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Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure
Cytotherapy, 2014; 16: 1207e1219
Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure
ZONGCAI LIU1,*, FANWEI MENG1,*, CHAN LI1, XIN ZHOU1, XIAOPING ZENG1, YIXIN HE1, RANDALL J. MRSNY3,4, MUYUN LIU1, XIANG HU1, JI-FAN HU2,3 & TAO LI1 1
Shenzhen Beike Cell Engineering Research Institute, Yuanxing Science and Technology Building, Nanshan, Shenzhen, PR China, 2VA Palo Alto Health Care System, Stanford University Medical School, Palo Alto, California, USA, 3 GMR Epigenetics, Palo Alto, California, USA, and 4Department of Pharmacy & Pharmacology, University of Bath, Bath, England Abstract Background aims. Acute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose. Methods. With the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cordederived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy. Results. Mechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-a, interleukin-6) and elevated serum levels of hepatocyte growth factor. Conclusions. Through these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF. Key Words: acute liver failure, anti-inflammation, antioxidants, apoptosis, hepatic regeneration, umbilical cord mesenchymal stromal cells
Introduction Acute liver failure (ALF) is a life-threatening disease characterized by the sudden loss of hepatic function as result of extensive hepatocyte death without compensating liver regeneration (1). The most common causes of ALF include viral hepatitis, side effects from certain prescription drugs, chronic liver diseases, mushroom-derived toxins, autoimmune hepatitis and acetaminophen-induced hepatotoxicity. The incidence of ALF, resulting from accidental or intentional acetaminophen overdose, is remarkably high in the United States and other western countries (2,3). Orthotopic liver transplantation followed by lifelong immunosuppressive treatments is currently the most effective therapy for ALF, but this procedure is limited by a severe shortage of donor organs and
exceptional expense (4). Because of its easier and less invasive procedure, hepatocyte transplantation has been proposed as a tangible alternative to liver transplantation for treatment of ALF. However, this procedure is also limited by a shortage of cadaveric livers required to obtain sufficient numbers of functional hepatocytes. Stem cellebased therapy, as a promising alternative approach, has attracted much attention in ALF treatment (5e7). The most commonly used cells for this therapy are mesenchymal stromal cells (MSCs). Therapeutic MSCs can be isolated from various tissues, including bone marrow, muscle, tooth, amniotic fluid, placenta, adipose tissue and umbilical cord blood (8e12). Isolated MSCs can be differentiated into multiple cell lineages that include osteoblasts, adipocytes,
*These authors contributed equally to this work. Correspondence: Tao Li, PhD, and Xiang Hu, MD, Shenzhen Beike Cell Engineering Research Institute, Yuanxing Science and Technology Building, 1 Songpingshan Rd, Nanshan, Shenzhen, Guangdong, PR China 518057. E-mail: [email protected]; Ji-Fan Hu, VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail: [email protected] (Received 21 October 2013; accepted 20 May 2014) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2014.05.018
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chondroblasts, myocytes, cardiomyocytes and hepatocytes and neurocytes (13e17). Several studies have reported that MSCs derived from different tissues could be administered directly to animals or humans as an alternative source of hepatocytes for treatment of ALF (12,18e22). Recent studies have focused on the use of human umbilical cordederived MSCs (hUCMSCs). As an alternative to MSCs, hUCMSCs have many clear advantages such as being obtained from a readily available source as well as having low immunogenic potency and high proliferative activity (23,24). Several studies have reported the therapeutic effects of undifferentiated stem cells in bone regeneration and liver cirrhosis models (25,26). Therapeutic and preventive effects of undifferentiated hUCMSCs in the context of ALF or liver injury, however, have not yet been fully explored (27,28). In this study, we isolated MSCs from human umbilical cord and examined their potential to prevent or treat liver damage in a mouse model of acetaminophen-induced ALF. Methods Isolation and expansion of hUCMSCs Umbilical cords were collected after full-term parturition in hospital after obtaining written parenteral consent. Isolation of hUCMSCs was performed as described previously (2,3). Briefly, Wharton’s jelly was isolated and cut into pieces, treated with collagenase type 1 (Sigma, St Louis, MO, USA), and then were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and antibiotics at 37 C in a 95% air and 5% CO2 humidified atmosphere. Cells that migrated out from the explants after 5e7 days were collected and cultured; their cellular morphology became homogenously spindleshaped in cultures after 2e3 passages. Passage 2 cells were cryopreserved for future transplantation use. Flow cytometry phenotyping of hUCMSCs hUCMSCs at passage 2 were trypsinized and suspended at a concentration of 1 106 cells/mL in phosphate-buffered saline containing 0.1% bovine serum albumin and then incubated at 4 C with antibodies against MSC cell markers (CD90, D73 and CD105), hematopoietic cell markers (CD45, CD34, CD14 and CD19) and extracellular matrix receptors (CD29 and CD44) and major histocompatibility elements (HLA-DR); all purchased from BD Biosciences, CA, USA. After 30 min, cells were washed and suspended in 300 mL of phosphate-buffered saline. Flow cytometry was performed with the use of FACSCAria (BD Biosciences).
Adipogenic and osteogenic differentiation Adipogenic and osteogenic differentiation of hUCMSCs was performed as described previously (29). After 4 weeks of induction, cells were stained with the use of oil red O or alizarin red to detect the presence of neutral lipid vacuoles in differentiated adipocytes or calcium deposition in osteocytes, respectively. Animal model and cell transplantation Male BALB/c mice (18e20 g body weight) were purchased from the Guangdong Laboratory Animal Center (Guangdong, China). All animals were maintained under standard conditions and received humane care in accordance with approved protocols. Acetaminophen (Aladdin, Shanghai, China) was dissolved immediately before use in sterile normal saline (NS). To establish an ALF model that is based on survival rate, animals received 750 mg/kg of acetaminophen by intraperitoneal injection (12 animals for each group). Additionally, mice were intoxicated with 600 mg/kg of acetaminophen to establish a model to assess changes in body weight, liver weight, serum parameters and cytology (seven animals for each group); cell death was evaluated by means of terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay, and real-time polymerase chain reaction (PCR) was used to follow effects on gene expression. Nonintoxicated control mice were treated with normal saline by the same route and dose volume as acetaminophen-treated mice. We varied the number of hUCMSC given by intravenous injection (1 105, 5 105, 1 106) in our ALF model of survival (supplementary Figure 1). On the basis of these data, we selected cell dose of 5 105 cells for hUCMSC intravenous administration in 200 mL of NS. In pre-ALF and post-ALF groups, hUCMSCs were injected intravenously 30 min before or after acetaminophen intoxication, respectively. In the NS group, mice received NS instead of hUCMSCs. Serum parameter detection Animals in all groups were weighed 72 h after acetaminophen intoxication. Blood samples were collected through orbital bleeds (2 h, 4 h, 8 h, 24 h and 72 h) after acetaminophen intoxication; serum was isolated and stored at 20 C until analyzed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels with the use of an automated biochemical analyzer (Mindray, Shenzhen, China). Total serum bilirubin (TBIL), malondialdehyde (MDA), superoxide dismutase (SOD),
UCMSC therapy of acute liver failure glutathione peroxidase (GSH-PX), glutathione (GSH) and total anti-oxidation capacity (T-AOC) were measured at the 4-h time point with the use of commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Inflammatory factors and cytokines, including interleukin (IL)-1b, IL-6, tumor necrosis factor-alpha (TNF-a) and hepatocyte growth factor (HGF), were determined by means of enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Dakewe, Shenzhen, China). Histological and immunohistochemical examination At study termination, livers were removed and weighed. Approximately 0.05 g of each liver was snap-frozen in N2(l) and stored at 80 C before
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real-time PCR analysis. The remainder of each liver sample was fixed in 4% paraformaldehyde for 24 h before processing for histological, immunohistochemical and TUNEL analyses. Fixed liver samples were cut into small pieces (<3 mm thick, three from each liver), dehydrated, paraffin-embedded, and cut into 5-mm-thick sections. Sections were stained with hematoxylin and eosin for pathological assessment. Cell apoptosis was assessed by means of TUNEL staining, with the use of a Cell Death Detection Kit (Roche, Germany). Proliferating cell nuclear antigen (PCNA)-positive cells were detected through the use of a specific antibody (AVIVA System Biology, Beijing, China). To quantify pathological changes, random microscopic views of each slide section were captured and used to calculate an average number of positive cells per field.
Figure 1. Characterization of hUCMSCs. (A) Morphology of hUCMSCs at day 10 of culture (40). (B) Adipogenic differentiation of hUCMSCs stained by oil red O (400). (C) Osteogenic differentiation of hUCMSCs shown by calcium deposits revealed after alizarin red staining (100). (D) MSC marker profile of hUCMSCs. Immunophenotype of hUCMSCs is determined by flow cytometry at passage 2 with the use of labeled antibodies specific for the indicated human surface antigens.
