (n = 6); group 3, FLF mice intravenously injected with 2 × 106 HLSCs (n = 9, 3.3 × 105 cells given six times for a total number of 2 × 106); group 4, FLF mice intravenously injected with 2 × 106 MSCs (n = 6, 3.3 × 105 cells given 6 times for a total number of 2 × 106); group 5, FLF mice intraperitoneally injected with 30 × 106 HLSCs (n = 9); group 6, FLF mice intraliver parenchyma (LP) injected (in the left lobe) with 0.5 × 106 (n = 5) or 0.2 × 106 HLSCs (n = 5); group 7, FLF mice intraperitoneally injected with concentrated HLSC-CM (n = 14); group 8, FLF mice intraperitoneally injected with concentrated MSC-CM (n = 5); group 9, FLF mice intraperitoneally injected with vehicle (PBS; n = 5); group 10, FLF mice intraperitoneally injected with concentrated HLSC-CM pretreated for 1 hour at 4°C with anti-HGF blocking antibody (100 mg/mouse; GeneTex, Aachen, Germany) (n = 5); group 11, FLF mice intraperitoneally injected with 25 ng/mouse of rhHGF (PreproTech, Rocky Hill, NJ,) (n = 5). The intravenous injection (120 mL) was performed in the vein of the tail within 30 seconds. In all experiments, cells cultured in T75 flasks until the 2-6 passage were detached by trypsin (0.5% w/v), washed, and resuspended in phosphate-buffered saline (PBS). Before in vivo injection, HLSCs were stained with the CellTrace CFSE Cell Proliferation Kit (Molecular Probes, Life Technologies, Paisley, UK).11
Morphologic Studies and Fluorescence In Situ Hybridization (FISH).
Full details are provided in the online Supporting Information.
Full details are provided in the online Supporting Information.
Apoptosis and Proliferation of Hepatocyte In Vitro.
Full details are provided in the online Supporting Information.
Data were analyzed using t tests, analysis of variance (ANOVA) with Newmann-Keuls' or ANOVA with Dunnett's multicomparison tests as appropriate. For survival experiments, a log-rank test was conducted. P < 0.05 was considered significant.
HLSCs Improved Survival in a Model of FLF in SCID Mice.
The intraperitoneal administration of 600 mg/kg of GalN and 125 ng of LPS in SCID mice induced FLF with a 100% mortality rate within 8 hours (Fig. 1A). To evaluate whether HLSCs might rescue mice with FLF, the cells were administered 30 minutes after GalN/LPS-induced injury in different protocols: single intraperitoneal injection (3 × 107 cells); six repeated intravenous injections (3.3 × 105 cells, total: 2 × 106 cells); LP injection at two concentrations (0.2 and 0.5 × 106). Survival rates were 77%, 100%, and 100% in the case of intraperitoneal, intravenous, and LP injections, respectively (Fig. 1A,B). An equal number of MSCs was intravenously injected six times (3.3 × 105 cells, total: 2 × 106 cells) using the same protocol. The survival rate was 33% (Fig. 1A). Cell injection timings were chosen on the basis of preliminary experiments performed at 30 minutes, 1 and 4 hours, which showed that cell administration after 30 minutes did not improve survival.
As paracrine mechanisms of adult stem cells were related to their secreted factors, we investigated and compared the effects of HLSC-CM and MSC-CM to that of HLSCs. As shown in Fig. 1C, the intraperitoneal injection of HLSC-CM induced an 80% survival. Conversely, MSC-CM did not protect mice from FLF. All controls treated with vehicle alone (PBS) died within 8 hours, suggesting that the effect on survival did not depend on mice hydration (Fig. 1C).
Histological analysis of mice treated with GalN/LPS showed extensive necrosis (Fig. 2A) and apoptosis (Fig. 3A) of the liver after 7 hours, along with a significant increase in liver enzyme levels measured in the peripheral blood (Fig. 2B,C). Treatment with intravenous injections of HLSCs led to a significant reduction of apoptosis and necrosis in surviving mice (Fig. 2A), despite the increase in liver enzymes at 7 hours. In mice surviving to injury, a significant decrease of liver enzymes was observed 3 days after HLSC injection, subsequently reaching normal values (Fig. 2B,C). A significantly lower concentration of ammonium was detected in serum of FLF mice (GalN/LPS) treated with HLSCs compared to vehicle alone (Fig. 2D). In HLSC-treated mice, normal liver morphology was reestablished after 7 to 21 days of treatment (not shown).
As shown in Fig. 3, HLSCs significantly inhibited liver apoptosis in FLF mice (GalN/LPS) compared to vehicle alone. Proliferating cell nuclear antigen (PCNA)-positive cells detected at 7 hours expressed human leukocyte antigen (HLA) or CFSE, indicating that they were derived from the injected HLSCs (Fig. 3C). However, after 3 days in mice treated with HLSCs, PCNA-positive cells were mainly negative for HLA and CFSE, indicating that most proliferating cells were of murine origin (Fig. 3C).