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Quantitative real-time PCR Total RNA was prepared with the use of the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was treated by DNase I master mix and converted to complementary DNA by use of MMLV RT Mix (Invitrogen). Aliquots of complementary DNA were evaluated in a real-time PCR assay with the use of SYBR Premix Ex Tq II (Takara, Japan) in a Bio-Rad CFX96 Real-Time System under standard cycling conditions of 2 min at 95 C, followed by 40 cycles of PCR with 20 sec at 95 C, 30 sec at 56 C and 30 sec at 72 C. This procedure was used to determine the relative expression of three genes (HGF, BCL-2 and BAX) relative to GAPDH, by use of the DDCt method (30). Three replicates were performed for every experimental condition, with each sample assayed in duplicate for each amplicon. The PCR primers used included. HGF: 50 -AAA GAT TGG ATC AGG ACC ATG TGA G and 50 -GAT GGC ACA TCC ACG ACC AGG AAC A; BCL-2: 50 -GCA TCT GCA CAC CTG GAT CCA GGA T and 50 -GAA ATC AAA CAG AGG TCG CAT GCT G; BAX: 50 -ACC ATC ATG GGC TGG ACA CTG GAC T and 50 -CAC AAA GAT GGT CAC TGT CTG CCA T; and GAPDH: 50 -TGA AGG TCG GTG TGA ACG GAT TTG and 50 -CAT GTA GGC CAT GAG GTC CAC CAC.
positive cell numbers and gene expression. Animal survival was analyzed by means of the KaplanMeier log rank method. A value of P < 0.05 was considered to be statistically significant. Data were analyzed with the use of SPSS statistical software. Results Characterization of hUCMSCs After 6e10 days of culture, isolated hUCMSCs began to grow as fibroblast-like cells (Figure 1A). The immunophenotype of these culture-expanded cells at passage 2 was analyzed by flow cytometry for specific cell surface markers. As expected, these cells were positive for known MSC markers (CD105, CD73 and CD90) and were negative for hematopoietic markers (CD45, CD34, CD14, CD19 and HLA-DR). The lineage potential of hUCMSCs was verified by the induction of osteogenic and adipogenic differentiation. Intracytoplasmic lipid droplets stained with oil red O (Figure 1B) and calcium deposits stained with alizarin red (Figure 1C) were observed, demonstrating the potential of adipogenic and osteogenic differentiation of the isolated hUCMSCs, respectively. Two cell surface markers that have affinity for extracellular matrix elements, CD29 and CD44, were also expressed by isolated hUCMSCs (Figure 1D).
Statistical analysis Data are presented as mean standard deviation (SD). The Student’s t-test was used to compare serum parameters, apoptotic cell numbers, PCNA-
hUCMSCs improve survival of ALF mice We first investigated whether pre- and post-injection of hUCMSCs could improve the survival in ALF
Figure 2. Survival rate, body weight and RLW in ALF mice induced by acetaminophen. (A) Percentage survival of mice after intoxication with 750 mg/kg of acetaminophen. Survival rates were compared over a 72-h period (no further deaths occurred in any group after that time). Acetaminophen-induced ALF without hUCMSC treatment functioned as a positive control group (NS). Data are presented as mean SD (n ¼ 12, treatments compared with NS control group; *P < 0.05 compared with the non-transplantation control group). Body weight (B) and RLW (C) of mice in different groups 72 h after intoxication with 600 mg/kg of acetaminophen. Acetaminophen-induced ALF without hUCMSC treatment functioned as a positive control group (NS). Data are presented as mean SD (n ¼ 6, #P < 0.05, ##P < 0.01 compared with control group; *P < 0.05, **P < 0.01 compared with the non-transplantation group).
UCMSC therapy of acute liver failure mice induced by a high acetaminophen dose (Figure 2A). By 72 h after acetaminophen injection (750 mg/kg), the survival rate of mice that received hUCMSCs was significantly higher than that of the NS control group (P < 0.05). ALF mice in the prehUCMSCs group had a survival rate of w60% and w40% in the post-hUCMSCs group compared with the NS group, in which there were no survivors. Most of the deaths in all groups occurred within 4w16 h after acetaminophen injection. These data suggest that hUCMSC injection, and presumably implantation into the liver, either before or after intoxication, was able to improve the survival of ALF mice.
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hUCMSC protect against relative liver weight loss in ALF mice High-dose treatment of mice with acetaminophen resulted in a significant decrease in their total body weight (P < 0.01), which was not corrected by hUCMSC injection either before or after ALF induction (Figure 2B). Relative liver weight (RLW) loss was also observed to occur in ALF mice, presumably caused by a sudden mass loss of hepatocytes (Figure 2C). Importantly, both the pre- and post-transplantation groups showed a significantly higher RLW compared with the untreated NS group (P < 0.05) (Figure 2C). Together, these data suggest
Figure 3. Serum levels of certain biochemical markers are altered in mice induced to have ALF by acetaminophen intoxication. (A) Serum levels of AST (A: a,c) and ALT (A: b,d) over a time course of 72 h. (B) Total bilirubin levels in different groups at time point 24 h after acetaminophen injection. Data are presented as mean SD (n ¼ 7, ##P < 0.01 compared with control group; *P < 0.05, **P < 0.01 compared with NS group).
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Figure 4. Analysis of liver morphological and apoptotic events after acetaminophen intoxication. (A) Liver morphology from control treated mice (A-a) and mice 24 h after receiving 600 mg/kg of acetaminophen (A: b). (B) Representative morphology of livers fixed in 4% paraformaldehyde for 24 h from normal, NS, pre-hUCMSC and post-hUCMSC groups, respectively, at 24 h. (C) Hematoxylin and eosin staining of liver sections from normal, non-transplantation, pre-hUCMSC and post-hUCMSC groups at 24 h after NS or 600 mg/kg of acetaminophen treatment (50). (D) Hepatocyte apoptosis demonstrated in liver sections obtained from negative, positive and normal control mice and three acetaminophen-treated mice at time point 4 h. (E) Percentage of necrosis area in liver sections from each group. Images were collected at original magnification (100); mean values of 10 randomly selected areas per sample were used to determine the percentage area of necrosis; ImageJ software was used to quantify the necrosis areas. (F) Number of apoptotic hepatocytes per single microscopic view of liver sections from three acetaminophen-treated groups after 4 h. Images were collected at original magnification (200). Data are presented as mean SD (n ¼ 6, *P < 0.05, **P < 0.01 compared with NS group).
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Figure 5. Immunohistochemical analysis of PCNA-positive cells in ALF mice 24 h after intoxication with 600 mg/kg of acetaminophen. (A) Liver tissue sections from mice not receiving acetaminophen (negative control) or receiving hUCMSCs or control injections (NS) 24 h after acetaminophen intoxication; all photographed at 50 except where noted. (B) Quantification of PCNA-positive cells per single microscopic view of liver sections. Images were collected at magnification (50), and eight randomly selected views in each section and six sections per group were evaluated. Data are presented as mean SD (n ¼ 6, **P < 0.01 compared with non-transplantation group).
that hUCMSC injection might attenuate injury by reducing the extent of liver cell loss in ALF mice. hUCMSCs attenuate liver damage in ALF mice The extent of liver damage in mice induced by intoxication with 600 mg/kg acetaminophen was monitored by measuring serum levels of ALT, AST and TBIL over the 72-h time course of the study (Figure 3A,B). Compared with control mice, ALT and AST levels in acetaminophen-treated groups started to increase at 4 h and continued to increase when measured at 8 h and 24 h (P < 0.01). Compared with the NS group, hUCMSC injection either before or after acetaminophen treatment reduced the extent of ALT and AST release compared with the control (NS) group at 24 h (P < 0.01). By 72 h, all three acetaminophen-treated groups had serum ALT and AST levels that were
comparable to pre-ALF values (Figure 3A). We also observed that the level of TBIL was markedly increased in acetaminophen-treated mice at the 24-h time point; injection of hUCMSCs suppressed the acetaminophen injuryeinduced increase of TBIL (P < 0.05) (Figure 3B). Taken together, injection of hUCMSC, presumably resulting in their transplantation, could attenuate liver damage as reflected by suppression of ALT, AST and TBIL level increases observed in this ALF model. hUCMSCs ameliorate hepatocyte cell death in ALF mice Cytochromes P450s metabolize acetaminophen to a reactive metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which reacts with and depletes GSH. This process initiates mitochondrial oxidative stress that leads to a mixture of necrosis and apoptosis events resulting from the same mitochondrial cell death
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pathway. On the basis of the time course of apparent liver damage and protection by hUCMSC injection in this acetaminophen-induced model of ALF, we chose the 4-h and 24-h time points to study liver damage and possible mechanisms of cell transplantation in mice. Compared with untreated mice, acetaminophen-treated mice had altered liver morphology that included a rougher surface, duller red color, severe internal bleeding and extensive areas of necrosis (Figure 4A). Injection of hUCMSCs dramatically attenuated the extent of liver necrosis compared with the NS group (Figure 4B,C). Injection of hUCMSCs either before or after acetaminophen-induced liver injury showed reduced levels of apoptosis and a lessened alteration in histological changes compared with the NS group. With the use of TUNEL analysis to examine apoptosis in acetaminophen-induced ALF mice, we observed more apoptotic hepatocytes in the NS group compared with control mice (Figure 4D). The extent of necrotic changes in the livers of mice in the different treatment groups is shown in Figure 4E; injection of hUCMSCs reduced the extent of these necrotic changes. Quantification of TUNEL-positive cells demonstrated that both pre- and post-injection of hUCMSCs attenuated hepatocyte apoptosis compared with the NS group (P < 0.05) at time point 4 h (Figure 4F). Thus, apoptosis may also contribute to
the mass loss of hepatocytes in acetaminophen-induced ALF of mice; the injection of hUCMSCs significantly reduced the extent of this type of cell death. hUCMSCs promote liver regeneration Liver tissue is noted for its remarkable capacity to regenerate after injury. Interestingly, liver regeneration is significantly inhibited in ALF non-survivors compared with survivors (31). Thus, we were interested in examining whether injected hUCMSCs could enhance the extent of hepatocyte proliferation at 24 h and used an immunohistochemistry analysis to compare the extent of PCNA-positive cells in the livers of mice from the different treatment groups (Figure 5A). Interestingly, we observed more PCNA-positive cells at 24 h in both pre- and postinjection groups compared with control and NS animals (P < 0.05) (Figure 5B). These data suggest that hUCMSC treatment, particularly in the pre-injection group, may facilitate liver regeneration by combating the loss of hepatocytes induced by acetaminophen. hUCMSCs enhance the antioxidant system in ALF mice Acetaminophen-induced liver injury is associated with oxidative stress, especially with GSH consumption;
Figure 6. Serum antioxidant indexes and MDA level changes in ALF mice 4 h after intoxication with 600 mg/kg of acetaminophen. Serum levels of GSH (A), of SOD (B), of MDA (C), of GSH-PX (D) and of T-AOC (E) in different control and treatment groups. Data are presented as mean SD (n ¼ 7, #P < 0.05, ##P < 0.01 compared with control group; *P < 0.05, **P <0.01 compared with NS group).
UCMSC therapy of acute liver failure we tested the influences of hUCMSC injection on the antioxidant system of ALF mice (Figure 6AeE). Four hours after treatment of acetaminophen, GSH levels and SOD activity in the serum were dramatically reduced. The lipid peroxidation marker MDA, on the other hand, was markedly increased in the NS group. After hUCMSC injection, animals in both the prehUCMSC and post-hUCMSC groups had restored serum GSH levels compared with the NS group (P < 0.01). However, only pre-injection of hUCMSCs was able to recover serum SOD activity and downregulate serum MDA levels (P < 0.05). Serum T-AOC was increased 4 h after acetaminophen treatment, suggesting a systemic host defensive response to this form of liver injury. Serum T-AOC, however, was not different between the three acetaminophen-treated groups (Figure 6E). Additionally, we did not observe a statistical differences in GSH-PX activity, which is known to promote GSH regeneration (Figure 6D). Thus, the preventative role of hUCMSCs in ALF may partly be due to an ability to reduce detrimental peroxidation events. hUCMSCs suppress inflammatory cytokines and promote HGF expression As reported previously, ALF induces cytokine expression and systemic inflammatory responses. Thus, we examined the serum cytokine changes in ALF mice with the use of a combination of ELISA methods (Figure 7). Our results showed that both pre- and post-hUCMSC injection attenuated
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pro-inflammatory cytokine and HGF expression induced by acetaminophen intoxication. In general, injection of hUCMSCs before acetaminopheninduced ALF was more effective at correcting alterations in cytokine and HGF expression compared with injection of hUCMSCs after this mode of liver injury (Figure 7). For example, hUCMSC injection downregulated the expression of TNF-a at 4 h and 24 h after ALF induction (Figure 7, P < 0.01). At time point 24 h, only the pre-hUCMSC group showed the downregulated expression of cytokine IL-6 (P < 0.01). Interestingly, IL-1b was not altered by the injection of hUCMSCs. These findings suggest that injection of hUCMSCs may suppress inflammatory reactions by decreasing serum levels of the pro-inflammatory cytokines TNF-a and IL-6. At the same time, hUCMSC injection may enhance hepatocyte survival and proliferation through an increase in HGF expression. Molecular evidence for the effects of cell transplantation Anti-apoptosis and regeneration of hepatocytes is quite important for preventing the toxicity in acetaminophen-induced ALF mice. We used real-time PCR analysis to measure messenger (m)RNA expression of the HGF, Bcl-2 and Bax genes in an effort to examine potential mechanisms of action induced by hUCMSC injection. We found that both pre- and post- hUCMSC injections upregulated the mRNA expression of HGF and Bcl-2 at 4 h but downregulated the expression of Bax (P < 0.01,
Figure 7. Serum cytokine expression changes in ALF mice after 600 mg/kg 4 h and 24 h after acetaminophen intoxication. Serum levels of pro-inflammatory cytokines TNF-a (A), IL-1b (B), IL-6 (C) and HGF (D). Data are presented as mean SD (n ¼ 7, **P < 0.01 compared with non-injection group).
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Figure 8. Quantitative real-time PCR analysis of mRNA for genes expressed in liver tissues obtained from mice in different control and treatment groups. (A) Hepatocyte growth factor; (B) Bcl-2; and (C) Bax. (D) Relative mRNA expression levels of HGF, Bcl-2 and Bax genes. Data are presented as mean SD (n ¼ 4, **P < 0.01 compared with NS group).
Figure 8). Together, the data demonstrating the regulation of these genes provides a potential molecular basis for the action of hUCMSC injection in regeneration and anti-apoptosis events in the liver. Discussion Extensive studies have used chemicals such as CCl4, lipopolysaccharides and D-gal to induce ALF or liver injury in animal models. In the present study, we used acetaminophen to induce an ALF model in mice to study the preventative and therapeutic potential of hUCMSCs. This model is simple and rapid. More importantly, it mimics the clinical condition of acetaminophen overdose prevalent in the United States and other Western countries (2,3). In the present study, we have demonstrated that hUCMSCs can improve the survival rate of acetaminophen-induced ALF in mice. In addition to the therapeutic role previously reported (27), we have now examined the preventative capacity of these stem cells in our ALF model. Whether hUCMSCs are able to completely prevent the occurrence of acetaminophen-induced ALF has not been fully examined previously. In the present study, we found that all animals in the NS group died of acetaminophen-induced
intoxication. However, pre- and post-injection of hUCMSCs significantly improved survival rates up to 60% and 40%, respectively. Similarly, liver mass loss was also improved significantly spared in the stem celletreatment groups. In parallel, serum levels of ALT, AST and TBIL that correlate with liver damage were significantly decreased in the hUCMSC injection groups. As early as 4 h after the acetaminophen treatment, mice died in the NS group, whereas no deaths were observed in cell hUCMSC injection groups, indicating that hUCMSCs may have a rapid impact. Coincidently, ALT and AST levels in the pre-transplantation group were significantly decreased when compared with the NS group at this early time point. Our studies also suggest that the time window of cell transplantation is critical for rescuing ALF animals. Most animals that received a high dose of acetaminophen died within 4w16 h, but animals administered hUCMSC 30 min after acetaminophen intoxication were spared from this acute death outcome. Stem cell dose was another key issue to be considered for this AFL model (supplementary Figure 1). We tested three doses of hUCMSCs (1 105, 5 105, 1 106 cells) in our model and found that the majority of animals died of ALF in the 1 105 cells group. No differences in animal survival
UCMSC therapy of acute liver failure were noticed between the 5 105 and the 1 106 cell groups. For this reason, we choose the dose of 5 105 cells for our study. In addition, we also compared the effect of stem cells from different sources, including UCMSC, chorion MSC, and decidua MSC (supplementary Figure S2); they had varied effects in protecting ALF animals. This current report focuses on hUCMSCs not only for its economical culturing, but also in that they have not previously been tested in the ALF model. Liver cell death can occur through distinct biochemical pathways, including apoptosis, necrosis and autophagic cell death (32). It is assumed that apoptotic alterations induce profound energy depletion and mitochondrial failure, diverting cell death to necrosis as the principal mode of acetaminopheninduced liver toxicity (33). Our data in Figure 4 show that hepatocytes undergo extensive necrosis in ALF mice compared with the control group. Both preand post-injection of hUCMSCs significantly attenuated the necrosis condition compared with the NS group. With the use of TUNEL and real-time PCR assays, we also confirmed that apoptosis plays a role
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in acetaminophen-induced ALF mice, at least at the 4-h time point. Again, both pre- and post-injection of hUCMSCs attenuated this condition compared with the NS control group. Liver regeneration after hepatocyte loss is a fundamental parameter of liver response to injury. Our results in Figure 5 show that both pre- and postinjection enhanced the proliferation of hepatocytes compared with the NS group. Together with the improvement of RLW described in Figure 2, it appears that hUCMSCs can promote liver regeneration in ALF mice induced by acetaminophen. HGF, a paracrine cellular growth, motility and morphogenic factor synthesized by nonparenchymal cells of the liver after liver injury or partial hepatectomy, is crucial in promoting liver regeneration. Its high mitogenic potency leads to acceleration of liver function and protection of liver cells from injury (34,35). With the use of ELISA and real-time PCR, we found that HGF was upregulated in cell therapy groups compared with the NS group. These data suggest that hUCMSCs may promote liver regeneration in ALF mice partially through activation of the HGF pathway.
Figure 9. Model proposed to illustrate how hUCMSC injection rescues liver injury. Acetaminophen can be oxidized by cytochrome P450 and form NAPQI, which is then conjugated to GSH and eliminated in the bile. Sustained NAPQI formation eventually causes depletion of GSH, which in turn leads to formation of protein adducts as well as reactive nitrogen and oxygen species. These events augment the level of protein and lipid peroxidation in hepatocytes. Reactive oxygen species (ROS) instigates the mitochondrial pathway of apoptosis involving Bax and Bcl-2. Increased ROS also causes mitochondrial membrane permeability (MPT) and hepatocyte necrosis that elicits Küpffer cells to secrete pro-inflammatory molecules such as IL-6 and TNF-a, which in turn amplify the necrosis of hepatocytes. Apoptosis and necrosis of hepatocytes together cause the observed liver injury. In ALF mice, injection of hUCMSCs can increase the level of serum GSH, accelerate the elimination of NAPQI, and reduce ROS levels. SOD function is improved and more ROS is eliminated, reducing liver injury outcomes caused by apoptosis or necrosis. Bax expression is downregulated and Bcl-2 is upregulated, leading to the amelioration of apoptosis. This, in turn, reduces levels of pro-inflammatory molecules such as IL-6 and TNF-a, leading to less necrosis. The level of HGF is increased, with the outcome of enhancing liver regeneration.