Intravenous- and LP-Injected HLSCs Localized in the Liver of FLF Mice.
Liver cell localization was evaluated by IVIS using DiD-labeled HLSCs.12, 13 As shown in Fig. 4A,B, after intravenous injection HLSCs preferentially accumulated in livers of mice with FLF but not in livers of healthy mice (Fig. 4A,B). Fluorescence signals, expressed as average radiance, increased until day 7 in livers of mice with FLF but not in those of healthy mice. In LP-injected mice, no difference in liver accumulation of HLSCs between FLF and healthy mice was observed (Supporting Fig. 1S,B).
By immunohistochemistry, CFSE-labeled HLSCs were mainly detected in large liver vessel walls after 24 hours and within the liver parenchyma after days 7 and 21 in intravenously injected surviving FLF mice (Fig. 5A,D,E). In these mice, CFSE-labeled HLSCs were transiently detected after 24 hours in lungs and spleens, decreasing thereafter (Fig. 5B,C; Fig. 2S). When HLSCs were intravenously injected in healthy mice treated with vehicle alone, liver accumulation was significantly lower than in FLF mice (Fig. 5A). In LP-injected mice, CFSE-labeled HLSCs were detected in the liver parenchyma at days 7 and 21 following injection (Fig. 1S,B), but there was no accumulation in lungs or spleens at any timepoint (not shown).
In Vivo Expression of Mature Hepatic Markers by HLSCs.
To assess whether HLSCs engrafted in the liver expressed mature hepatic markers, we investigated coexpression of human antigen HLA and mature hepatic markers such as cytokeratin 8 and 18 by confocal analysis (Fig. 6). At day 7 the majority of HLA-positive cells expressed cytokeratin 8 and 18 (Fig. 6A,B). At day 21, ∼50% of HLA-positive cells expressed cytokeratin 8/18 (Fig. 6A,B). FISH analysis with the human pan-centromeric probe showed that at 24 hours positive cells were pan-cytokeratin-negative, whereas at days 7 and 21 they coexpressed pan-cytokeratin and human chromosome (Fig. 6B). At day 21 several pan-centromeric-positive cells did not express pan-cytokeratin, suggesting that some undifferentiated HLSCs were still present in liver parenchyma (Fig. 6B).
HLSC-CM Inhibited Liver Necrosis and Apoptosis and Enhanced Liver Regeneration.
In the GalN/LPS model of FLF, HLSC-CM showed a similar protective activity on liver function and morphology compared to HLSCs (Figs. 1C, 7A,B). The increased presence of PCNA-positive cells demonstrated liver regeneration in mice treated with HLSC-CM (Fig. 7C) and apoptosis was significantly reduced compared to mice treated with vehicle alone (Fig. 7D).
In vitro studies confirmed that HLSC-CM at doses as low as 29 µg protein/mL protected human hepatocytes from GalN-induced apoptosis (Fig. 7E). Moreover, increasing concentrations of HLSC-CM enhanced proliferation of human hepatocytes (Fig. 7F). Similar results were obtained with murine hepatocytes isolated from both normal and FLF mice (Fig. 3S)
As shown in Table 1 and Fig. 4S, HLSC-CM contained several growth factors/cytokines potentially involved in liver protection and regeneration, the most represented of which were interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and MSP, reconfirmed by single ELISA. The concentration of HGF in HLSC-CM was ∼50-fold higher than in MSC-CM. Treatment of HLSC-CM with neutralizing antihuman HGF antibody abrogated the protective effect of HLSC-CM (Fig. 1C), and the survival rate of mice treated with rhHGF was ∼40%, suggesting a relevant role of HGF in the hepatoprotective effect of HLSC-CM (Fig. 1C).
Table 1. Concentration of Cytokines (ng/mL)
119 ± 56
119 ± 79
158 ± 5
158 ± 7
52 ± 12
52 ± 17
38 ± 4
37.9 ± 6.2
30 ± 3
30.4 ± 4.9
207 ± 15
207 ± 20.5
228 ± 13
228 ± 17.7
5.7 ± 0.2
5.68 ± 0.27
5.5 ± 0.5
5.49 ± 0.66
34.4 ± 28
34.4 ± 39.6
We also evaluated the amount of human and murine VEGF, IL-6, and HGF in SCID mice with FLF after intravenous injection with HLSCs. Human HGF and IL-6 levels in mouse serum peaked at 7 hours after HLSC injection (hIL6 = 596 ± 390 pg/mL; hHGF = 482 ± 370 pg/mL), decreased after 24 hours (hIL6 = 46 ± 19 pg/mL; hHGF = 2 ± 1.6 pg/mL), and were undetectable after day 7. Human VEGF was undetectable. In mice injected with HLSCs, murine HGF was not significantly different from untreated controls (respectively 101 ± 49 pg/mL versus 107 ± 19 pg/mL). An increase of murine HGF (159 ± 106 pg/mL) was observed after 24 hours, decreasing thereafter. Levels of murine VEGF and IL-6 did not increase at any time after HLSC administration.