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Acetaminophen is metabolized by the cytochrome P450 enzyme system located predominantly in perivenous hepatocytes (36). Acetaminophen can be oxidized by cytochrome P450 and form NAPQI, which is then conjugated to GSH and eliminated in the bile (Figure 9). Sustained NAPQI formation, however, eventually causes depletion of GSH, which leads to formation of protein adducts as well as reactive nitrogen and oxygen species that leads to increased protein and lipid peroxidation in liver (33). In a previous study, transplantation of hUCMSCs increased catalase activity in mice with CCl4-induced injury (28). In the present study, we found both preand post-transplantation of hUCMSCs increased serum levels GSH that were reduced by acetaminophen treatment. Pre-injection of hUCMSCs increased the activity of serum SOD and suppressed serum MDA levels. Acetaminophen-induced injury is closely related to oxidative stress and is especially linked to GSH consumption. Thus, improvement in antioxidative ability of hUCMSCs might be important for its anti-necrosis and anti-apoptosis properties. Previous studies found that the action of MSCs is primarily through their paracrine function rather than direct tissue replacement (27). MSC-derived molecules attenuated ALF in mice or rats by inhibition of apoptosis, stimulation of hepatocyte proliferation and minimization of the inflammatory response (37). The paracrine action of MSC was also corroborated by the amelioration of systemic inflammation induced by lipopolysaccharides or burns (38). The death of animals in our ALF model was probably directly related to severe liver necrosis induced by acetaminophen. Liver necrosis induces oxidative and inflammatory responses that can be monitored by a variety of serum components that can be detected in the systemic circulation. In contrast, HGF and hepatic regeneration requires intact stem cells to function in the liver. Thus, in this acute model, the direct anti-necrotic effect of paracrine factors delivered by hUCMSC may play a key role in animal survival through liver-specific actions. We found that hUCMSC injection suppressed the release of pro-inflammatory cytokines such as TNFa and IL-6. Thus, inhibition of pro-inflammatory cytokines and reduced liver cell necrosis or apoptosis resulting from hUCMSC transplantation may have been due to local paracrine events in the liver. In summary, this is the first evidence to demonstrate the preventative and therapeutic potential of transplanted hUCMSCs in treating mice with ALF induced by acetaminophen. Intravenous injection and presumably implantation of hUCMSCs attenuated liver injury by anti-apoptosis, anti-necrosis and regeneration-promoting properties. The protection from ALF-related death by injected hUCMSCs may
involve the participation of multiple mechanisms, including stabilization of the antioxidant system, anti-inflammatory actions and elevated HGF expression. This work suggests that intravenous injection of hUCMSCs could be a practical cell therapy to prevent or treat ALF. Acknowledgments This study was supported by Shenzhen International Collaboration grant (GJ200807210024A) to XH; California Institute for Regenerative Medicine (CIRM) grant (RT2-01942), National Institutes of Health grant (1R43 CA103553-01), Department of Defense grant (W81XWH-04-1-0597) to JFH. RJM acknowledges support from the National Institutes of Health (RO1 DK056754-11A1). Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology. 2006;43(2 Suppl 1): S31e44. 2. Reuben A, Koch DG, Lee WM. Drug-induced acute liver failure: results of a U.S. multicenter, prospective study. Hepatology. 2010;52:2065e76. 3. Lee WM, Stravitz RT, Larson AM. Introduction to the revised American Association for the Study of Liver Diseases Position Paper on acute liver failure 2011. Hepatology. 2012;55: 965e7. 4. Fiegel HC, Lange C, Kneser U, Lambrecht W, Zander AR, Rogiers X, et al. Fetal and adult liver stem cells for liver regeneration and tissue engineering. J Cell Mol Med. 2006;10: 577e87. 5. Stutchfield BM, Forbes SJ, Wigmore SJ. Prospects for stem cell transplantation in the treatment of hepatic disease. Liver Transplant. 2010;16:827e36. 6. Enns GM, Millan MT. Cell-based therapies for metabolic liver disease. Mol Genet Metab. 2008;95:3e10. 7. Yuan S, Jiang T, Sun L, Zheng R, Ahat N, Zhang Y. The role of bone marrow mesenchymal stem cells in the treatment of acute liver failure. BioMed Res Int. 2013;2013:251846. 8. Buhring HJ, Treml S, Cerabona F, de Zwart P, Kanz L, Sobiesiak M. Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci. 2009; 1176:124e34. 9. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins K, et al. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec. 2001;264:51e62. 10. Wang X, Sha XJ, Li GH, Yang FS, Ji K, Wen LY, et al. Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol. 2012;57:1231e40. 11. Zagoura DS, Roubelakis MG, Bitsika V, Trohatou O, Pappa KI, Kapelouzou A, et al. Therapeutic potential of a
UCMSC therapy of acute liver failure
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure. Gut. 2012;61:894e906. Cao H, Yang J, Yu J, Pan Q, Li J, Zhou P, et al. Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure. BMC Med. 2012;10:56. Karaoz E, Okcu A, Unal ZS, Subasi C, Saglam O, Duruksu G. Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulin-producing cells in pancreatic islet microenvironment both in vitro and in vivo. Cytotherapy. 2013;15:557e70. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41e9. Bonanno G, Mariotti A, Procoli A, Corallo M, Rutella S, Pessina G, et al. Human cord blood CD133þ cells immunoselected by a clinical-grade apparatus differentiate in vitro into endothelial- and cardiomyocyte-like cells. Transfusion. 2007;47:280e9. Rowlands AS, George PA, Cooper-White JJ. Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation. Am J Physiol Cell Physiol. 2008;295:C1037e44. Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112:295e307. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, et al. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells. 2008;26:2705e12. de Freitas Souza BS, Nascimento RC, de Oliveira SA, Vasconcelos JF, Kaneto CM, de Carvalho LF, et al. Transplantation of bone marrow cells decreases tumor necrosis factor-alpha production and blood-brain barrier permeability and improves survival in a mouse model of acetaminophen-induced acute liver disease. Cytotherapy. 2012;14:1011e21. Peng L, Xie DY, Lin BL, Liu J, Zhu HP, Xie C, et al. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: shortterm and long-term outcomes. Hepatology. 2011;54:820e8. El-Ansary M, Abdel-Aziz I, Mogawer S, Abdel-Hamid S, Hammam O, Teaema S, et al. Phase II trial: undifferentiated versus differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis. Stem Cell Rev. 2012;8:972e81. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al. Mesenchymal stem cellderived molecules reverse fulminant hepatic failure. PloS One. 2007;2:e941. Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, et al. Immune properties of human umbilical cord Wharton’s jelly-derived cells. Stem Cells. 2008;26:2865e74.
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24. Cho PS, Messina DJ, Hirsh EL, Chi N, Goldman SN, Lo DP, et al. Immunogenicity of umbilical cord tissue derived cells. Blood. 2008;111:430e8. 25. Byeon YE, Ryu HH, Park SS, Koyama Y, Kikuchi M, Kim WH, et al. Paracrine effect of canine allogenic umbilical cord blood-derived mesenchymal stromal cells mixed with beta-tricalcium phosphate on bone regeneration in ectopic implantations. Cytotherapy. 2010;12:626e36. 26. Wang J, Zhou X, Cui L, Yan L, Liang J, Cheng X, et al. The significance of CD14þ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis. Cytotherapy. 2010; 12:1022e34. 27. Zhang S, Chen L, Liu T, Zhang B, Xiang D, Wang Z, et al. Human umbilical cord matrix stem cells efficiently rescue acute liver failure through paracrine effects rather than hepatic differentiation. Tissue Eng Part A. 2012;18:1352e64. 28. Burra P, Arcidiacono D, Bizzaro D, Chioato T, Di Liddo R, Banerjee A, et al. Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury. BMC Gastroenterol. 2012;12:88. 29. Chen M, Zhang H, Wu J, Xu L, Xu D, Sun J, et al. Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway. Biomaterials. 2012;33:5514e23. 30. Li T, Hu JF, Qiu X, Ling J, Chen H, Wang S, et al. CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop. Mol Cell Biol. 2008;28:6473e82. 31. Rutherford A, Chung RT. Acute liver failure: mechanisms of hepatocyte injury and regeneration. Semin Liver Dis. 2008; 28:167e74. 32. Bantel H, Schulze-Osthoff K. Mechanisms of cell death in acute liver failure. Frontiers Physiol. 2012;3:79. 33. Christ B, Bruckner S. Rodent animal models for surrogate analysis of cell therapy in acute liver failure. Frontiers Physiol. 2012;3:78. 34. Vargas GA, Hoeflich A, Jehle PM. Hepatocyte growth factor in renal failure: promise and reality. Kidney Int. 2000;57: 1426e36. 35. Kaido T, Seto S, Yamaoka S, Yoshikawa A, Imamura M. Perioperative continuous hepatocyte growth factor supply prevents postoperative liver failure in rats with liver cirrhosis. J Surg Res. 1998;74:173e8. 36. Gebhardt R, Hovhannisyan A. Organ patterning in the adult stage: the role of Wnt/beta-catenin signaling in liver zonation and beyond. Dev Dyn. 2010;239:45e55. 37. van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, et al. Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology. 2008;47:1634e43. 38. Yagi H, Soto-Gutierrez A, Kitagawa Y, Tilles AW, Tompkins RG, Yarmush ML. Bone marrow mesenchymal stromal cells attenuate organ injury induced by LPS and burn. Cell Transplant. 2010;19:823e30.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcyt.2014.05.018.
Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure
ZONGCAI LIU1,*, FANWEI MENG1,*, CHAN LI1, XIN ZHOU1, XIAOPING ZENG1, YIXIN HE1, RANDALL J. MRSNY3,4, MUYUN LIU1, XIANG HU1, JI-FAN HU2,3 & TAO LI1 1
Shenzhen Beike Cell Engineering Research Institute, Yuanxing Science and Technology Building, Nanshan, Shenzhen, PR China, 2VA Palo Alto Health Care System, Stanford University Medical School, Palo Alto, California, USA, 3 GMR Epigenetics, Palo Alto, California, USA, and 4Department of Pharmacy & Pharmacology, University of Bath, Bath, England Abstract Background aims. Acute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose. Methods. With the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cordederived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy. Results. Mechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-a, interleukin-6) and elevated serum levels of hepatocyte growth factor. Conclusions. Through these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF. Key Words: acute liver failure, anti-inflammation, antioxidants, apoptosis, hepatic regeneration, umbilical cord mesenchymal stromal cells
Introduction Acute liver failure (ALF) is a life-threatening disease characterized by the sudden loss of hepatic function as result of extensive hepatocyte death without compensating liver regeneration (1). The most common causes of ALF include viral hepatitis, side effects from certain prescription drugs, chronic liver diseases, mushroom-derived toxins, autoimmune hepatitis and acetaminophen-induced hepatotoxicity. The incidence of ALF, resulting from accidental or intentional acetaminophen overdose, is remarkably high in the United States and other western countries (2,3). Orthotopic liver transplantation followed by lifelong immunosuppressive treatments is currently the most effective therapy for ALF, but this procedure is limited by a severe shortage of donor organs and
exceptional expense (4). Because of its easier and less invasive procedure, hepatocyte transplantation has been proposed as a tangible alternative to liver transplantation for treatment of ALF. However, this procedure is also limited by a shortage of cadaveric livers required to obtain sufficient numbers of functional hepatocytes. Stem cellebased therapy, as a promising alternative approach, has attracted much attention in ALF treatment (5e7). The most commonly used cells for this therapy are mesenchymal stromal cells (MSCs). Therapeutic MSCs can be isolated from various tissues, including bone marrow, muscle, tooth, amniotic fluid, placenta, adipose tissue and umbilical cord blood (8e12). Isolated MSCs can be differentiated into multiple cell lineages that include osteoblasts, adipocytes,
*These authors contributed equally to this work. Correspondence: Tao Li, PhD, and Xiang Hu, MD, Shenzhen Beike Cell Engineering Research Institute, Yuanxing Science and Technology Building, 1 Songpingshan Rd, Nanshan, Shenzhen, Guangdong, PR China 518057. E-mail: [email protected]; Ji-Fan Hu, VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail: [email protected] (Received 21 October 2013; accepted 20 May 2014) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2014.05.018
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chondroblasts, myocytes, cardiomyocytes and hepatocytes and neurocytes (13e17). Several studies have reported that MSCs derived from different tissues could be administered directly to animals or humans as an alternative source of hepatocytes for treatment of ALF (12,18e22). Recent studies have focused on the use of human umbilical cordederived MSCs (hUCMSCs). As an alternative to MSCs, hUCMSCs have many clear advantages such as being obtained from a readily available source as well as having low immunogenic potency and high proliferative activity (23,24). Several studies have reported the therapeutic effects of undifferentiated stem cells in bone regeneration and liver cirrhosis models (25,26). Therapeutic and preventive effects of undifferentiated hUCMSCs in the context of ALF or liver injury, however, have not yet been fully explored (27,28). In this study, we isolated MSCs from human umbilical cord and examined their potential to prevent or treat liver damage in a mouse model of acetaminophen-induced ALF. Methods Isolation and expansion of hUCMSCs Umbilical cords were collected after full-term parturition in hospital after obtaining written parenteral consent. Isolation of hUCMSCs was performed as described previously (2,3). Briefly, Wharton’s jelly was isolated and cut into pieces, treated with collagenase type 1 (Sigma, St Louis, MO, USA), and then were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and antibiotics at 37 C in a 95% air and 5% CO2 humidified atmosphere. Cells that migrated out from the explants after 5e7 days were collected and cultured; their cellular morphology became homogenously spindleshaped in cultures after 2e3 passages. Passage 2 cells were cryopreserved for future transplantation use. Flow cytometry phenotyping of hUCMSCs hUCMSCs at passage 2 were trypsinized and suspended at a concentration of 1 106 cells/mL in phosphate-buffered saline containing 0.1% bovine serum albumin and then incubated at 4 C with antibodies against MSC cell markers (CD90, D73 and CD105), hematopoietic cell markers (CD45, CD34, CD14 and CD19) and extracellular matrix receptors (CD29 and CD44) and major histocompatibility elements (HLA-DR); all purchased from BD Biosciences, CA, USA. After 30 min, cells were washed and suspended in 300 mL of phosphate-buffered saline. Flow cytometry was performed with the use of FACSCAria (BD Biosciences).
Adipogenic and osteogenic differentiation Adipogenic and osteogenic differentiation of hUCMSCs was performed as described previously (29). After 4 weeks of induction, cells were stained with the use of oil red O or alizarin red to detect the presence of neutral lipid vacuoles in differentiated adipocytes or calcium deposition in osteocytes, respectively. Animal model and cell transplantation Male BALB/c mice (18e20 g body weight) were purchased from the Guangdong Laboratory Animal Center (Guangdong, China). All animals were maintained under standard conditions and received humane care in accordance with approved protocols. Acetaminophen (Aladdin, Shanghai, China) was dissolved immediately before use in sterile normal saline (NS). To establish an ALF model that is based on survival rate, animals received 750 mg/kg of acetaminophen by intraperitoneal injection (12 animals for each group). Additionally, mice were intoxicated with 600 mg/kg of acetaminophen to establish a model to assess changes in body weight, liver weight, serum parameters and cytology (seven animals for each group); cell death was evaluated by means of terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay, and real-time polymerase chain reaction (PCR) was used to follow effects on gene expression. Nonintoxicated control mice were treated with normal saline by the same route and dose volume as acetaminophen-treated mice. We varied the number of hUCMSC given by intravenous injection (1 105, 5 105, 1 106) in our ALF model of survival (supplementary Figure 1). On the basis of these data, we selected cell dose of 5 105 cells for hUCMSC intravenous administration in 200 mL of NS. In pre-ALF and post-ALF groups, hUCMSCs were injected intravenously 30 min before or after acetaminophen intoxication, respectively. In the NS group, mice received NS instead of hUCMSCs. Serum parameter detection Animals in all groups were weighed 72 h after acetaminophen intoxication. Blood samples were collected through orbital bleeds (2 h, 4 h, 8 h, 24 h and 72 h) after acetaminophen intoxication; serum was isolated and stored at 20 C until analyzed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels with the use of an automated biochemical analyzer (Mindray, Shenzhen, China). Total serum bilirubin (TBIL), malondialdehyde (MDA), superoxide dismutase (SOD),
UCMSC therapy of acute liver failure glutathione peroxidase (GSH-PX), glutathione (GSH) and total anti-oxidation capacity (T-AOC) were measured at the 4-h time point with the use of commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Inflammatory factors and cytokines, including interleukin (IL)-1b, IL-6, tumor necrosis factor-alpha (TNF-a) and hepatocyte growth factor (HGF), were determined by means of enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Dakewe, Shenzhen, China). Histological and immunohistochemical examination At study termination, livers were removed and weighed. Approximately 0.05 g of each liver was snap-frozen in N2(l) and stored at 80 C before
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real-time PCR analysis. The remainder of each liver sample was fixed in 4% paraformaldehyde for 24 h before processing for histological, immunohistochemical and TUNEL analyses. Fixed liver samples were cut into small pieces (<3 mm thick, three from each liver), dehydrated, paraffin-embedded, and cut into 5-mm-thick sections. Sections were stained with hematoxylin and eosin for pathological assessment. Cell apoptosis was assessed by means of TUNEL staining, with the use of a Cell Death Detection Kit (Roche, Germany). Proliferating cell nuclear antigen (PCNA)-positive cells were detected through the use of a specific antibody (AVIVA System Biology, Beijing, China). To quantify pathological changes, random microscopic views of each slide section were captured and used to calculate an average number of positive cells per field.
Figure 1. Characterization of hUCMSCs. (A) Morphology of hUCMSCs at day 10 of culture (40). (B) Adipogenic differentiation of hUCMSCs stained by oil red O (400). (C) Osteogenic differentiation of hUCMSCs shown by calcium deposits revealed after alizarin red staining (100). (D) MSC marker profile of hUCMSCs. Immunophenotype of hUCMSCs is determined by flow cytometry at passage 2 with the use of labeled antibodies specific for the indicated human surface antigens.