The results of our study can be summarized as follows: (1) HLSCs and HLSC-CM strikingly improved survival and reduced plasma levels of liver enzymes and ammonium in mice with FLF, and (2) the protective effect of HLSCs and HLSC-CM was due to reduced apoptosis, necrosis, and enhanced proliferation.
Cell therapy based on hepatocyte transplantation is a potential treatment option in patients with acute liver failure.14 Transplanted hepatocytes may replace liver functions allowing endogenous regeneration. However, the availability of a suitable cellular source is a limiting factor, but stem cells could represent an ideal cell source for liver regeneration. The capacity of stem cells to reverse liver injury has been shown in different experimental models of liver failure, e.g., in a liver fibrosis model induced by carbon tetrachloride (CCl4), mice treated with MSCs presented less fibrosis, better liver function, and a significant improvement of survival compared to untreated mice.15, 16 In addition, MSCs were shown to protect the liver against hepatocyte apoptosis induced by ischemia-reperfusion injury, and to enhance liver regeneration.9
Recently, Parekkadan et al.6 demonstrated that intravenous injection of MSC-CM or extracorporeal perfusion in a bioreactor containing MSCs had a significant survival benefit in treatment of FLF in rats. Zagoura et al.17 reported that human amniotic fluid-derived MSCs also led to liver repair in a model of CCl4-induced acute hepatic injury. In particular, repair was increased when MSCs were initially differentiated in vitro into hepatic progenitor-like cells. Despite the observation of engraftment of the injected cells, a relevant role for secreted molecules was suggested.17
In the present study we showed that HLSCs may have a therapeutic potential in a lethal model of FLF in SCID mice. Enhanced survival and the improved histopathological findings were associated with significantly lower plasma serum transaminases and ammonium levels. HLSCs are liver-resident MSCs that are already committed to a hepatic lineage, thus do not require in vitro differentiation.7 Immunofluorescence tracing as well as FISH analyses using a human pan-centromeric probe showed some HLSC engraftment within the liver. Coexpression of pan-cytokeratin and human centromere indicated that at day 7 the majority of engrafted HLSCs expressed pan-cytokeratin, with a significantly reduced expression at day 21. This suggests the persistence of an undifferentiated, small population of human HLSCs. However, our hypothesis is that recovery by HLSCs is attributed to a paracrine mechanism, and not by the substitution of the injured parenchyma. In fact, repopulation of the liver was mainly dependent on proliferation of murine hepatocytes. Crucially, HLSC-CM, containing several cytokines with proproliferative and antiapoptotic properties mimicked the effect of HLSCs. Contrary to other experimental models of liver injury, MSC-CM was totally ineffective in the present model. Comparing the composition of HLSC-CM and MSC-CM, HGF was found to be ∼50-fold higher in HLSC-CM than in MSC-CM, which prompted us to perform in vivo experiments using rhHGF or HGF blockade, demonstrating that the beneficial effect of HLSC-CM depended, at least partly, on HGF.
These results suggest that in liver regeneration, hepatocyte proliferation is sustained by soluble factors. In this context, Strick-Marchand et al.18 recently showed a beneficial crosstalk between the immune system and liver stem cells that operates through the release of cytokines that could promote tissue regeneration following acute liver damage. Moreover, Van Poll et al.19 demonstrated a direct antiapoptotic and promitotic effect of MSC-CM in vitro and Parekkadan et al.6 demonstrated that the MSC-induced improvement in survival was attributed to antiinflammatory chemokine release in a rat model of GalN-induced FLF.
In the present study we found that some HLSCs, unlike MSCs, persisted in the liver after days 7 and 21. However, the observation that cell-free HLSC-CM mimicked the HLSC effects suggests a paracrine action by the release of cytokines and growth factors. Interestingly, HLSC-CM inhibited in vitro hepatocyte death and stimulated proliferation, and ELISA analysis of the HLSCs-CM showed the presence of several growth factors/cytokines, potentially involved in liver regeneration. In particular, HLSC-CM contained liver protective factors, such as IL-10 (an antiinflammatory cytokine, recently identified as a mediator of the hepatoprotective action of amniotic fluid-derived hepatic progenitor cells), IL-1ra, MCP-1, and IL-1 beta.6, 17 HLSCs also secrete growth factors such as VEGF, which facilitates angiogenesis and is involved in tissue repair,21, 22 and IL-8, a chemokine with proangiogenic activity.23 In HLSC-CM, we also found growth factors with known hepatoprotective properties, such as HGF, IGF-1, and IL-6.24 However, the growth factor of greatest relevance is HGF in HLSC-CM (only expressed at low levels in MSC-CM), as blocking HGF significantly prevented the protective effect of HLSC-CM, and stimulation with rhHGF improved survival.
In conclusion, HLSCs and HLSC-CM may improve survival in a lethal mouse model mainly by paracrine mechanisms, and HLSCs may therefore represent a new cell source for FLF treatment.
We thank Federica Antico for technical assistance.