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Quantitative real-time PCR Total RNA was prepared with the use of the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was treated by DNase I master mix and converted to complementary DNA by use of MMLV RT Mix (Invitrogen). Aliquots of complementary DNA were evaluated in a real-time PCR assay with the use of SYBR Premix Ex Tq II (Takara, Japan) in a Bio-Rad CFX96 Real-Time System under standard cycling conditions of 2 min at 95 C, followed by 40 cycles of PCR with 20 sec at 95 C, 30 sec at 56 C and 30 sec at 72 C. This procedure was used to determine the relative expression of three genes (HGF, BCL-2 and BAX) relative to GAPDH, by use of the DDCt method (30). Three replicates were performed for every experimental condition, with each sample assayed in duplicate for each amplicon. The PCR primers used included. HGF: 50 -AAA GAT TGG ATC AGG ACC ATG TGA G and 50 -GAT GGC ACA TCC ACG ACC AGG AAC A; BCL-2: 50 -GCA TCT GCA CAC CTG GAT CCA GGA T and 50 -GAA ATC AAA CAG AGG TCG CAT GCT G; BAX: 50 -ACC ATC ATG GGC TGG ACA CTG GAC T and 50 -CAC AAA GAT GGT CAC TGT CTG CCA T; and GAPDH: 50 -TGA AGG TCG GTG TGA ACG GAT TTG and 50 -CAT GTA GGC CAT GAG GTC CAC CAC.
positive cell numbers and gene expression. Animal survival was analyzed by means of the KaplanMeier log rank method. A value of P < 0.05 was considered to be statistically significant. Data were analyzed with the use of SPSS statistical software. Results Characterization of hUCMSCs After 6e10 days of culture, isolated hUCMSCs began to grow as fibroblast-like cells (Figure 1A). The immunophenotype of these culture-expanded cells at passage 2 was analyzed by flow cytometry for specific cell surface markers. As expected, these cells were positive for known MSC markers (CD105, CD73 and CD90) and were negative for hematopoietic markers (CD45, CD34, CD14, CD19 and HLA-DR). The lineage potential of hUCMSCs was verified by the induction of osteogenic and adipogenic differentiation. Intracytoplasmic lipid droplets stained with oil red O (Figure 1B) and calcium deposits stained with alizarin red (Figure 1C) were observed, demonstrating the potential of adipogenic and osteogenic differentiation of the isolated hUCMSCs, respectively. Two cell surface markers that have affinity for extracellular matrix elements, CD29 and CD44, were also expressed by isolated hUCMSCs (Figure 1D).
Statistical analysis Data are presented as mean standard deviation (SD). The Student’s t-test was used to compare serum parameters, apoptotic cell numbers, PCNA-
hUCMSCs improve survival of ALF mice We first investigated whether pre- and post-injection of hUCMSCs could improve the survival in ALF
Figure 2. Survival rate, body weight and RLW in ALF mice induced by acetaminophen. (A) Percentage survival of mice after intoxication with 750 mg/kg of acetaminophen. Survival rates were compared over a 72-h period (no further deaths occurred in any group after that time). Acetaminophen-induced ALF without hUCMSC treatment functioned as a positive control group (NS). Data are presented as mean SD (n ¼ 12, treatments compared with NS control group; *P < 0.05 compared with the non-transplantation control group). Body weight (B) and RLW (C) of mice in different groups 72 h after intoxication with 600 mg/kg of acetaminophen. Acetaminophen-induced ALF without hUCMSC treatment functioned as a positive control group (NS). Data are presented as mean SD (n ¼ 6, #P < 0.05, ##P < 0.01 compared with control group; *P < 0.05, **P < 0.01 compared with the non-transplantation group).
UCMSC therapy of acute liver failure mice induced by a high acetaminophen dose (Figure 2A). By 72 h after acetaminophen injection (750 mg/kg), the survival rate of mice that received hUCMSCs was significantly higher than that of the NS control group (P < 0.05). ALF mice in the prehUCMSCs group had a survival rate of w60% and w40% in the post-hUCMSCs group compared with the NS group, in which there were no survivors. Most of the deaths in all groups occurred within 4w16 h after acetaminophen injection. These data suggest that hUCMSC injection, and presumably implantation into the liver, either before or after intoxication, was able to improve the survival of ALF mice.
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hUCMSC protect against relative liver weight loss in ALF mice High-dose treatment of mice with acetaminophen resulted in a significant decrease in their total body weight (P < 0.01), which was not corrected by hUCMSC injection either before or after ALF induction (Figure 2B). Relative liver weight (RLW) loss was also observed to occur in ALF mice, presumably caused by a sudden mass loss of hepatocytes (Figure 2C). Importantly, both the pre- and post-transplantation groups showed a significantly higher RLW compared with the untreated NS group (P < 0.05) (Figure 2C). Together, these data suggest
Figure 3. Serum levels of certain biochemical markers are altered in mice induced to have ALF by acetaminophen intoxication. (A) Serum levels of AST (A: a,c) and ALT (A: b,d) over a time course of 72 h. (B) Total bilirubin levels in different groups at time point 24 h after acetaminophen injection. Data are presented as mean SD (n ¼ 7, ##P < 0.01 compared with control group; *P < 0.05, **P < 0.01 compared with NS group).
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Figure 4. Analysis of liver morphological and apoptotic events after acetaminophen intoxication. (A) Liver morphology from control treated mice (A-a) and mice 24 h after receiving 600 mg/kg of acetaminophen (A: b). (B) Representative morphology of livers fixed in 4% paraformaldehyde for 24 h from normal, NS, pre-hUCMSC and post-hUCMSC groups, respectively, at 24 h. (C) Hematoxylin and eosin staining of liver sections from normal, non-transplantation, pre-hUCMSC and post-hUCMSC groups at 24 h after NS or 600 mg/kg of acetaminophen treatment (50). (D) Hepatocyte apoptosis demonstrated in liver sections obtained from negative, positive and normal control mice and three acetaminophen-treated mice at time point 4 h. (E) Percentage of necrosis area in liver sections from each group. Images were collected at original magnification (100); mean values of 10 randomly selected areas per sample were used to determine the percentage area of necrosis; ImageJ software was used to quantify the necrosis areas. (F) Number of apoptotic hepatocytes per single microscopic view of liver sections from three acetaminophen-treated groups after 4 h. Images were collected at original magnification (200). Data are presented as mean SD (n ¼ 6, *P < 0.05, **P < 0.01 compared with NS group).
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Figure 5. Immunohistochemical analysis of PCNA-positive cells in ALF mice 24 h after intoxication with 600 mg/kg of acetaminophen. (A) Liver tissue sections from mice not receiving acetaminophen (negative control) or receiving hUCMSCs or control injections (NS) 24 h after acetaminophen intoxication; all photographed at 50 except where noted. (B) Quantification of PCNA-positive cells per single microscopic view of liver sections. Images were collected at magnification (50), and eight randomly selected views in each section and six sections per group were evaluated. Data are presented as mean SD (n ¼ 6, **P < 0.01 compared with non-transplantation group).
that hUCMSC injection might attenuate injury by reducing the extent of liver cell loss in ALF mice. hUCMSCs attenuate liver damage in ALF mice The extent of liver damage in mice induced by intoxication with 600 mg/kg acetaminophen was monitored by measuring serum levels of ALT, AST and TBIL over the 72-h time course of the study (Figure 3A,B). Compared with control mice, ALT and AST levels in acetaminophen-treated groups started to increase at 4 h and continued to increase when measured at 8 h and 24 h (P < 0.01). Compared with the NS group, hUCMSC injection either before or after acetaminophen treatment reduced the extent of ALT and AST release compared with the control (NS) group at 24 h (P < 0.01). By 72 h, all three acetaminophen-treated groups had serum ALT and AST levels that were
comparable to pre-ALF values (Figure 3A). We also observed that the level of TBIL was markedly increased in acetaminophen-treated mice at the 24-h time point; injection of hUCMSCs suppressed the acetaminophen injuryeinduced increase of TBIL (P < 0.05) (Figure 3B). Taken together, injection of hUCMSC, presumably resulting in their transplantation, could attenuate liver damage as reflected by suppression of ALT, AST and TBIL level increases observed in this ALF model. hUCMSCs ameliorate hepatocyte cell death in ALF mice Cytochromes P450s metabolize acetaminophen to a reactive metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which reacts with and depletes GSH. This process initiates mitochondrial oxidative stress that leads to a mixture of necrosis and apoptosis events resulting from the same mitochondrial cell death
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pathway. On the basis of the time course of apparent liver damage and protection by hUCMSC injection in this acetaminophen-induced model of ALF, we chose the 4-h and 24-h time points to study liver damage and possible mechanisms of cell transplantation in mice. Compared with untreated mice, acetaminophen-treated mice had altered liver morphology that included a rougher surface, duller red color, severe internal bleeding and extensive areas of necrosis (Figure 4A). Injection of hUCMSCs dramatically attenuated the extent of liver necrosis compared with the NS group (Figure 4B,C). Injection of hUCMSCs either before or after acetaminophen-induced liver injury showed reduced levels of apoptosis and a lessened alteration in histological changes compared with the NS group. With the use of TUNEL analysis to examine apoptosis in acetaminophen-induced ALF mice, we observed more apoptotic hepatocytes in the NS group compared with control mice (Figure 4D). The extent of necrotic changes in the livers of mice in the different treatment groups is shown in Figure 4E; injection of hUCMSCs reduced the extent of these necrotic changes. Quantification of TUNEL-positive cells demonstrated that both pre- and post-injection of hUCMSCs attenuated hepatocyte apoptosis compared with the NS group (P < 0.05) at time point 4 h (Figure 4F). Thus, apoptosis may also contribute to
the mass loss of hepatocytes in acetaminophen-induced ALF of mice; the injection of hUCMSCs significantly reduced the extent of this type of cell death. hUCMSCs promote liver regeneration Liver tissue is noted for its remarkable capacity to regenerate after injury. Interestingly, liver regeneration is significantly inhibited in ALF non-survivors compared with survivors (31). Thus, we were interested in examining whether injected hUCMSCs could enhance the extent of hepatocyte proliferation at 24 h and used an immunohistochemistry analysis to compare the extent of PCNA-positive cells in the livers of mice from the different treatment groups (Figure 5A). Interestingly, we observed more PCNA-positive cells at 24 h in both pre- and postinjection groups compared with control and NS animals (P < 0.05) (Figure 5B). These data suggest that hUCMSC treatment, particularly in the pre-injection group, may facilitate liver regeneration by combating the loss of hepatocytes induced by acetaminophen. hUCMSCs enhance the antioxidant system in ALF mice Acetaminophen-induced liver injury is associated with oxidative stress, especially with GSH consumption;
Figure 6. Serum antioxidant indexes and MDA level changes in ALF mice 4 h after intoxication with 600 mg/kg of acetaminophen. Serum levels of GSH (A), of SOD (B), of MDA (C), of GSH-PX (D) and of T-AOC (E) in different control and treatment groups. Data are presented as mean SD (n ¼ 7, #P < 0.05, ##P < 0.01 compared with control group; *P < 0.05, **P <0.01 compared with NS group).
UCMSC therapy of acute liver failure we tested the influences of hUCMSC injection on the antioxidant system of ALF mice (Figure 6AeE). Four hours after treatment of acetaminophen, GSH levels and SOD activity in the serum were dramatically reduced. The lipid peroxidation marker MDA, on the other hand, was markedly increased in the NS group. After hUCMSC injection, animals in both the prehUCMSC and post-hUCMSC groups had restored serum GSH levels compared with the NS group (P < 0.01). However, only pre-injection of hUCMSCs was able to recover serum SOD activity and downregulate serum MDA levels (P < 0.05). Serum T-AOC was increased 4 h after acetaminophen treatment, suggesting a systemic host defensive response to this form of liver injury. Serum T-AOC, however, was not different between the three acetaminophen-treated groups (Figure 6E). Additionally, we did not observe a statistical differences in GSH-PX activity, which is known to promote GSH regeneration (Figure 6D). Thus, the preventative role of hUCMSCs in ALF may partly be due to an ability to reduce detrimental peroxidation events. hUCMSCs suppress inflammatory cytokines and promote HGF expression As reported previously, ALF induces cytokine expression and systemic inflammatory responses. Thus, we examined the serum cytokine changes in ALF mice with the use of a combination of ELISA methods (Figure 7). Our results showed that both pre- and post-hUCMSC injection attenuated
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pro-inflammatory cytokine and HGF expression induced by acetaminophen intoxication. In general, injection of hUCMSCs before acetaminopheninduced ALF was more effective at correcting alterations in cytokine and HGF expression compared with injection of hUCMSCs after this mode of liver injury (Figure 7). For example, hUCMSC injection downregulated the expression of TNF-a at 4 h and 24 h after ALF induction (Figure 7, P < 0.01). At time point 24 h, only the pre-hUCMSC group showed the downregulated expression of cytokine IL-6 (P < 0.01). Interestingly, IL-1b was not altered by the injection of hUCMSCs. These findings suggest that injection of hUCMSCs may suppress inflammatory reactions by decreasing serum levels of the pro-inflammatory cytokines TNF-a and IL-6. At the same time, hUCMSC injection may enhance hepatocyte survival and proliferation through an increase in HGF expression. Molecular evidence for the effects of cell transplantation Anti-apoptosis and regeneration of hepatocytes is quite important for preventing the toxicity in acetaminophen-induced ALF mice. We used real-time PCR analysis to measure messenger (m)RNA expression of the HGF, Bcl-2 and Bax genes in an effort to examine potential mechanisms of action induced by hUCMSC injection. We found that both pre- and post- hUCMSC injections upregulated the mRNA expression of HGF and Bcl-2 at 4 h but downregulated the expression of Bax (P < 0.01,
Figure 7. Serum cytokine expression changes in ALF mice after 600 mg/kg 4 h and 24 h after acetaminophen intoxication. Serum levels of pro-inflammatory cytokines TNF-a (A), IL-1b (B), IL-6 (C) and HGF (D). Data are presented as mean SD (n ¼ 7, **P < 0.01 compared with non-injection group).
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Figure 8. Quantitative real-time PCR analysis of mRNA for genes expressed in liver tissues obtained from mice in different control and treatment groups. (A) Hepatocyte growth factor; (B) Bcl-2; and (C) Bax. (D) Relative mRNA expression levels of HGF, Bcl-2 and Bax genes. Data are presented as mean SD (n ¼ 4, **P < 0.01 compared with NS group).
Figure 8). Together, the data demonstrating the regulation of these genes provides a potential molecular basis for the action of hUCMSC injection in regeneration and anti-apoptosis events in the liver. Discussion Extensive studies have used chemicals such as CCl4, lipopolysaccharides and D-gal to induce ALF or liver injury in animal models. In the present study, we used acetaminophen to induce an ALF model in mice to study the preventative and therapeutic potential of hUCMSCs. This model is simple and rapid. More importantly, it mimics the clinical condition of acetaminophen overdose prevalent in the United States and other Western countries (2,3). In the present study, we have demonstrated that hUCMSCs can improve the survival rate of acetaminophen-induced ALF in mice. In addition to the therapeutic role previously reported (27), we have now examined the preventative capacity of these stem cells in our ALF model. Whether hUCMSCs are able to completely prevent the occurrence of acetaminophen-induced ALF has not been fully examined previously. In the present study, we found that all animals in the NS group died of acetaminophen-induced
intoxication. However, pre- and post-injection of hUCMSCs significantly improved survival rates up to 60% and 40%, respectively. Similarly, liver mass loss was also improved significantly spared in the stem celletreatment groups. In parallel, serum levels of ALT, AST and TBIL that correlate with liver damage were significantly decreased in the hUCMSC injection groups. As early as 4 h after the acetaminophen treatment, mice died in the NS group, whereas no deaths were observed in cell hUCMSC injection groups, indicating that hUCMSCs may have a rapid impact. Coincidently, ALT and AST levels in the pre-transplantation group were significantly decreased when compared with the NS group at this early time point. Our studies also suggest that the time window of cell transplantation is critical for rescuing ALF animals. Most animals that received a high dose of acetaminophen died within 4w16 h, but animals administered hUCMSC 30 min after acetaminophen intoxication were spared from this acute death outcome. Stem cell dose was another key issue to be considered for this AFL model (supplementary Figure 1). We tested three doses of hUCMSCs (1 105, 5 105, 1 106 cells) in our model and found that the majority of animals died of ALF in the 1 105 cells group. No differences in animal survival
UCMSC therapy of acute liver failure were noticed between the 5 105 and the 1 106 cell groups. For this reason, we choose the dose of 5 105 cells for our study. In addition, we also compared the effect of stem cells from different sources, including UCMSC, chorion MSC, and decidua MSC (supplementary Figure S2); they had varied effects in protecting ALF animals. This current report focuses on hUCMSCs not only for its economical culturing, but also in that they have not previously been tested in the ALF model. Liver cell death can occur through distinct biochemical pathways, including apoptosis, necrosis and autophagic cell death (32). It is assumed that apoptotic alterations induce profound energy depletion and mitochondrial failure, diverting cell death to necrosis as the principal mode of acetaminopheninduced liver toxicity (33). Our data in Figure 4 show that hepatocytes undergo extensive necrosis in ALF mice compared with the control group. Both preand post-injection of hUCMSCs significantly attenuated the necrosis condition compared with the NS group. With the use of TUNEL and real-time PCR assays, we also confirmed that apoptosis plays a role
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in acetaminophen-induced ALF mice, at least at the 4-h time point. Again, both pre- and post-injection of hUCMSCs attenuated this condition compared with the NS control group. Liver regeneration after hepatocyte loss is a fundamental parameter of liver response to injury. Our results in Figure 5 show that both pre- and postinjection enhanced the proliferation of hepatocytes compared with the NS group. Together with the improvement of RLW described in Figure 2, it appears that hUCMSCs can promote liver regeneration in ALF mice induced by acetaminophen. HGF, a paracrine cellular growth, motility and morphogenic factor synthesized by nonparenchymal cells of the liver after liver injury or partial hepatectomy, is crucial in promoting liver regeneration. Its high mitogenic potency leads to acceleration of liver function and protection of liver cells from injury (34,35). With the use of ELISA and real-time PCR, we found that HGF was upregulated in cell therapy groups compared with the NS group. These data suggest that hUCMSCs may promote liver regeneration in ALF mice partially through activation of the HGF pathway.
Figure 9. Model proposed to illustrate how hUCMSC injection rescues liver injury. Acetaminophen can be oxidized by cytochrome P450 and form NAPQI, which is then conjugated to GSH and eliminated in the bile. Sustained NAPQI formation eventually causes depletion of GSH, which in turn leads to formation of protein adducts as well as reactive nitrogen and oxygen species. These events augment the level of protein and lipid peroxidation in hepatocytes. Reactive oxygen species (ROS) instigates the mitochondrial pathway of apoptosis involving Bax and Bcl-2. Increased ROS also causes mitochondrial membrane permeability (MPT) and hepatocyte necrosis that elicits Küpffer cells to secrete pro-inflammatory molecules such as IL-6 and TNF-a, which in turn amplify the necrosis of hepatocytes. Apoptosis and necrosis of hepatocytes together cause the observed liver injury. In ALF mice, injection of hUCMSCs can increase the level of serum GSH, accelerate the elimination of NAPQI, and reduce ROS levels. SOD function is improved and more ROS is eliminated, reducing liver injury outcomes caused by apoptosis or necrosis. Bax expression is downregulated and Bcl-2 is upregulated, leading to the amelioration of apoptosis. This, in turn, reduces levels of pro-inflammatory molecules such as IL-6 and TNF-a, leading to less necrosis. The level of HGF is increased, with the outcome of enhancing liver regeneration.
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Acetaminophen is metabolized by the cytochrome P450 enzyme system located predominantly in perivenous hepatocytes (36). Acetaminophen can be oxidized by cytochrome P450 and form NAPQI, which is then conjugated to GSH and eliminated in the bile (Figure 9). Sustained NAPQI formation, however, eventually causes depletion of GSH, which leads to formation of protein adducts as well as reactive nitrogen and oxygen species that leads to increased protein and lipid peroxidation in liver (33). In a previous study, transplantation of hUCMSCs increased catalase activity in mice with CCl4-induced injury (28). In the present study, we found both preand post-transplantation of hUCMSCs increased serum levels GSH that were reduced by acetaminophen treatment. Pre-injection of hUCMSCs increased the activity of serum SOD and suppressed serum MDA levels. Acetaminophen-induced injury is closely related to oxidative stress and is especially linked to GSH consumption. Thus, improvement in antioxidative ability of hUCMSCs might be important for its anti-necrosis and anti-apoptosis properties. Previous studies found that the action of MSCs is primarily through their paracrine function rather than direct tissue replacement (27). MSC-derived molecules attenuated ALF in mice or rats by inhibition of apoptosis, stimulation of hepatocyte proliferation and minimization of the inflammatory response (37). The paracrine action of MSC was also corroborated by the amelioration of systemic inflammation induced by lipopolysaccharides or burns (38). The death of animals in our ALF model was probably directly related to severe liver necrosis induced by acetaminophen. Liver necrosis induces oxidative and inflammatory responses that can be monitored by a variety of serum components that can be detected in the systemic circulation. In contrast, HGF and hepatic regeneration requires intact stem cells to function in the liver. Thus, in this acute model, the direct anti-necrotic effect of paracrine factors delivered by hUCMSC may play a key role in animal survival through liver-specific actions. We found that hUCMSC injection suppressed the release of pro-inflammatory cytokines such as TNFa and IL-6. Thus, inhibition of pro-inflammatory cytokines and reduced liver cell necrosis or apoptosis resulting from hUCMSC transplantation may have been due to local paracrine events in the liver. In summary, this is the first evidence to demonstrate the preventative and therapeutic potential of transplanted hUCMSCs in treating mice with ALF induced by acetaminophen. Intravenous injection and presumably implantation of hUCMSCs attenuated liver injury by anti-apoptosis, anti-necrosis and regeneration-promoting properties. The protection from ALF-related death by injected hUCMSCs may
involve the participation of multiple mechanisms, including stabilization of the antioxidant system, anti-inflammatory actions and elevated HGF expression. This work suggests that intravenous injection of hUCMSCs could be a practical cell therapy to prevent or treat ALF. Acknowledgments This study was supported by Shenzhen International Collaboration grant (GJ200807210024A) to XH; California Institute for Regenerative Medicine (CIRM) grant (RT2-01942), National Institutes of Health grant (1R43 CA103553-01), Department of Defense grant (W81XWH-04-1-0597) to JFH. RJM acknowledges support from the National Institutes of Health (RO1 DK056754-11A1). Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References 1. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology. 2006;43(2 Suppl 1): S31e44. 2. Reuben A, Koch DG, Lee WM. Drug-induced acute liver failure: results of a U.S. multicenter, prospective study. Hepatology. 2010;52:2065e76. 3. Lee WM, Stravitz RT, Larson AM. Introduction to the revised American Association for the Study of Liver Diseases Position Paper on acute liver failure 2011. Hepatology. 2012;55: 965e7. 4. Fiegel HC, Lange C, Kneser U, Lambrecht W, Zander AR, Rogiers X, et al. Fetal and adult liver stem cells for liver regeneration and tissue engineering. J Cell Mol Med. 2006;10: 577e87. 5. Stutchfield BM, Forbes SJ, Wigmore SJ. Prospects for stem cell transplantation in the treatment of hepatic disease. Liver Transplant. 2010;16:827e36. 6. Enns GM, Millan MT. Cell-based therapies for metabolic liver disease. Mol Genet Metab. 2008;95:3e10. 7. Yuan S, Jiang T, Sun L, Zheng R, Ahat N, Zhang Y. The role of bone marrow mesenchymal stem cells in the treatment of acute liver failure. BioMed Res Int. 2013;2013:251846. 8. Buhring HJ, Treml S, Cerabona F, de Zwart P, Kanz L, Sobiesiak M. Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci. 2009; 1176:124e34. 9. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins K, et al. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec. 2001;264:51e62. 10. Wang X, Sha XJ, Li GH, Yang FS, Ji K, Wen LY, et al. Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol. 2012;57:1231e40. 11. Zagoura DS, Roubelakis MG, Bitsika V, Trohatou O, Pappa KI, Kapelouzou A, et al. Therapeutic potential of a
UCMSC therapy of acute liver failure
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure. Gut. 2012;61:894e906. Cao H, Yang J, Yu J, Pan Q, Li J, Zhou P, et al. Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure. BMC Med. 2012;10:56. Karaoz E, Okcu A, Unal ZS, Subasi C, Saglam O, Duruksu G. Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulin-producing cells in pancreatic islet microenvironment both in vitro and in vivo. Cytotherapy. 2013;15:557e70. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41e9. Bonanno G, Mariotti A, Procoli A, Corallo M, Rutella S, Pessina G, et al. Human cord blood CD133þ cells immunoselected by a clinical-grade apparatus differentiate in vitro into endothelial- and cardiomyocyte-like cells. Transfusion. 2007;47:280e9. Rowlands AS, George PA, Cooper-White JJ. Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation. Am J Physiol Cell Physiol. 2008;295:C1037e44. Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112:295e307. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, et al. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells. 2008;26:2705e12. de Freitas Souza BS, Nascimento RC, de Oliveira SA, Vasconcelos JF, Kaneto CM, de Carvalho LF, et al. Transplantation of bone marrow cells decreases tumor necrosis factor-alpha production and blood-brain barrier permeability and improves survival in a mouse model of acetaminophen-induced acute liver disease. Cytotherapy. 2012;14:1011e21. Peng L, Xie DY, Lin BL, Liu J, Zhu HP, Xie C, et al. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: shortterm and long-term outcomes. Hepatology. 2011;54:820e8. El-Ansary M, Abdel-Aziz I, Mogawer S, Abdel-Hamid S, Hammam O, Teaema S, et al. Phase II trial: undifferentiated versus differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis. Stem Cell Rev. 2012;8:972e81. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al. Mesenchymal stem cellderived molecules reverse fulminant hepatic failure. PloS One. 2007;2:e941. Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, et al. Immune properties of human umbilical cord Wharton’s jelly-derived cells. Stem Cells. 2008;26:2865e74.
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24. Cho PS, Messina DJ, Hirsh EL, Chi N, Goldman SN, Lo DP, et al. Immunogenicity of umbilical cord tissue derived cells. Blood. 2008;111:430e8. 25. Byeon YE, Ryu HH, Park SS, Koyama Y, Kikuchi M, Kim WH, et al. Paracrine effect of canine allogenic umbilical cord blood-derived mesenchymal stromal cells mixed with beta-tricalcium phosphate on bone regeneration in ectopic implantations. Cytotherapy. 2010;12:626e36. 26. Wang J, Zhou X, Cui L, Yan L, Liang J, Cheng X, et al. The significance of CD14þ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis. Cytotherapy. 2010; 12:1022e34. 27. Zhang S, Chen L, Liu T, Zhang B, Xiang D, Wang Z, et al. Human umbilical cord matrix stem cells efficiently rescue acute liver failure through paracrine effects rather than hepatic differentiation. Tissue Eng Part A. 2012;18:1352e64. 28. Burra P, Arcidiacono D, Bizzaro D, Chioato T, Di Liddo R, Banerjee A, et al. Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury. BMC Gastroenterol. 2012;12:88. 29. Chen M, Zhang H, Wu J, Xu L, Xu D, Sun J, et al. Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway. Biomaterials. 2012;33:5514e23. 30. Li T, Hu JF, Qiu X, Ling J, Chen H, Wang S, et al. CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop. Mol Cell Biol. 2008;28:6473e82. 31. Rutherford A, Chung RT. Acute liver failure: mechanisms of hepatocyte injury and regeneration. Semin Liver Dis. 2008; 28:167e74. 32. Bantel H, Schulze-Osthoff K. Mechanisms of cell death in acute liver failure. Frontiers Physiol. 2012;3:79. 33. Christ B, Bruckner S. Rodent animal models for surrogate analysis of cell therapy in acute liver failure. Frontiers Physiol. 2012;3:78. 34. Vargas GA, Hoeflich A, Jehle PM. Hepatocyte growth factor in renal failure: promise and reality. Kidney Int. 2000;57: 1426e36. 35. Kaido T, Seto S, Yamaoka S, Yoshikawa A, Imamura M. Perioperative continuous hepatocyte growth factor supply prevents postoperative liver failure in rats with liver cirrhosis. J Surg Res. 1998;74:173e8. 36. Gebhardt R, Hovhannisyan A. Organ patterning in the adult stage: the role of Wnt/beta-catenin signaling in liver zonation and beyond. Dev Dyn. 2010;239:45e55. 37. van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, et al. Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology. 2008;47:1634e43. 38. Yagi H, Soto-Gutierrez A, Kitagawa Y, Tilles AW, Tompkins RG, Yarmush ML. Bone marrow mesenchymal stromal cells attenuate organ injury induced by LPS and burn. Cell Transplant. 2010;19:823e30.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcyt.2014.05.018.